Abstract: BACKGROUND: Burkholderia pseudomallei is intrinsically resistant to aminoglycosides and macrolides, mostly due to AmrAB-OprA efflux pump expression. We investigated the molecular mechanisms of aminoglycoside susceptibility exhibited by Thai strains 708a, 2188a, and 3799a. METHODOLOGY/PRINCIPAL FINDINGS: qRT-PCR revealed absence of amrB transcripts in 708a and greatly reduced levels in 2188a and 3799a. Serial passage on increasing gentamicin concentrations yielded 2188a and 3799a mutants that became simultaneously resistant to other aminoglycosides and macrolides, whereas such mutants could not be obtained with 708a. Transcript analysis showed that the resistance of the 2188a and 3799a mutants was due to upregulation of amrAB-oprA expression by unknown mechanism(s). Use of a PCR walking strategy revealed that the amrAB-oprA operon was missing in 708a and that this loss was associated with deletion of more than 70 kb of genetic material. Rescue of the amrAB-oprB region from a 708a fosmid library and sequencing showed the presence of a large chromosome 1 deletion (131 kb and 141 kb compared to strains K96243 and 1710b, respectively). This deletion not only removed the amrAB-oprA operon, but also the entire gene clusters for malleobactin and cobalamin synthesis. Other genes deleted included the anaerobic arginine deiminase pathway, putative type 1 fimbriae and secreted chitinase. Whole genome sequencing and PCR analysis confirmed absence of these genes from 708a. Despite missing several putative virulence genes, 708a was fully virulent in a murine melioidosis model. CONCLUSIONS/SIGNIFICANCE: Strain 708a may be a natural candidate for genetic manipulation experiments that use Select Agent compliant antibiotics for selection and validates the use of laboratory-constructed Delta(amrAB-oprA) mutants in such experiments.
Abstract: Francisella tularensis is a fastidious Gram-negative bacterium responsible for the zoonotic disease tularemia. Investigation of the biology and molecular pathogenesis of F. tularensis has been limited by the difficulties in manipulating such a highly pathogenic organism and by a lack of genetic tools. However, recent advances have substantially improved the ability of researchers to genetically manipulate this organism. To expand the molecular toolbox we have developed two systems to stably integrate genetic elements in single-copy into the F. tularensis genome. The first system is based upon the ability of transposon Tn7 to insert in both a site- and orientation-specific manner at high frequency into the attTn7 site located downstream of the highly conserved glmS gene. The second system consists of a sacB-based suicide plasmid used for allelic exchange of unmarked elements with the blaB gene, encoding a beta-lactamase, resulting in the replacement of blaB with the element and the loss of ampicillin resistance. To test these new tools we used them to complement a novel d-glutamate auxotroph of F. tularensis LVS, created using an improved sacB-based allelic exchange plasmid. These new systems will be helpful for the genetic manipulation of F. tularensis in studies of tularemia biology, especially where the use of multi-copy plasmids or antibiotic markers may not be suitable.
Abstract: Transposon mini-Tn7 vectors insert into the chromosome of several Gram-negative bacteria in a site-specific manner. Here, we showed the application of mini-Tn7 as single copy site-specific integration vector system for Xanthomonas campestris pv. campestris. The transposition of the mini-Tn7 into the bacterial genome was detected at a Tn7 attachment (attTn7) site located downstream of glmS1. Furthermore, using a newly constructed vector pBBR1FLP2 containing the flipase (FLP) recombinase for site-specific excision of the sequence between the FLP recombinase target (FRT) sites, and a sacB counter selection marker, an unmarked mini-Tn7 insertion mutant was created. Mini-Tn7 insertion did not affect bacterial virulence on the tested plant. The mini-Tn7 and FLP-FRT systems also work well in Xanthomonas oryzae pv. oryzae.
Abstract: Burkholderia pseudomallei is the etiologic agent of melioidosis, a rare but serious tropical disease. In the United States, genetic research with this select agent bacterium is strictly regulated. Although several select agent compliant methods have been developed for allelic replacement, all of them suffer from some drawbacks, such as a need for specific host backgrounds or use of minimal media. Here we describe a versatile select agent compliant allele replacement system for B. pseudomallei based on a mobilizable vector, pEXKm5, which contains (i) a multiple cloning site within a lacZalpha gene for facile cloning of recombinant DNA fragments, (ii) a constitutively expressed gusA indicator gene for visual detection of merodiploid formation and resolution, and (iii) elements required for resolution of merodiploids using either I-SceI homing endonuclease-stimulated recombination or sacB-based counterselection. The homing endonuclease-based allele replacement system is completed by pBADSce, which contains an araC-P(BAD)-I-sceI expression cassette for arabinose-inducible I-SceI expression and a temperature-sensitive pRO1600 replicon for facile plasmid curing. Complementing these systems is the improved Deltaasd Escherichia coli mobilizer strain RHO3. This strain is susceptible to commonly used antibiotics and allows nutritional counterselection on rich media because of its diaminopimelic acid auxotrophy. The versatility of the I-SceI- and sacB-based methods afforded by pEXKm5 in conjunction with E. coli RHO3 was demonstrated by isolation of diverse deletion mutants in several clinical, environmental, and laboratory B. pseudomallei strains. Finally, sacB-based counterselection was employed to isolate a defined chromosomal fabD(Ts) allele that causes synthesis of a temperature-sensitive FabD, an essential fatty acid biosynthesis enzyme.
Abstract: Although Pseudomonas aeruginosa is an opportunistic pathogen that does not often naturally infect alternate hosts, such as plants, the plant-P. aeruginosa model has become a widely recognized system for identifying new virulence determinants and studying the pathogenesis of the organism. Here, we examine how both host factors and P. aeruginosa PAO1 gene expression are affected in planta after infiltration into incompatible and compatible cultivars of tobacco (Nicotiana tabacum L.). N. tabacum has a resistance gene (N) against tobacco mosaic virus, and although resistance to PAO1 infection is correlated with the presence of a dominant N gene, our data suggest that it is not a factor in resistance against PAO1. We did observe that the resistant tobacco cultivar had higher basal levels of salicylic acid and a stronger salicylic acid response upon infiltration of PAO1. Salicylic acid acts as a signal to activate defense responses in plants, limiting the spread of the pathogen and preventing access to nutrients. It has also been shown to have direct virulence-modulating effects on P. aeruginosa. We also examined host effects on the pathogen by analyzing global gene expression profiles of bacteria removed from the intracellular fluid of the two plant hosts. We discovered that the availability of micronutrients, particularly sulfate and phosphates, is important for in planta pathogenesis and that the amounts of these nutrients made available to the bacteria may in turn have an effect on virulence gene expression. Indeed, there are several reports suggesting that P. aeruginosa virulence is influenced in mammalian hosts by the availability of micronutrients, such as iron and nitrogen, and by levels of O(2).
Abstract: The gram-negative bacillus Burkholderia pseudomallei is a saprophyte and the cause of melioidosis. Natural infection is most commonly reported in northeast Thailand and northern Australia but also occurs in other parts of Asia, South America, and the Caribbean. Melioidosis develops after bacterial inoculation or inhalation, often in relation to occupational exposure in areas where the disease is endemic. Clinical infection has a peak incidence between the fourth and fifth decades; with diabetes mellitus, excess alcohol consumption, chronic renal failure, and chronic lung disease acting as independent risk factors. Most affected adults ( approximately 80%) in northeast Thailand, northern Australia, and Malaysia have >/=1 underlying diseases. Symptoms of melioidosis may be exhibited many years after exposure, commonly in association with an alteration in immune status. Manifestations of disease are extremely broad ranging and form a spectrum from rapidly life-threatening sepsis to chronic low-grade infection. A common clinical picture is that of sepsis associated with bacterial dissemination to distant sites, frequently causing concomitant pneumonia and liver and splenic abscesses. Infection may also occur in bone, joints, skin, soft tissue, or the prostate. The clinical symptoms of melioidosis mimic those of many other diseases; thus, differentiating between melioidosis and other acute and chronic bacterial infections, including tuberculosis, is often impossible. Confirmation of the diagnosis relies on good practices for specimen collection, laboratory culture, and isolation of B. pseudomallei. The overall mortality rate of infected persons is 50% in northeast Thailand (35% in children) and 19% in Australia.
Abstract: Genetic tools are required to take full advantage of the wealth of information generated by genome sequencing efforts and ensuing global gene and protein expression analyses. Bacterial genetics was originally developed and refined in Escherichia coli. As a consequence, elegant plasmid, cloning, expression, and mutagenesis systems were developed over the years and a good number of them are commercially available. This is not true for other bacteria. Although the development of genetic tools has generally not kept up with the sequencing pace, substantial progress has been made in this arena with many bacterial species. This short review highlights selected topics and achievements in the field over the past 25 years and presents some strategies that may help address future challenges. BioTechniques has played an integral part in the publication of important technological advances in the field over the first 25 years of its existence and it can be anticipated that it will continue to do so in the future.
Abstract: The contribution of the MexXY multidrug efflux system to aminoglycoside resistance was investigated in 18 clinical isolates of Pseudomonas aeruginosa obtained from dairy cows with Pseudomonas mastitis. All of the isolates expressed MexXY as determined by reverse transcription-PCR. The loss of mexXY resulted in increased susceptibility (two- to 16-fold decline in MIC) to aminoglycosides, confirming the contribution of this system in aminoglycoside resistance in these strains. As the impact of DeltamexXY varied, overexpression of MexXY alone is not sufficient for aminoglycoside resistance. Expression of mexXY also varied and did not strictly correlate with aminoglycoside insusceptibility. Transcription levels of mexY were independent on mutations in mexZ, suggesting the existence of additional regulatory mechanisms other than mexZ.
Abstract: MexXY is an active efflux system that contributes to intrinsic resistance to aminoglycosides in Pseudomonas aeruginosa. MexXY can function in combination with OprM in aminoglycoside efflux but may also functionally associate with another as yet unidentified outer membrane channel. The possible role of OpmG as a third component of MexXY in aminoglycoside efflux was investigated by construction of unmarked opmG mutants. Loss of OpmG did not have any impact on minimum inhibitory concentrations for aminoglycosides regardless of the presence of oprM, indicating that MexXY does not interact with OpmG in aminoglycoside efflux. In a clinical isolate PAJ010, (mexXY) enhanced streptomycin susceptibility but neither oprM nor opmG could, suggesting that MexXY functionally associates with an unidentified outer membrane protein for aminoglycoside efflux. Expression of an opmG-lacZ transcriptional fusion revealed that OpmG expression was neither constitutive nor inducible by gentamicin. Growth rates of wildtype P. aeruginosa and opmG mutant derivatives were not different, indicating that expression of opmG is not essential for P. aeruginosa growth.
Abstract: Because of Burkholderia pseudomallei's classification as a select agent in the United States, genetic manipulation of this bacterium is strictly regulated. Only a few antibiotic selection markers, including gentamicin, kanamycin, and zeocin, are currently approved for use with this bacterium, but wild-type strains are highly resistant to these antibiotics. To facilitate routine genetic manipulations of wild-type strains, several new tools were developed. A temperature-sensitive pRO1600 broad-host-range replicon was isolated and used to construct curable plasmids where the Flp and Cre recombinase genes are expressed from the rhamnose-regulated Escherichia coli P(BAD) promoter and kanamycin (nptI) and zeocin (ble) selection markers from the constitutive Burkholderia thailandensis ribosomal P(S12) or synthetic bacterial P(EM7) promoter. Flp and Cre site-specific recombination systems allow in vivo excision and recycling of nptII and ble selection markers contained on FRT or loxP cassettes. Finally, expression of Tn7 site-specific transposase from the constitutive P1 integron promoter allowed development of an efficient site-specific chromosomal integration system for B. pseudomallei. In conjunction with a natural transformation method, the utility of these new tools was demonstrated by isolating an unmarked delta(amrRAB-oprA) efflux pump mutant. Exploiting natural transformation, chromosomal DNA fragments carrying this mutation marked with zeocin resistance were transferred between the genomes of two different B. pseudomallei strains. Lastly, the deletion mutation was complemented by a chromosomally integrated mini-Tn7 element carrying the amrAB-oprA operon. The new tools allow routine select-agent-compliant genetic manipulations of B. pseudomallei and other Burkholderia species.
Abstract: We report the construction of a series of Escherichia-Pseudomonas broad-host-range expression vectors utilizing the P(BAD) promoter and the araC regulator for routine cloning, conditional expression, and analysis of tightly controlled and/or toxic genes in pseudomonads.
Abstract: To facilitate the discovery of new therapeutics for Burkholderia pseudomallei infections, we have developed cellular reporter screens for inhibitors of B. pseudomallei targets in the surrogate host Pseudomonas aeruginosa. Pseudomonas aeruginosa strains carrying deletions of essential genes were engineered to be dependent on the isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated expression of their B. pseudomallei orthologues on a broad-host-range plasmid. Pseudomonas aeruginosa genes which are upregulated in response to depletion of each target gene product, were fused to the Photorhabdus luminescens luxCDABE operon via pGSV3-lux-Sp(R) to generate reporter strains with increased bioluminescence upon target inhibition. A total of 11 of 19 B. pseudomallei genes complemented deletions of their orthologues in P. aeruginosa. The dependence of growth on IPTG levels varied from complete dependence (ftsQ, gyrA, glmU, secA) to slower growth in the absence of IPTG (coaD, efp, mesJ), to apparently normal growth in the absence of IPTG (ligA, lpxA, folA, ipk). Reporter screening strains have been constructed for three gene targets (gyrA, glmU, secA), and one (gyrA) has been applied to 68,000 compounds resulting in a primary hit rate of 0.5% and a confirmed hit rate of 0.06%, including several fluoroquinolones. These results provide proof of principle for surrogate cellular reporter screens as a useful approach to identify inhibitors of essential gene products.
