Jean-Pierre Claverys

The genetic transformation machinery: composition, localization, and mechanism

Natural genetic transformation is widely distributed in bacteria. It is a genetically programmed process that is inherent to the species. Transformation requires a specialized membrane-associated machinery for uptake of exogenous double-stranded DNA. It also requires dedicated cytosolic proteins, some of which have been characterized only recently, for the processing of internalized single-stranded DNA fragments into recombination products. A series of observations made in Bacillus subtilis and Streptococcus pneumoniae led to the recent emergence of a picture of a unique, highly integrated machine localized at the cell poles. This dynamic machine, which we propose to name the transformasome, involves both membrane and cytosolic proteins, to internalize, protect, and process transforming DNA. This review attempts to summarize these recent observations with special emphasis on the early stages in DNA processing.

Interplay of the Serine/Threonine-Kinase StkP and the Paralogs DivIVA and GpsB in Pneumococcal Cell Elongation and Division

Despite years of intensive research, much remains to be discovered to understand the regulatory networks coordinating bacterial cell growth and division. The mechanisms by which Streptococcus pneumoniae achieves its characteristic ellipsoid-cell shape remain largely unknown. In this study, we analyzed the interplay of the cell division paralogs DivIVA and GpsB with the ser/thr kinase StkP. We observed that the deletion of divIVA hindered cell elongation and resulted in cell shortening and rounding. By contrast, the absence of GpsB resulted in hampered cell division and triggered cell elongation. Remarkably, ΔgpsB elongated cells exhibited a helical FtsZ pattern instead of a Z-ring, accompanied by helical patterns for DivIVA and peptidoglycan synthesis. Strikingly, divIVA deletion suppressed the elongated phenotype of ΔgpsB cells. These data suggest that DivIVA promotes cell elongation and that GpsB counteracts it. Analysis of protein-protein interactions revealed that GpsB and DivIVA do not interact with FtsZ but with the cell division protein EzrA, which itself interacts with FtsZ. In addition, GpsB interacts directly with DivIVA. These results are consistent with DivIVA and GpsB acting as a molecular switch to orchestrate peripheral and septal PG synthesis and connecting them with the Z-ring via EzrA. The cellular co-localization of the transpeptidases PBP2x and PBP2b as well as the lipid-flippases FtsW and RodA in ΔgpsB cells further suggest the existence of a single large PG assembly complex. Finally, we show that GpsB is required for septal localization and kinase activity of StkP, and therefore for StkP-dependent phosphorylation of DivIVA. Altogether, we propose that the StkP/DivIVA/GpsB triad finely tunes the two modes of peptidoglycan (peripheral and septal) synthesis responsible for the pneumococcal ellipsoid cell shape.

A Type IV Pilus Mediates DNA Binding during Natural Transformation in Streptococcus pneumoniae

Natural genetic transformation is widely distributed in bacteria and generally occurs during a genetically programmed differentiated state called competence. This process promotes genome plasticity and adaptability in Gram-negative and Gram-positive bacteria. Transformation requires the binding and internalization of exogenous DNA, the mechanisms of which are unclear. Here, we report the discovery of a transformation pilus at the surface of competent Streptococcus pneumoniae cells. This Type IV-like pilus, which is primarily composed of the ComGC pilin, is required for transformation. We provide evidence that it directly binds DNA and propose that the transformation pilus is the primary DNA receptor on the bacterial cell during transformation in S. pneumoniae. Being a central component of the transformation apparatus, the transformation pilus enables S. pneumoniae, a major Gram-positive human pathogen, to acquire resistance to antibiotics and to escape vaccines through the binding and incorporation of new genetic material.

