Acinetobacter sp. strain ADP1 is a nutritionally versatile soil bacterium closely related to representatives of the well-characterized Pseudomonas aeruginosa and Pseudomonas putida. Unlike these bacteria, the Acinetobacter ADP1 is highly competent for natural transformation which affords extraordinary convenience for genetic manipulation. The circular chromosome of the Acinetobacter ADP1, presented here, encodes 3325 predicted coding sequences, of which 60% have been classified based on sequence similarity to other documented proteins. The close evolutionary proximity of Acinetobacter and Pseudomonas species, as judged by the sequences of their 16S RNA genes and by the highest level of bidirectional best hits, contrasts with the extensive divergence in the GC content of their DNA (40 versus 62%). The chromosomes also differ significantly in size, with the Acinetobacter ADP1 chromosome <60% of the length of the Pseudomonas counterparts. Genome analysis of the Acinetobacter ADP1 revealed genes for metabolic pathways involved in utilization of a large variety of compounds. Almost all of these genes, with orthologs that are scattered in other species, are located in five major 'islands of catabolic diversity', now an apparent 'archipelago of catabolic diversity', within one-quarter of the overall genome. Acinetobacter ADP1 displays many features of other aerobic soil bacteria with metabolism oriented toward the degradation of organic compounds found in their natural habitat. A distinguishing feature of this genome is the absence of a gene corresponding to pyruvate kinase, the enzyme that generally catalyzes the terminal step in conversion of carbohydrates to pyruvate for respiration by the citric acid cycle. This finding supports the view that the cycle itself is centrally geared to the catabolic capabilities of this exceptionally versatile organism.

We have constructed a collection of single-gene deletion mutants for all dispensable genes of the soil bacterium Acinetobacter baylyi ADP1. A total of 2594 deletion mutants were obtained, whereas 499 (16%) were not, and are therefore candidate essential genes for life on minimal medium. This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection profiled for growth on minimal medium, while 80% of the orthologous genes described as essential in Pseudomonas aeruginosa are also essential in ADP1. Several strategies were undertaken to investigate ADP1 metabolism by (1) searching for discrepancies between our essentiality data and current metabolic knowledge, (2) comparing this essentiality data set to those from other organisms, (3) systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2-3 butanediol, glucose, etc.). This collection provides a new resource for the study of gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches.

Many microorganisms live in anaerobic environments. Most of these microorganisms have not yet been cultivated. Here, we present, from a metagenomic analysis of an anaerobic digester of a municipal wastewater treatment plant, a reconstruction of the complete genome of a bacterium belonging to the WWE1 candidate division. In silico proteome analysis indicated that this bacterium might derive most of its carbon and energy from the fermentation of amino acids, and hence, it was provisionally classified as "Candidatus Cloacamonas acidaminovorans." "Candidatus Cloacamonas acidaminovorans" is probably a syntrophic bacterium that is present in many anaerobic digesters. This report highlights how environmental sequence data might provide genomic and functional information about a new bacterial clade whose members are involved in anaerobic digestion.The use of molecular techniques over the past few decades has shown the extent of microbial diversity and that the majority of archaeal and bacterial phyla still lack a cultivable representative (18,24). The recent advent of whole-community genome sequencing, or metagenomics, is rapidly changing this view and will revolutionize our understanding of the functional diversity of complex environments. For instance, a recent study has surprisingly revealed that ammonia-oxidizing Crenarchaeota are very abundant in soils (20). However, apart from the pioneering work in the Sargasso Sea (31) and the recent addition of millions of sequences from the Global Ocean Sampling expedition that revealed the extent of the ocean microbial diversity (25), most of the community sequencing programs have focused on relatively simple ecosystems. However, most microbial ecosystems are complex. Anaerobic digestion is a complex biological process that involves several metabolic pathways for the decomposition of organic matter into methane and carbon dioxide. The overall reactions-depolymerization, primary and secondary fermentation, acidogenesis, acetogenesis, and methanogenesis-are performed by a complex microbial community. Despite the industrial, technological, economic, and ecological importance of this community, little is known about the roles and activities of the microorganisms that inhabit anaerobic niches. The anaerobic digestion of organic matter involved in wastewater processing represents a good example of a complex and active microflora. During exploration of the bacterial diversity of an anaerobic mesophilic digester, a new bacterial candidate division called WWE1 was discovered (8). It was found that WWE1 bacteria could represent up to 10% of the bacterial microflora and thus could be a subdominant group. Using metagenomic sequence data and a specific genome assembly procedure, we were able to reconstruct the genome of a representative bacterium of the WWE1 division. We have for the first time obtained the complete genome sequence from a complex environment and from a bacterial candidate division with no cultivated representative. Because the metabolic pathways of anaerobic bacteria are ...

