Evolutionary and functional insights into the Ski2-like helicase family in Archaea: a comparison of Thermococcales ASH-Ski2 and Hel308 activities.

RNA helicases perform essential housekeeping and regulatory functions in all domains of life by binding and unwinding RNA molecules. The Ski2-like proteins are primordial helicases that play an active role in eukaryotic RNA homeostasis pathways, with multiple homologs having specialized functions. The significance of the expansion and diversity of Ski2-like proteins in Archaea, the third domain of life, has not yet been established. Here, by studying the phylogenetic diversity of Ski2-like helicases among archaeal genomes and the enzymatic activities of those in Thermococcales, we provide further evidence of the function of this protein family in archaeal metabolism of nucleic acids. We show that, in the course of evolution, ASH-Ski2 and Hel308-Ski2, the two main groups of Ski2-like proteins, have diverged in their biological functions. Whereas Hel308 has been shown to mainly act on DNA, we show that ASH-Ski2, previously described to be associated with the 5'-3' aRNase J exonuclease, acts on RNA by supporting an efficient annealing activity, but also an RNA unwinding with a 3'-5' polarity. To gain insights into the function of Ski2, we also analyse the transcriptome of Thermococcus barophilus ΔASH-Ski2 mutant strain and provide evidence of the importance of ASH-Ski2 in cellular metabolism pathways related to translation.

The NagY regulator: A member of the BglG/SacY antiterminator family conserved in Enterococcus faecalis and involved in virulence.

Enterococcus faecalis is a commensal bacterium of the gastrointestinal tract but also a major nosocomial pathogen. This bacterium uses regulators like BglG/SacY family of transcriptional antiterminators to adapt its metabolism during host colonization. In this report, we investigated the role of the BglG/SacY family antiterminator NagY in the regulation of the nagY-nagE operon in presence of N-acetylglucosamine, with nagE encoding a transporter of this carbohydrate, as well as the expression of the virulence factor HylA. We showed that this last protein is involved in biofilm formation and glycosaminoglycans degradation that are important features in bacterial infection, confirmed in the Galleria mellonella model. In order to elucidate the evolution of these actors, we performed phylogenomic analyses on E. faecalis and Enterococcaceae genomes, identified orthologous sequences of NagY, NagE, and HylA, and we report their taxonomic distribution. The study of the conservation of the upstream region of nagY and hylA genes showed that the molecular mechanism of NagY regulation involves ribonucleic antiterminator sequence overlapping a rho-independent terminator, suggesting a regulation conforming to the canonical model of BglG/SacY family antiterminators. In the perspective of opportunism understanding, we offer new insights into the mechanism of host sensing thanks to the NagY antiterminator and its targets expression.

Phylogenetic Diversity of Lhr Proteins and Biochemical Activities of the Thermococcales aLhr2 DNA/RNA Helicase.

Helicase proteins are known to use the energy of ATP to unwind nucleic acids and to remodel protein-nucleic acid complexes. They are involved in almost every aspect of DNA and RNA metabolisms and participate in numerous repair mechanisms that maintain cellular integrity. The archaeal Lhr-type proteins are SF2 helicases that are mostly uncharacterized. They have been proposed to be DNA helicases that act in DNA recombination and repair processes in Sulfolobales and Methanothermobacter. In Thermococcales, a protein annotated as an Lhr2 protein was found in the network of proteins involved in RNA metabolism. To investigate this, we performed in-depth phylogenomic analyses to report the classification and taxonomic distribution of Lhr-type proteins in Archaea, and to better understand their relationship with bacterial Lhr. Furthermore, with the goal of envisioning the role(s) of aLhr2 in Thermococcales cells, we deciphered the enzymatic activities of aLhr2 from Thermococcus barophilus (Tbar). We showed that Tbar-aLhr2 is a DNA/RNA helicase with a significant annealing activity that is involved in processes dependent on DNA and RNA transactions.

J-Domain Proteins in Bacteria and Their Viruses.

Molecular chaperones maintain cellular protein homeostasis by acting at almost every step in protein biogenesis pathways. The DnaK/HSP70 chaperone has been associated with almost every known essential chaperone functions in bacteria. To act as a bona fide chaperone, DnaK strictly relies on essential co-chaperone partners known as the J-domain proteins (JDPs, DnaJ, Hsp40), which preselect substrate proteins for DnaK, confer its specific cellular localization, and stimulate both its weak ATPase activity and substrate transfer. Remarkably, genome sequencing has revealed the presence of multiple JDP/DnaK chaperone/co-chaperone pairs in a number of bacterial genomes, suggesting that certain pairs have evolved toward more specific functions. In this review, we have used representative sets of bacterial and phage genomes to explore the distribution of JDP/DnaK pairs. Such analysis has revealed an unexpected reservoir of novel bacterial JDPs co-chaperones with very diverse and unexplored function that will be discussed.

