Abstract: The bacterial pathogen Agrobacterium tumefaciens produces the quorum-sensing (QS) signal 3-oxo-octanoylhomoserine lactone (OC8HSL) for controlling horizontal transfer of its tumor inducing (Ti) plasmid that carries both the T-DNA and the virulence genes. Over-accumulation of OC8HSL also increases severity of plant symptoms (number of emerging tumors at infection site) by an unknown mechanism. A. tumefaciens strain C58 expresses two lactonases, AttM (BlcC) and AiiB, that cleave OC8HSL and are potential modulators of QS. Recent data highlight the direct contribution of lactonases AttM and AiiB in the control of OC8HSL level and QS-regulated functions such as conjugation of Ti plasmid and seriousness of plant symptoms. Expression of the two lactonases is regulated by different plant signals. A working model of QS in the course of the A. tumefaciens-plant host interaction is proposed and discussed.
Abstract: Bacterial periplasmic binding proteins (PBPs) and eukaryotic PBP-like domains (also called as Venus Flytrap modules) of G-protein coupled receptors are involved in extracellular GABA perception. We investigated the structural and functional basis of ligand specificity of the PBP Atu2422 which is implicated in virulence and transport of GABA in the plant pathogen Agrobacterium tumefaciens. Five high-resolution X-ray structures of Atu2422 liganded to GABA, proline, alanine, valine and that of the point mutant Atu2422-F77A liganded to a leucine were determined. Structural analysis of the ligand binding site revealed two essential residues Phe77 and Tyr275, of which implication in GABA-signalling and virulence was confirmed using A. tumefaciens cells expressing corresponding Atu2422 mutants. Phe77 residue restricts ligand specificity to alpha-amino-acids with a short lateral chain which act as antagonists of the GABA-signalling in A. tumefaciens. Tyr275 specifically interacts with the GABA gama-amino group. Conservation of these two key-residues in proteins phylogenetically related to Atu2422 brought to light a sub-family of PBPs in which all members could bind GABA and short alpha-amino-acids. This work led to the identification of a fingerprint sequence and structural features for defining PBPs that bind GABA and its competitors, and revealed their occurrence among host-interacting proteobacteria.
Abstract: This paper will exemplify molecular communications in the rhizosphere, especially between plants and bacteria, and between bacteria and bacteria. More specifically, we describe signalling pathways that allow bacteria to sense a wide diversity of plant signals, plants to respond to bacterial infection, and bacteria to coordinate gene expression at population and community level. Thereafter, we focus on mechanisms evolved by bacteria and plants to disturb bacterial signalling, and by bacteria to modulate hormonal signalling in plants. Finally, the dynamics of signal exchange and its biological significance we elaborate on the cases of Rhizobium symbiosis and Agrobacterium pathogenesis.
Abstract: The phytopathogen Agrobacterium tumefaciens C58 expresses two lactonases, AttM and AiiB. We showed that expression of the aiiB gene was controlled by agrocinopines A and B and required the agrocinopine-ABC transporter Acc, but was not affected by the level of quorum-sensing (QS) signal 3-oxo-octanoylhomoserine lactone (OC8-HSL). In the presence of agrocinopines, a constructed aiiB mutant accumulated OC8-HSL at a level 10-fold higher than that of the wild-type strain, and showed an exacerbated expression of a key QS-regulated function, conjugation of Ti plasmid (in vitro and in planta), as well as an increase of the number of emerging tumors on the host plant. The expression and acyl-HSL-degrading activity of AttM were evident in the presence of wounded tissues; however, in unwounded plant tumors, the QS-regulated functions were weakly affected in an attM mutant. By contrast, we observed that attM conferred a selective advantage in the course of colonization of plant tumors. Finally, polymerase chain reaction survey of genes attM and aiiB showed that they were not strictly conserved in the genus Agrobacterium. This work proved that the lactonases AttM and AiiB are regulated by different plant signals and are implicated in different functions in the course of the A. tumefaciens C58-host interaction.
Abstract: Plants accumulate free L-proline (Pro) in response to abiotic stresses (drought and salinity) and presence of bacterial pathogens, including the tumor-inducing bacterium Agrobacterium tumefaciens. However, the function of Pro accumulation in host-pathogen interaction is still unclear. Here, we demonstrated that Pro antagonizes plant GABA-defense in the A. tumefaciens C58-induced tumor by interfering with the import of GABA and consequently the GABA-induced degradation of the bacterial quorum-sensing signal, 3-oxo-octanoylhomoserine lactone. We identified a bacterial receptor Atu2422, which is implicated in the uptake of GABA and Pro, suggesting that Pro acts as a natural antagonist of GABA-signaling. The Atu2422 amino acid sequence contains a Venus flytrap domain that is required for trapping GABA in human GABA(B) receptors. A constructed atu2422 mutant was more virulent than the wild type bacterium; moreover, transgenic plants with a low level of Pro exhibited less severe tumor symptoms than did their wild-type parents, revealing a crucial role for Venus flytrap GABA-receptor and relative abundance of GABA and Pro in host-pathogen interaction.