Abstract: Burkholderia pseudomallei is the aetiological agent of melioidosis. Therapy for this disease is lengthy and limited to only a few antibiotics because of this bacterium's intrinsic antibiotic resistance to many clinically useful antibiotics. These properties of B. pseudomallei may partially be due to expression of efflux pumps of the resistance-nodulation-cell-division (RND) family. The patterns and magnitude of RND efflux pump expression in commonly used strains and clinical isolates of B. pseudomallei from the Royal Darwin Hospital, Darwin, Australia, were assessed in cells grown to late exponential phase using quantitative real-time PCR (qRT-PCR). Expression of the three previously identified RND efflux pumps AmrAB-OprA, BpeAB-OprB and BpeEF-OprC, as well as four other yet uncharacterized pumps, was found to be widespread in the clinical isolates. In 45 of 50 isolates (90%), mRNA was detected for at least one of the seven RND pumps. Of these 45 isolates, 41 (82%) expressed multiple pumps with nine strains expressing all seven pumps tested. While these studies revealed no striking correlation between RND efflux pump expression and clinically significant antibiotic resistance, the data support the notion that RND pumps probably play important roles in this bacterium's physiology, defence against toxic compounds, and perhaps virulence.
Abstract: Genetic research into the select agents Burkholderia pseudomallei and B. mallei is currently hampered by a paucity of approved antimicrobial drug-selection markers. The strict regulations imposed on researchers in the United States but not in other parts of the world lead to discrepancies in practice, hinder distribution of genetically modified strains, and impede progress in the field. Deliberation and decisions regarding alternative selection markers (antimicrobial and nonantimicrobial drugs) by the international community, regulatory authorities, and funding agencies are needed.
Abstract: Burkholderia psedudomallei is the etiologic agent of melioidosis, and the bacterium is listed as a potential agent of bioterrorism because of its low infectious dose, multiple infectious routes, and intrinsic antibiotic resistance. To further accelerate research with this understudied bacterium, we developed a Himar1-based random mutagenesis system for B. pseudomallei (HimarBP). The transposons contain a Flp recombinase-excisable, approved kanamycin resistance selection marker and an R6K origin of replication for transposon rescue. In vivo mutagenesis of virulent B. pseudomallei strain 1026b was highly efficient, with up to 44% of cells transformed with the delivery plasmid harboring chromosomal HimarBP insertions. Southern analyses revealed single insertions with no evidence of delivery plasmid maintenance. Sequence analysis of rescued HimarBP insertions revealed random insertions on both chromosomes within open reading frames and intergenic regions and that the orientation of insertions was largely unbiased. Auxotrophic mutants were obtained at a frequency of 0.72%, and nutritional supplementation experiments supported the functional assignment of genes within the respective biosynthetic pathways. HimarBP insertions were stable in the absence of selection and could be readily transferred between naturally transformable strains. Experiments with B. thailandensis suggest that the newly developed HimarBP transposons can also be used for random mutagenesis of other Burkholderia spp., especially the closely related species B. mallei. Our results demonstrate that comprehensive transposon libraries of B. pseudomallei can be generated, providing additional tools for the study of the biology, pathogenesis, and antibiotic resistance of this pathogen.
Abstract: Protection from NO gas, a toxic byproduct of anaerobic respiration in Pseudomonas aeruginosa, is mediated by nitric oxide (NO) reductase (NOR), the norCB gene product. Nevertheless, a norCB mutant that accumulated approximately 13.6 microM NO paradoxically survived anaerobic growth. Transcription of genes encoding nitrate and nitrite reductases, the enzymes responsible for NO production, was reduced >50- and 2.5-fold in the norCB mutant. This was due, in part, to a predicted compromise of the [4Fe-4S](2+) cluster in the anaerobic regulator ANR by physiological NO levels, resulting in an inability to bind to its cognate promoter DNA sequences. Remarkably, two O(2)-dependent dioxygenases, homogentisate-1,2-dioxygenase (HmgA) and 4-hydroxyphenylpyruvate dioxygenase (Hpd), were derepressed in the norCB mutant. Electron paramagnetic resonance studies showed that HmgA and Hpd bound NO avidly, and helped protect the norCB mutant in anaerobic biofilms. These data suggest that protection of a P. aeruginosa norCB mutant against anaerobic NO toxicity occurs by both control of NO supply and reassignment of metabolic enzymes to the task of NO sequestration.
Abstract: A homogeneous, sensitive, cellular bioluminescent high-throughput screen was developed for inhibitors of gyrase and other DNA-damaging agents in Pseudomonas aeruginosa. The screen is based on a Photorhabdus luminescens luciferase operon transcriptional fusion to a promoter that responds to DNA damage caused by reduced gyrase levels and fluoroquinolone inhibition. This reporter strain is sensitive to levels of ciprofloxacin as low as one-fourth minimum inhibitory concentration (MIC) with Z' scores greater than 0.5, indicating the assay is suitable for high-throughput screening. This screen combines the benefits of a whole-cell assay with a sensitivity and target specificity superior to those of traditional cell-based screens for inhibitors of viability or growth. In duplicate pilot screens of 2000 known bioactive compounds, 13 compounds generated reproducible signals >50% of that of the control (ciprofloxacin at one-half MIC) using bioluminescence readings after 7 h of incubation. Ten are fluoroquinolones known to cause accumulation of cleaved DNA-enzyme complexes in bacterial cells; the other 3 are known to create DNA adducts. Therefore, all 13 hits inhibit DNA synthesis but by a variety of different DNA-damaging mechanisms. This convenient, inexpensive screen will be useful for rapidly identifying DNA gyrase inhibitors and other DNA-damaging agents, which may lead to potent new antibacterials.
Abstract: Pseudomonas aeruginosa achieves high-level (MIC>1 mg/ml) triclosan resistance either by constitutive expression of MexAB-OprM, an efflux pump of the resistance nodulation cell division (RND) family, or expression of MexCD-OprJ, MexEF-OprN, and MexJK-OpmH in regulatory mutants. A triclosan-resistant target enzyme and perhaps other mechanisms probably act synergistically with efflux. To probe this notion, we exposed the susceptible Delta(mexAB-oprM) Delta(mexCD-oprJ) Delta(mexEF-oprN) Delta(mexJK) Delta(mexXY) strain PAO509 to increasing triclosan concentrations and derived a resistant strain, PAO509.5. This mutant overexpressed the PA0156-PA0157-PA0158 pump, which only effluxed triclosan, but not closely related compounds, antibiotics, and divalent cations, and was therefore renamed TriABC. Constitutive expression of the triABC operon was due to a single promoter-up mutation. Deletion of two adjacent genes, pcaR and PA0159, encoding transcriptional regulators had no effect on expression of this operon. TriABC is the only P. aeruginosa RND pump which contains two membrane fusion proteins, TriA and TriB, and both are required for efflux pump function. Probably owing to tight transcriptional coupling of the triABC genes, complementation of individual mutations was only partially achievable. Full complementation was only observed when a complete triABC operon was provided in trans, either in single or multiple copies. TriABC associated with OpmH, but not OprM, for assembly of a functional triclosan efflux pump. TriABC is the fifth RND pump in P. aeruginosa shown to efficiently efflux triclosan, supporting the notion that efflux is the primary mechanism responsible for this bacterium's high intrinsic and acquired triclosan resistance.
Abstract: The acyl-homoserine lactone molecular species (AHLs) produced by the Yersinia pestis AHL synthase YspI were identified by biochemical and physical/chemical techniques. Bioassays of extracts from culture supernatants of the recombinant YspI and wild-type Yersinia pestis showed similar profiles of AHLs. Analysis by liquid chromatography-mass spectrometry revealed that the predominant AHLs were N-3-oxooctanoyl-L-homoserine lactone and N-3-oxo-hexanoyl-L-homoserine lactone.
Abstract: A rapid microcentrifuge-based method is described for preparation of Pseudomonas aeruginosa electrocompetent cells with up to 10,000-fold increased transformation efficiencies over existing procedures. This increased efficiency now enables the use of transformation for all applications requiring DNA transfer. These include transfer of chromosomal mutations marked with antibiotic resistance genes between P. aeruginosa strains, which solves the riddle of not having an efficient and reliable transduction procedure for this bacterium. Not surprisingly, the method also allows for very efficient transformation with replicative plasmids, with transformation efficiencies ranging from 10(7) to >10(11) transformants per microgram of DNA. Lastly, with efficiencies of up to >10(3) transformants per microgram of DNA the method replaces in most instances conjugation for the transfer of non-replicative plasmids used in gene replacement, site-specific gene integration and transposon mutagenesis experiments.
Abstract: The double bond in anaerobic unsaturated fatty acid (UFA) biosynthesis is introduced by the FabA dehydratase/isomerase of the bacterial type II fatty acid biosynthetic pathway. A DeltafabA mutant of Pseudomonas aeruginosa grew aerobically, but required a UFA supplement for anaerobic growth. Wild-type cells produced 18:1Delta11 as the principal UFA, whereas the DeltafabA strain produced only 16:1Delta9. The double bond in the 16:1Delta9 was introduced after phospholipid formation and was localized in the sn-2 position. Two predicted membrane proteins, DesA and DesB, possessed the conserved histidine clusters characteristic of fatty acid desaturases. The DeltafabADeltadesA double mutant required exogenous fatty acids for growth but the DeltafabAdesB double mutant did not. Exogenous stearate was converted to 18:1Delta9 and supported the growth of DeltafabADeltadesA double mutant. A DeltafabADeltadesAdesB triple mutant was unable to desaturate exogenous stearate and was an UFA auxotroph. We detected a 2.5-fold increase in desA expression in DeltafabA mutants, whereas desB expression was derepressed by the deletion of the gene encoding a transcriptional repressor DesT. These data add two aerobic desaturases to the enzymes used for fatty acid metabolism in proteobacteria: DesA, a 2-position phospholipid Delta9-desaturase that supplements the anaerobic FabA pathway, and DesB, an inducible acyl-CoA Delta9-desaturase whose expression is repressed by DesT.
Abstract: The mini-Tn7 vectors are universally applicable in gram-negative bacteria and thereby facilitate the manipulation of many organisms for which few genetic systems are available. These vectors, when provided with only the Tn7 site-specific transposition machinery, insert site and orientation specifically in the bacterial chromosome at an attTn7 site downstream of the essential glmS gene. A few bacteria, including Burkholderia spp., contain multiple glmS genes and therefore several attTn7 sites. Here we provide a protocol for application of the mini-Tn7 system in B. mallei as an example of bacteria with multiple glmS sites. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into B. mallei by conjugation, followed by selection of insertion-containing strains; and last, PCR verification of mini-Tn7 insertions. B. mallei possesses two glmS genes on chromosome 1 and Tn7 transposes to both sites, although transposition to attTn7-1 associated with glmS1 occurs in more than 90% of the clones examined. Transposition is efficient and the whole procedure from start to verification of insertion events can be done in less than 5 d. This first chromosome integration system in B. mallei provides an important contribution to the genetic tools emerging for Burkholderia spp. Vectors are available for gene complementation and expression, and gene fusion analyses.
Abstract: Construction and integration of recombinant mini-Tn7 expression vectors into the chromosome of a surrogate, efflux-sensitized, and biosafe Pseudomonas aeruginosa host was validated as a generally applicable method for studies of uncharacterized bacterial efflux pumps. Using this method, the Burkholderia pseudomallei bpeEF-oprC operon was shown to encode a chloramphenicol and trimethoprim efflux pump.
Abstract: We previously constructed a series of mini-Tn7 chromosome integration vectors that, when provided only with the site-specific transposition machinery, generally transpose to a naturally evolved, neutral attTn7 site that is located 25-bp downstream of the glmS gene. Here we provide a protocol for application of the mini-Tn7 system in Proteus mirabilis as an example of a bacterium with a secondary attTn7 site that is not linked to glmS but, in this case, located in the carAB operon. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into P. mirabilis by transformation, followed by selection of insertion-containing strains; third, PCR verification of mini-Tn7 insertions; and last, optional Flp-mediated excision of the antibiotic-resistance selection marker present on the chromosomally integrated mini-Tn7 element. When transposon-containing cells are selected on rich medium, insertions occur at both attTn7 sites with equal efficiency and frequency. Because carA mutants are arginine and pyrimidine auxotrophs, single-site insertions at the glmS attTn7 sites can be obtained by selection on minimal medium. From start to verification of the insertion events, the whole procedure takes 5 d. This chromosome integration system in P. mirabilis provides an important tool for animal and biofilm studies based on this bacterium. Vectors are available for gene complementation and expression, gene fusion analyses and tagging with a green fluorescent protein (GFP)-encoding reporter gene.
