Thermus thermophilus HB27 is an extremely thermophilic, halotolerant bacterium, which was originally isolated from a natural thermal environment in Japan. This organism has considerable biotechnological potential; many thermostable proteins isolated from members of the genus Thermus are indispensable in research and in industrial applications. We present here the complete genome sequence of T. thermophilus HB27, the first for the genus Thermus. The genome consists of a 1,894,877 base pair chromosome and a 232,605 base pair megaplasmid, designated pTT27. The 2,218 identified putative genes were compared to those of the closest relative sequenced so far, the mesophilic bacterium Deinococcus radiodurans. Both organisms share a similar set of proteins, although their genomes lack extensive synteny. Many new genes of potential interest for biotechnological applications were found in T. thermophilus HB27. Candidates include various proteases and key enzymes of other fundamental biological processes such as DNA replication, DNA repair and RNA maturation.

For the uropathogenic Escherichia coli strain 536 (O6:K15:H31), the DNA sequences of three pathogenicity islands (PAIs) (PAI I 536 to PAI III 536 ) and their flanking regions (about 270 kb) were determined to further characterize the virulence potential of this strain. PAI I 536 to PAI III 536 exhibit features typical of PAIs, such as (i) association with tRNA-encoding genes; (ii) G؉C content differing from that of the host genome; (iii) flanking repeat structures; (iv) a mosaic-like structure comprising a multitude of functional, truncated, and nonfunctional putative open reading frames (ORFs) with known or unknown functions; and (v) the presence of many fragments of mobile genetic elements. PAI I 536 to PAI III 536 range between 68 and 102 kb in size. Although these islands contain several ORFs and known virulence determinants described for PAIs of other extraintestinal pathogenic E. coli (ExPEC) isolates, they also consist of as-yet-unidentified ORFs encoding putative virulence factors. The genetic structure of PAI IV 536 , which represents the core element of the so-called high-pathogenicity island encoding a siderophore system initially identified in pathogenic yersiniae, was further characterized by sample sequencing. For the first time, multiple PAI sequences (PAI I 536 to PAI IV 536 ) in uropathogenic E. coli were studied and their presence in several wild-type E. coli isolates was extensively investigated. The results obtained suggest that these PAIs or at least large fragments thereof are detectable in other pathogenic E. coli isolates. These results support our view that the acquisition of large DNA regions, such as PAIs, by horizontal gene transfer is an important factor for the evolution of bacterial pathogens.Pathogenicity islands (PAIs), as a distinct type of genetic element, were described for the first time for uropathogenic Escherichia coli strain 536 (O6:K15:H31) (2, 17), which is one of the model organisms of extraintestinal pathogenic E. coli (ExPEC) used for studies on ExPEC pathogenesis and the evolution of bacterial pathogens. The PAI type of genetic elements is characterized by a large size (Ͼ10 kb), the presence of virulence-associated genes, frequent association with tRNAencoding genes or other att sites for temperate bacteriophages, and a GϩC content different from that of the rest of the chromosome. These elements are frequently flanked by repeat structures and carry many fragments of other mobile and accessory genetic elements, such as bacteriophages, plasmids, and insertion sequence (IS) elements.

Intraclonal genome diversity of Pseudomonas aeruginosa was studied in one of the most diverse mosaic regions of the P. aeruginosa chromosome. The ca. 110-kb large hypervariable region located near the lipH gene in two members of the predominant P. aeruginosa clone C, strain C and strain SG17M, was sequenced. In both strains the region consists of an individual strain-specific gene island of 111 (strain C) or 106 (SG17M) open reading frames (ORFs) and of a 7-kb stretch of clone C-specific sequence of 9 ORFs. The gene islands are integrated into conserved tRNAGly genes and have a bipartite structure. The first part adjacent to the tRNA gene consists of strain-specific ORFs encoding metabolic functions and transporters, the majority of which have homologs of known function in other eubacteria, such as hemophores, cytochrome c biosynthesis, or mercury resistance. The second part is made up mostly of ORFs of yet-unknown function. Forty-seven of these ORFs are mutual homologs with a pairwise amino acid sequence identity of 35 to 88% and are arranged in the same order in the two gene islands. We hypothesize that this novel type of gene island derives from mobile elements which, upon integration, endow the recipient with strain-specific metabolic properties, thus possibly conferring on it a selective advantage in its specific habitat.Genetic variability within bacterial species can be the result of nucleotide substitutions, intragenomic reshuffling, and acquisition of DNA sequences from another organism (3). The considerable impact of the last strategy, termed horizontal gene transfer, on microbial evolution and its integral role in the diversification and speciation of the bacteria has become apparent from recent analyses based on the growing pool of genomic sequence information (7,18,23,28). Prominent examples are the pathogenicity islands of many obligatory pathogens (14). These chromosomally encoded regions typically contain large clusters of virulence genes not present in closely related nonpathogenic strains and can, upon integration, transform a benign organism into a pathogen. Whereas the molecular mechanism of chromosomal integration has been resolved for some conjugative transposons and bacteriophages and details about the transmissibility of conjugative plasmids are well known, the evolution and mobility of gene islands remain obscure (14). Often these DNA blocks are integrated adjacent to or within tRNA genes, and some contain a phage-related integrase gene near one end, suggesting that gene islands may have been generated by a phage or by a plasmid with integrative functions (14, 42). Nevertheless, the comparative sequence analysis of gene islands so far have not pointed to any common genetic repertoire that confers transmission and acquisition.The gram-negative bacterium Pseudomonas aeruginosa is ubiquitously distributed in aquatic and soil habitats, and it is an opportunistic pathogen for plants, animals, and humans (38). No correlation between certain P. aeruginosa clones and disease habitats or environm...