Abstract: A metagenomic library of 10,121 clones, generated from bacteria inhabiting a pasture soil from France, was screened for the presence of fosmids conferring either N-acylhomoserine lactone (NAHL) synthesis or NAHL degradation ability upon their Escherichia coli host. No clone producing NAHLs was identified whereas one, containing a 31 972 bp insert in fosmid p2H8, allowed NAHL degradation. This led to the cloning and identification of a gene, qlcA, encoding an NAHL-lactonase activity, as judged by lactone-ring closure and HPLC/MS analyses of NAHL degradation products. The qlcA gene efficiently quenched quorum-sensing regulated pathogenic functions when expressed in Pectobacterium carotovorum. The QlcA peptide belongs to the family of zinc-dependent metallohydrolases and appears to be distantly related to other NAHL-lactonases discovered in Agrobacterium, Bacillus, Photorhabdus and Rhizobium. In-silico analysis of the metagenomic insert revealed the occurrence of 20 orf, with a constant GC% and codon usage, suggesting a unique bacterial origin. Nine out of these 20 orf were homologous to genes encoding biosynthesis of arginine; they were clustered with an unusual succession argFJADBCRGH. The fosmid p2H8 is able to complement the argA, argB and argC mutants in E. coli. Phylogenetic analysis showed that 9 orf out of 20 were related to sequences from members of the Acidobacteria, supporting the hypothesis that the analysed insert might be originated from an organism related to this phylum.
Abstract: Quorum sensing (QS) is a signal mediated cell-cell communication system that couples bacterial cell density to a synchronized gene expression (Fuqua et al., 1994). Mostly, in Gram negative bacteria QS signals are N-acylhomoserine lactones (NAHLs) that coordinate important functions such as virulence and pathogenicity. QS signals or the elements involved in their production or perception could be targeted to disrupt QS, a phenomenon called Quorum quenching (QQ). QQ properties (chemicals and enzymes) are naturally found in various Living organisms, like bacteria (Rhodococcus and Commamonas), plants (carrot, soybean, pea seedling, chilli, garlic etc), and animals (human sera, pork kidney tissues). Consequently, various bacterial genes encoding for NAHL degrading enzymes, like NAHL lactonases (AiiA in Bacillus, AiiB and AttM in Agrobacterium tumefaciens) and acylase/-amidohydrolase (AiiD in Ralstonia) were identified (Givskov et al., 2006). In Pectobacterium carotovorum (causal agent of soft rot diseases) production of various virulence factors and cell wall maceration enzymes is QS dependant, and relies upon successful production, stability, emission and perception of NAHLs (C-8, oxo-C8 and C-10). Disruption of QS signalling by NAHL degrading bacteria, modified bacteria or plants expressing NAHL lactonases resulted in the reduced virulence of the pathogen (Faure et al., 2007). Until recently, investigations on QQ enzymes were carried out mostly on cultivable bacteria, that represent a tiny fraction of soil and root-associated bacteria. In this study, a metagenomics approach (Handelsman, 2004) was employed to access the hidden diversity of uncultivable soil bacteria that revealed a QQ enzyme, an NAHL lactonase, in these bacteria (Riaz et al., 2008).
Abstract: A gene involved in N-acyl homoserine lactone (N-AHSL) degradation was identified by screening a genomic library of Rhodococcus erythropolis strain W2. This gene, named qsdA (for quorum-sensing signal degradation), encodes an N-AHSL lactonase unrelated to the two previously characterized N-AHSL-degrading enzymes, i.e., the lactonase AiiA and the amidohydrolase AiiD. QsdA is related to phosphotriesterases and constitutes the reference of a novel class of N-AHSL degradation enzymes. It confers the ability to inactivate N-AHSLs with an acyl chain ranging from C(6) to C(14), with or without substitution at carbon 3. Screening of a collection of 15 Rhodococcus strains and strains closely related to this genus clearly highlighted the relationship between the ability to degrade N-AHSLs and the presence of the qsdA gene in Rhodococcus. Bacteria harboring the qsdA gene interfere very efficiently with quorum-sensing-regulated functions, demonstrating that qsdA is a valuable tool for developing quorum-quenching procedures.
Abstract: Agrobacterium has evolved sophisticated strategies to perceive and transduce plant-derived cues. Recent studies have found that numerous plant signals, including salicylic acid (SA), indole-3-acetic acid (IAA) and gamma-amino butyric acid (GABA), profoundly affect Agrobacterium-plant interactions. Here we determine and compare the transcriptome profiles of Agrobacterium in response to these three plant signals. Collectively, the transcription of 103, 115 and 95 genes was significantly altered by SA, IAA and GABA respectively. Both distinct cellular responses and overlapping signalling pathways were elicited by these three plant signals. Interestingly, these three plant compounds function additively to shut off the Agrobacterium virulence programme and activate the quorum-quenching machinery. Moreover, the repression of the virulence programme by SA and IAA and the inactivation of quorum-sensing signals by SA and GABA are regulated through independent pathways. Our data indicate that these plant signals, while cross-talk in plant signalling networks, also act as cross-kingdom signals and play redundant roles in tailoring Agrobacterium regulatory pathways, resulting in intensive signalling cross-talk in Agrobacterium. Our results support the notion that Agrobacterium has evolved the ability to hijack plant signals for its own benefit. The complex signalling interplay between Agrobacterium and its plant hosts reflects an exquisite co-evolutionary balance.