Abstract: Broad host-range mini-Tn7 vectors facilitate integration of single-copy genes into bacterial chromosomes at a neutral, naturally evolved site. Here we present a protocol for employing the mini-Tn7 system in bacteria with single attTn7 sites, using the example Pseudomonas aeruginosa. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into P. aeruginosa by either transformation or conjugation, followed by selection of insertion-containing strains; third, PCR verification of mini-Tn7 insertions; and last, optional Flp-mediated excision of the antibiotic-resistance selection marker present on the chromosomally integrated mini-Tn7 element. From start to verification of the insertion events, the procedure takes as little as 4 d and is very efficient, yielding several thousand transformants per microgram of input DNA or conjugation mixture. In contrast to existing chromosome integration systems, which are mostly based on species-specific phage or more-or-less randomly integrating transposons, the mini-Tn7 system is characterized by its ready adaptability to various bacterial hosts, its site specificity and its efficiency. Vectors have been developed for gene complementation, construction of gene fusions, regulated gene expression and reporter gene tagging.
Abstract: The Pseudomonas aeruginosa mexJK efflux operon is constitutively expressed in mutants with defects in the upstream mexL gene, which encodes a repressor of the TetR family. MexL and a MexL(A47D) mutant protein were purified from Escherichia coli as fusion proteins with carboxy-terminal hexahistidine tags. Native polyacrylamide gel electrophoresis and size exclusion chromatography revealed that MexL is a tetramer in solution. MexL and MexL(A47D) oligomerization was confirmed using a genetic approach, and the MexL(A47D) mutant protein was not impaired in multimerization. Gel mobility shift and footprinting assays demonstrated that MexL, but not MexL(A47D), binds specifically to the 94-bp mexL-mexJ intergenic region to sequences located between positions -84 and -20 from the mexJ initiation codon. MexL protected about 60 nucleotides on each strand, and the protected regions overlapped almost perfectly, a finding consistent with MexL regulating the expression of both mexL and mexJK, which was ascertained by gene fusion analyses. The protected region contains predicted -10 and -35 promoter sequences for both mexL and mexJ, with partially overlapping -10 regions. The mexL promoter assignment was verified by mapping the mexL transcription start site, and the mexJ promoter was localized to the predicted regions using lacZ fusions. The MexL-protected region contains two inverted GTATTT repeats, and their location in the protected region and overlap with the mexL and mexJ promoter sequences strongly support a role in MexL binding.
Abstract: MexJK requires OprM for erythromycin efflux but not for triclosan efflux. Deletion of 15 OprM family outer membrane proteins (OMPs) revealed that only the TolC homolog OpmH functions with MexJK for triclosan efflux. This is the first report of natural utilization of multiple OMPs by a given resistance nodulation cell division transporter/membrane fusion protein pair.
Abstract: Several bacteria that are pathogenic to animals also infect plants. Mechanistic studies have proven that some human/animal pathogenic bacteria employ a similar subset of virulence determinants to elicit disease in animals, invertebrates and plants. Therefore, the results of plant infection studies are relevant to animal pathogenesis. This discovery has resulted in the development of convenient, cost-effective, and reliable plant infection models to study the molecular basis of infection by animal pathogens. Plant infection models provide a number of advantages in the study of animal pathogenesis. Using a plant model, mutations in animal pathogenic bacteria can easily be screened for putative virulence factors, a process which if done using existing animal infection models would be time-consuming and tedious. High-throughput screening of plants also provides the potential for unravelling the mechanisms by which plants resist animal pathogenic bacteria, and provides a means to discover novel therapeutic agents such as antibiotics and anti-infective compounds. In this review, we describe the developing technique of using plants as a model system to study Pseudomonas aeruginosa, Enterococcus faecalis and Staphylococcus aureus pathogenesis, and discuss ways to use this new technology against disease warfare and other types of bioterrorism.
Abstract: Antibiotic resistance of bacterial pathogens is a fast emerging global crisis and an understanding of the underlying resistance mechanisms is paramount for design and development of new therapeutic strategies. Permeability barriers for and active efflux of drug molecules are two resistance mechanisms that have been implicated in various infectious outbreaks of antibiotic-resistant pathogens, suggesting that these mechanisms may be good targets for new drugs. The synergism of reduced uptake and efflux is most evident in the multiplicative action of the outer membrane permeability barrier and active efflux, which results in high-level intrinsic and/or acquired resistance in many clinically important Gram-negative bacteria. This review summarizes the current knowledge of these two important resistance mechanisms and potential strategies to overcome them. Recent advances in understanding the physical structures, function and regulation of efflux systems will facilitate exploitation of pumps as new drug targets.
Abstract: Salicylic acid (SA) is a phenolic metabolite produced by plants and is known to play an important role in several physiological processes, such as the induction of plant defense responses against pathogen attack. Here, using the Arabidopsis thaliana-Pseudomonas aeruginosa pathosystem, we provide evidence that SA acts directly on the pathogen, down regulating fitness and virulence factor production of the bacteria. Pseudomonas aeruginosa PA14 showed reduced attachment and biofilm formation on the roots of the Arabidopsis mutants lox2 and cpr5-2, which produce elevated amounts of SA, as well as on wild-type Arabidopsis plants primed with exogenous SA, a treatment known to enhance endogenous SA concentration. Salicylic acid at a concentration that did not inhibit PA14 growth was sufficient to significantly affect the ability of the bacteria to attach and form biofilm communities on abiotic surfaces. Furthermore, SA down regulated three known virulence factors of PA14: pyocyanin, protease, and elastase. Interestingly, P. aeruginosa produced more pyocyanin when infiltrated into leaves of the Arabidopsis transgenic line NahG, which accumulates less SA than wild-type plants. This finding suggests that endogenous SA plays a role in down regulating the synthesis and secretion of pyocyanin in vivo. To further test if SA directly affects the virulence of P. aeruginosa, we used the Caenorhabditis elegans-P. aeruginosa infection model. The addition of SA to P. aeruginosa lawns significantly diminished the bacterium's ability to kill the worms, without affecting the accumulation of bacteria inside the nematodes' guts, suggesting that SA negatively affects factors that influence the virulence of P. aeruginosa. We employed microarray technology to identify SA target genes. These analyses showed that SA treatment affected expression of 331 genes. It selectively repressed transcription of exoproteins and other virulence factors, while it had no effect on expression of housekeeping genes. Our results indicate that in addition to its role as a signal molecule in plant defense responses, SA works as an anti-infective compound by affecting the physiology of P. aeruginosa and ultimately attenuating its virulence.
Abstract: BACKGROUND: Traditional gene replacement procedures are still time-consuming. They usually necessitate cloning of the gene to be mutated, insertional inactivation of the gene with an antibiotic resistance cassette and exchange of the plasmid-borne mutant allele with the bacterial chromosome. PCR and recombinational technologies can be exploited to substantially accelerate virtually all steps involved in the gene replacement process. RESULTS: We describe a method for rapid generation of unmarked P. aeruginosa deletion mutants. Three partially overlapping DNA fragments are amplified and then spliced together in vitro by overlap extension PCR. The resulting DNA fragment is cloned in vitro into the Gateway vector pDONR221 and then recombined into the Gateway-compatible gene replacement vector pEX18ApGW. The plasmid-borne deletions are next transferred to the P. aeruginosa chromosome by homologous recombination. Unmarked deletion mutants are finally obtained by Flp-mediated excision of the antibiotic resistance marker. The method was applied to deletion of 25 P. aeruginosa genes encoding transcriptional regulators of the GntR family. CONCLUSION: While maintaining the key features of traditional gene replacement procedures, for example, suicide delivery vectors, antibiotic resistance selection and sucrose counterselection, the method described here is considerably faster due to streamlining of some of the key steps involved in the process, especially plasmid-borne mutant allele construction and its transfer into the target host. With appropriate modifications, the method should be applicable to other bacteria.
Abstract: Pseudomonas aeruginosa biofilms are extremely recalcitrant to antibiotic treatment. Treatment of cystic fibrosis patients with azithromycin (AZM) has shown promise. We used DNA microarrays to identify differentially expressed transcripts in developing P. aeruginosa biofilms exposed to 2 mug/ml AZM. We report that transcripts for multiple restriction-nodulation-cell division (RND) efflux pumps, known to be involved in planktonic antibiotic resistance, and transcripts involved in type III secretion were upregulated in the resistant biofilms that developed in the presence of AZM. Interestingly, the MexAB-OprM and MexCD-OprJ efflux pumps, but not type III secretion, appear to be integral to biofilm formation in the presence of AZM, as evidenced by the fact that a mutant deleted in both mexAB-oprM and mexCD-oprJ was unable to form a biofilm in the presence of AZM. A mutant deleted in type III secretion was still able to form biofilms in the presence of drug. Furthermore, single mexAB-oprM- and mexCD-oprJ-null mutants were able to form a biofilm in the presence of drug, indicating that either of the pumps can confer resistance to AZM during biofilm development. In contrast to planktonically grown cells, where no mexC expression was detectable regardless of the presence of AZM, biofilms exhibited induction of mexC expression from the outset of their formation, but only in the presence of AZM. mexA, which is constitutively expressed in planktonic cells, was uniformly expressed in biofilms regardless of the presence of AZM. These data indicate that the MexCD-OprJ pump acts as a biofilm-specific mechanism for AZM resistance.
Abstract: For many bacteria, cloning and expression systems are either scarce or nonexistent. We constructed several mini-Tn7 vectors and evaluated their potential as broad-range cloning and expression systems. In bacteria with a single chromosome, including Pseudomonas aeruginosa, Pseudomonas putida and Yersinia pestis, and in the presence of a helper plasmid encoding the site-specific transposition pathway, site- and orientation-specific Tn7 insertions occurred at a single attTn7 site downstream of the glmS gene. Burkholderia thailandensis contains two chromosomes, each containing a glmS gene and an attTn7 site. The Tn7 system allows engineering of diverse genetic traits into bacteria, as demonstrated by complementing a biofilm-growth defect of P. aeruginosa, establishing expression systems in P. aeruginosa and P. putida, and 'GFP-tagging' Y. pestis. This system will thus have widespread biomedical and environmental applications, especially in environments where plasmids and antibiotic selection are not feasible, namely in plant and animal models or biofilms.
Abstract: In Gram-negative bacteria, intercellular communication and virulence regulation is mediated by the diffusible chemical signal acyl-homoserine-L-lactone (AHL). The AHL synthase enzymes produce a variety of AHLs from the substrates S-adenosyl-L-methionine and acyl-acyl carrier protein. LasI, the AHL synthase from Pseudomonas aeruginosa, has low solubility and has failed to crystallize despite extensive crystallization trials. Based on the previously determined structure of the AHL synthase EsaI, active soluble LasI was produced by re-engineering residues in a tight turn to produce a type I' beta-turn. The resulting protein is active, more stable than the wild-type LasI and has been crystallized in the cubic space group F23, with unit-cell parameters a = b = c = 154.90 A.
Abstract: Pseudomonas aeruginosa is an opportunistic human pathogen capable of forming a biofilm under physiological conditions that contributes to its persistence despite long-term treatment with antibiotics. Here, we report that pathogenic P. aeruginosa strains PAO1 and PA14 are capable of infecting the roots of Arabidopsis and sweet basil (Ocimum basilicum), in vitro and in the soil, and are capable of causing plant mortality 7 d postinoculation. Before plant mortality, PAO1 and PA14 colonize the roots of Arabidopsis and sweet basil and form a biofilm as observed by scanning electron microscopy, phase contrast microscopy, and confocal scanning laser microscopy. Upon P. aeruginosa infection, sweet basil roots secrete rosmarinic acid (RA), a multifunctional caffeic acid ester that exhibits in vitro antibacterial activity against planktonic cells of both P. aeruginosa strains with a minimum inhibitory concentration of 3 microg mL(-1). However, in our studies RA did not attain minimum inhibitory concentration levels in sweet basil's root exudates before P. aeruginosa formed a biofilm that resisted the microbicidal effects of RA and ultimately caused plant mortality. We further demonstrated that P. aeruginosa biofilms were resistant to RA treatment under in vivo and in vitro conditions. In contrast, induction of RA secretion by sweet basil roots and exogenous supplementation of Arabidopsis root exudates with RA before infection conferred resistance to P. aeruginosa. Under the latter conditions, confocal scanning laser microscopy revealed large clusters of dead P. aeruginosa on the root surface of Arabidopsis and sweet basil, and biofilm formation was not observed. Studies with quorum-sensing mutants PAO210 (DeltarhlI), PAO214 (DeltalasI), and PAO216 (DeltalasI DeltarhlI) demonstrated that all of the strains were pathogenic to Arabidopsis, which does not naturally secrete RA as a root exudate. However, PAO214 was the only pathogenic strain toward sweet basil, and PAO214 biofilm appeared comparable with biofilms formed by wild-type strains of P. aeruginosa. Our results collectively suggest that upon root colonization, P. aeruginosa forms a biofilm that confers resistance against root-secreted antibiotics.