Role of the Single-Stranded DNA–Binding Protein SsbB in Pneumococcal Transformation: Maintenance of a Reservoir for Genetic Plasticity

Bacteria encode a single-stranded DNA (ssDNA) binding protein (SSB) crucial for genome maintenance. In Bacillus subtilis and Streptococcus pneumoniae, an alternative SSB, SsbB, is expressed uniquely during competence for genetic transformation, but its precise role has been disappointingly obscure. Here, we report our investigations involving comparison of a null mutant (ssbB −) and a C-ter truncation (ssbBΔ7) of SsbB of S. pneumoniae, the latter constructed because SSBs acidic tail has emerged as a key site for interactions with partner proteins. We provide evidence that SsbB directly protects internalized ssDNA. We show that SsbB is highly abundant, potentially allowing the binding of ∼1.15 Mb ssDNA (half a genome equivalent); that it participates in the processing of ssDNA into recombinants; and that, at high DNA concentration, it is of crucial importance for chromosomal transformation whilst antagonizing plasmid transformation. While the latter observation explains a long-standing observation that plasmid transformation is very inefficient in S. pneumoniae (compared to chromosomal transformation), the former supports our previous suggestion that SsbB creates a reservoir of ssDNA, allowing successive recombination cycles. SsbBΔ7 fulfils the reservoir function, suggesting that SsbB C-ter is not necessary for processing protein(s) to access stored ssDNA. We propose that the evolutionary raison dêtre of SsbB and its abundance is maintenance of this reservoir, which contributes to the genetic plasticity of S. pneumoniae by increasing the likelihood of multiple transformation events in the same cell.

Expression and maintenance of ComD–ComE, the two-component signal-transduction system that controls competence of Streptococcus pneumoniae

A secreted competence‐stimulating peptide (CSP), encoded by comC, constitutes, together with the two‐component system ComD‐ComE, the master switch for competence induction in Streptococcus pneumoniae. Interaction between CSP and its membrane‐bound histidine‐kinase receptor, ComD, is believed to lead to autophosphorylation of ComD, which then transphosphorylates the ComE response regulator to activate transcription of a limited set of genes, including the comCDE operon. This generates a positive feedback loop, amplifying the signal and co‐ordinating competence throughout the population. On the other hand, the promoter(s) and proteins important for basal comCDE expression have not been defined. We now report that CSP‐induced and basal comCDE transcription both initiate from the same promoter, PE; that basal expression necessitates the presence of both ComD and a phosphate‐accepting form of ComE, but not CSP; and that overexpression of ComER120S triggers ComD‐dependent transformation in the absence of CSP. These observations suggest that self‐activation of ComD is required for basal comCDE expression. We also establish that transcriptional readthrough occurs across the tRNAArg5 terminator and contributes significantly to comCDE expression. Finally, we demonstrate by various means, including single‐cell competence analysis with GFP, that readthrough is crucial to avoid the stochastic production of CSP non‐responsive cells lacking ComD or ComE.

Induction of competence for genetic transformation by antibiotics: convergent evolution of stress responses in distant bacterial species lacking SOS?

Bacterial transformation is a programmed process resulting in genetic transfer and diversity. It relies on the development of competence via regulatory circuits which are diverse and tailored to the particular lifestyle of each species. Despite this diversity, some species have been reported to trigger competence in response to antibiotics. Here, we review these recent findings, which reinforce the view that competence is a stress response and can substitute for SOS in bacteria lacking it.

ComE/ComE∼P interplay dictates activation or extinction status of pneumococcal X‐state (competence)

Since 1996, induction of competence for genetic transformation of Streptococcus pneumoniae is known to be controlled by the ComD/ComE two‐component regulatory system. The mechanism of induction is generally described as involving ComD autophosphorylation, transphosphorylation of ComE and transcriptional activation by ComE∼P of the early competence (com) genes, including comX which encodes the competence‐specific σX. However, none of these features has been experimentally established. Here we document the autokinase activity of ComD proteins in vitro, and provide an estimate of the stoichiometry of ComD and ComE in vivo. We report that a phosphorylmimetic mutant, ComED58E, constructed because of the failure to detect transphosphorylation of purified ComE in vitro, displays full spontaneous competence in ΔcomD cells, an that in vitro ComED58E exhibits significantly improved binding affinity for PcomCDE. We also provide evidence for a differential transcriptional activation and repression of PcomCDE and PcomX. Altogether, these data support the model of ComE∼P‐dependent activation of transcription. Finally, we establish that ComE antagonizes expression of the early com genes and propose that the rapid deceleration of transcription from PcomCDE observed even in cells lacking σX is due to the progressive accumulation of ComE, which outcompetes ComE∼P.