RNA processing machineries in Archaea: the 5'-3' exoribonuclease aRNase J of the ß-CASP family is engaged specifically with the helicase ASH-Ski2 and the 3'-5' exoribonucleolytic RNA exosome machinery.

A network of RNA helicases, endoribonucleases and exoribonucleases regulates the quantity and quality of cellular RNAs. To date, mechanistic studies focussed on bacterial and eukaryal systems due to the challenge of identifying the main drivers of RNA decay and processing in Archaea. Here, our data support that aRNase J, a 5'-3' exoribonuclease of the ß-CASP family conserved in Euryarchaeota, engages specifically with a Ski2-like helicase and the RNA exosome to potentially exert control over RNA surveillance, at the vicinity of the ribosome. Proteomic landscapes and direct protein-protein interaction analyses, strengthened by comprehensive phylogenomic studies demonstrated that aRNase J interplay with ASH-Ski2 and a cap exosome subunit. Finally, Thermococcus barophilus whole-cell extract fractionation experiments provide evidences that an aRNase J/ASH-Ski2 complex might exist in vivo and hint at an association of aRNase J with the ribosome that is emphasised in absence of ASH-Ski2. Whilst aRNase J homologues are found among bacteria, the RNA exosome and the Ski2-like RNA helicase have eukaryotic homologues, underlining the mosaic aspect of archaeal RNA machines. Altogether, these results suggest a fundamental role of ß-CASP RNase/helicase complex in archaeal RNA metabolism.

Dynamic Modeling of Streptococcus pneumoniae Competence Provides Regulatory Mechanistic Insights Into Its Tight Temporal Regulation.

In the human pathogen Streptococcus pneumoniae, the gene regulatory circuit leading to the transient state of competence for natural transformation is based on production of an auto-inducer that activates a positive feedback loop. About 100 genes are activated in two successive waves linked by a central alternative sigma factor ComX. This mechanism appears to be fundamental to the biological fitness of S. pneumoniae. We have developed a knowledge-based model of the competence cycle that describes average cell behavior. It reveals that the expression rates of the two competence operons, comAB and comCDE, involved in the positive feedback loop must be coordinated to elicit spontaneous competence. Simulations revealed the requirement for an unknown late com gene product that shuts of competence by impairing ComX activity. Further simulations led to the predictions that the membrane protein ComD bound to CSP reacts directly to pH change of the medium and that blindness to CSP during the post-competence phase is controlled by late DprA protein. Both predictions were confirmed experimentally.

Single-strand DNA processing: phylogenomics and sequence diversity of a superfamily of potential prokaryotic HuH endonucleases.

BACKGROUND: Some mobile genetic elements target the lagging strand template during DNA replication. Bacterial examples are insertion sequences IS608 and ISDra2 (IS200/IS605 family members). They use obligatory single-stranded circular DNA intermediates for excision and insertion and encode a transposase, TnpAIS200, which recognizes subterminal secondary structures at the insertion sequence ends. Similar secondary structures, Repeated Extragenic Palindromes (REP), are present in many bacterial genomes. TnpAIS200-related proteins, TnpAREP, have been identified and could be responsible for REP sequence proliferation. These proteins share a conserved HuH/Tyrosine core domain responsible for catalysis and are involved in processes of ssDNA cleavage and ligation. Our goal is to characterize the diversity of these proteins collectively referred as the TnpAY1 family. RESULTS: A genome-wide analysis of sequences similar to TnpAIS200 and TnpAREP in prokaryotes revealed a large number of family members with a wide taxonomic distribution. These can be arranged into three distinct classes and 12 subclasses based on sequence similarity. One subclass includes sequences similar to TnpAIS200. Proteins from other subclasses are not associated with typical insertion sequence features. These are characterized by specific additional domains possibly involved in protein/DNA or protein/protein interactions. Their genes are found in more than 25% of species analyzed. They exhibit a patchy taxonomic distribution consistent with dissemination by horizontal gene transfers followed by loss. The tnpAREP genes of five subclasses are flanked by typical REP sequences in a REPtron-like arrangement. Four distinct REP types were characterized with a subclass specific distribution. Other subclasses are not associated with REP sequences but have a large conserved domain located in C-terminal end of their sequence. This unexpected diversity suggests that, while most likely involved in processing single-strand DNA, proteins from different subfamilies may play a number of different roles. CONCLUSIONS: We established a detailed classification of TnpAY1 proteins, consolidated by the analysis of the conserved core domains and the characterization of additional domains. The data obtained illustrate the unexpected diversity of the TnpAY1 family and provide a strong framework for future evolutionary and functional studies. By their potential function in ssDNA editing, they may confer adaptive responses to host cell physiology and metabolism.

