Abstract: A new cloning system is described, which allows the construction of large-insert fosmid libraries in Escherichia coli and the transfer of the recombinant libraries to the extreme thermophile Thermus thermophilus via natural transformation. Libraries are established in the thermophilic host by site-specific chromosomal insertion of the recombinant fosmids via single crossover or double crossover recombination at the T. thermophilus pyr locus. Comparative screening of a fosmid library constructed from genomic DNA from the thermophilic spirochaete, Spirochaeta thermophila, for clones expressing thermoactive xylanase activity revealed that 50% of the fosmids that conferred xylanase activity upon the corresponding T. thermophilus transformants did not give rise to xylanase-positive E. coli clones, indicating that significantly more S. thermophila genes are functionally expressed in T. thermophilus than in E. coli. The novel T. thermophilus host/vector system may be of value for the construction and functional screening of recombinant DNA libraries from individual thermophilic or extremely thermophilic organisms as well as from complex metagenomes isolated from thermophilic microbial communities.
Abstract: Thermoacidophiles are prokaryotic microorganisms with the stunning capability to survive and multiply at extremely low pH and simultaneously at high temperatures. The mechanisms by which these organisms, exclusively members of the Archaea, cope with their harsh surroundings are poorly understood. The genome sequences of several representatives of the thermoacidophilic genera Picrophilus, Thermoplasma and Sulfolobus have recently become available. Genome-wide comparison has revealed a number of features as possible facets of the overall acidophilic survival strategy of the most thermoacidophilic organisms known, such as a high ratio of secondary over primary transport systems, the composition of the respiratory chain, and the frequent genetic input via lateral gene transfer (LGT) during evolution.
Abstract: The genes encoding a putative alpha-glucosidase (aglA) and an alpha-mannosidase (manA) appear to be physically clustered in the genome of the extreme acidophile Picrophilus torridus, a situation not found previously in any other organism possessing aglA or manA homologs. While archaeal alpha-glucosidases have been described, no alpha-mannosidase enzymes from the archaeal kingdom have been reported previously. Transcription start site mapping and Northern blot analysis revealed that despite their colinear orientation and the small intergenic space, the genes are independently transcribed, both producing leaderless mRNA. aglA and manA were cloned and overexpressed in Escherichia coli, and the purified recombinant enzymes were characterized with respect to their physicochemical and biochemical properties. AglA displayed strict substrate specificity and hydrolyzed maltose, as well as longer alpha-1,4-linked maltooligosaccharides. ManA, on the other hand, hydrolyzed all possible linkage types of alpha-glycosidically linked mannose disaccharides and was able to hydrolyze alpha3,alpha6-mannopentaose, which represents the core structure of many triantennary N-linked carbohydrates in glycoproteins. The probable physiological role of the two enzymes in the utilization of exogenous glycoproteins and/or in the turnover of the organism's own glycoproteins is discussed.
Abstract: In Picrophilus torridus, a euryarchaeon that grows optimally at 60 degrees C and pH 0.7 and thus represents the most acidophilic thermophile known, glucose oxidation is the first proposed step of glucose catabolism via a nonphosphorylated variant of the Entner-Doudoroff pathway, as deduced from the recently completed genome sequence of this organism. The P. torridus gene for a glucose dehydrogenase was cloned and expressed in Escherichia coli, and the recombinant enzyme, GdhA, was purified and characterized. Based on its substrate and coenzyme specificity, physicochemical characteristics, and mobility during native PAGE, GdhA apparently resembles the main glucose dehydrogenase activity present in the crude extract of P. torridus DSM 9790 cells. The glucose dehydrogenase was partially purified from P. torridus cells and identified by MS to be identical with the recombinant GdhA. P. torridus GdhA preferred NADP+ over NAD+ as the coenzyme, but was nonspecific for the configuration at C-4 of the sugar substrate, oxidizing both glucose and its epimer galactose (Km values 10.0 and 4.5 mM, respectively). Detection of a dual-specific glucose/galactose dehydrogenase points to the possibility that a 'promiscuous' Entner-Doudoroff pathway may operate in P. torridus, similar to the one recently postulated for the crenarchaeon Sulfolobus solfataricus. Based on Zn2+ supplementation and chelation experiments, the P. torridus GdhA appears to contain structurally important zinc, and conserved metal-binding residues suggest that the enzyme also contains a zinc ion near the catalytic site, similar to the glucose dehydrogenase enzymes from yeast and Thermoplasma acidophilum. Strikingly, NADPH, one of the products of the GdhA reaction, is unstable under the conditions thought to prevail in Picrophilus cells, which have been reported to maintain the lowest cytoplasmic pH known (pH 4.6). At the optimum growth temperature for P. torridus, 60 degrees C, the half-life of NADPH at pH 4.6 was merely 2.4 min, and only 1.7 min at 65 degrees C (maximum growth temperature). This finding suggests a rapid turnover of NADPH in Picrophilus.
Abstract: The euryarchaea Picrophilus torridus and Picrophilus oshimae are able to grow around pH 0 at up to 65 degrees C, thus they represent the most thermoacidophilic organisms known. Several features that may contribute to the thermoacidophilic survival strategy of P. torridus were deduced from analysis of its 1.55-megabase genome. P. torridus has the smallest genome among nonparasitic aerobic microorganisms growing on organic substrates and simultaneously the highest coding density among thermoacidophiles. An exceptionally high ratio of secondary over ATP-consuming primary transport systems demonstrates that the high proton concentration in the surrounding medium is extensively used for transport processes. Certain genes that may be particularly supportive for the extreme lifestyle of P. torridus appear to have been internalized into the genome of the Picrophilus lineage by horizontal gene transfer from crenarchaea and bacteria. Finally, it is noteworthy that the thermoacidophiles from phylogenetically distant branches of the Archaea apparently share an unexpectedly large pool of genes.