Abstract: In response to infection by the pathogen Agrobacterium tumefaciens, plants synthesize several stress amino acids, including gamma-aminobutyric acid (GABA), which modulates the expression of bacterial virulence factors. GABA penetrates into the bacterial cytoplasm via an ABC transporter that is associated with the periplasmic receptor Atu2422. Mature receptor Atu2422 (without its signal peptide) was overexpressed in Escherichia coli, purified and crystallized. A complete data set was collected to 1.35 A resolution at 100 K. The crystals belonged to the monoclinic space group C2 and contained one molecule in the asymmetric unit. Molecular replacement was performed and the initial electron-density maps revealed a closed form of this Venus flytrap (VFT) receptor, suggesting the presence of an endogenous E. coli ligand.
Abstract: Among 17 molecules structurally related to N-acylhomoserine lactone (NAHL), gamma-caprolactone (GCL), 6-caprolactone (6CL) and 4-heptanolide (HTN) were found to stimulate the degradation of NAHL by bacterial communities recovered from bulk and rhizospheric soils. In the 6CL-, GCL- and HTN-treated bacterial consortia, the NAHL-degrading bacteria were more abundant than in control (mannitol-treated) consortia. Moreover, the GCL- and HTN-consortia showed a biocontrol activity against Pectobacterium atrosepticum in soft rot assays with tubers of Solanum tuberosum. When GCL was applied to hydroponic cultures of S. tuberosum, a significant increase of the ratio of NAHL-degrading bacteria among total cultivable bacteria was observed in several independent experiments. Most of these bacteria, the growth of which was stimulated by GCL amendment, were also able to use GCL as a sole carbon source. They belong to the Rhodococcus and Delftia genera. DGGE analysis revealed that GCL treatments affected the structure of bacterial communities. This work highlights the possibility to manage the NAHL-degrading bacteria in a complex environment such as rhizosphere.
Abstract: Quorum sensing is a regulatory mechanism that connects gene expression to cell density in bacteria. Amongst proteobacteria, numerous functions are regulated in this way, including pathogenicity in the Enterobacteriaceae genus Pectobacterium. In Pectobacterium, the signalling molecules involved in this regulatory process belong to the N-acyl-homoserine lactone class. Over the last 6 years, various studies have shown that these signal molecules could be degraded by other bacteria or by plant and animal cells, opening the path to innovative biocontrol strategies. This review explores the various determinants of pathogenicity in Pectobacterium and describes approaches that have been developed to quench the quorum-sensing-dependent pathogenicity in Pectobacterium. These approaches range from signal degradation by physicochemical constraints to the identification of signal-sensing inhibitors and from the identification of enzymes degrading acyl-homoserine lactones to the construction of transgenic plants tolerant to Pectobacterium.
Abstract: Forty Azospirillum strains were tested for their ability to synthesize N-acyl-homoserine lactones (AHLs). AHL production was detected for four strains belonging to the lipoferum species and isolated from a rice rhizosphere. AHL molecules were structurally identified for two strains: Azospirillum lipoferum TVV3 produces 3O,C(8)-HSL (N-3-oxo-octanoyl-homoserine-lactone), C(8)-HSL (N-3-octanoyl-homoserine-lactone), 3O,C(10)-HSL (N-3-oxo-decanoyl-homoserine-lactone), 3OH,C(10)-HSL (N-3-hydroxy-decanoyl-homoserine-lactone) and C(10)-HSL (N-3-decanoyl-homoserine-lactone), whereas A. lipoferum B518 produced 3O,C(6)-HSL (N-3-oxo-hexanoyl-homoserine-lactone), C(6)-HSL (N-3-hexanoyl-homoserine-lactone), 3O,C(8)-HSL, 3OH,C(8)-HSL and C(8)-HSL. Genes involved in AHL production were characterized for A. lipoferum TVV3 by generating a genomic library and complementing an AHL-deficient strain with sensor capabilities. Those genes, designated alpI and alpR, were found to belong to the luxI and luxR families, respectively. When cloned in a suitable heterologous host, alpI and alpR could direct the synthesis of the five cognate AHLs present in A. lipoferum TVV3. These two adjacent genes were found to be located on a 85 kb plasmid. Southern hybridization experiments with probes alpI/R indicated that genes involved in AHL production in the three other AHL-producing strains were not closely related to alpI and alpR. This study demonstrates that AHL-based quorum-sensing is not widespread among the genus Azospirillum and could be found only in some A. lipoferum strains.
