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Author(s): Yibei Xiao [1]; Sherwin Ng [1]; Ki Hyun Nam [2]; Ailong Ke (corresponding author) [1] CRISPR (clustered regularly interspaced short palindromic repeats) and the nearby Cas (CRISPR-associated) operon establish [...]

Regular spacing of short runs of A or T nucleotides in DNA sequences with a period close to the helical period of the DNA double helix has been associated with intrinsic DNA bending and nucleosome positioning in eukaryotes. Analogous periodic signals were also observed in prokaryotic genomes. While the exact role of this periodicity in prokaryotes is not known, it has been proposed to facilitate the DNA packaging in the prokaryotic nucleoid and/or to promote negative or positive supercoiling. We developed a methodology for assessments of intragenomic heterogeneity of these periodic patterns and applied it in analysis of 1,025 prokaryotic chromosomes. This technique allows more detailed analysis of sequence periodicity than previous methods where sequence periodicity was assessed in an integral form across the whole chromosome. We found that most genomes have the periodic signal confined to several chromosomal segments while most of the chromosome lacks a strong sequence periodicity. Moreover, there are significant differences among different prokaryotes in both the intensity and persistency of sequence periodicity related to DNA curvature. We proffer that the prokaryotic nucleoid consists of relatively rigid sections stabilized by short intrinsically bent DNA segments and characterized by locally strong periodic patterns alternating with regions featuring a weak periodic signal, which presumably permits higher structural flexibility. This model applies to most bacteria and archaea. In genomes with an exceptionally persistent periodic signal, highly expressed genes tend to concentrate in aperiodic sections, suggesting that structural heterogeneity of the nucleoid is related to local differences in transcriptional activity. doi: 10.1128/JB.00149-10

The Abi protein family consists of putative membrane-bound metalloproteases. While they are involved in membrane anchoring of proteins in eukaryotes, little is known about their function in prokaryotes. In some known bacteriocin loci, Abi genes have been found downstream of bacteriocin structural genes (e.g., pln locus from Lactobacillus plantarum and sag locus from Streptococcus pyogenes), where they probably are involved in self-immunity. By modifying the profile hidden Markov model used to select Abi proteins in the Pfam protein family database, we show that this family is larger than presently recognized. Using bacteriocin-associated Abi genes as a means to search for novel bacteriocins in sequenced genomes, seven new bacteriocin-like loci were identified in Gram-positive bacteria. One such locus, from Lactobacillus sakei 23K, was selected for further experimental study, and it was confirmed that the bacteriocin-like genes (skkAB) exhibited antimicrobial activity when expressed in a heterologous host and that the associated Abi gene (skkI) conferred immunity against the cognate bacteriocin. Similar investigation of the Abi gene plnI and the Abi-like gene plnL from L. plantarum also confirmed their involvement in immunity to their cognate bacteriocins (PlnEF and PlnJK, respectively). Interestingly, the immunity genes from these three systems conferred a high degree of cross-immunity against each other's bacteriocins, suggesting the recognition of a common receptor. Site-directed mutagenesis demonstrated that the conserved motifs constituting the putative proteolytic active site of the Abi proteins are essential for the immunity function of SkkI, and to our knowledge, this represents a new concept in self-immunity. doi: 10.1128/JB.01553-09

Horizontal gene transfer (HGT) is one of the most dominant forces molding prokaryotic gene repertoires. These repertoires can be as small as [approximately equal to]200 genes in intracellular organisms or as large as [approximately equal to]9,000 genes in large, free-living bacteria. In this article we ask what is the impact of HGT from phylogenetically distant sources, relative to the size of the gene repertoire. Using different approaches for HGT detection and focusing on both cumulative and recent evolutionary histories, we find a surprising pattern of nonlinear enrichment of long-distance transfers in large genomes. Moreover, we find a strong positive correlation between the sizes of the donor and recipient genomes. Our results also show that distant horizontal transfers are biased toward those functional groups that are enriched in large genomes, showing that the trends in functional gene content and the impact of distant transfers are interdependent. These results highlight the intimate relationship between environmental and genomic complexity in microbes and suggest that an ecological, as opposed to phylogenetic, signal in gene content gains relative importance in large-genomed bacteria. functional gene content | microbial genomes | scaling | lateral gene transfer doi/10.1073/pnas.0907584106

DNA-DNA hybridization (DDH) has been used for nearly 50 years as the gold standard for prokaryotic species circumscriptions at the genomic level. It has been the only taxonomic method that offered a numerical and relatively stable species boundary, and its use has had a paramount influence on how the current classification has been constructed. However, now, in the era of genomics, DDH appears to be an outdated method for classification that needs to be substituted. The average nucleotide identity (ANI) between two genomes seems the most promising method since it mirrors DDH closely. Here we examine the work package JSpecies as a user-friendly, biologist-oriented interface to calculate ANI and the correlation of the tetranucleotide signatures between pairwise genomic comparisons. The results agreed with the use of ANI to substitute DDH, with a narrowed boundary that could be set at [approximately equal to]95-96%. In addition, the JSpecies package implemented the tetranucleotide signature correlation index, an alignment-free parameter that generally correlates with ANI and that can be of help in deciding when a given pair of organisms should be classified in the same species. Moreover, for taxonomic purposes, the analyses can be produced by simply randomly sequencing at least 20% of the genome of the query strains rather than obtaining their full sequence. average nucleotide identity | DNA-DNA hybridization | genome-based taxonomy | tetranucleotide regression www.pnas.org/cgi/doi/10.1073/pnas.0906412106