Abstract: Fatty acid synthases (primary metabolism), non-ribosomal peptide synthases and polyketide synthases (secondary metabolism) contain phosphopantetheinyl (Ppant)-dependent carrier proteins that must be made functionally active by transfer of the 4'-Ppant moiety from coenzyme A. These reactions are usually catalysed by dedicated Ppant transferases. Although rich in Ppant-dependent carrier proteins, it was previously shown that Pseudomonas aeruginosa possesses only one Ppant transferase, encoded by pcpS, which functions in both primary and secondary metabolism. Consistent with this notion are our findings that pcpS can genetically complement mutations in the Escherichia coli acpS and entD genes, encoding the apo-acyl carrier protein (ACP) synthase of fatty acid synthesis and a Ppant transferase of enterobactin synthesis, respectively. It also complements a Bacillus subtilis sfp mutation affecting a gene encoding a Ppant transferase essential for surfactin synthesis. A pcpS insertion mutant could only be constructed in a strain carrying the E. coli acpS gene on a chromosomally integrated element in trans, implying that the in vitro essentiality of pcpS is due to its requirement for activation of apo-ACP of fatty acid synthesis. The conditional pcpS mutant is non-fluorescent, does not produce pyoverdine and pyochelin, and does not grow in the presence of iron chelators. The data presented here for the first time confirm that PcpS plays an essential role in both fatty acid and siderophore metabolism.
Abstract: The LasI/LasR quorum-sensing system plays a pivotal role in virulence gene regulation of the opportunistic human pathogen, Pseudomonas aeruginosa. Here we report the crystal structure of the acyl-homoserine lactone (AHL) synthase LasI that produces 3-oxo-C12-AHL from the substrates 3-oxo-C12-acyl-carrier protein (acyl-ACP) and S-adenosyl-L-methionine. The LasI six-stranded beta sheet platform, buttressed by three alpha helices, forms a V-shaped substrate-binding cleft that leads to a tunnel passing through the enzyme that can accommodate the acyl-chain of acyl-ACP. This tunnel places no apparent restriction on acyl-chain length, in contrast to a restrictive hydrophobic pocket seen in the AHL-synthase EsaI. Interactions of essential conserved N-terminal residues, Arg23, Phe27 and Trp33, suggest that the N-terminus forms an enclosed substrate-binding pocket for S-adenosyl-L-methionine. Analysis of AHL-synthase surface residues identified a binding site for acyl-ACP, a role that was supported by in vivo reporter assay analysis of the mutated residues, including Arg154 and Lys150. This structure and the novel explanation of AHL-synthase acyl-chain-length selectivity promise to guide the design of Pseudomonas aeruginosa-specific quorum-sensing inhibitors as antibacterial agents.
Abstract: BACKGROUND: Pseudomonas aeruginosa is intrinsically resistant to high levels of triclosan. METHODS: Using the agar incorporation method and defined mutant strains, the contribution of multidrug efflux pumps to high-level (>1,000 microg/mL) triclosan resistance in P aeruginosa was assessed. RESULTS: The results showed that the ability of P aeruginosa to survive in the presence of triclosan concentrations in excess of 1,000 microg/mL is solely attributable to the expression of efflux pumps.
Abstract: The las and rhl quorum sensing (QS) systems regulate the expression of several genes in response to cell density changes in Pseudomonas aeruginosa. Many of these genes encode surface-associated or secreted virulence factors. Proteins from stationary phase culture supernatants were collected from wild-type and P. aeruginosa PAO1 mutants deficient in one or more of the lasRI, rhlRI and vfr genes and analysed using two-dimensional gel electrophoresis. All mutants released significantly lower amounts of protein than the wild-type. Protein spot patterns from each strain were compared using image analysis and visible spot differences were identified using mass spectrometry. Several previously unknown QS-regulated proteins were characterized, including an aminopeptidase (PA2939), an endoproteinase (PrpL) and a unique 'hypothetical' protein (PA0572), which could not be detected in the culture supernatants of Deltalas mutants, although they were unaffected in Deltarhl mutants. Chitin-binding protein (CbpD) and a hypothetical protein (PA4944) with similarity to host factor I (HF-I) could not be detected when any of the lasRI or rhlRI genes were disrupted. Fourteen proteins were present at significantly greater levels in the culture supernatants of QS mutants, suggesting that QS may also negatively control the expression of some genes. Increased levels of two-partner secretion exoproteins (PA0041 and PA4625) were observed and may be linked to increased stability of their cognate transporters in a QS-defective background. Known QS-regulated extracellular proteins, including elastase (lasB), LasA protease (lasA) and alkaline metalloproteinase (aprA) were also detected.
Abstract: Pseudomonas aeruginosa is an opportunistic human pathogen exhibiting innate resistance to multiple antimicrobial agents. This intrinsic multidrug resistance is caused by synergy between a low-permeability outer membrane and expression of a number of broadly-specific multidrug efflux (Mex) systems, including MexAB-OprM and MexXY-OprM. In addition to this intrinsic resistance, these and three additional systems, MexCD-OprJ, MexEF-OprN and MexJK-OprM promote acquired multidrug resistance as a consequence of hyper-expression of the efflux genes by mutational events. In addition to antibiotics, these pumps export biocides, dyes, detergents, metabolic inhibitors, organic solvents and molecules involved in bacterial cell-cell communication. Homologues of the resistance-nodulation-division systems of P. aeruginosa have been found in Burkholderia cepacia, B. pseudomallei, Stenotrophomonas maltophilia, and the nonpathogen P. putida, where they play roles in resistance to antimicrobials and/or organic solvents. Despite intensive studies of these multidrug efflux systems over the past several years, their precise molecular architectures, their modes of regulation of expression and their natural functions remain largely unknown.
Abstract: The Flp-FRT site-specific recombination system from Saccharomyces cerevisiae is a powerful and efficient tool for high-throughput genetic analysis of bacteria in the postgenomic era. This review highlights the features of the Flp-FRT system, describes current bacterial genetic methods incorporating this technology and, finally, suggests potential future uses of this system. In combination with improved allele replacement methods, recyclable FRT mutagenesis cassettes, whose antibiotic resistance markers can be excised from the chromosome in vivo, are useful for the rapid construction of multiple, unmarked mutations in the same chromosome, and thus aid in the generation of live vaccine strains or food-safe bacteria. The high-specificity of the Flp-FRT system makes it also applicable for manipulation of whole genomes, including in vivo cloning of large genomic segments. Integration-proficient vectors, from which antibiotic resistance markers and replication functions can be evicted after integration of the desired sequences into the chromosome, are useful for the construction of strains destined for environmental release, e.g. strains used as biosensors or for bioremediation. Although the Flp-FRT system is extremely efficient and easy to use, its true potential in bacterial genetics has not yet been fully exploited. On the contrary, in many instances this technology is probably greatly underutilized, especially in gram-positive bacteria.
Abstract: Using the biocide triclosan as a selective agent, several triclosan-resistant mutants of a susceptible Pseudomonas aeruginosa strain were isolated. Cloning and characterization of a DNA fragment conferring triclosan resistance from one of these mutants revealed a hitherto uncharacterized efflux system of the resistance nodulation cell division (RND) family, which was named MexJK and which is encoded by the mexJK operon. Expression of this operon is negatively regulated by the product of mexL, a gene located upstream of and transcribed divergently from mexJK. The triclosan-resistant mutant contained a single nucleotide change in mexL, which caused an amino acid change in the putative helix-turn-helix domain of MexL. The MexL protein belongs to the TetR family of repressor proteins. The MexJK system effluxed tetracycline and erythromycin but only in the presence of the outer membrane protein channel OprM; OprJ and OprN did not function with MexJK. Triclosan efflux required neither of the outer membrane protein channels tested but necessitated the MexJ membrane fusion protein and the MexK inner membrane RND transporter. The results presented in this study suggest that MexJK may function as a two-component RND pump for triclosan efflux but must associate with OprM to form a tripartite antibiotic efflux system. Furthermore, the results confirm that triclosan is an excellent tool for the study of RND multidrug efflux systems and that this popular biocide therefore readily selects mutants which are cross-resistant with antibiotics.
Abstract: The two acyl-homoserine lactones (AHLs) N-(butyryl)-L-homoserine lactone and N-[3-oxododecanoyl]-L-homoserine lactone (3-oxo-C(12)-HSL) are required for quorum sensing in Pseudomonas aeruginosa. These AHLs derive their invariant lactone rings from S-adenosylmethionine and their variable acyl chains from the cellular acyl-acyl carrier protein (ACP) pool. This reaction is catalysed by specific AHL synthases, which exhibit acyl chain specificity. Culture supernatants of P. aeruginosa contain multiple 3-oxo-AHLs that differ in their acyl chain lengths but their physiological role, if any, remains unknown. An in vitro fatty acid-3-oxo-AHL synthesis system was established utilizing purified P. aeruginosa Fab proteins, ACP and the LasI 3-oxo-AHL synthase. In the presence of excess protein, substrates and cofactors, this system produced almost exclusively 3-oxo-C(12)-HSL. When the beta-ketoacyl-ACP reductase (FabG) catalysed step was made rate-limiting by switching from the preferred NADPH cofactor to NADH, increased levels of short chain 3-oxo-AHLs were produced, presumably because shorter-chain ketoacyl-ACPs accumulated and thus became LasI substrates. Consistent with these in vitro observations, a fabG(Ts) mutant produced increased amounts of 3-oxo-AHLs in vivo. Thus, in vitro and in vivo evidence indicated that modulation of FabG activity of the fatty acid biosynthetic pathway may determine the acyl chain lengths of these 3-oxo-AHLs and that the LasI 3-oxo-AHL synthase is sufficient for their synthesis.
Abstract: The contribution of efflux pumps to multidrug resistance in 12 Pseudomonas aeruginosa isolates from various animal sources was assessed. Western immunoblot analyses demonstrated that all twelve isolates expressed significant levels of the MexAB OprM efflux system whereas two isolates simultaneously expressed the MexEF OprN or MexXY systems, respectively. One strain contained a single mutation in mexR, a regulator of mexAB-oprM expression, that did not adversely affect the MexR amino acid sequence, and three isolates contained the same, single base change in the mexA-mexR intergenic region. The MexXY-expressing strain contained two base substitutions in its mexZ regulatory gene which did not alter the MexR sequence.
Abstract: Triclosan is an antiseptic frequently added to items as diverse as soaps, lotions, toothpaste, and many commonly used household fabrics and plastics. Although wild-type Pseudomonas aeruginosa expresses the triclosan target enoyl-acyl carrier protein reductase, it is triclosan resistant due to expression of the MexAB-OprM efflux system. Exposure of a susceptible Delta(mexAB-oprM) strain to triclosan selected multidrug-resistant bacteria at high frequencies. These bacteria hyperexpressed the MexCD-OprJ efflux system due to mutations in its regulatory gene, nfxB. The MICs of several drugs for these mutants were increased up to 500-fold, including the MIC of ciprofloxacin, which was increased 94-fold. Whereas the MexEF-OprN efflux system also participated in triclosan efflux, this antimicrobial was not a substrate for MexXY-OprM.
Abstract: Triclosan is the active ingredient in a multitude of health care and consumer products with germicidal properties, which have flooded the market in recent years in response to the public's fear of communicable bacteria. Although originally thought to kill bacteria by attacking multiple cellular targets, triclosan was recently shown to target a specific bacterial fatty acid biosynthetic enzyme, enoyl-[acyl-carrier protein] reductase, in Gram-negative and Gram-positive bacteria, as well as in the Mycobacteria. Triclosan resistance mechanisms include target mutations, increased target expression, active efflux from the cell, and enzymatic inactivation/degradation. These are the same types of mechanisms involved in antibiotic resistance and some of them account for the observed cross-resistance with antibiotics in laboratory isolates. Therefore, there is a link between triclosan and antibiotics, and the widespread use of triclosan-containing antiseptics and disinfectants may indeed aid in development of microbial resistance, in particular cross-resistance to antibiotics.
Abstract: Improved tools for Pseudomonas research include small, broad-host-range vectors that allow regulated expression from the lac operon and T7 promoters whose biology is well understood and adaptable to many bacteria. To facilitate studies on gene regulation, tracking and monitoring of bacteria in diverse environments, and the construction of biosensors, various reporter genes with versatile assay formats have been developed that can be delivered on plasmid, transposon and integration-proficient vectors.
Abstract: An improved method for integration of exogenous DNA fragments at a defined site within the genome of Pseudomonas aeruginosa was developed. The method relies on two integration-proficient vectors, mini-CTX1 and mini-CTX2. These two vectors contain (1) a tetracycline (tet) selectable marker, (2) an oriT for conjugation-mediated plasmid transfer, (3) the pMB1-derived origin of replication, (4) a modified φCTX integrase (int) gene, (5) a versatile multiple cloning site (MCS) flanked by T4 transcriptional termination sequences (Omega elements), and (6) the φCTX attachment site. The MCS and Omega elements are flanked by yeast Flp recombinase target sites that allow in vivo excision of unwanted plasmid backbone sequences, including tet and int, from the genome of integrants by Flp recombinase. In the mini-CTX2 vector int transcription is driven from the strong trc promoter, which is regulated by the Lac repressor that is encoded by lacI(q) also contained on the plasmid. Upon conjugal transfer, mini-CTX1 and mini-CTX2 integrated at frequencies of 10(-8) and 10(-7), respectively. The usefulness of the integration vectors for gene fusion analyses was demonstrated by chromosomal insertion of autoinducer (AI)-regulated lasB-lacZ and rhlA-lacZ fusions into wild-type and AI synthase mutants. In wild-type, the fusions responded in a cell density-dependent manner and expression of both fusions was either greatly reduced or abolished in AI synthase mutants. Finally, an expression cassette containing the T7 polymerase gene under Lac repressor control was constructed, integrated into the P. aeruginosa chromosome, and used to express the hexahistidine-tagged P. aeruginosa AI synthase RhlI.