Insights into RNA-processing pathways and associated RNA-degrading enzymes in Archaea.

RNA-processing pathways are at the centre of regulation of gene expression. All RNA transcripts undergo multiple maturation steps in addition to covalent chemical modifications to become functional in the cell. This includes destroying unnecessary or defective cellular RNAs. In Archaea, information on mechanisms by which RNA species reach their mature forms and associated RNA-modifying enzymes are still fragmentary. To date, most archaeal actors and pathways have been proposed in light of information gathered from Bacteria and Eukarya. In this context, this review provides a state of the art overview of archaeal endoribonucleases and exoribonucleases that cleave and trim RNA species and also of the key small archaeal proteins that bind RNAs. Furthermore, synthetic up-to-date views of processing and biogenesis pathways of archaeal transfer and ribosomal RNAs as well as of maturation of stable small non-coding RNAs such as CRISPR RNAs, small C/D and H/ACA box guide RNAs, and other emerging classes of small RNAs are described. Finally, prospective post-transcriptional mechanisms to control archaeal messenger RNA quality and quantity are discussed.

Evolution of Mycolic Acid Biosynthesis Genes and Their Regulation during Starvation in Mycobacterium tuberculosis.

UNLABELLED: Mycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information is available on the expression and regulation of the biosynthesis genes. In this study, we report that the hadA, hadB, and hadC genes, which code for the mycolate biosynthesis dehydratase enzymes, are coexpressed with three genes that encode proteins of the translational apparatus. Consistent with the well-established control of the translation potential by nutrient availability, starvation leads to downregulation of the hadABC genes along with most of the genes required for the synthesis, modification, and transport of mycolates. The downregulation of a subset of the biosynthesis genes is partially dependent on RelMtb, the key enzyme of the stringent response. We also report the phylogenetic evolution scenario that has shaped the current genetic organization, characterized by the coregulation of the hadABC operon with genes of the translational apparatus and with genes required for the modification of the mycolates. IMPORTANCE: Mycobacterium tuberculosis infects one-third of the human population worldwide, and despite the available therapeutic arsenal, it continues to kill millions of people each year. There is therefore an urgent need to identify new targets and develop a better understanding of how the bacterium is adapting itself to host defenses during infection. A prerequisite of this understanding is knowledge of how this adaptive skill has been implanted by evolution. Nutrient scarcity is an environmental condition the bacterium has to cope with during infection. In many bacteria, adaptation to starvation relies partly on the stringent response. M. tuberculosis's unique outer membrane layer, the mycomembrane, is crucial for its viability and virulence. Despite its being the target of the major antituberculosis drugs, only scattered information exists on how the genes required for biosynthesis of the mycomembrane are expressed and regulated during starvation. This work has addressed this issue as a step toward the identification of new targets in the fight against M. tuberculosis.

Bacterial transformation: distribution, shared mechanisms and divergent control.

Natural bacterial transformation involves the internalization and chromosomal integration of DNA and has now been documented in ~80 species. Recent advances have established that phylogenetically distant species share conserved uptake and processing proteins but differ in the inducing cues and regulatory mechanisms that are involved. In this Review, we highlight divergent and common principles that govern the transformation process in different bacteria. We discuss how this cumulative knowledge enables the prediction of new transformable species and supports the idea that the main role of internalized DNA is in the generation of genetic diversity or in chromosome repair rather than in nutrition.

Diversity of ß-lactam resistance mechanisms in cystic fibrosis isolates of Pseudomonas aeruginosa: a French multicentre study.