Abstract: The concentration of GABA increases rapidly in wounded plant tissues, but the implication of this GABA pulse for plant-bacteria interactions is not known. Here we reveal that GABA stimulated the inactivation of the N-(3-oxooctanoyl)homoserine lactone (OC8-HSL) quorum-sensing signal (or "quormone") by the Agrobacterium lactonase AttM. GABA induced the expression of the attKLM operon, which was correlated to a decrease in OC8-HSL concentration in Agrobacterium tumefaciens cultures. The Agrobacterium GABA transporter Bra was required for this GABA-signaling pathway. Furthermore, transgenic tobacco plants with elevated GABA levels were less sensitive to A. tumefaciens C58 infection than were wild-type plants. These findings indicate that plant GABA may modulate quorum sensing in A. tumefaciens, thereby affecting its virulence on plants. Whereas GABA is an essential cell-to-cell signal in eukaryotes, here we provide evidence of GABA acting as a signal between eukaryotes and pathogenic bacteria. The GABA signal represents a potential target for the development of a strategy to control the virulence of bacterial pathogens.
Abstract: A Gram-negative, non-sporulating, rod-shaped, motile bacterium, with a single polar flagellum, designated strain PsJN(T), was isolated from surface-sterilized onion roots. This isolate proved to be a highly effective plant-beneficial bacterium, and was able to establish rhizosphere and endophytic populations associated with various plants. Seven related strains were recovered from Dutch soils. Based on 16S rRNA gene sequence data, strain PsJN(T) and the Dutch strains were identified as representing a member of the genus Burkholderia, as they were closely related to Burkholderia fungorum (98.7 %) and Burkholderia phenazinium (98.5 %). Analysis of whole-cell protein profiles and DNA-DNA hybridization experiments confirmed that all eight strains belonged to a single species. Strain PsJN(T) had a DNA G+C content of 61.0 mol%. Only low levels of DNA-DNA hybridization to closely related species were found. Qualitative and quantitative differences in fatty acid composition between strain PsJN(T) and closely related species were identified. The predominant fatty acids in strain PsJN(T) were 16 : 0, 18 : 1omega7c and summed feature 3 (comprising 16 : 1omega7c and/or iso-15 : 0 2-OH). Isolate PsJN(T) showed high 1-aminocyclopropane-1-carboxylate deaminase activity and is therefore able to lower the ethylene level in a developing or stressed plant. Production of the quorum-sensing signal compound 3-hydroxy-C8-homoserine lactone was detected. Based on the results of this polyphasic taxonomic study, strain PsJN(T) and the seven Dutch isolates are considered to represent a single, novel species, for which the name Burkholderia phytofirmans sp. nov. is proposed. The type strain is strain PsJN(T) (=LMG 22146(T) = CCUG 49060(T)).
Abstract: In Gram-negative bacteria, quorum-sensing (QS) communication is mostly mediated by N-acyl homoserine lactones (N-AHSL). The diversity of bacterial populations that produce or inactivate the N-AHSL signal in soil and tobacco rhizosphere was investigated by restriction fragment length polymorphism (RFLP) analysis of amplified 16S DNA and DNA sequencing. Such analysis indicated the occurrence of N-AHSL-producing strains among the alpha-, beta- and gamma-proteobacteria, including genera known to produce N-AHSL (Rhizobium, Sinorhizobium and Pseudomonas) and novel genera with no previously identified N-AHSL-producing isolates (Variovorax, Sphingomonas and Massilia). The diversity of N-AHSL signals was also investigated in relation to the genetic diversity of the isolates. However, N-AHSL-degrading strains isolated from soil samples belonged to the Bacillus genus, while strains isolated from tobacco rhizospheres belonged to both the Bacillus genus and to the alpha subgroup of proteobacteria, suggesting that diversity of N-AHSL-degrading strains may be modulated by the presence of the tobacco plant. Among these rhizospheric isolates, novel N-AHSL-degrading genera have been identified (Sphingomonas and Bosea). As the first simultaneous analysis of both N-AHSL-degrading and -producing bacterial communities in a complex environment, this study revealed the coexistence of bacterial isolates, belonging to the same genus or species that may produce or degrade N-AHSL.
Abstract: The half-life of N-hexanoyl-l-homoserine lactone (C6-HSL) was determined under various pH and temperature conditions, and in several plant environments. C6-HSL was sensitive to alkaline pH, a process that was also temperature-dependent. In addition, C6-HSL disappeared from plant environments, i.e. axenic monocot and dicot plants cultivated under gnotobiotic, hydroponic conditions, albeit with variable kinetics. The disappearance was rapid at the root system of legume plants such as clover or Lotus, and slow or non-existent at the root system of monocots such as wheat or corn. These variable kinetics were not dependent upon pH changes that may have affected the growth media of the plants. Furthermore, C6-HSL did not accumulate in the plant, and the plant did not produce inhibitors of the C6-HSL signal. HPLC analyses revealed that C6-HSL disappeared from the media, and hence, Lotus exhibited a natural C6-HSL inactivating ability. This ability was not specific for C6-HSL and allowed the degradation of other N-acyl-homoserine lactones such as 3-oxo-C6-HSL, 3-oxo-octanoyl-HSL and 3-oxo-decanoyl-HSL. Preliminary investigation revealed that the inactivating ability is temperature-dependant and possibly of enzymatic origin.