It has been reported that the number of transcription factors encoded in prokaryotic genomes scales approximately quadratically with their total number of genes. We propose a conceptual explanation of this finding and illustrate it using a simple model in which metabolic and regulatory networks of prokaryotes are shaped by horizontal gene transfer of coregulated metabolic pathways. Adapting to a new environmental condition monitored by a new transcription factor (e.g., learning to use another nutrient) involves both acquiring new enzymes and reusing some of the enzymes already encoded in the genome. As the repertoire of enzymes of an organism (its toolbox) grows larger, it can reuse its enzyme tools more often and thus needs to get fewer new ones to master each new task. From this observation, it logically follows that the number of functional tasks and their regulators increases faster than linearly with the total number of genes encoding enzymes. Genomes can also shrink, e.g., because of a loss of a nutrient from the environment, followed by deletion of its regulator and all enzymes that become redundant. We propose several simple models of network evolution elaborating on this toolbox argument and reproducing the empirically observed quadratic scaling. The distribution of lengths of pathway branches in our model agrees with that of the real-life metabolic network of Escherichia coli. Thus, our model provides a qualitative explanation for broad distributions of regulon sizes in prokaryotes. functional genome analysis | horizontal gene transfer | transcriptional regulatory networks

Adenosine kinase (ADK) is a purine salvage enzyme and a typical housekeeping enzyme in eukaryotes which catalyzes the phosphorylation of adenosine to form AMP. Since prokaryotes synthesize purines de novo and no endogenous ADK activity is detectable in Escherichia coli, ADK has long been considered to be rare in bacteria. To date, only two prokaryotes, both of which are gram-positive bacteria, have been reported to contain ADK. Here we report that the gram-negative bacterium Xanthomonas campestris pathovar campestris, the causal agent of black rot of crucifers, possesses a gene (designated [adk.sub.Xcc]) encoding an ADK (named [ADK.sub.Xcc]), and we demonstrate genetically that the [ADK.sub.Xcc] is involved in extracellular polysaccharide (EPS) production, cell motility, and pathogenicity of X. campestris pv. campestris, [adk.sub.Xcc] was overexpressed as a [His.sub.6]-tagged protein in E. coli, and the purified [His.sub.6]-tagged protein exhibited ADK activity. Mutation of [adk.sub.Xcc] did not affect bacterial growth in rich and minimal media but led to an accumulation of intracellular adenosine and diminutions of intracellular ADK activity and ATP level, as well as EPS. The [adk.sub.Xcc] mutant displayed significant reductions in bacterial growth and virulence in the host plant.

The specific and tightly controlled transport of numerous nutrients and metabolites across cellular membranes is crucial to all forms of life. However, many of the transporter proteins involved have yet to be identified, including the vitamin transporters in various human pathogens, whose growth depends strictly on vitamin uptake. Comparative analysis of the ever-growing collection of microbial genomes coupled with experimental validation enables the discovery of such transporters. Here, we used this approach to discover an abundant class of vitamin transporters in prokaryotes with an unprecedented architecture. These transporters have energy-coupling modules comprised of a conserved transmembrane protein and two nucleotide binding proteins similar to those of ATP binding cassette (ABC) transporters, but unlike ABC transporters, they use small integral membrane proteins to capture specific substrates. We identified 21 families of these substrate capture proteins, each with a different specificity predicted by genome context analyses. Roughly half of the substrate capture proteins (335 cases) have a dedicated energizing module, but in 459 cases distributed among almost 100 gram-positive bacteria, including numerous human pathogens, different and unrelated substrate capture proteins share the same energy-coupling module. The shared use of energy-coupling modules was experimentally confirmed for folate, thiamine, and riboflavin transporters. We propose the name energy-coupling factor transporters for the new class of membrane transporters.

In order to explore microevolutionary trends in bacteria and archaea, we constructed a data set of 41 alignable tight genome clusters (ATGCs). We show that the ratio of the medians of nonsynonymous to synonymous substitution rates (dN/dS) that is used as a measure of the purifying selection pressure on protein sequences is a stable characteristic of the ATGCs. In agreement with previous findings, parasitic bacteria, notwithstanding the sometimes dramatic genome shrinkage caused by gene loss, are typically subjected to relatively weak purifying selection, presumably owing to relatively small effective population sizes and frequent bottlenecks. However, no evidence of genome streamlining caused by strong selective pressure was found in any of the ATGCs. On the contrary, a significant positive correlation between the genome size, as well as gene size, and selective pressure was observed, although a variety of free-living prokaryotes with very close selective pressures span nearly the entire range of genome sizes. In addition, we examined the connections between the sequence evolution rate and other genomic features. Although gene order changes much faster than protein sequences during the evolution of prokaryotes, a strong positive correlation was observed between the 'rearrangement distance' and the amino acid distance, suggesting that at least some of the events leading to genome rearrangement are subjected to the same type of selective constraints as the evolution of amino acid sequences.