OBJECTIVES: To investigate the resistance mechanisms of ß-lactam-resistant Pseudomonas aeruginosa isolated from cystic fibrosis (CF) patients in France. METHODS: Two-hundred-and-four P. aeruginosa CF isolates were collected in 10 French university hospitals in 2007. Their susceptibility to 14 antibiotics and their resistance mechanisms to ß-lactams were investigated. Their ß-lactamase contents were characterized by isoelectric focusing, PCR and enzymatic assays. Expression levels of efflux pumps and the intrinsic ß-lactamase AmpC were quantified by reverse transcription real-time quantitative PCR. Genotyping was performed using multiple-locus variable number of tandem repeats analysis (MLVA). The oprD genes were sequenced and compared with those of reference P. aeruginosa strains. To assess deficient OprD production, western blotting experiments were carried out on outer membrane preparations. RESULTS: MLVA typing discriminated 131 genotypes and 47 clusters. One-hundred-and-twenty-four isolates (60.8%) displayed a susceptible phenotype to ß-lactams according to EUCAST breakpoints. In the 80 remaining isolates, resistance to ß-lactams resulted from derepression of intrinsic cephalosporinase AmpC (61.3%) and/or acquisition of secondary ß-lactamases (13.8%). Efflux pumps were up-regulated in 88.8% of isolates and porin OprD was lost in 53.8% of isolates due to frameshifting or nonsense mutations in the oprD gene. CONCLUSIONS: ß-Lactam resistance rates are quite high in CF strains of P. aeruginosa isolated in France and not really different from those reported for nosocomial strains. Development of ß-lactam resistance is correlated with patient age. It results from intrinsic mechanisms sequentially accumulated by bacteria isolated from patients who have undergone repeated courses of chemotherapy.

Direct involvement of DprA, the transformation-dedicated RecA loader, in the shut-off of pneumococcal competence.

Natural bacterial transformation is a genetically programmed process allowing genotype alterations that involves the internalization of DNA and its chromosomal integration catalyzed by the universal recombinase RecA, assisted by its transformation-dedicated loader, DNA processing protein A (DprA). In Streptococcus pneumoniae, the ability to internalize DNA, known as competence, is transient, developing suddenly and stopping as quickly. Competence is induced by the comC-encoded peptide, competence stimulating peptide (CSP), via a classic two-component regulatory system ComDE. Upon CSP binding, ComD phosphorylates the ComE response-regulator, which then activates transcription of comCDE and the competence-specific σ(X), leading to a sudden rise in CSP levels and rendering all cells in a culture competent. However, how competence stops has remained unknown. We report that DprA, under σ(X) control, interacts with ComE∼P to block ComE-driven transcription, chiefly impacting σ(X) production. Mutations of dprA specifically disrupting interaction with ComE were isolated and shown to map mainly to the N-terminal domain of DprA. Wild-type DprA but not ComE interaction mutants affected in vitro binding of ComE to its promoter targets. Once introduced at the dprA chromosomal locus, mutations disrupting DprA interaction with ComE altered competence shut-off. The absence of DprA was found to negatively impact growth following competence induction, highlighting the importance of DprA for pneumococcal physiology. DprA has thus two key roles: ensuring production of transformants via interaction with RecA and competence shut-off via interaction with ComE, avoiding physiologically detrimental consequences of prolonged competence. Finally, phylogenetic analyses revealed that the acquisition of a new function by DprA impacted its evolution in streptococci relying on ComE to regulate comX expression.

Centromere binding and evolution of chromosomal partition systems in the Burkholderiales.

How split genomes arise and evolve in bacteria is poorly understood. Since each replicon of such genomes encodes a specific partition (Par) system, the evolution of Par systems could shed light on their evolution. The cystic fibrosis pathogen Burkholderia cenocepacia has three chromosomes (c1, c2, and c3) and one plasmid (pBC), whose compatibility depends on strictly specific interactions of the centromere sequences (parS) with their cognate binding proteins (ParB). However, the Par systems of B. cenocepacia c2, c3, and pBC share many features, suggesting that they arose within an extended family. Database searching revealed seven subfamilies of Par systems like those of B. cenocepacia. All are from plasmids and secondary chromosomes of the Burkholderiales, which reinforces the proposal of an extended family. The subfamily of the Par system of B. cenocepacia c3 includes plasmid variants with parS sequences divergent from that of c3. Using electrophoretic mobility shift assay (EMSA), we found that ParB-c3 binds specifically to centromeres of these variants, despite high DNA sequence divergence. We suggest that the Par system of B. cenocepacia c3 has preserved the features of an ancestral system. In contrast, these features have diverged variably in the plasmid descendants. One such descendant is found both in Ralstonia pickettii 12D, on a free plasmid, and in Ralstonia pickettii 12J, on a plasmid integrated into the main chromosome. These observations suggest that we are witnessing a plasmid-chromosome interaction from which a third chromosome will emerge in a two-chromosome species.