Abstract: The genomes of the two clonally derived Pseudomonas aeruginosa prototypic strains PAO1 and DSM-1707 differ by the presence of a 2. 19-Mb inversion including oriC. Integration of two Flp recombinase target sites near the rrn operons containing the inversion endpoints in PAO1 led to Flp-catalyzed inversion of the intervening 1.59-Mb fragment, including oriC, at high frequencies (83%), favoring the chromosome configuration found in DSM-1707. The results indicate that the oriC-containing region of the P. aeruginosa chromosome can readily undergo and tolerate large inversions.
Abstract: The Pseudomonas aeruginosa fabI structural gene, encoding enoyl-acyl carrier protein (ACP) reductase, was cloned and sequenced. Nucleotide sequence analysis revealed that fabI is probably the last gene in a transcriptional unit that includes a gene encoding an ATP-binding protein of an ABC transporter of unknown function. The FabI protein was similar in size and primary sequence to other bacterial enoyl-ACP reductases, and it contained signature motifs for the FAD-dependent pyridine nucleotide reductase and glucose/ribitol dehydrogenase families, respectively. The chromosomal fabI gene was disrupted, and the resulting mutant was viable but possessed only 62% of the total enoyl-ACP reductase activity found in wild-type cell extracts. The fabI-encoded enoyl-ACP reductase activity was NADH dependent and inhibited by triclosan; the residual activity in the fabI mutant was also NADH dependent but not inhibited by triclosan. An polyhistidine-tagged FabI protein was purified and characterized. Purified FabI (i) could use NADH but not NADPH as a cofactor; (ii) used both crotonyl-coenzyme A and crotonyl-ACP as substrates, although it was sixfold more active with crotonyl-ACP; and (iii) was efficiently inhibited by low concentrations of triclosan. A FabI Gly95-to-Val active-site amino acid substitution was generated by site-directed mutagenesis, and the mutant protein was purified. The mutant FabI protein retained normal enoyl-ACP reductase activity but was highly triclosan resistant. When coupled to FabI, purified P. aeruginosa N-butyryl-L-homoserine lactone (C4-HSL) synthase, RhlI, could synthesize C4-HSL from crotonyl-ACP and S-adenosylmethionine. This reaction was NADH dependent and inhibited by triclosan. The levels of C4-HSL and N-(3-oxo)-dodecanoyl-L-homoserine lactones were reduced 50% in a fabI mutant, corroborating the role of FabI in acylated homoserine lactone synthesis in vivo.
Abstract: The role of the two known catalases in Pseudomonas aeruginosa in protecting planktonic and biofilm cells against hydrogen peroxide (H(2)O(2)) was investigated. Planktonic cultures and biofilms formed by the wild-type strain PAO1 and the katA and katB catalase mutants were compared for their susceptibility to H(2)O(2). Over the course of 1 h, wild-type cell viability decreased steadily in planktonic cells exposed to a single dose of 50 mM H(2)O(2), whereas biofilm cell viability remained at approximately 90% when cells were exposed to a flowing stream of 50 mM H(2)O(2). The katB mutant, lacking the H(2)O(2)-inducible catalase KatB, was similar to the wild-type strain with respect to H(2)O(2) resistance. The katA mutant possessed undetectable catalase activity. Planktonic katA mutant cultures were hypersusceptible to a single dose of 50 mM H(2)O(2), while biofilms displayed a 10-fold reduction in the number of culturable cells after a 1-h exposure to 50 mM H(2)O(2). Catalase activity assays, activity stains in nondenaturing polyacrylamide gels, and lacZ reporter genes were used to characterize the oxidative stress responses of planktonic cultures and biofilms. Enzyme assays and catalase activity bands in nondenaturing polyacrylamide gels showed significant KatB catalase induction occurred in biofilms after a 20-min exposure to H(2)O(2), suggesting that biofilms were capable of a rapid adaptive response to the oxidant. Reporter gene data obtained with a katB::lacZ transcriptional reporter strain confirmed katB induction and that the increase in total cellular catalase activity was attributable to KatB. Biofilms upregulated the reporter in the constant presence of 50 mM H(2)O(2), while planktonic cells were overwhelmed by a single 50 mM dose and were unable to make detectable levels of beta-galactosidase. The results of this study demonstrated the following: the constitutively expressed KatA catalase is important for resistance of planktonic and biofilm P. aeruginosa to H(2)O(2), particularly at high H(2)O(2) concentrations; KatB is induced in both planktonic and biofilm cells in response to H(2)O(2) insult, but plays a relatively small role in biofilm resistance; and KatB is important to either planktonic cells or biofilm cells for acquired antioxidant resistance when initial levels of H(2)O(2) are sublethal.
Abstract: A DNA fragment containing the Pseudomonas aeruginosa fabD (encoding malonyl-coenzyme A [CoA]:acyl carrier protein [ACP] transacylase), fabG (encoding beta-ketoacyl-ACP reductase), acpP (encoding ACP), and fabF (encoding beta-ketoacyl-ACP synthase II) genes was cloned and sequenced. This fab gene cluster is delimited by the plsX (encoding a poorly understood enzyme of phospholipid metabolism) and pabC (encoding 4-amino-4-deoxychorismate lyase) genes; the fabF and pabC genes seem to be translationally coupled. The fabH gene (encoding beta-ketoacyl-ACP synthase III), which in most gram-negative bacteria is located between plsX and fabD, is absent from this gene cluster. A chromosomal temperature-sensitive fabD mutant was obtained by site-directed mutagenesis that resulted in a W258Q change. A chromosomal fabF insertion mutant was generated, and the resulting mutant strain contained substantially reduced levels of cis-vaccenic acid. Multiple attempts aimed at disruption of the chromosomal fabG gene were unsuccessful. We purified FabD as a hexahistidine fusion protein (H6-FabD) and ACP in its native form via an ACP-intein-chitin binding domain fusion protein, using a novel expression and purification scheme that should be applicable to ACP from other bacteria. Matrix-assisted laser desorption-ionization spectroscopy, native polyacrylamide electrophoresis, and amino-terminal sequencing revealed that (i) most of the purified ACP was properly modified with its 4'-phosphopantetheine functional group, (ii) it was not acylated, and (iii) the amino-terminal methionine was removed. In an in vitro system, purified ACP functioned as acyl acceptor and H(6)-FabD exhibited malonyl-CoA:ACP transacylase activity.
Abstract: Purification of proteins from Escherichia coli under native conditions is often hampered by inclusion-body formation after overexpression from T7 promoter-based expression vectors. This is probably due to the relatively high copy number of the ColE1-based expression vectors. To circumvent these problems, the low-copy-number pViet and pNam expression vectors were constructed. These vectors contain the pSC101 origin of replication and allow the expression of oligohistidine and intein chitin-binding domain fusion proteins, respectively. Since pViet and pNam do not replicate in E. coli B strains, an E. coli K-12 host strain [SA1503(DE3)] was constructed. This strain is defective in the Lon and OmpT proteases and allows IPTG-inducible expression of recombinant proteins from the T7 promoter. The new vectors were successfully tested by purification of three very insoluble proteins (RmlD, LasI and RhlI) under non-denaturing conditions, and all three proteins retained enzymatic activity. The purified hexahistidine (His6)-tagged Pseudomonas aeruginosa RhlI protein was subjected to more detailed analyses, which indicated that (1) only butyryl-acyl carrier protein (ACP) and S-adenosylmethionine (SAM) were required for synthesis of N-butyryl-L-homoserine lactone; (2) when present at physiological concentrations, butyryl-coenzyme A and NADPH were not substrates for RhlI; (3) RhlI was able to synthesize N-hexanoyl-L-homoserine lactone from hexanoyl-ACP and SAM; (4) RhlI was able to direct synthesis of N-butyryl-L-homoserine lactone from crotonyl-ACP in a reaction coupled to purified P. aeruginosa FabI (enoyl-ACP reductase).
Abstract: We describe the construction of promoter probe vectors designed for identification of bacterial genes induced in vitro and/or in vivo and for measurement of gene expression levels for in vivo expression technology. These plasmids use the Pseudomonas aeruginosa aspartate beta-semialdehyde dehydrogenase (asd) gene as a selectable marker and beta-galactosidase (pIVPRO, 10.88 kb) or mutant green fluorescent protein with enhanced fluorescence properties (mut3GFP, pIVET-GFP, 5.48 kb) as reporter gene systems. The proposed strategies can be adapted for use in most Gram-negative bacteria.
Abstract: An improved method for gene replacement in Pseudomonas aeruginosa was developed. The method employs several new gene replacement vectors that incorporate (1) the counterselectable sacB marker, (2) a lacZ alpha-allele for blue-white screening, (3) the pUC18/19 vectors multiple cloning site with 10 unique restriction sites, (4) an oriT for conjugation-mediated plasmid transfer and (5) carbenicillin, gentamicin (Gm) and tetracycline selectable markers. A cassette was constructed that contains a GmR selectable marker next to the green fluorescent protein structural gene, with both markers being flanked by Flp recombinase target (FRT) sites. The FRT cassette was used to insertionally inactivate the cloned P. aeruginosa pabC gene encoding aminodeoxychorismate lyase. After conjugal transfer into P. aeruginosa, plasmid integrants were selected, and deletion of unwanted DNA sequences was promoted by sucrose counterselection. The FRT cassette was excised with high frequencies (close to 100%) from the chromosome after conjugal transfer of a Flp recombinase-expressing plasmid; this sacB-containing plasmid was subsequently cured by sucrose counterselection, resulting in an unmarked P. aeruginosa delta pabC strain.
Abstract: Many strains of Pseudomonas aeruginosa are resistant to the antibiotics cerulenin and thiolactomycin, potent inhibitors of bacterial fatty acid biosynthesis. A novel yeast Flp recombinase-based technique was used to isolate an unmarked mexAB-oprM deletion encoding an efflux system mediating resistance to multiple antibiotics in P. aeruginosa. The experiments showed that the MexAB-OprM system is responsible for the intrinsic resistance of this bacterium to cerulenin and thiolactomycin. Whereas thiolactomycin was not a substrate of the MexCD-OprJ pump expressed in a delta(mexAB-oprM) nfxB mutant, cerulenin was efficiently effluxed by the MexCD-OprJ system. It was also found that the MexAB-OprM system is capable of efflux of irgasan, a broad-spectrum antimicrobial compound used in media selective for Pseudomonas.
Abstract: asd mutants of Gram-negative and some Gram-positive bacteria have an obligate requirement for diaminopimelic acid (DAP), an essential constituent of the cell wall of these organisms. In environments deprived of DAP, for example mammalian tissues, they will undergo lysis. This was previously exploited to develop vaccine strains of Salmonella typhimurium and cloning vectors containing asd as an in vivo selectable marker. As a first step for development of such systems for Pseudomonas aeruginosa, the asd gene from wild-type strain PAO1 was cloned by a combined approach of PCR amplification from chromosomal DNA, construction of mini-libraries and by complementation of an Escherichia coli delta asd mutant. The nucleotide sequence of a 2433 bp Smal-Nsil fragment was determined. This fragment contained the C-terminal 47 nucleotides of leuB, encoding 3-isopropylmalate dehydrogenase; asd, encoding aspartate-beta-semialdehyde dehydrogenase (Asd); and orfA, whose product showed similarity to the Asd proteins from Vibrio spp. By subcloning, asd was localized to a 1.24 kb DNA fragment which in an E. coli T7 expression system strongly expressed a 40,000 Da protein. The amino acid sequence was deduced from the DNA sequence. A comparison of the Asd proteins from P. aeruginosa, E. coli and Haemophilus influenzae revealed greater than 63% identity, demonstrating the conserved nature of Asd in Gram-negative bacteria, and defined the active-site-containing consensus sequence GGNCTVXMLMXXXLGLF as a possible signature motif. Chromosomal delta asd mutants were isolated. They were auxotrophic for DAP, lysine, methionine and threonine, and lysed in the absence of DAP. Genetic analyses indicated that orfA probably is naturally frame-shifted and does not contribute to the Asd phenotype. By PFGE, the asd gene was mapped to between coordinates 1.89 and 2.15 Mbp, or 37-40 min, on the 5.9 Mbp P. aeruginosa chromosome.
Abstract: The Pseudomonas aeruginosa leuB gene, encoding 3-isopropylmalate dehydrogenase, was identified upstream of asd, encoding aspartate-beta-semialdehyde dehydrogenase. Genetic analysis indicated that leuB is identical to the previously mapped gene defined by the leu-10 allele. The chromosomal leuB locus was inactivated by gene replacement. The insertions had no adverse effect on expression of the downstream asd gene but resulted in leucine auxotrophy. The leuB gene encodes a protein containing 360 amino acids (with a molecular weight of 39153), which was expressed in Escherichia coli as a M, 42000 protein. The results suggested that, in contrast to the situation in other bacteria (E. coli, Salmonella typhimurium and Bacillus subtilis) the P. aeruginosa leuB gene is physically separated from the genes encoding the other enzymes of the isopropylmalate pathway.