Ser/Thr/Tyr kinases, which together comprise a major class of regulatory proteins in eukaryotes, were not believed to play an important role in prokaryotes until recently. However, our analysis of 626 prokaryotic genomes reveals that eukaryotic-like protein kinases (ELKs) are found in nearly two-thirds of the sequenced strains. We have identified 2697 ELKs, most of which are encoded by multicellular strains of the phyla Proteobacteria (Myxococcales), Actinobacteria, Cyanobacteria, and Chloroflexi, and 2 Acidobacteria and 1 Planctomycetes. Astonishingly, 7 myxobacterial strains together encode 892 ELKs, with 4 of the strains exhibiting a genomic ELK density similar to that observed in eukaryotes. Most myxobacterial ELKs show a modular organization in which the kinase domain is located at the N terminus. The C-terminal portion of the ELKs is highly diverse and often contains sequences with similarity to characterized domains, most of them involved in signaling mechanisms or in protein-protein interactions. However, many of these architectures are unique to the myxobacteria, an observation that suggests that this group exploits sophisticated and novel signal transduction systems. Phylogenetic reconstruction using the kinase domains revealed many orthologous sequence pairs and a huge number of gene duplications that probably occurred after speciation. Furthermore, studies of the microsynteny in the ELK-encoding regions reveal only low levels of synteny among Myxococcus xanthus, Plesiocystis pacifica, and Sorangium cellulosum. However, extensive similarities between M. xanthus, Stigrnatella aurantiaca, and 3 Anaeromyxobacter strains were observed, indicating that they share regulatory pathways involving various ELKs. Anaeromyxobacter | Myxococcus | Plesiocystis | Sorangium | Stigmatella

Several prokaryotic expression studies are conducted to explain the impact of ALAS2 gene deletions in different families. Results prove that the C-terminal deletions of the gene lead to the gain of function, which then leads to X-linked dominant protoporphyria without anemia or iron overload in humans.

A variant of the diaminopimelate (DAP)-lysine biosynthesis pathway uses an LL-DAP aminotransferase (DapL, EC 2.6.1.83) to catalyze the direct conversion of L-2,3,4,5-tetrahydrodipicolinate to LL-DAP. Comparative genomic analysis and experimental verification of DapL candidates revealed the existence of two diverged forms of DapL (DapL! and DapL2). DapL orthologs were identified in eubacteria and archaea. In some species the corresponding dapL gene was found to lie in genomic contiguity with other dap genes, suggestive of a polycistronic structure. The DapL candidate enzymes were found to cluster into two classes sharing approximately 30% amino acid identity. The function of selected enzymes from each class was studied. Both classes were able to functionally complement Escherichia coli dapD and dapE mutants and to catalyze LL-DAP transamination, providing functional evidence for a role in DAP/lysine biosynthesis. In all cases the occurrence of dapL in a species correlated with the absence of genes for dapD and dapE representing the acyl DAP pathway variants, and only in a few cases was dapL coincident with ddh encoding meso-DAP dehydrogenase. The results indicate that the DapL pathway is restricted to specific lineages of eubacteria including the Cyanobacteria, Desulfuromonadales, Firmicutes, Bacteroidetes, Chlamydiae, Spirochaeta, and Chloroflexi and two archaeal groups, the Methanobacteriaceae and Archaeoglobaceae.

Dissimilatory adenosine-5'-phosphosulfate (APS) reductase (AprBA) is a key enzyme of the dissimilatory sulfate-reduction pathway. Homologues have been found in photo- and chemotrophic sulfur-oxidizing prokaryotes (SOP), in which they are postulated to operate in the reverse direction, oxidizing sulfite to APS. Newly developed PCR assays allowed the amplification of 92-93% (2.1-2.3 kb) of the APS reductase locus aprBA. PCR-based screening of t 16 taxonomically divergent SOP reference strains revealed a distribution of aprBA restricted to photo- and chemotrophs with strict anaerobic or at least facultative anaerobic lifestyles, including Chlorobiaceae, Chromatiaceae, Thiobacillus, Thiothrix and invertebrate symbionts. In the AprBA-based tree, the SOP diverge into two distantly related phylogenetic lineages, Apr lineages I and II, with the proteins of lineage II (Chlorobiaceae and others) in closer affiliation to the enzymes of the sulfate-reducing prokaryotes (SRP). This clustering is discordant with the dissimilatory sulfite reductase (DsrAB) phylogeny and indicates putative lateral aprBA gene transfer from SRP to the respective SOB lineages. In support of lateral gene transfer (LGT), several beta- and gammaproteobacterial species harbour both aprBA homologues, the DsrAB-congruent 'authentic' and the SRP-related, LGT-derived gene loci, while some relatives possess exclusively the SRP-related apr genes as a possible result of resident gene displacement by the xenologue. The two-gene state might be an intermediate in the replacement of the resident essential gene. Collected genome data demonstrate the correlation between the AprBA tree topology and the composition/arrangement of the apr gene loci (occurrence of qmoABC or aprM genes) from SRP and SOP of lineages I and II. The putative functional role of the SRP-related APS reductases in photo- and chemotrophic SOP is discussed.