Structuring the bacterial genome: Y1-transposases associated with REP-BIME sequences.

REPs are highly repeated intergenic palindromic sequences often clustered into structures called BIMEs including two individual REPs separated by short linker of variable length. They play a variety of key roles in the cell. REPs also resemble the sub-terminal hairpins of the atypical IS200/605 family of insertion sequences which encode Y1 transposases (TnpA(IS200/IS605)). These belong to the HUH endonuclease family, carry a single catalytic tyrosine (Y) and promote single strand transposition. Recently, a new clade of Y1 transposases (TnpA(REP)) was found associated with REP/BIME in structures called REPtrons. It has been suggested that TnpA(REP) is responsible for REP/BIME proliferation over genomes. We analysed and compared REP distribution and REPtron structure in numerous available E. coli and Shigella strains. Phylogenetic analysis clearly indicated that tnpA(REP) was acquired early in the species radiation and was lost later in some strains. To understand REP/BIME behaviour within the host genome, we also studied E. coli K12 TnpA(REP) activity in vitro and demonstrated that it catalyses cleavage and recombination of BIMEs. While TnpA(REP) shared the same general organization and similar catalytic characteristics with TnpA(IS200/IS605) transposases, it exhibited distinct properties potentially important in the creation of BIME variability and in their amplification. TnpA(REP) may therefore be one of the first examples of transposase domestication in prokaryotes.

The cyst-dividing bacterium Ramlibacter tataouinensis TTB310 genome reveals a well-stocked toolbox for adaptation to a desert environment.

Ramlibacter tataouinensis TTB310(T) (strain TTB310), a betaproteobacterium isolated from a semi-arid region of South Tunisia (Tataouine), is characterized by the presence of both spherical and rod-shaped cells in pure culture. Cell division of strain TTB310 occurs by the binary fission of spherical "cyst-like" cells ("cyst-cyst" division). The rod-shaped cells formed at the periphery of a colony (consisting mainly of cysts) are highly motile and colonize a new environment, where they form a new colony by reversion to cyst-like cells. This unique cell cycle of strain TTB310, with desiccation tolerant cyst-like cells capable of division and desiccation sensitive motile rods capable of dissemination, appears to be a novel adaptation for life in a hot and dry desert environment. In order to gain insights into strain TTB310's underlying genetic repertoire and possible mechanisms responsible for its unusual lifestyle, the genome of strain TTB310 was completely sequenced and subsequently annotated. The complete genome consists of a single circular chromosome of 4,070,194 bp with an average G+C content of 70.0%, the highest among the Betaproteobacteria sequenced to date, with total of 3,899 predicted coding sequences covering 92% of the genome. We found that strain TTB310 has developed a highly complex network of two-component systems, which may utilize responses to light and perhaps a rudimentary circadian hourglass to anticipate water availability at the dew time in the middle/end of the desert winter nights and thus direct the growth window to cyclic water availability times. Other interesting features of the strain TTB310 genome that appear to be important for desiccation tolerance, including intermediary metabolism compounds such as trehalose or polyhydroxyalkanoate, and signal transduction pathways, are presented and discussed.

The PprA-PprB two-component system activates CupE, the first non-archetypal Pseudomonas aeruginosa chaperone-usher pathway system assembling fimbriae.

The opportunistic pathogen Pseudomonas aeruginosa has redundant molecular systems that contribute to its pathogenicity. Those assembling fimbrial structures promote complex organized community lifestyle. We characterized a new 5.8 kb genetic locus, cupE, that includes the conserved usher- and chaperone-encoding genes. This locus, widely conserved in different bacterial species, contains four additional genes encoding non-archetypal fimbrial subunits. We first evidenced that the cupE gene cluster was specifically expressed in biofilm conditions and was responsible for fibre assembly containing at least CupE1 protein, at the bacterial cell surface. These fimbriae not only played a significant role in the early stages (microcolony and macrocolony formation) but also in shaping 3D mushrooms during P. aeruginosa biofilm development. Using wide-genome transposon mutagenesis, we identified the PprAB two-component system (TCS) as a regulator of cupE expression, and further demonstrated the involvement of the PprAB TCS in direct CupE fimbrial assembly activation. Thus, this TCS represents a new regulatory element controlling the transition between planktonic and community lifestyles in P. aeruginosa.