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BackgroundClostridium sticklandii belongs to a cluster of non-pathogenic proteolytic clostridia which utilize amino acids as carbon and energy sources. Isolated by T.C. Stadtman in 1954, it has been generally regarded as a "gold mine" for novel biochemical reactions and is used as a model organism for studying metabolic aspects such as the Stickland reaction, coenzyme-B12- and selenium-dependent reactions of amino acids. With the goal of revisiting its carbon, nitrogen, and energy metabolism, and comparing studies with other clostridia, its genome has been sequenced and analyzed.ResultsC. sticklandii is one of the best biochemically studied proteolytic clostridial species. Useful additional information has been obtained from the sequencing and annotation of its genome, which is presented in this paper. Besides, experimental procedures reveal that C. sticklandii degrades amino acids in a preferential and sequential way. The organism prefers threonine, arginine, serine, cysteine, proline, and glycine, whereas glutamate, aspartate and alanine are excreted. Energy conservation is primarily obtained by substrate-level phosphorylation in fermentative pathways. The reactions catalyzed by different ferredoxin oxidoreductases and the exergonic NADH-dependent reduction of crotonyl-CoA point to a possible chemiosmotic energy conservation via the Rnf complex. C. sticklandii possesses both the F-type and V-type ATPases. The discovery of an as yet unrecognized selenoprotein in the D-proline reductase operon suggests a more detailed mechanism for NADH-dependent D-proline reduction. A rather unusual metabolic feature is the presence of genes for all the enzymes involved in two different CO2-fixation pathways: C. sticklandii harbours both the glycine synthase/glycine reductase and the Wood-Ljungdahl pathways. This unusual pathway combination has retrospectively been observed in only four other sequenced microorganisms.ConclusionsAnalysis of the C. sticklandii genome and additional experimental procedures have improved our understanding of anaerobic amino acid degradation. Several specific metabolic features have been detected, some of which are very unusual for anaerobic fermenting bacteria. Comparative genomics has provided the opportunity to study the lifestyle of pathogenic and non-pathogenic clostridial species as well as to elucidate the difference in metabolic features between clostridia and other anaerobes.

SummaryThe hyperthermophilic euryarchaeon Pyrococcus abyssi and the related species Pyrococcus furiosus and Pyrococcus horikoshii , whose genomes have been completely sequenced, are presently used as model organisms in different laboratories to study archaeal DNA replication and gene expression and to develop genetic tools for hyperthermophiles. We have performed an extensive re-annotation of the genome of P. abyssi to obtain an integrated view of its phylogeny, molecular biology and physiology. Many new functions are predicted for both informational and operational proteins. Moreover, several candidate genes have been identified that might encode missing links in key metabolic pathways, some of which have unique biochemical features. The great majority of Pyrococcus proteins are typical archaeal proteins and their phylogenetic pattern agrees with its position near the root of the archaeal tree. However, proteins probably from bacterial origin, including some from mesophilic bacteria, are also present in the P. abyssi genome.

The metC gene of Escherichia coli K-12 was cloned and the nucleotide sequence of the metC gene and its flanking regions was determined. The translation initiation codon was identified by sequencing the NH2-terminal part of 13-cystathionase, the MetC gene product. The meIC gene (1185 nucleotides) encodes a protein having 395 amino acid residues.The 5' noncoding region was found to contain a "Met box" homologous to sequences suggestive of operator structures upstream from other methionine genes that are controlled by the product of the pleiotropic regulatory metJ gene. The deduced amino acid sequence of (3-cystathionase showed extensive homology with that of the MetB protein (cystathionine y-synthase) that catalyzes the preceding step in methionine biosynthesis. The homology strongly suggests that the structural genes for the MetB and MetC proteins evolved from a common ancestral gene.

That the entry of organic substrates into bacmeases" has been suggested for these systems. That cel entry of or les inoee-Although this designation may be criticized, it terial cells may be mediated by more or less selechas the overwhelming advantage that its general tive permeation systems has been suggested primeaning and scope are immediately understood. manly by two kinds of observations concerning, The object of the present review is to discuss respectively: (a) the capacity of certain cells to critically the recent evidence from different labo-accumulate internally certain nutrilites; (b) the state of "crypticity" of certain cells toward cer-' The work performed in the Service de Bio-tain substrates, i.e., their incapacity to metabochimie Cellulaire of the Institut Pasteur, has been lize a given substrate, even though they posses supported by grants from the Rockefeller Foundation, the Jane Coffin Childs Memorial Fund for the relevant enzyme system. Medical Research and the Commissariat A l'Ener-Let us see why both accumulation and crypgie Atomique. ticity phenomena were strongly suggestive, yet 2 A stereospecific system is one whose activity is inconclusive, as evidence of the operation of selecprimarily dependent upon the spacial configura-tive permeation systems. tion of the reacting molecules.