Magnetotactic bacteria (MTB) are a heterogeneous group of aquatic prokaryotes with a unique intracellular organelle, the magnetosome, which orients the cell along magnetic field lines. Magnetotaxis is a complex phenotype, which depends on the coordinate synthesis of magnetosomes and the ability to swim and orient along the direction caused by the interaction with the Earth's magnetic field. Although a number of putative magnetotaxis genes were recently identified within a conserved genomic magnetosome island (MAI) of several MTB, their functions have remained mostly unknown, and it was speculated that additional genes located outside the MAI might be involved in magnetosome formation and magnetotaxis. In order to identify genes specifically associated with the magnetotactic phenotype, we conducted comparisons between four sequenced magnetotactic Alphaproteobacteria including the nearly complete genome of Magnetospirillum gryphiswaidense strain MSR-1, the complete genome of Magnetospirillum magneticum strain AMB-1, the complete genome of the magnetic coccus MC-1, and the comparative-ready preliminary genome assembly of Magnetospirillum magnetotacticum strain MS-1 against an in-house database comprising 426 complete bacterial and archaeal genome sequences. A magnetobacterial core genome of about 891 genes was found shared by all four MTB. In addition to a set of approximately 152 genus-specific genes shared by the three Magnetospirillum strains, we identified 28 genes as group specific, i.e., which occur in all four analyzed MTB but exhibit no (MTB-specific genes) or only remote (MTB-related genes) similarity to any genes from nonmagnetotactic organisms and which besides various novel genes include nearly all mam and mms genes previously shown to control magnetosome formation. The MTB-specific and MTB-related genes to a large extent display synteny, partially encode previously unrecognized magnetosome membrane proteins, and are either located within (18 genes) or outside (10 genes) the MAI of M. gryphiswaldense. These genes, which represent less than 1% of the 4,268 open reading frames of the MSR-1 genome, as yet are mostly of unknown functions but are likely to be specifically involved in magnetotaxis and, thus, represent prime targets for future experimental analysis.

In prokaryotes, homologous recombination is essential for the repair of genomic DNA damage and for the integration of DNA taken up during horizontal gene transfer. In Escherichia coli, the exonucleases RecJ (specific for 5' single-stranded DNA) and RecBCD (degrades duplex DNA) play important roles in recombination and recombinational double-strand break (DSB) repair by the RecF and RecBCD pathways, respectively. The cloned recJ of Acinetobacter baylyi partially complemented an E. coli recJ mutant, suggesting functional similarity of the enzymes. A [DELTA]recJ mutant of A. baylyi was only slightly altered in transformability and was not affected in UV survival. In contrast, a [DELTA]recBCD mutant was UV-sensitive, and had a low viability and altered transformation. Compared to wild-type, transformation with large chromosomal DNA fragments was decreased about 5-ford, while transformation with 1.5 kbp DNA fragments was increased 3.3- to 7-fold. A [DELTA]recD mutation did not affect transformation, viability or UV resistance. However, double mutants recJ recBCD and recJ recD were non-viable, suggesting that the RecJ DNase or the RecBCD DNase (presumably absent in recD) becomes essential for the recombinational repair of spontaneously inactivated replication forks if the other DNase is absent. A model of recombination during genetic transformation is discussed in which the two ends of the single-stranded donor DNA present in the cytoplasm frequently integrate separately and often with a time difference. If replication runs through that genomic region before both ends of the donor DNA are ligated to recipient DNA, a double-strand break (DSB) is formed. In these cases, transformation becomes dependent on DSB repair.