Abstract: Agrobacterium tumefaciens C58 communicates using N-acyl-homoserine lactones (acyl-HSL) and contains two lactonase-encoding genes, attM and aiiB, the products of which are capable of inactivating the acyl-HSL signal. In A. tumefaciens A6, the expression of the attKLM operon is controlled by the transcriptional repressor encoded by an adjacent gene, attJ. An attJ::Tn5 mutant does not accumulate acyl-HSL because of the constitutive expression of the lactonase AttM, the activity of which inactivates acyl-HSL. In this work, the attKLM operon of A. tumefaciens C58 was shown to be involved in an assimilative pathway of gamma-butyrolactone (GBL), gamma-hydroxybutyrate (GHB), and succinate semialdehyde (SSA), in which AttM and AttL are key enzymes for GBL and GHB assimilation. The expression of the attKLM promoter was activated in the presence of GBL, GHB, and SSA. Under these conditions, A. tumefaciens C58 did not accumulate the acyl-HSL that it naturally synthesizes, and also became able to inactivate exogenous acyl-HSL signals. Therefore, in A. tumefaciens C58, the assimilative pathway of gamma-butyrolactone interferes with the acyl-HSL signaling.
Abstract: INTRODUCTION: Soil micro-organisms have evolved functions that allow them to withstand the strong competition for survival that characterizes most of their habitats. The production of antibiotic or antifungal compounds is one of these mechanisms. The relevant molecules often exhibit valuable therapeutic properties. EXEGESIS: Chromobacterium violaceum is a soil-borne bacterium producing a characteristic antibiotic termed violacein. It is part of a series of compounds released by C. violaceum to oppose competitors and predators in the soil and water environments. Violacein, and one of these compounds, i.e. structure FR901228, exhibit antiparasitic and antitumoral activities of potential medical interest. Genes involved in the synthesis of these compounds are available, the genome sequence of C. violaceum (strain ATCC 12472) being published. CONCLUSIONS: The above example, involving Chromobacterium, is not an exception: soil constitutes a reservoir of molecules, enzymatic activities and micro-organisms of biological interest, the study of which will undoubtedly lead to developments in fields as diverse as agronomy or animal and human therapeutics.
Abstract: A tobacco line genetically modified to produce two N-acyl homoserine lactones and its non-transformed parental line were grown in non-sterile soil. Microbial populations inhabiting the bulk soil, and those colonizing the root system of the two tobacco lines, were analyzed using cultivation-independent (phospholipid fatty acid and denaturing gradient gel electrophoresis) and cultivation-based assays. The cell density of total cultivable bacteria, fluorescent pseudomonads, sporulated, and thermotolerant bacteria was also determined in a time-course experiment (15 weeks). A possible "rhizosphere effect" related to the development of the plant was seen. However, no dissimilarities in cell population densities or population ratios of the microbial groups were detected in the rhizosphere of the two plant lines. Similarly, bacterial communities that either produced N-acyl homoserine lactone or degraded the signal hexanoyl homoserine lactone were enumerated from the two plant lines. No noticeable differences were evidenced from one plant genotype to the other. Whilst the transgenic plants released detectable amounts of the quorum-sensing signal molecules and efficiently cross-talked with the surrounding microbial populations, the bias generated by these signals in the reported experimental conditions therefore appears to remain weak, if not non-existent.
Abstract: Erwinia carotovora subsp. atroseptica is responsible for potato blackleg disease in the field and tuber soft rot during crop storage. The process leading to the disease occurs in two phases: a primary invasion step followed by a maceration step. Bacteria-to-bacteria communication is associated with a quorum-sensing (QS) process based on the production of N-acylhomoserine lactones (HSL). The role of HSL throughout plant infection was analyzed. To this purpose, HSL produced by a specific E. carotovora subsp. atroseptica wild-type strain, which was particularly virulent on potato, were identified. A derivative of this strain that expressed an HSL lactonase gene and produced low amounts of HSL was generated. The comparison of these strains allowed the evaluation of the role of HSL and QS in disease establishment and development. Bacterial growth and motility; activity of proteins secreted by type I, II, and III systems; and hypersensitive and maceration reactions were evaluated. Results indicated that HSL production and QS regulate only those traits involved in the second stage of the host plant infection (i.e., tissue maceration) and hypersensitive response in nonhost tobacco plants. Therefore, the use of QS quenching strategies for biological control in E. carotovora subsp. atroseptica cannot prevent initial infection and multiplication of this pathogen.
Abstract: Genomic scans of clones isolated from long-term stab cultures of Escherichia coli K-12 showed the loss of two large segments of the genome, with each lost segment being approximately 20 kb long. A detailed analysis of one of the deletions, located between 5.4 and 5.9 min, revealed that similar deletions had arisen in several other stab cultures. All deletions of this type exhibited a right terminus ending precisely at an IS5A element and a left terminus that varied over an approximately 5-kb range but was bordered in all but two cases by sequences belonging to the preferred consensus target sequence for IS5, YTAR. The ubiquity of such deletions in independent stab cultures and the increase in their frequency over time argue that they have a selective advantage. It is speculated that the loss of the crl locus is responsible for the selective advantage of the deletions.