Abstract: The Pseudomonas aeruginosa fabA and fabB genes, encoding beta-hydroxyacyl-acyl carrier protein dehydratase and beta-ketoacyl-acyl carrier protein synthase I, respectively, were cloned, sequenced, and expressed in Escherichia coli. Northern analysis demonstrated that fabA and fabB are cotranscribed and most probably form a fabAB operon. The FabA and FabB proteins were similar in size and amino acid composition to their counterparts from Escherichia coli and to the putative homologs from Haemophilus influenzae. Chromosomal fabA and fabB mutants were isolated; the mutants were auxotrophic for unsaturated fatty acids. A temperature-sensitive fabA mutant was obtained by site-directed mutagenesis of a single base that induced a G101D change; this mutant grew normally at 30 degrees C but not at 42 degrees C, unless the growth medium was supplemented with oleate. By physical and genetic mapping, the fabAB genes were localized between 3.45 and 3.6 Mbp on the 5.9-Mbp chromosome, which corresponds to the 58- to 59.5-min region of the genetic map.
Abstract: The glycerol facilitator is one of the few known examples of bacterial solute transport proteins that catalyse facilitated diffusion across the cytoplasmic membrane. A second protein, glycerol kinase, is involved in entry of external glycerol into cellular metabolism by trapping glycerol in the cytoplasm as sn-glycerol 3-phosphate. Evidence is presented that glycerol transport in Pseudomonas aeruginosa is mediated by a similar transport system. The genes encoding the glycerol facilitator, glpF, and glycerol kinase, glpK, were isolated on a 4.5 kb EcoRI fragment from a chromosomal mini-library by functional complementation of an Escherichia coli glpK mutant after establishing a map of the chromosomal glpFK region with the help of a PCR-amplified glpK segment. The nucleotide sequence revealed that glpF is the promoter-proximal gene of the glpFK operon. The glycerol facilitator and glycerol kinase were identified in a T7 expression system as proteins with apparent molecular masses of 25 and 56 kDa, respectively. The identities of the glycerol facilitator and glycerol kinase amino acid sequences with their counterparts from Escherichia coli were 70 and 81%, respectively; this similarity extended to two homologues in the genome sequence of Haemophilus influenzae. A chromosomal delta glpFK mutant was isolated by gene replacement. This mutant no longer transported glycerol and could no longer utilize it as sole carbon and energy source. Two ORFs, orfX and orfY, encoding a putative regulatory protein and a carbohydrate kinase of unknown function, were located upstream of the glpFK operon.
Abstract: The plcHR operon of Pseudomonas aeruginosa includes the structural gene for the hemolytic phospholipase C,plcH (previously known as plcS), and two overlapping, in-phase, genes designated plcR1 and plcR2. Hemolytic and phospholipase C (PLC) activities produced by Escherichia coli and P. aeruginosa T7 expression systems were measured in strains carrying both plcH and plcR genes and in strains carrying each gene separately. When plcH was expressed by itself in the E. coli T7 system, the area of the hemolytic zone on blood agar was less than twice the area of growth. By contrast, when plcR was coexpressed with plcH in this system, the area of the hemolytic zone was approximately 10 times that of the area of the growth on blood agar. Native polyacrylamide gel electrophoretic analyses of PlcH activity expressed in either the E. coli or the P. aeruginosa T7 system carrying plcH alone, or along with the plcR genes, suggest that PlcR either posttranslationally alters the physical or biochemical nature of PlcH or releases PlcH from a complex in the cell so that it can be secreted. The hypothesis that PlcR is involved in the secretion of PlcH is supported by the observation that the ratio of extracellular to cell-associated PlcH activity produced by P. aeruginosa strains containing an in-frame deletion in the chromosomal plcR genes is significantly reduced in comparison with this ratio seen with the wild-type parental strain. This defect in the secretion of PlcH can be complemented by the plcR genes in trans. Additional data suggest that PlcR does not directly affect the secretion of the nonhemolytic phospholipase C (PlcN). PlcR is highly similar to a calcium-binding protein (CAB) from Streptomyces erythraeus. PlcR and CAB contain typical motifs (EF hands) characteristic of eucaryotic calcium-binding proteins, including calmodulin. P. aeruginosa naturally produces membrane vesicles (MVs) containing extracellular proteins including PLC. MVs from the PAO1WT strain contained at least 10-fold more PLC specific activity than those isolated from a strain carrying a deletion of plcR (PAO1 deltaR). Immunogold electron microscopy of PAO1WT and PAO1 deltaR whole cells revealed a distribution of PlcH in these strains consistent with the hypothesis that PlcR is required for the secretion of PlcH.
Abstract: Pseudomonas aeruginosa is considered a strict aerobe that possesses several enzymes important in the disposal of toxic oxygen reduction products including iron- and manganese-cofactored superoxide dismutase and catalase. At present, the nature of the regulation of these enzymes in P. aeruginosa Is not understood. To address these issues, we used two mutants called A4 and C6 which express altered Fur (named for ferric uptake regulation) proteins and constitutively produce the siderophores pyochelin and pyoverdin. Both mutants required a significant lag phase prior to log-phase aerobic growth, but this lag was not as apparent when the organisms were grown under microaerobic conditions. The addition of iron salts to mutant A4 and, to a greater extent, C6 cultures allowed for an increased growth rate under both conditions relative to that of bacteria without added iron. Increased manganese superoxide dismutase (Mn-SOD) and decreased catalase activities were also apparent in the mutants, although the second catalase, KatB, was detected in cell extracts of each fur mutant. Iron deprivation by the addition of the iron chelator 2,2'-dipyridyl to wild-type bacteria produced an increase in Mn-SOD activity and a decrease in total catalase activity, similar to the fur mutant phenotype. Purified wild-type Fur bound more avidly than mutant Fur to a PCR product containing two palindromic 19-bp "iron box" regions controlling expression of an operon containing the sodA gene that encodes Mn-SOD. All mutants were defective in both ferripyochelin- and ferripyoverdin-mediated iron uptake. Two mutants of strain PAO1, defective in pyoverdin but not pyochelin biosynthesis, produced increased Mn-SOD activity. Sensitivity to both the redox-cycling agent paraquat and hydrogen peroxide was greater in each mutant than in the wild-type strain. In summary, the results indicate that mutations in the P. aeruginosa fur locus affect aerobic growth and SOD and catalase activities in P. aeruginosa. We postulate that reduced siderophore-mediated iron uptake, especially that by pyoverdin, may be one possible mechanism contributing to such effect.
Abstract: The Pseudomonas aeruginosa chromosome was fractionated with the enzymes SpeI and DpnI, and genomic fragments were separated by PFGE and used for mapping a collection of 40 genes. This permitted the localization of 8 genes previously mapped and of 32 genes which had not been mapped. We showed that a careful search of databases and identification of sequences that were homologous to known genes could be used to design and synthesize DNA probes for the mapping of P. aeruginosa homologues by Southern hybridization with genomic fragments, resulting in definition of the locations of the aro-2, dapB, envA, mexA, groEL, oprH, oprM, oprP, ponA, rpoB and rpoH genetic markers. In addition, a combination of distinct DNA sources were utilized as radioactively labelled probes, including specific restriction fragments of the cloned genes (glpD, opdE, oprH, oprO, oprP, phoS), DNA fragments prepared by PCR, and single-stranded DNA prepared from phagemid libraries that had been randomly sequenced. We used a PCR approach to clone fragments of the putative yhhF, sucC, sucD, cypH, pbpB, murE, pbpC, soxR, ftsA, ftsZ and envA genes. Random sequencing of P. aeruginosa DNA from phagemid libraries and database searching permitted the cloning of sequences from the acoA, catR, hemD, pheS, proS, oprD, pyo and rpsB gene homologues. The described genomic methods permit the rapid mapping of the P. aeruginosa genome without linkage analysis.
Abstract: The operons of the glp regulon encoding the glycerol metabolic enzymes of Pseudomonas aeruginosa were hitherto believed to be positively regulated by the product of the glpR regulatory gene. During nucleotide sequence analysis of the region located upstream of the previously characterized glpD gene, encoding sn-glycerol-3-phosphate dehydrogenase, an open reading frame (glpR) was identified which encodes a protein of 251 amino acids that is 59% identical to the Glp repressor from Escherichia coli and could be expressed as a 28-kDa protein in a T7 expression system. Inactivation of chromosomal glpR by gene replacement resulted in constitutive expression of glycerol transport activity and glpD activity. These activities were strongly repressed after introduction of a multicopy plasmid containing the glpR gene; the same plasmid also efficiently repressed expression of a glpT-lacZ+ transcriptional fusion in an E. coli glpR mutant. Analysis of the glpD and glpF upstream region identified conserved palindromic sequences which were 70% identical to the E. coli glp operator consensus sequence. The results suggest that the operons of the glp regulon in P. aeruginosa are negatively regulated by the action of a glp repressor.
Abstract: Previous work from our laboratory has shown that cosmid clone pFV100, containing a 26 kb insert, is able to restore O-antigen synthesis in serotype O5 rough mutants of Pseudomonas aeruginosa. Mobilization of pFV100 into two P. aeruginosa semi-rough (SR) mutants, AK14O1 and rd7513, resulted in O-antigen expression, indicating that pFV100 may contain an O-polymerase (rfc) gene. pFV.TK6, a subclone of pFV100 that contains a 5.6 kb chromosomal insert, was able to complement O-antigen expression in these SR mutants. Mutagenesis of pFV.TK6 using Tn1000 exposed a 1.5 kb region that was essential for complementing O-antigen expression in AK14O1. A 2.0 kb XhoI-HindIII fragment, containing this region, was cloned into vector pUCP26 and the resulting plasmid called pFV.TK8. In Southern analysis of the 20 P. aeruginosa serotypes using a probe generated from the 1.5 kb XhoI fragment of pFV.TK8, the rfc probe hybridized to a common fragment of the cross-reactive O2-O5-O16-O18-O20 serogroup, suggesting that these serotypes may share a common O-polymerase gene. In functional studies of the rfc gene, the PAO1 (serotype O5) chromosomal rfc was mutated using a gene-replacement strategy. These knockout mutants expressed the SR lipopolysaccharide (LPS) phenotype, which indicated that they were no longer producing a functional O-polymerase enzyme. Nucleotide sequence analysis of the insert DNA of pFV.TK8 revealed one open reading frame (ORF), designated ORF48.9, which could code for a 48.9 kDa protein. In comparisons of the P. aeruginosa rfc nucleotide and amino acid sequences with DNA and protein databases, no significant homology was found. However, the deduced structure of the P. aeruginosa Rfc protein indicated that it is very hydrophobic and contains 11 putative membrane-spanning domains. Therefore, the predicted structure is similar to that of other reported Rfc proteins. Furthermore, comparison of the amino acid composition and codon usage of the P. aeruginosa Rfc with other Rfc proteins revealed significant similarity between them.
Abstract: A novel pUC19-based gene replacement vector has been developed. This vector incorporates (i) the counterselectable sacB marker, (ii) a lacZ alpha allele for blue-white screening, (iii) an oriT for conjugation-mediated plasmid transfer and (iv) unique cloning sites for SmaI and the rare-cutting meganuclease I-SceI. These rare restriction sites are also present on the helper plasmid pUC19Sce. The replacement vector is engineered to contain few restriction sites to gain greater access to restriction sites within cloned DNA fragments, thus facilitating their genetic manipulation. The usefulness of the system was demonstrated by chromosomal integration of a newly constructed xylE::GmR fusion cassette into the glpD gene of Pseudomonas aeruginosa.
Abstract: In a mucB (algN) genetic background, insertion of an omega element approximately 200 bp downstream of glpD, encoding sn-glycerol-3-phosphate dehydrogenase from Pseudomonas aeruginosa, had an adverse effect on alginate biosynthesis from various carbon sources. The insertion inactivated glpM, a gene encoding a 12,040-M(r) hydrophobic protein containing 109 amino acids. This protein, which was expressed in a T7 RNA polymerase expression system, appears to be a cytoplasmic membrane protein.
Abstract: The consumption of molecular oxygen by Pseudomonas aeruginosa can lead to the production of reduced oxygen species, including superoxide, hydrogen peroxide, and the hydroxyl radical. As a first line of defense against potentially toxic levels of endogenous superoxide, P. aeruginosa possesses an iron- and manganese-cofactored superoxide dismutase (SOD) to limit the damage evoked by this radical. In this study, we have generated mutants which possess an interrupted sodA (encoding manganese SOD) or sodB (encoding iron SOD) gene and a sodA sodB double mutant. Mutagenesis of sodA did not significantly alter the aerobic growth rate in rich medium (Luria broth) or in glucose minimal medium in comparison with that of wild-type bacteria. In addition, total SOD activity in the sodA mutant was decreased only 15% relative to that of wild-type bacteria. In contrast, sodB mutants grew much more slowly than the sodA mutant or wild-type bacteria in both media, and sodB mutants possessed only 13% of the SOD activity of wild-type bacteria. There was also a progressive decrease in catalase activity in each of the mutants, with the sodA sodB double mutant possessing only 40% of the activity of wild-type bacteria. The sodA sodB double mutant grew very slowly in rich medium and required approximately 48 h to attain saturated growth in minimal medium. There was no difference in growth of either strain under anaerobic conditions. Accordingly, the sodB but not the sodA mutant demonstrated marked sensitivity to paraquat, a superoxide-generating agent. P. aeuroginosa synthesizes a blue, superoxide-generating antibiotic similar to paraquat in redox properties which is called pyocyanin, the synthesis of which is accompanied by increased iron SOD and catalase activities (D.J. Hassett, L. Charniga, K. A. Bean, D. E. Ohman, and M. S. Cohen, Infect. Immun. 60:328-336, 1992). Pyocyanin production was completely abolished in the sodB and sodA sodB mutants and was decreased approximately 57% in sodA mutants relative to that of the wild-type organism. Furthermore, the addition of sublethal concentrations of paraquat to wild-type bacteria caused a concentration-dependent decrease in pyocyanin production, suggesting that part of the pyocyanin biosynthetic cascade is inhibited by superoxide. These results suggest that iron SOD is more important than manganese SOD for aerobic growth, resistance to paraquat, and optimal pyocyanin biosynthesis in P. aeruginosa.