Functional dichotomy of ribosomal proteins during the synthesis of mammalian 40S ribosomal subunits.

Our knowledge of the functions of metazoan ribosomal proteins in ribosome synthesis remains fragmentary. Using siRNAs, we show that knockdown of 31 of the 32 ribosomal proteins of the human 40S subunit (ribosomal protein of the small subunit [RPS]) strongly affects pre-ribosomal RNA (rRNA) processing, which often correlates with nucleolar chromatin disorganization. 16 RPSs are strictly required for initiating processing of the sequences flanking the 18S rRNA in the pre-rRNA except at the metazoan-specific early cleavage site. The remaining 16 proteins are necessary for progression of the nuclear and cytoplasmic maturation steps and for nuclear export. Distribution of these two subsets of RPSs in the 40S subunit structure argues for a tight dependence of pre-rRNA processing initiation on the folding of both the body and the head of the forming subunit. Interestingly, the functional dichotomy of RPS proteins reported in this study is correlated with the mutation frequency of RPS genes in Diamond-Blackfan anemia.

SpxA1, a novel transcriptional regulator involved in X-state (competence) development in Streptococcus pneumoniae.

Streptococcus pneumoniae is a naturally transformable human pathogen. Genome and phylogenetic analyses uncovered two Spx-like global transcriptional regulators, SpxA1 and SpxA2, encoded by S. pneumoniae. spxA1 and spxA2 are not essential, but their simultaneous inactivation is lethal. SpxA1 represses transcription of the early competence operon comCDE and thereby negatively regulates the initiation of the X-state (competence). The molecular basis of this repression could be similar to that of SpxA of Bacillus subtilis, involving a specific interaction with the alpha subunit of RNA polymerase. S. pneumoniae lacks an SOS-like stress response and the X-state is proposed to be a general stress response mechanism in this species. In light of this, SpxA1-dependent repression could act to sense environmental or metabolic stresses and prevent launching of the X-state in the absence of stress.

Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources?

BACKGROUND: The availability of hundreds of bacterial genomes allowed a comparative genomic study of the Type VI Secretion System (T6SS), recently discovered as being involved in pathogenesis. By combining comparative and phylogenetic approaches using more than 500 prokaryotic genomes, we characterized the global T6SS genetic structure in terms of conservation, evolution and genomic organization. RESULTS: This genome wide analysis allowed the identification of a set of 13 proteins constituting the T6SS protein core and a set of conserved accessory proteins. 176 T6SS loci (encompassing 92 different bacteria) were identified and their comparison revealed that T6SS-encoded genes have a specific conserved genetic organization. Phylogenetic reconstruction based on the core genes showed that lateral transfer of the T6SS is probably its major way of dissemination among pathogenic and non-pathogenic bacteria. Furthermore, the sequence analysis of the VgrG proteins, proposed to be exported in a T6SS-dependent way, confirmed that some C-terminal regions possess domains showing similarities with adhesins or proteins with enzymatic functions. CONCLUSION: The core of T6SS is composed of 13 proteins, conserved in both pathogenic and non-pathogenic bacteria. Subclasses of T6SS differ in regulatory and accessory protein content suggesting that T6SS has evolved to adapt to various microenvironments and specialized functions. Based on these results, new functional hypotheses concerning the assembly and function of T6SS proteins are proposed.

A key presynaptic role in transformation for a widespread bacterial protein: DprA conveys incoming ssDNA to RecA.

Natural transformation is a mechanism for genetic exchange in many bacterial genera. It proceeds through the uptake of exogenous DNA and subsequent homology-dependent integration into the genome. In Streptococcus pneumoniae, this integration requires the ubiquitous recombinase, RecA, and DprA, a protein of unknown function widely conserved in bacteria. To unravel the role of DprA, we have studied the properties of the purified S. pneumoniae protein and its Bacillus subtilis ortholog (Smf). We report that DprA and Smf bind cooperatively to single-stranded DNA (ssDNA) and that these proteins both self-interact and interact with RecA. We demonstrate that DprA-RecA-ssDNA filaments are produced and that these filaments catalyze the homology-dependent formation of joint molecules. Finally, we show that while the Escherichia coli ssDNA-binding protein SSB limits access of RecA to ssDNA, DprA lowers this barrier. We propose that DprA is a new member of the recombination-mediator protein family, dedicated to natural bacterial transformation.