Abstract: A phylogenetic analysis of the glycoside hydrolases of family 3 (GH3s) was conducted in order to infer particular trends in its evolution: functional specialization, gene transfer events, gene duplications and paralogous evolution, and gene deletions. The phylogenetic analysis of GH3s revealed six clusters, i.e., A, B, C, D, E, and F that could fit the definition of 3 sub-families, i.e., AB, AB' and AB". While the sub-families AB' and AB" contain a single cluster, F and E, respectively, the AB sub-family is sub-divided into four clusters. Global analysis of the GH3 phylogenetic tree suggests a primary burst of amplification of the GH3s that might have led to these sub-families. Specializations, gene transfers, and gene duplications among each of these sub-families and phylogenetic clusters might then have occurred and have been inferred. The fine comparison of the enzyme properties and phylogenetic relationships of GH3s allowed to detect common functional groups that belong to the same cluster (D, E or F), or sub-cluster (A1, A2 or B2). The prokaryotic and eukaryotic beta-xylosidases and beta-glucosidases belong to the AB and AB' sub-families, and the N-acetylglucosaminidases are in sub-family AB" (in cluster E). In some instances (B1, B2, C1, C2, and C3), the lack of data and/or the high heterogeneity of the hydrolytic properties did not allow to infer a particular link between an enzyme functional group and a phylogenetic cluster, suggesting the emergence of some highly specialized GH3s.
Abstract: Bacteria degrading the quorum-sensing (QS) signal molecule N-hexanoylhomoserine lactone were isolated from a tobacco rhizosphere. Twenty-five isolates degrading this homoserine lactone fell into six groups according to their genomic REP-PCR and rrs PCR-RFLP profiles. Representative strains from each group were identified as members of the genera Pseudomonas, Comamonas, Variovorax and Rhodococcus: all these isolates degraded N-acylhomoserine lactones other than the hexanoic acid derivative, albeit with different specificity and kinetics. One of these isolates, Rhodococcus erythropolis strain W2, was used to quench QS-regulated functions of other microbes. In vitro, W2 strongly interfered with violacein production by Chromobacterium violaceum, and transfer of pathogenicity in Agrobacterium tumefaciens. In planta, R. erythropolis W2 markedly reduced the pathogenicity of Pectobacterium carotovorum subsp. carotovorum in potato tubers. These series of results reveal the diversity of the QS-interfering bacteria in the rhizosphere and demonstrate the validity of targeting QS signal molecules to control pathogens with natural bacterial isolates.
Abstract: The Agrobacterium tumefaciens C58 genome contains three putative N-acyl homoserine lactone (acyl-HSL) hydrolases, which are closely related to the lactonase AiiA of BACILLUS: When expressed in Escherichia coli, two of the putative acyl-HSL hydrolases, AttM and AiiB, conferred the ability to degrade acyl-HSLs on the host. In Erwinia strain 6276, the lactonases reduced the endogenous acyl-HSL level and the bacterial virulence in planta.
Abstract: The CelA beta-glucosidase of Azospirillum irakense, belonging to glycosyl hydrolase family 3 (GHF3), preferentially hydrolyzes cellobiose and releases glucose units from the C(3), C(4), and C(5) oligosaccharides. The growth of a DeltacelA mutant on these cellobiosides was affected. In A. irakense, the GHF3 beta-glucosidases appear to be functional alternatives for the GHF1 beta-glucosidases in the assimilation of beta-glucosides by other bacteria.
Abstract: The presence of two insertion sequences, IS406 and IS407, was tested by polymerase chain reaction (PCR) amplification in 25 strains representing 15 Burkholderia species and the close relative Ralstonia pickettii. A total of 50% of the 25 strains contained at least one of the two insertion sequences (ISs) and a statistically significant correlation was found between the occurrences of IS406 and IS407. Moreover, PCR-RFLP studies of the amplified fragments showed that IS406 is largely conserved among all the strains tested, whereas IS407 is rather polymorphic. Transposition activity was studied in Burkholderia vietnamiensis TVV75, using the pGBG1 target plasmid. This entrapping plasmid permitted the isolation and characterization of three active IS, able to activate the plasmid-borne tetA gene after transposition. Sequencing permitted the identification of these mobile genetic elements as isoforms of IS402, IS407 and IS1416. PCR amplification products provided IS probes, which were used to determine the copy-numbers of IS402, IS407 and IS1416 in the genome of B. vietnamiensis TVV75, by Southern blotting. Copy numbers are 12, 3 and 11 respectively. To our knowledge, this is the first description of active insertion sequences in B. vietnamiensis.
Abstract: Some bacteria of the soil microflora or of the digestive systems of mammals can grow on aryl beta-glucosides as sole carbon sources. The bgl operon of E. coli is the paradigm for such a catabolic pathway. In Azospirillum irakense, the two adjacent genes salAB encode beta-glucosidases which are required for salicin assimilation. In this report, we analyse the sequence of salC, the last gene to be identified in the salCAB operon and investigate the phylogenetic origins of the sal and bgl genes. The results suggest an alternative model for the assimilation of aryl beta-glucosides in bacteria.