Newly developed PCR assays were used to PCR-amplify and sequence fragments of the dissimilatory adenosine-5'-phosphosulfate (APS) reductase genes (aprBA) comprising nearly the entire gene locus (2.2-2-4 kb, equal to 92-94% of the protein coding sequence) from 75 sulfate-reducing prokaryotes (SRP) of a taxonomically wide range. Comparative phylogenetic analysis included all determined and publicly available AprBA sequences from SRP and selected homologous sequences of sulfur-oxidizing bacteria (SOB). The almost identical AprB and AprA tree topologies indicated a shared evolutionary path for the aprBA among the investigated SRP by vertical inheritance and concomitant lateral gene transfer (LGT). The topological comparison of AprB/A- and 16S rRNA gene-based phylogenetic trees revealed novel LGT events across the SRP divisions. Compositional gene analysis confirmed Thermacetogenium phaeum to be the first validated strain affected by a recent lateral transfer of aprBA as a putative effect of long-term co-cultivation with a Thermodesulfovibrio species. Interestingly, the Apr proteins of SRP and SOB diverged into two phylogenetic lineages, with the SRP affiliated with the green sulfur bacteria, e.g. Chlorobaculum tepidum, while the Aflochromatium vinosum-related sequences formed a distinct group. Analysis of genome data indicated that this phylogenetic separation is also reflected in the differing presence of the putative proteins functionally associated with Apr, QmoABC complex (quinone-interacting membrane-bound oxidoreductase) and AprM (transmembrane protein). Scenarios for the origin and evolution of the dissimilatory APS reductase are discussed within the context of the dissimilatory sulfite reductase (DsrAB) phylogeny, the appearance of QmoABC and AprM in the SRP and SOB genomes, and the geochemical setting of Archean Earth.

The article reviews physiological roles of bacterial nonhomologous end-joining and double-strand breaks repair pathway in prokaryotic organisms.

The article presents RNA degradosome in mRNA degradation of Escherichia coli and the alternative forms that modulate the activity.

The protein pirin, which is involved in a variety of biological processes, is conserved from prokaryotic microorganisms, fungi, and plants to mammals. It acts as a transcriptional cofactor or an apoptosis-related protein in mammals and is involved in seed germination and seedling development in plants. In prokaryotes, while pirin is stress induced in cyanobacteria and may act as a quercetinase in Escherichia coli, the functions of pirin orthologs remain mostly uncharacterized. We show that the Serratia marcescens pirin (pirins,.) gene encodes an ortholog of pirin protein. Protein pull-down and bacterial two-hybrid assays followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization-tandem mass spectrometry analyses showed the pyruvate dehydrogenase (PDH) El subunit as a component interacting with the pirins, gene. Functional analyses showed that both PDH E1 subunit activity and PDH enzyme complex activity are inhibited by the [pirins.sub.Sm] gene in S. marcescens CH-1. The S. marcescens CH-1 [pirin.sub.Sm] gene was subsequently mutated by insertion-deletion homologous recombination. Accordingly, the PDH E1 and PDH enzyme complex activities and cellular ATP concentration increased up to 250%, 140%, and 220%, respectively, in the S. marcescens CH-1 [pirin.sub.Sm] mutant. Concomitantly, the cellular NADH/NA[D.sup.+] ratio increased in the [pirins.sub.Sm] mutant, indicating increased tricarboxylic acid (TCA) cycle activity. Our results show that the [pirin.sub.Sm] gene plays a regulatory role in the process of pyruvate catabolism to acetyl coenzyme A through interaction with the PDH E1 subunit and inhibiting PDH enzyme complex activity in S. marcescens CH-1, and they suggest that [pirin.sub.Sm] is an important protein involved in determining the direction of pyruvate metabolism towards either the TCA cycle or the fermentation pathways.

A study is presented which proposes that evolution of complex genomic architecture was driven primarily by non-adaptive stochastic forces, rather than by adaptive evolution. A general negative relationship between selection efficiency and genome complexity provides a strong support for the hypothesis provided by the study.

ABSTRACT Two-component regulation systems (TCRSs) are the dominant type of signal transduction system in prokaryotes that are used to inform the cellular trancriptional machinery (and additional targets for regulation, like [...]

Protein modifiers are involved in diverse biological processes and regulate the activity or function of target proteins by covalently conjugating to them. Although ubiquitin and a number of ubiquitin-like protein modifiers (Ubls) in eukaryotes have been identified, no protein modifier has been found in prokaryotes; thus, their evolutionary origin remains a puzzle. To infer the evolutionary relationships between the protein modifiers and sulfur carrier proteins, we solved the solution NMR structure of the Urm1 (ubiquitin-related modifier-1) protein from Saccharomyces cerevisiae. Both structural comparison and phylogenetic analysis of the ubiquitin superfamily, with emphasis on the Urm1 family, indicate that Urm1 is the unique 'molecular fossil' that has the most conserved structural and sequence features of the common ancestor of the entire superfamily. The similarities of 3D structure and hydrophobic and electrostatic surface features between Urm1 and MoaD (molybdopterin synthase small subunit) suggest that they may interact with partners in a similar manner, and similarities between Urm1-Uba4 and MoaD-MoeB establish an evolutionary link between ATP-dependent protein conjugation in eukaryotes and ATP-dependent cofactor sulfuration. evolution | NMR structure

The complete modification map for SSU rRNA from Thermus thermophilus, determined primarily by HPLC/ electrospray ionization MS-based methods is reported. The results provide an example of the potential of LC/ MS for extensive modification mapping in large RNAs.