Abstract: The fur gene, encoding an iron-regulatory protein in Neisseria meningitidis strain B16B6, has been cloned and sequenced. Its product showed a high degree of homology to known Fur sequences.
Abstract: The nucleotide sequence of the 1.9-kb PstI fragment from pRO1614, that allows stable maintenance of pMB1 (ColE1)-based cloning vectors in Pseudomonas, was determined. This fragment encodes a putative origin of replication (ori), a replication-controlling protein, and the C terminus of the Tn3 beta-lactamase-encoding gene. Improved versions of the broad-host-range plasmid vectors, pUCP18 and pUCP19, were constructed by deletion of nonessential DNA or replacement of nonessential DNA with an antibiotic-resistance cassette.
Abstract: Exoenzyme S is an ADP-ribosyltransferase produced by Pseudomonas aeruginosa. Synthesis of exoenzyme S depends on an intact trans-regulatory locus encoding three protein products, ExsC, ExsB, and ExsA. To identify the phenotype of ExsC, -B, and -A mutants in exoenzyme S production, specific insertional mutations with the streptomycin resistance-encoding omega interposon were introduced into cloned DNA and returned to the chromosomes of P. aeruginosa PA103, PAO1, and PAK. Southern blot analysis was used to confirm insertion of omega and resolution of vector sequences. Exoenzyme S expression was measured in parental and mutant derivatives by Western blot (immunoblot) analysis and ADP-ribosyltransferase activity measurement. A complete set of mutations were obtained in strains PAK and PAO1, but in strain PA103, only an insertion in the exsA coding region was identified. Southern blot analysis demonstrated that extensive duplication and rearrangement of the PA103 chromosomal trans-regulatory locus occurred when exsC::omega or exsB::omega recombination events were attempted. Exoenzyme S antigen was not detectable in the supernatant or lysate fractions of mutant strains by Western blot analysis. ADP-ribosyltransferase activity was detected in the lysate but not in the supernatant fractions of mutant derivatives. The general secretion pathway appeared to function normally in mutant strains, as elastase, exotoxin A, and phospholipase C were measured in the supernatants of parental and mutant strains. Several differences were noted when the extracellular protein profiles of parental strains were compared with similar samples from the insertional mutant strains. Some of these differences appeared to be unrelated to exoenzyme S. These data suggest that insertional inactivation of the exoenzyme S trans-regulatory locus may affect a subset of other extracellular proteins.
Abstract: Nitrosoguanidine-induced Pseudomonas aeruginosa mutants which were unable to utilize glycerol as a carbon source were isolated. By utilizing PAO104, a mutant defective in glycerol transport and sn-glycerol-3-phosphate dehydrogenase (glpD), the glpD gene was cloned by a phage mini-D3112-based in vivo cloning method. The cloned gene was able to complement an Escherichia coli glpD mutant. Restriction analysis and recloning of DNA fragments located the glpD gene to a 1.6-kb EcoRI-SphI DNA fragment. In E. coli, a single 56,000-Da protein was expressed from the cloned DNA fragments. An in-frame glpD'-'lacZ translational fusion was isolated and used to determine the reading frame of glpD by sequencing across the fusion junction. The nucleotide sequence of a 1,792-bp fragment containing the glpD region was determined. The glpD gene encodes a protein containing 510 amino acids and with a predicted molecular weight of 56,150. Compared with the aerobic sn-glycerol-3-phosphate dehydrogenase from E. coli, P. aeruginosa GlpD is 56% identical and 69% similar. A similar comparison with GlpD from Bacillus subtilis reveals 21% identity and 40% similarity. A flavin-binding domain near the amino terminus which shared the consensus sequence reported for other bacterial flavoproteins was identified.
Abstract: A lac-based alpha-complementation and expression system was developed for use in molecular cloning in Pseudomonas aeruginosa. A bacteriophage D3112-based mini-Dlac transposable element, containing the lacIq-regulated lacZ delta M15 gene next to a selectable marker, was constructed. Mixed D3112 lysates were used to transduce P. aeruginosa PAO1, and derivatives containing randomly inserted chromosomal copies of the mini-Dlac element were obtained. Transformation of the PAO1::mini-Dlac transductants with the broad-host-range vector, pUCP19, led to the formation of blue colonies on indicator medium in the presence of inducer. In contrast, transformants harboring the pUCP19 derivative pCDO, containing the catechol-2,3-dioxygenase (C23O)-encoding xylE gene under lac promoter control, were white on the same medium. Expression of xylE was tightly controlled by single-copy mini-Dlac-encoded lac repressor and in induced cultures was increased more than 100-fold over that observed in uninduced cultures. The usefulness of the system for molecular cloning in P. aeruginosa was demonstrated by ligating size-fractionated PAO1 chromosomal fragments into pUCP19, followed by transformation of the newly isolated PAO1::mini-Dlac host. All randomly chosen white colonies contained recombinant plasmids, with inserts of the correct size range, while blue colonies contained pUCP19 alone. The functionality of the system was also shown in another frequently studied strain, PA103.
Abstract: The asd mutants of Gram- and some Gram+ bacteria have an obligate requirement for diaminopimelic acid (DAP), an essential constituent of the cell wall of these organisms. In environments deprived of DAP, i.e., mammalian tissues, they will undergo lysis. This has previously been exploited to develop vaccine strains of Salmonella typhimurium and Streptococcus mutans. As a first step for the development of a biosafe Neisseria meningitidis laboratory strain, we have cloned the asd from wild-type strain B16B6 by complementation of an Escherichia coli asd mutant. By subcloning and insertion mutagenesis, the N. meningitidis asd was localized to a 1.5-kb DNA fragment. In a T7 RNA polymerase-T7 promoter expression system, a 38-kDa protein was strongly expressed from this DNA fragment. The N-terminal amino acid (aa) sequence was deduced from the nucleotide sequence, which was determined with the help of an in-frame Asd'::'LacZ protein fusion. A comparison of the N-terminal aa of the Asd proteins from N.meningitidis and E. coli revealed 70% identity, suggesting that the Asd protein may be highly conserved among Gram- bacteria.
Abstract: Two plasmids, X1918 and Z1918, were constructed which contain the promoter-less xylE and lacZ reporter genes flanked symmetrically by the multiple cloning site (MCS) from pUC19. These cassettes can easily be derived from the multicopy plasmid, pUC1918.
Abstract: An improved method for allele replacement in Pseudomonas aeruginosa was developed. The two main ingredients of the method are: (i) novel ColE1-type cloning vectors derived from pBR322 and pUC19; and (ii) a family of cassettes containing a portable oriT, the sacB gene from Bacillus subtilis as a counter-selectable marker, and a chloramphenicol-resistance gene allowing positive selection of both oriT and sacB. Introduction of plasmid-borne DNA into the chromosome was achieved in several steps. The DNA to be exchanged was first cloned into the new ColE1-type vectors. After insertion of the oriT and sacB sequences, these plasmid were conjugally transferred into P. aeruginosa and plasmid integrants were selected. Plating on sucrose-containing medium allowed positive selection for both plasmid excision and curing since Pseudomonas aeruginosa strains containing the sacB gene in single- or multiple copy were highly sensitive to 5% sucrose in rich medium. This procedure was successfully used to introduce an agmR mutation into P. aeruginosa wild-type strain PAO1 and should allow the exchange of any DNA segment into any non-essential regions of the P. aeruginosa chromosome.
Abstract: Several new broad-host-range vectors for the construction of protein fusions to the Escherichia coli lacZ gene have been developed. In all of the constructs, a multiple cloning site (MCS) containing unique restriction sites is located upstream of lac operon segments whose lacZ genes lack translational start signals. Some of the vectors (pPZ10, pPZ20 and pPZ30) also contain transcriptional terminators upstream of the MCS. The new vectors allow the fusion of genes to lacZ in all translational reading frames. Due to a higher copy number they allow direct screening in E. coli for weakly expressed foreign promoters. Their usefulness for gene analysis in Pseudomonas aeruginosa was demonstrated by construction and expression of a regA'::'lacZ-encoded protein fusion.
Abstract: Two new broad-host-range plasmid vectors, pUCP18 and pUCP19, which are stably maintained in Escherichia coli and Pseudomonas aeruginosa have been constructed. The plasmids are based on the E. coli pUC18 and pUC19 vectors and possess all their features: (i) convenient direct screening of recombinants; (ii) versatile multiple cloning site; (iii) use as sequencing and expression vectors; (iv) small size; and (v) intermediate to high copy number.
Abstract: The physical and genetic structure of 37 kilobases of DNA encompassing the tdc region at 68.3 min of the Escherichia coli chromosome was determined by DNA sequence analysis and restriction mapping. Re-examination of new data concerning the direction of transcription of the tdc operon revealed that in strain W3110 the tdc region is located on a transposable segment of DNA.
Abstract: The gene products of the ugp operon of Escherichia coli are responsible for the uptake of sn-glycerol-3-phosphate and certain glycerophosphodiesters. The regulation of ugp is mainly phoBR-dependent. Significant expression, however, can be observed even in the presence of high concentrations of phosphate, a condition which normally completely represses pho expression. Pho-independent ugp expression was found to be derepressed during the late logarithmic growth phase due to carbon starvation. Among different carbon sources tested, glucose caused the most complete repression. Addition of cAMP prevented glucose repression, indicating that a cAMP-CRP control mechanism may be directly or indirectly involved in the carbon-starvation response. This conclusion is supported by the fact that pho-independent ugp expression correlated with the presence of the cya and crp gene products.
Abstract: The genes for the peripheral glycerol carbon metabolic pathway (glp) in Pseudomonas aeruginosa are postulated to be positively regulated by GlpR. A gene complementing the glpR2 allele, affecting expression of the putative activator, was cloned by a bacteriophage mini-D3112-based in vivo cloning method. Mini-D3112 replicons were isolated by transfecting glpR2 strain PRP406 and selecting clones able to grow on minimal medium containing glycerol as the sole carbon and energy source. Preliminary biochemical characterization indicated that the cloned activator gene for glycerol metabolism (agmR) may not be allelic to glpR. Restriction analysis and recloning of DNA fragments located the agmR gene to a 2.3-kb EcoRV-SstI DNA fragment. In a T7 RNA polymerase expression system, a single 26,000-Da protein was expressed from this DNA fragment. The amino acid sequence of this protein, deduced from the nucleotide sequence reported here, demonstrates its homology to the effector (or regulator) proteins of the environmentally responsive two-component regulators. The carboxy-terminal region of AgmR contains a possible helix-turn-helix DNA-binding motif and resembles sequences found in transcriptional regulators of the LuxR family.
Abstract: A novel L-threonine transport system is induced in Escherichia coli cells when incubated in amino acid-rich medium under anaerobic conditions. Genetic and biochemical analyses with plasmids harboring mutations in the anaerobically expressed tdcABC operon indicated that the tdcC gene product was responsible for L-threonine uptake. Competition experiments revealed that the L-threonine transport system is also involved in L-serine uptake and is partially shared for L-leucine transport; L-alanine, L-valine, and L-isoleucine did not affect L-threonine uptake. Transport of L-threonine was inhibited by the respiratory chain inhibitors KCN and carbonyl cyanide m-chlorophenylhydrazone and was Na+ independent. These results identify for the first time an E. coli gene encoding a permease specific for L-threonine-L-serine transport that is distinct from the previously described threonine-serine transport systems. A two-dimensional topological model predicted from the amino acid composition and hydropathy plot showed that the TdcC polypeptide appears to be an integral membrane protein with several membrane-spanning domains exhibiting a striking similarity with other bacterial permeases.
Abstract: The ugp promoter (pugp) responsible for expression of the binding-protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli was cloned into a small multicopy plasmid pTER5, a derivative of pBR322, between the transcription terminators rpoCt and tL1. The resulting expression vector, pPH3, permits convenient insertion of structural genes containing their own translational-initiation regions, into the multiple-cloning site derived from the pUC19 plasmid. The efficiency and regulatory properties of pugp were measured using xylE and lacZ as reporter genes, which code for the corresponding enzymes catechol-2,3-dioxygenase (C23O) and beta-galactosidase (beta Gal), respectively. Enzyme activities were virtually completely repressed in the presence of excess inorganic phosphates (Pi) and high concentrations of glucose. Maximal induction was observed at limiting Pi (less than 0.1 mM) and normal levels of glucose (0.2-0.4%). The maximum expression of the pugp-directed beta Gal synthesis was approx. 80% of that directed by strong ptac. When the xylE gene was maximally expressed, the induced enzyme constituted approx. 50% of total cellular protein as judged by laser densitometry following sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. These results suggest the usefulness of the pugp in expression vectors for strong, but controlled, expression of cloned genes in E. coli. This Pi controlled vector can be adapted to large-scale fermentation by using Pi-limiting growth conditions.