Abstract: The salAB genes of Azospirillum irakense KBC1, which encode two aryl-beta-glucosidases, are required for growth on salicin. In the 4-kb region upstream of the salAB genes, two additional genes, salC and salR, were identified. SalC shows characteristics of the outer membrane receptors in the FepA/FhuA family. The salC AB genes are transcribed as a polycistronic mRNA. The salR gene encodes a protein homologous to the LacI/GalR family of transcriptional repressors. Expression of the sal operon, measured by means of a salC-gusA translational fusion in A. irkense KBC1, requires the presence of aryl-beta-glucosides such as arbutin and salicin. Expression is markedly enhanced when a simple carbon source, like glucose, cellobiose or malate, is added to the medium. In a salR mutant, expression of the salC-gusA fusion does not require an aryl-beta-glucoside inducer. Expression of a salR-gusA fusion is constitutive. The product of arbutin hydrolysis (hydroquinone) partly inhibits the expression of a salC-gusA fusion in arbutin- or salicin-containing minimal medium. This effect is independent of SalR. Salicylalcohol, the hydrolysis product of salicin, also partly inhibits salC expression in a SalR-independent fashion, but only in salicin-containing minimal medium.
Abstract: Plasmid pGBG1 was constructed to isolate mobile genetic elements in a wide variety of gram-negative bacteria. The mutation target, carried on a broad-host-range vector, allows positive selection for tetracycline resistance. In tests using several gram-negative bacteria we could detect transposition events of either insertion sequences or transposons. A new insertion sequence (IS) element was identified in Ralstonia eutropha.
Abstract: The rhizosphere nitrogen-fixing bacterium Azospirillum irakense KBC1 is able to grow on pectin and beta-glucosides such as cellobiose, arbutin, and salicin. Two adjacent genes, salA and salB, conferring beta-glucosidase activity to Escherichia coli, have been identified in a cosmid library of A. irakense DNA. The SalA and SalB enzymes preferentially hydrolyzed aryl beta-glucosides. A Delta(salA-salB) A. irakense mutant was not able to grow on salicin but could still utilize arbutin, cellobiose, and glucose for growth. This mutant could be complemented by either salA or salB, suggesting functional redundancy of these genes in salicin utilization. In contrast to this functional homology, the SalA and SalB proteins, members of family 3 of the glycosyl hydrolases, show a low degree of amino acid similarity. Unlike SalA, the SalB protein exhibits an atypical truncated C-terminal region. We propose that SalA and SalB are representatives of the AB and AB' subfamilies, respectively, in glycosyl hydrolase family 3. This is the first genetic implication of this beta-glucosidase family in the utilization of beta-glucosides for microbial growth.
Abstract: In order to develop a reliable and specific tool for the detection of Azospirillum lipoferum CRT1, randomly cloned DNA fragments from this strain were used as hybridization probes to differentiate A. lipoferum CRT1 from 29 closely related Azospirillum strains. Two cloned fragments hybridizing only with DNA from A. lipoferum CRT1 (CRT1-5 and CRT1-7) were considered as specific probes of this strain. CRT1-7 fragment (1.4 kb) was further tested for purity control of the inoculant Azogreen-m by colony hybridization. The sequence of the CRT1-7 fragment has been determined and compared with those present in databases: no significant similarity with other sequences was detected. This probe permitted us to count specifically A. lipoferum CRT1 cells on maize roots during a field trial. During the first two weeks, A. lipoferum CRT1 remained at 107 CFU plant−1. Afterwards, bacterial concentration sharply decreased. We could not detect any CRT1 cells on maize roots 28 days after sowing. Concurrently, three plant parameters were estimated (plant height, primary root length and root fresh weight). The results showed that A. lipoferum CRT1 growth promotion effect began early on (from day 14) in plant development and increased in spite of a rapid decrease of bacterial density.
Abstract: A new, simple method for isolating Azospirillum strains from the roots and the rhizosphere of rice is described. The method is based on the capacity of Azospirillum, a nitrogen-fixing bacterial genus, to grow in nutrient-deficient liquid media such as distilled water, KCl (8.5 g/L), or soil extract medium. The enrichment efficiency of the deficient media was compared with classical N-free malate medium. Serial dilutions from 10−1 to 10−10 of rice root macerates and rhizosphere soil were incubated at 28 °C in these media without agitation for 15 days. Pellicle formation was regularly examined under a microscope to detect the spiral motility of Azospirillum cells. Tubes with putative azospirilla were plated on nutrient agar. Most probable numbers of Azospirillum in root macerates were 2 × 103/g dry root weight in KCl, 2 × 104/g dry root weight in H2O, 2 × 106/g dry root weight in malate, and 3 × 106/g dry root weight in the soil extract medium. Soil extract medium repressed the most abundant bacterial populations and facilitated isolation of azospirilla from a population representing less than 0.001% of the total microflora. The isolates had vibrioid to S-shaped cells with one polar flagellum when grown in liquid medium and fixed nitrogen according to the acetylene reduction assay. Their identification as Azospirillum lipoferum was based on biochemical tests and hybridization with a 16S rRNA probe specific for this species and was confirmed by the detection of a 150-MDa plasmid.