Amino acid racemases catalyze the stereoinversion of the chiral [C.sup.[alpha]] to produce the D-enantiomers that participate in biological processes, such as cell wall construction in prokaryotes. Within this large protein family, bacterial proline racemases have been extensively studied as a model of enzymes acting with a pyridoxalphosphate-independent mechanism. Here we report the crystal structure of the proline racemase from the human parasite Trypanosoma cruzi (TcPRACA), a secreted enzyme that triggers host B cell polyclonal activation, which prevents specific humoral immune responses and is crucial for parasite evasion and fate. The enzyme is a homodimer, with each monomer folded in two symmetric [alpha]/[beta] subunits separated by a deep crevice. The structure of TcPRACA in complex with a transition-state analog, pyrrole-2-carboxylic acid, reveals the presence of one reaction center per monomer, with two Cys residues optimally located to perform acid/base catalysis through a carbanion stabilization mechanism. Mutation of the catalytic Cys residues abolishes the enzymatic activity but preserves the mitogenic properties of the protein. In contrast, inhibitor binding promotes the closure of the interdomain crevice and completely abrogates B cell proliferation, suggesting that the mitogenic properties of TcPRACA depend on the exposure of transient epitopes in the ligand-free enzyme. B cell mitogen | pyridoxal phosphate-independent proline racemase | epimerases | enzyme-inhibitor complex | titration calorimetry

Nucleotide excision repair (NER) in prokaryotes is a DNA repair system, wherein insertion of short stretches of new DNA into repair patches takes place, along with dimer removal. The UvrABC system, comprising of uvrA, uvrB and uvrC genes responsible for NER, is a complex network that responds to stress placed on the cell.

The [Mn.sup.2} transport in prokaryotes and the regulation of prokaryotic [Mn.sup.2} transport are discussed. The [Mn.sup.2} transport is regulated by a [Mn.sup.2}-specific transcription factor (MntR) and the degree to which the prokaryotic [Mn.sup.2} transporters are important for virulence depends on whether or not the host organism expresses Nramp1.

The effects of increasing concentrations of 2,2,2-trifluoroethanol (TFL) in detergent solution on the structure of potassium channel KcsA, a predominantly alpha-helical membrane protein is studied. The results are interpreted in terms of a protein and TFE concentration-dependent reversible equilibrium between the folded tetrameric protein and partly unfolded monomeric subunits in which folding and refolding are seemingly coupled processes.

We study the evolution of transcription factor-binding sites in prokaryotes, using an empirically grounded model with point mutations and genetic drift. Selection acts on the site sequence via its binding affinity to the corresponding transcription factor. Calibrating the model with populations of functional binding sites, we verify this form of selection and show that typical sites are under substantial selection pressure for functionality: for cAMP response protein sites in Escherichia coil, the product of fitness difference and effective population size takes values 2N[DELTA]F of order 10. We apply this model to cross-species comparisons of binding sites in bacteria and obtain a prediction method for binding sites that uses evolutionary information in a quantitative way. At the same time, this method predicts the functional histories of orthologous sites in a phylogeny, evaluating the likelihood for conservation or loss or gain of function during evolution. We have performed, as an example, a cross-species analysis of E. coil, Salmonella typhimurium, and Yersinia pseudotuberculosis. Detailed lists of predicted sites and their functional phylogenies are available.

The dissimilatory reduction of sulfate is an ancient metabolic process central to today's biogeochemical cycling of sulfur and carbon in marine sediments. Until now its polyphyletic distribution was most parsimoniously explained by multiple horizontal transfers of single genes rather than by a not-yet-identified 'metabolic island.' Here we provide evidence that the horizontal transfer of a gene cluster may indeed be responsible for the patchy distribution of sulfate-reducing prokaryotes (SRP) in the phylogenetic tree. We isolated three DNA fragments (32 to 41 kb) from uncultured, closely related SRP from DNA directly extracted from two distinct marine sediments. Fosmid ws39f7, and partially also fosmids ws7f8 and hr42c9, harbored a core set of essential genes for the dissimilatory reduction of sulfate, including enzymes for the reduction of sulfur intermediates and synthesis of the prosthetic group of the dissimilatory sulfite reductase. Genome comparisons suggest that encoded membrane proteins universally present among SRP are critical for electron transfer to cytoplasmic enzymes. In addition, novel, conserved hypothetical proteins that are likely involved in dissimilatory sulfate reduction were identified. Based on comparative genomics and previously published experimental evidence, a more comprehensive model of dissimilatory sulfate reduction is presented. The observed clustering of genes involved in dissimilatory sulfate reduction has not been previously found. These findings strongly support the hypothesis that genes responsible for dissimilatory sulfate reduction were concomitantly transferred in a single event among prokaryotes. The acquisition of an optimized gene set would enormously facilitate a successful implementation of a novel pathway.

Numerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats (CRISPRs), composed of 25-50 bp repeats separated by unique sequence spacers of similar length. CRISPR structures are found in the vicinity of four genes named cas 1 to cas4. In silico analysis revealed another cluster of three genes associated with CRISPR structures in many bacterial species, named here as cas1B, cas5 and cas6, and also revealed a certain number of spacers that have homology with extant genes, most frequently derived from phages, but also derived from other extrachromosomal elements. Sequence analysis of CRISPR structures from 24 strains of Streptococcus thermophilus and Streptococcus vestibularis confirmed the homology of spacers with extrachromosomal elements. Phage sensitivity of S. thermophilus strains appears to be correlated with the number of spacers in the CRISPR locus the strain carries. The authors suggest that the spacer elements are the traces of past invasions by extrachromosomal elements, and hypothesize that they provide the cell immunity against phage infection, and more generally foreign DNA expression, by coding an anti-sense RNA. The presence of gene fragments in CRISPR structures and the nuclease motifs in cas genes of both cluster types suggests that CRISPR formation involves a DNA degradation step.

The use of molecular techniques to assess prokaryotic diversity independent of the need for enrichment culture has profoundly changed the way microbial diversity is studied. Analysis of the geographic locations and habitat types of environmental isolates at the American Type Culture Collection (ATCC) showed that there were significant disparities between both locations and habitats that were represented in the culture collection.

The committed biosynthetic reaction to benzoyl-coenzyme A in the marine bacterium 'Streptomyces maritimus' is carried out by the novel prokaryotic phenylalanine ammonia lyase (PAL) EncP, which converts the primary amino acid L-phenylalanine to trans-cinnamic acid. Recombinant EncP is specific for L-phenylalanine and shares many biochemical features with eukaryotic PALs, which are substantially larger proteins by ~200 amino acid residues.

Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertion elements from the IS110/IS492 family and shows no similarity to the site-specific reeombinases of the tyrosine- or serine-recombinase families. Piv tertiary structure is predicted to include the RNase H-like fold that typically encompasses the catalytic site of the recombinases or nucleases of the retroviral integrase superfamily, including transposases and RuvC-like Holliday junction resolvases. Analogous to the DDE and DEDD catalytic motifs of transposases and RuvC, respectively, four Piv acidic residues Dg, E89, D101, and D104 appear to be positioned appropriately within the RNase H fold to coordinate two divalent metal cations. This suggests mechanistic similarity between site-specific inversion mediated by Piv and transposition or endonucleolytic reactions catalyzed by enzymes of the retroviral integrase superfamily. The role of the DEDD motif in Ply catalytic activity was addressed using Piv variants that are substituted individually or multiply at these acidic residues and assaying for in vivo inversion, intermolecular recombination, and DNA binding activities. The results indicate that all four residues of the DEDD motif are required for Piv catalytic activity. The DEDD residues are not essential for inv recombination site recognition and binding, but this acidic tetrad does appear to contribute to the stability of Piv-inv interactions. On the basis of these results, a working model for Piv-mediated inversion that includes resolution of a Holliday junction is presented.

A full-cycle molecular approach is used to show that the endosymbionts of the larger gut flagellates of Reticulitermes santonesis belong to the termite group 1 (TG-1) bacteria. The members of the TG-1 phylum, named as 'Endomicrobia', are phylogenetically extremely diverse and are present in and also restricted to the guts of all lower termites and wood-feeding cockroaches of the genus Cryptocercus, the only insects that are associated with such unique cellulose-fermenting protists.

To help advance the species definition for prokaryotes, we have compared the gene content of 70 closely related and fully sequenced bacterial genomes to identify whether species boundaries exist, and to determine the role of the organism's ecology on its shared gene content. We found the average nucleotide identity (ANI) of the shared genes between two strains to be a robust means to compare genetic relatedness among strains, and that ANI values of [approximately equal to] 94% corresponded to the traditional 70% DNA-DNA reassociation standard of the current species definition. At the 94% ANI cutoff, current species includes only moderately homogeneous strains, e.g., most of the >4-Mb genomes share only 65-90% of their genes, apparently as a result of the strains having evolved in different ecological settings. Furthermore, diagnostic genetic signatures (boundaries) are evident between groups of strains of the same species, and the intergroup genetic similarity can be as high as 98-99% ANI, indicating that justifiable species might be found even among organisms that are nearly identical at the nucleotide level. Notably, a large fraction, e.g., up to 65%, of the differences in gene content within species is associated with bacteriophage and transposase elements, revealing an important role of these elements during bacterial speciation. Our findings are consistent with a definition for species that would include a more homogeneous set of strains than provided by the current definition and one that considers the ecology of the strains in addition to their evolutionary distance. prokaryotic diversity | species concept | nucleotide identity | comparative genomics | evolution

To study the roles of translational accuracy, translational efficiency, and the Hill-Robertson effect in codon usage bias, we studied the intragenic spatial distribution of synonymous codon usage bias in four prokaryotic (Escherichia coli, Bacillus subtilis, Sulfolobus tokodaii, and Theonotoga maritima) and two eukaryotic (Saccharomyces cerevisiae and Drosophila melanogaster) genomes. We generated supersequences at each codon position across genes in a genome and computed the overall bias at each codon position. By quantitatively evaluating the trend of spatial patterns using isotonic regression, we show that in yeast and prokaryotic genomes, codon usage bias increases along translational direction, which is consistent with purifying selection against nonsense errors. Fruit fly genes show a nearly symmetric M-shaped spatial pattern of codon usage bias, with less bias in the middle and both ends. The low codon usage bias in the middle region is best explained by interference (the Hill-Robertson effect) between selections at different codon positions. In both yeast and fruit fly, spatial patterns of codon usage bias are characteristically different from patterns of GC-content variations. Effect of expression level on the strength of codon usage bias is more conspicuous than its effect on the shape of the spatial distribution.