Abstract: Efficient in vivo expression of the biodegradative threonine dehydratase (tdc) operon of Escherichia coli is dependent on a regulatory gene, tdcR. The tdcR gene is located 198 base pairs upstream of the tdc operon and is transcribed divergently from this operon. The nucleotide sequence of tdcR and two unrelated reading frames has been determined. The deduced amino acid sequence of TdcR indicates that it is a polypeptide of Mr 12,000 with 99 amino acid residues and contains a potential helix-turn-helix DNA binding motif. Deletion analysis and minicell expression of the tdcR gene suggest that TdcR may serve as a trans-acting positive activator for the tdc operon.
Abstract: The nucleotide sequence of a 2-kilobase DNA fragment of the tdc region of Escherichia coli K-12, previously cloned in this laboratory, revealed two open reading frames, tdcC and ORFX, downstream from the tdcB gene (formerly designated tdc) encoding biodegradative threonine dehydratase. A 24-base-pair sequence separated tdcC from the dehydratase coding region, and an untranslated region of 60 nucleotides, which contains a recognizable -10 consensus sequence, was found between tdcC and ORFX. The deduced amino acid sequence of tdcC showed it to be a large hydrophobic polypeptide of 431 amino acid residues, whereas ORFX coded for a small 135-residue polypeptide lacking glutamine and tryptophan. A computer-assisted sequence analysis revealed no similarity among the tdcB, tdcC, and ORFX polypeptides, and a search of the GenBank database failed to detect similarity with any other known proteins. The tdc genes and ORFX showed similar codon usage and, in analogy with other bacterial genes, showed codon usage typical for genes expressed at an intermediate level. Transcriptional analysis with S1 nuclease indicated two distinct transcription start sites upstream of the tdcB gene in regions previously identified as promoterlike elements P1 and P2. Interestingly, expression of tdcB and tdcC, but not ORFX, was contingent upon the presence of P1. These results taken together tend to suggest that the biodegradative threonine dehydratase is the second gene in a polycistronic transcription unit constituting a novel operon (tdcABC) in E. coli implicated in anaerobic threonine metabolism.
Abstract: The biodegradative threonine dehydratase (tdc) operon was mapped at 68 min on the Escherichia coli K-12 chromosome. The order of markers in the clockwise direction was dnaG uxaA tdc argG. A tdc deletion was isolated and mapped to this region of the chromosome. By using a tdcB-lacZ fusion the clockwise direction of transcription of tdc was determined.
Abstract: The glpD gene encoding aerobic sn-glycerol-3-phosphate dehydrogenase of Escherichia coli K-12 was cloned into pACYC177 from a lambda glpD transducing phage. The recombinant plasmid, designated pSH55, carried a 7.4-kilobase-pair HindIII fragment containing the glpD and glpR genes. The glpD gene was subcloned into pACYC177 on a 4.4-kilobase-pair BamHI-HindIII fragment. Expression of the cloned glpD gene was regulated in the manner previously described for the chromosomal glpD gene. The position of glpD on this plasmid was determined by Tn1000 insertional inactivation experiments. The glpD gene product, a polypeptide of Mr 55,000, was detected in a maxicell system. Truncated polypeptides replaced the 55,000-molecular-weight polypeptide when plasmid derivatives harboring Tn1000 insertions that inactivate glpD were used as templates. The sizes of these polypeptides confirmed the previously determined direction of transcription and allowed estimation of the translation start site. Determination of the apparent Mr of a hybrid protein encoded by a glpD'-'lacZ fusion provided additional evidence for the position of the glpD control region. The amino-terminal 30 to 60 amino acids of this hybrid protein (provided by glpD) were sufficient for efficient membrane localization of glpD'-'lacZ-encoded beta-galactosidase activity. The glpD3 mutation was mapped within the glpD gene, providing additional evidence that glpD is the structural gene for aerobic sn-glycerol-3-phosphate dehydrogenase.
Abstract: The glpTQ operon and the glpA and glpB genes are located adjacent to one another near min 49 of the linkage map of Escherichia coli K-12. The positions and directions of transcription of the glpA and glpB genes with respect to the glpTQ operon were determined in the present work. Strains harboring Mu d1(Ap lac) fusions in either glpA or glpB were converted to the respective lambda p1(209) lysogens. Induction of these lysogens with mitomycin C resulted in production of Lac+ phage progeny which carried adjacent chromosomal DNA. Genetic crosses with a collection of glpT mutant strains were performed with several such phage lines. A fine-structure deletion map of the glpT gene was thus constructed. All phages used for this mapping carried DNA starting with the promoter-proximal end of glpT. This indicated that the glpTQ operon and the glpA and glpB genes are transcribed divergently. Additional evidence supporting this conclusion was obtained by physical mapping of restriction endonuclease cleavage sites in plasmids carrying these genes and in plasmids carrying glpA-lacZ or glpB-lacZ fusions. A new designation (glpC) for the gene encoding the 41,000-Mr subunit of the anaerobic sn-glycerol-3-phosphate dehydrogenase was proposed to distinguish it from the glpA gene, which encodes the 62,000-Mr subunit of the dehydrogenase, and the glpB gene, which encodes a membrane anchor subunit of the dehydrogenase. These three genes were present in an operon transcribed in the order glpA glpC glpB in the clockwise direction on the linkage map of E. coli.
Abstract: The glpR gene encoding the repressor for the sn-glycerol 3-phosphate regulon of Escherichia coli was cloned downstream from the strong pL promoter of bacteriophage lambda. This allowed overproduction of the repressor upon thermal induction of a cryptic lambda lysogen harboring the cI857 gene. The repressor was purified 40-fold to homogeneity from an induced strain. The purification scheme utilized polyethyleneimine and ammonium sulfate fractionation, followed by phosphocellulose and DEAE-Sephadex chromatography. Purification was monitored by measuring the binding of radiolabeled inducer (sn-glycerol 3-phosphate) to the repressor. The purified repressor migrated as a single band exhibiting a subunit molecular weight of 30,000 assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the repressor under nondenaturing conditions was 100,000-130,000 suggesting the repressor is a tetramer under native conditions. Interaction of the repressor with sn-glycerol 3-phosphate was studied using flow dialysis. Scatchard analysis of the data indicated four binding sites/repressor tetramer and a dissociation constant of 31 microM. Interaction of the repressor with DNA was studied using band-shift electrophoresis. The repressor specifically bound DNA fragments containing the control regions for the glpD, glpK, and glpT-A genes. Binding of DNA by the repressor was diminished in the presence of sn-glycerol 3-phosphate.
Abstract: A transducing lambda phage carrying glpD''lacZ, glpR, and malT was isolated from a strain harboring a glpD''lacZ fusion. Comparison of restriction endonuclease cleavage patterns of DNA isolated from this phage with that of the previously cloned malT region (Raibaud and Schwartz 1980) facilitated the construction of recombinant plasmids carrying different portions of the glpD-malT region. Results of minicell analysis and complementation studies showed that this region of the chromosome encodes at least five polypeptides. These included the previously identified glpD, glpR, and malT gene products. In addition, two new structural genes of the glp regulon (glpE and glpG) located between the glpD and glpR genes were identified. Hybrid plasmids carrying glpD''lacZ and glpR''lacZ fusions were constructed. Restriction endonuclease cleavage analysis of these two plasmids demonstrated that glpD and glpR are divergently transcribed.
Abstract: The glpR gene encoding the repressor for the glp regulon of Escherichia coli was cloned from a library of HindIII DNA fragments established in bacteriophage lambda. Phages harboring glpR were isolated by selection for sn-glycerol-3-phosphate dehydrogenase function encoded by glpD, which is adjacent to glpR on the E. coli linkage map. Restriction endonuclease analysis and recloning of DNA fragments localized glpR to a 3-kilobase-pair EcoRI-SalI segment of DNA. Strains exhibiting constitutive expression of the glp operons were strongly repressed after introduction of multicopy plasmids containing the glpR gene. Analysis of proteins labeled in minicells harboring either glpR+ recombinant plasmids or a glpR::Tn5 derivative showed that the glpR gene product is a protein with an apparent molecular weight of 33,000.
Abstract: The expression of the ugp-dependent sn-glycerol-3-phosphate transport system that is part of the pho regulon was studied in mutants of Escherichia coli K-12 containing regulatory mutations of the pho regulon. The phoR and phoST gene products exerted a negative control on the expression of ugp. Induction of the system was positively controlled by the phoB, phoM, and phoR gene products. Using a ugp-lacZ operon fusion, we showed that the ugp and phoA genes were coordinately derepressed and repressed.
Abstract: The ugp structural genes, coding for the pho regulon dependent sn-glycerol-3-phosphate transport system, were cloned in pBR322 and characterized. The expression of the cloned ugp system was phoB dependent. Cells containing the ugp plasmid overproduced the G3P binding protein upon phosphate starvation. Tn5 mutagenesis of the cloned DNA revealed that the ugp genes are organized in two separate operons which comprise at least four genes: ugpB and ugpD constitute one operon, ugpA and ugpC constitute the other. The structural gene for the G3P binding protein (G3PBP) is ugpB. The ugpC gene product was also synthesized in minicells as a polypeptide, with an apparent molecular weight of 40,000. No gene products could be assigned to the ugpA and ugpD genes. Hybridization experiments allowed the physical characterization of 20 kb of DNA adjacent to the ugp genes on the E. coli chromosome including the liv genes.
Abstract: Discrepancies between our mapping data concerning the cdd and gat marker of Escherichia coli and data obtained by Josephsen et al. (1983) as well as with the current linkage map of Escherichia coli (Bachmann 1983) led us to reinvestigate the mapping of markers in the gyrA-his region by P1 transduction. In addition, we isolated Hfr strains by integrating the temperature-sensitive F'ts114lac+ episome via lac homology of cir'lacZ and mgl'lacZ fusions into the chromosome. From the results of the P1 transductions and using these Hfr strains as donors in crosses it became clear that the transcription of cir and mgl is the same and counterclockwise on the chromosome, with cdd and gat as the counterclockwise markers to mgl and cir. We conclude that our previously published sequence of markers was incorrect and is in fact inverted. The present data indicate the following sequence of markers in clockwise order: his gat cdd mgl cir fpk gyrA.
Abstract: Using a novel positive selection method for G3P transport activity, lambda phages that carry either all or part of ugp, the genes of the pho regulon-dependent G3P transport system of Escherichia coli were isolated from a library of EcoRI fragments of Escherichia coli established in lambda gt7. By subcloning EcoRI fragments carried by the different phages into the multicopy plasmids pACYC184 and pUR222, it was shown that two chromosomal fragments of 6.0 and 6.6 kb are required for the expression of ugp, whereas all the structural information is located on the 6.6 kb EcoRI fragment. A restriction map of the cloned DNA was established and the extent of ugp genes determined by Tn5 insertions. Using ugp-lacZ fusions, it could be shown that the ugp region consists of at least two different operons that are transcribed in the same direction (counterclockwise) on the E. coli chromosome.
Abstract: To facilitate construction of mutants harboring delta lac for use in gene fusion studies, strains were constructed that carry the transposon Tn10 next to the well defined lac deletion U169. This deletion can now be moved to other Escherichia coli strains in transductional or conjugational crosses by selecting resistance to tetracycline.
Abstract: Two genes, ugpA and ugpB, coding for a binding protein-dependent sn-glycerol-3-phosphate transport system, were mapped at 75.3 min on the Escherichia coli chromosome. A Tn10 insertion in ugpA resulted in loss of transport activity but still allowed the synthesis of the sn-glycerol-3-phosphate-binding protein. This Tn10 insertion was found to be linked by P1 transduction to pit, aroB, malA, asd, and livH with 2.5, 2.8, 25, 63.5, and 83% cotransduction frequency. An insertion of Mud (Ampr lac) in ugpB resulted in the loss of the binding protein. ugpB is closely linked to ugpA. It is either the structural gene for the binding protein or located proximal to it. The analysis of the crosses allowed the ordering of the markers in the clockwise direction as follows: aroB, malA, asd, ugpA, ugpB, livH, pit.
Abstract: The ugp-dependent transport system for sn-glycerol-3-phosphate has been characterized. The system is induced under conditions of phosphate starvation and in mutants that are constitutive for the pho regulon. The system does not operate in membrane vesicles and is highly sensitive toward osmotic shock. The participation of a periplasmic binding protein in the transport process can be deduced from the isolation of transport mutants that lack the binding protein. As with other binding protein-dependent transport systems, this protein appears to be necessary but not sufficient for transport activity. The isolation of mutants has become possible by selection for resistance against the toxic analog 3,4-dihydroxybutyl-1-phosphonate that is transported by the system. sn-Glycerol-3-phosphate transported via ugp cannot be used as the sole carbon source. Strains have been constructed that lack alkaline phosphatase and glycerol kinase. In addition, they are constitutive for the glp regulon and contain high levels of glycerol-3-phosphate dehydrogenase. Despite the fact that these strains exhibit high ugp-dependent transport activity for sn-glycerol-3-phosphate they are unable to grow on it as a sole source of carbon. However, when cells are grown on an alternate carbon source, (14)C label from [(14)C]sn-glycerol-3-phosphate appears in phospholipids as well as in trichloroacetic acid-precipitable material. The incorporation of (14)C label is strongly reduced when sn-glycerol-3-phosphate is the only carbon source. In the presence of an alternate carbon source, this inhibition is relieved, and sn-glycerol-3-phosphate transported by ugp can be used as the sole source of phosphate.