The F 420 H 2 dehydrogenase is part of the energy conserving electron transport system of the methanogenic archaeon Methanosarcina mazei Gö 1. Here it is shown that cofactor F 420 H 2 -dependent reduction of 2-hydroxyphenazine as catalyzed by the membrane-bound enzyme is coupled to proton translocation across the cytoplasmic membrane, exhibiting a stoichiometry of 0.9 H Methanosarcina mazei strain Gö1 is a strictly anaerobic methanogenic archaeon that converts a limited number of simple substrates (H 2 ϩ CO 2 , methanol, methylamines, and acetate) to methane. 2-methylthioethanesulfonate is the central intermediate in all methanogenic pathways and is reductively demethylated to methane catalyzed by the 2-methylthioethanesulfonate reductase. The two electrons required for the reduction are derived from 7-mercaptoheptanoylthreonine phosphate, resulting in the formation of a heterodisulfide (CoB-S-S-CoM) 1 of 2-mercaptoethanesulfonate (HS-CoM) and 7-mercaptoheptanoylthreonine phosphate (HS-CoB) (1). An energyconserving step in the metabolism of methanogens is the reduction of CoB-S-S-CoM with either molecular hydrogen or reduced coenzyme F 420 . In recent years, the membrane-bound electron transfer of M. mazei Gö1 has been analyzed in detail, resulting in the discovery of two proton translocating systems referred to as H 2 :heterodisulfide oxidoreductase and F 420 H 2 : heterodisulfide oxidoreductase, respectively (2).During growth on methylated substrates, part of the methyl groups of the substrates is oxidized to CO 2 , and reducing equivalents are transferred to F 420 . The reduced cofactor (F 420 H 2 ) is reoxidized by the above-mentioned membrane-bound electron transport system consisting of an F 420 H 2 dehydrogenase and a heterodisulfide reductase. The transfer of electrons between the enzymes is most likely mediated by methanophenazine, a hydrophobic cofactor that has been isolated from the cytoplasmic membrane of M. mazei Gö1. The overall process has been shown to be competent in driving proton translocation across the cytoplasmic membrane (3). The resulting electrochemical proton gradient is the driving force for ATP synthesis from ADP ϩ P i catalyzed by an A 1 A 0 -type ATP synthase (2, 4).The F 420 H 2 dehydrogenase with a molecular mass of 115 kDa has been purified from M. mazei Gö1 and contains iron-sulfur clusters and FAD (5). The isolated enzyme is very similar to the corresponding protein from Methanolobus tindarius (6) and is composed of five different subunits with molecular masses of 40, 37, 22, 20, and 17 kDa. A F 420 H 2 dehydrogenase has also been purified form the sulfate-reducing archaeon Archaeoglobus fulgidus (7).In this report the gene locus encoding the F 420 H 2 dehydrogenase on the M. mazei genome is described. Furthermore, it is shown that the corresponding enzyme is a novel proton pump ¶ To whom correspondence should be addressed. Fax: 49-551-393793; E-mail: udeppen@gwdg.de. 1 The abbreviations used are: CoB-S-S-CoM, heterodisulfide of HSCoM and HS-CoB; HS-CoM, 2-mercaptoethansulfona...

Enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular level and to be explored as potential sources for biotechnology processes. We have used a combined approach of enrichment culture and direct cloning to construct cosmid libraries with large (>30-kb) inserts from microbial consortia. Enrichment cultures were inoculated with samples from five environments, and high amounts of avidin were added to the cultures to favor growth of biotin-producing microbes. DNA was extracted from three of these enrichment cultures and used to construct cosmid libraries; each library consisted of between 6,000 and 35,000 clones, with an average insert size of 30 to 40 kb. The inserts contained a diverse population of genomic DNA fragments isolated from the consortia organisms. These three libraries were used to complement the Escherichia coli biotin auxotrophic strain ATCC 33767 ⌬(bio-uvrB). Initial screens resulted in the isolation of seven different complementing cosmid clones, carrying biotin biosynthesis operons. Biotin biosynthesis capabilities and growth under defined conditions of four of these clones were studied. Biotin measured in the different culture supernatants ranged from 42 to 3,800 pg/ml/optical density unit. Sequencing the identified biotin synthesis genes revealed high similarities to bio operons from gram-negative bacteria. In addition, random sequencing identified other interesting open reading frames, as well as two operons, the histidine utilization operon (hut), and the cluster of genes involved in biosynthesis of molybdopterin cofactors in bacteria (moaABCDE).