Haloviruses HF1 and HF2 were isolated from the same saltern pond and are adapted to hypersaline conditions, where they infect a broad range of haloarchaeal species. The HF2 genome has previously been reported. The complete sequence of the HF1 genome has now been determined, mainly by PCR and primer walking. It was 75,898 bp in length and was 94.4% identical to the HF2 genome but about 1.8 kb shorter. A total of 117 open reading frames and five tRNA-like genes were predicted, and their database matches and characteristics were similar to those found in HF2. A comparison of the predicted restriction digest patterns based on nucleotide sequence with the observed restriction digest patterns of viral DNA showed that, unlike the case for HF2, some packaged HF1 DNA had cohesive termini. Except for a single base change, HF1 and HF2 were identical in sequence over the first 48 kb, a region that includes the early and middle genes. The remaining 28 kb of HF1 showed many differences from HF2, and the similarity of the two genomes over this late gene region was 87%. The abrupt shift in sequence similarity around 48 kb suggests a recent recombination event between either HF1 or HF2 and another HF-like halovirus that has swapped most of the right-end 28 kb. This example indicates there is a high level of recombination among viruses that live in this extreme environment.

The availability of an ever increasing number of complete genome sequences of diverse prokaryotic taxa has led to the introduction of novel approaches to infer phylogenetic relationships among bacteria. In the present study the sequences of the 16S rRNA gene and nine housekeeping genes were compared with the fraction of shared putative orthologous protein-encoding genes, conservation of gene order, dinucteotide relative abundance and codon usage among 11 genomes of species belonging to the lactic acid bacteria. In general there is a good correlation between the results obtained with various approaches, although it is clear that there is a stronger phylogenetic signal in some datasets than in others, and that different parameters have different taxonomic resolutions. It appears that trees based on different kinds of information derived from whole-genome sequencing projects do not provide much additional information about the phylogenetic relationships among bacterial taxa compared to more traditional alignment-based methods. Nevertheless, it is expected that the study of these novel forms of information will have its value in taxonomy, to determine which genes are shared, when genes or sets of genes were lost in evolutionary history, to detect the presence of horizontally transferred genes and/or confirm or enhance the phylogenetic signal derived from traditional methods. Although these conclusions are based on a relatively small dataset, they are largely in agreement with other studies and it is anticipated that similar trends will be observed when comparing other genomes.

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes with important structural and signalling functions. Although many prokaryotes lack PC, it can be found in significant amounts in membranes of rather diverse bacteria. Two pathways for PC biosynthesis are known in bacteria, the methylation pathway and the phosphatidylcholine synthase (PCS) pathway. In the methylation pathway, phosphatidylethanolamine is methylated three times to yield PC, in reactions catalysed by one or several phospholipid N-methyltransferases (PMTs). In the PCS pathway, choline is condensed directly with CDP-diacylglyceride to form PC in a reaction catalysed by PCS. Using cell-free extracts, it was demonstrated that Sinorhizobium meliloti, Agrobacterium tumefaciens, Rhizobium leguminosarum, Bradyrhizobium japonicum, Mesorhizobium Ioti and Legionella pneumophila have both PMT and PCS activities. In addition, Rhodobacter sphaeroides has PMT activity and Brucella melitensis, Pseudomonas aeruginosa and Borrelia burgdorferi have PCS activities. Genes from M. Ioti and L. pneumophila encoding a Pmt or a Pcs activity and the genes from P. aeruginosa and Borrelia burgdorferi responsible for Pcs activity have been identified. Based on these functional assignments and on genomic data, one might predict that if bacteria contain PC as a membrane lipid, they usually possess both bacterial pathways for PC biosynthesis. However, important pathogens such as Brucella melitensis, P. aeruginosa and Borrelia burgdorferi seem to be exceptional as they possess only the PCS pathway for PC formation.

The ability of each of the nine Escherichia coli division proteins (FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsL, FtsW, Ftsl, FtsN) to interact with itself and with each of the remaining eight proteins was studied in 43 possible combinations of protein pairs by the two-hybrid system previously developed by the authors' group. Once the presumed interactions between the division proteins were determined, a model showing their temporal sequence of assembly was developed. This model agrees with that developed by other authors, based on the co-localization sequence in the septum of the division proteins fused with GFP. In addition, this paper shows that the authors' assay, which has already proved to be very versatile in the study of prokaryotic and eukaryotic protein interaction, is also a powerful instrument for an in vivo study of the interaction and assembly of proteins, as in the case of septum division formation.