Abstract: Abstract: Azospirillum lipoferum 4B and non-motile A. lipoferum 4T have been simultaneously isolated from rice rhizosphere at the same frequency. A. lipoferum 4T showed stable morphological and metabolic traits which are atypical for A. lipoferum species such as lack of motility, carbohydrate metabolism and laccase activity. Inoculation experiments showed that A. lipoferum 4T, but not A. lipoferum 4B, needed rice roots to stabilize in sterile soil. Both strains were able to colonize efficiently rice roots (108 cfu g−1 fresh roots) but motile form 4B remained dominant. In spite of their phenotypical differences, A. lipoferum 4B and 4T co-existed without exclusion in sterile soil (planted or not) and rice rhizosphere. Inoculation of rice roots with A. lipoferum 4B showed that rice rhizosphere enhanced the frequency of appearance of stable non-motile forms (40%). This percentage was weaker in plantlet growth medium (4%). However, these non-motile bacteria kept the same biochemical traits than the motile parental strain 4B (carbohydrates metabolism, laccase activity).
Abstract: Activity of bacterial Azospirillum lipoferum laccase on phenolic derivatives was studied by spectrophotometry, HPLC and GC/MS. Phenolic compounds of the syringic type (aldehyde, acid or acetophenone) were transformed into 2,6-dimethoxy-1,4-benzoquinone (2,6-DMBQ). Comparison has been made with the fungal Pyricularia oryzae laccase. Transformation of other phenolic acid derivatives, related to lignin metabolism, have been studied with both bacterial and fungal enzymes, using spectrophotometric analysis.
Abstract: Partial sequences of the 16S rRNA molecules of nine strains belonging to four Azospirillum species were used to design species-specific oligonucleotide probes. Azospirillum strains sequences were analyzed and three homologous fragments containing 16 nucleotides were determined. These three probes were found to be characteristic of A. lipoferum (Al), A. irakense (Ai), and A. brasilense/amazonense species (Aba) and of few nontarget organisms. The specificity of these three probes was tested both against sequences in the GenBank data base and in numerous colony hybridization experiments. As a few non-target organisms hyridized with the different Azospirillum probes, the use of these probes in bulk soil hybridization is not permitted. However, their use together with specific isolation techniques is validated.
Abstract: Azospirillum lipoferum and Pyricularia oryzae laccases were compared, using several substrates and inhibitors. Sixteen phenolic or nonphenolic compounds were found to be substrates of both fungal and bacterial laccases. In the presence of different phenol oxidase inhibitors, P. oryzae and A. lipoferum laccase activities had similar properties.
Abstract: DNA/DNA hybridization, plasmid content and partial 16S rDNA sequence were determined to confirm the relationship between two Azospirillum strains, 4B and 4T, isolated from the same rice rhizosphere. The partial 16S rDNA sequence was determined for 9 strains belonging to 5 Azospirillum species which included Azospirillum lipoferum strains 4B and 4T, and was compared to a set of ribosomal sequences from other bacteria of the alpha subdivision of the Proteobacteria. Four Azospirillum species sequences were found to form a coherent group, whereas A. halopraeferens was found to be more divergent. The five Azospirillum species were closely related to Magnetospirillum and to a lesser extent to Rhodospirillum, with which they share some morphological features. The partial 16S rDNA sequences of the two strains 4B and 4T were identical and confirmed the closeness of these two strains. Plasmid content and DNA/DNA hybridization data (S1 nuclease and delta Tm) grouped Azospirillum strains 4B and 4T with the other strains of species Azospirillum lipoferum (USA5a, 59b, BR17). Other biochemical and genetic characters confirmed the grouping of Azospirillum strains 4B and 4T together with A. lipoferum.
Abstract: Azospirillum lipoferum 4T has original properties such as nonmotility, melanin synthesis, and laccase activity. Following random Tn5 mutagenesis in A. lipoferum 4T, we obtained 10 mutants which were affected in melanization and laccase activity. The class 1 mutants, with intermediate levels of laccase activity, showed some coloration; the class 2 mutants, which were completely negative for laccase activity, were also colorless. The Tn5 localization on the chromosome or on the cryptic 300-MDa plasmid of A. lipoferum 4T was proven by hybridization for all class 1 mutants or for most class 2 mutants, respectively.
Abstract: Abstract In non-motile forms of Azospirillum lipoferum isolated from the rhizosphere of rice, polyphenol oxidase activity was observed which correlated with production of a dark-brown pigment. Using a combination of substrate/inhibitor specificity tests, intracellular enzyme extracts of non-motile strains were clearly demonstrated to have a laccase activity by oxidising various o- and p-diphenols. This work is the first report on laccase activity in Azospirillum.