The global nutritional regulator CodY is an essential protein in the human pathogen Streptococcus pneumoniae

CodY is a global regulator highly conserved in low‐G+C Gram‐positive bacteria. It plays a key role in the adaptation of Bacillus subtilis to nutritional limitation through repression of a large gene set during exponential growth and relief of repression upon starvation. In several pathogenic bacteria, CodY regulates major virulence genes. Our interest in Streptococcus pneumoniae CodY originates from our observations that the oligopeptide permease Ami was involved in repression of competence for genetic transformation. We hypothesized that peptide uptake through Ami feeds amino acid pools, which are sensed by CodY to repress competence. As our initial attempts at inactivating codY failed, we launched an in‐depth analysis into the question of the essentiality of codY. We report that codY cannot be inactivated unless a complementing ectopic copy is present. We obtained genetic evidence that a recently published D39 codY knock‐out contains additional mutations allowing survival of codY mutant cells. Whole‐genome sequencing revealed mutations in fatC, which encodes a ferric iron permease, and amiC. This combination of mutations was confirmed to allow tolerance of codY inactivation. The amiC mutation is in itself sufficient to account for the strong derepression of competence development observed in D39 codY cells.

Transforming DNA Uptake Gene Orthologs Do Not Mediate Spontaneous Plasmid Transformation in Escherichia coli

Spontaneous plasmid transformation of Escherichia coli occurs on nutrient-containing agar plates. E. coli has also been reported to use double-stranded DNA (dsDNA) as a carbon source. The mechanism(s) of entry of exogenous dsDNA that allows plasmid establishment or the use of DNA as a nutrient remain(s) unknown. To further characterize plasmid transformation, we first documented the stimulation of transformation by agar and agarose. We provide evidence that stimulation is not due to agar contributing a supplement of Ca(2+), Fe(2+), Mg(2+), Mn(2+), or Zn(2+). Second, we undertook to inactivate the E. coli orthologues of Haemophilus influenzae components of the transformation machine that allows the uptake of single-stranded DNA (ssDNA) from exogenous dsDNA. The putative outer membrane channel protein (HofQ), transformation pseudopilus component (PpdD), and transmembrane pore (YcaI) are not required for plasmid transformation. We conclude that plasmid DNA does not enter E. coli cells as ssDNA. The finding that purified plasmid monomers transform E. coli with single-hit kinetics supports this conclusion; it establishes that a unique monomer molecule is sufficient to give rise to a transformant, which is not consistent with the reconstitution of an intact replicon through annealing of partially overlapping complementary ssDNA, taken up from two independent monomers. We therefore propose that plasmid transformation involves internalization of intact dsDNA molecules. Our data together, with previous reports that HofQ is required for the use of dsDNA as a carbon source, suggest the existence of two routes for DNA entry, at least across the outer membrane of E. coli.

Natural Genetic Transformation Generates a Population of Merodiploids in Streptococcus pneumoniae

Partial duplication of genetic material is prevalent in eukaryotes and provides potential for evolution of new traits. Prokaryotes, which are generally haploid in nature, can evolve new genes by partial chromosome duplication, known as merodiploidy. Little is known about merodiploid formation during genetic exchange processes, although merodiploids have been serendipitously observed in early studies of bacterial transformation. Natural bacterial transformation involves internalization of exogenous donor DNA and its subsequent integration into the recipient genome by homology. It contributes to the remarkable plasticity of the human pathogen Streptococcus pneumoniae through intra and interspecies genetic exchange. We report that lethal cassette transformation produced merodiploids possessing both intact and cassette-inactivated copies of the essential target gene, bordered by repeats (R) corresponding to incomplete copies of IS861. We show that merodiploidy is transiently stimulated by transformation, and only requires uptake of a ∼3-kb DNA fragment partly repeated in the chromosome. We propose and validate a model for merodiploid formation, providing evidence that tandem-duplication (TD) formation involves unequal crossing-over resulting from alternative pairing and interchromatid integration of R. This unequal crossing-over produces a chromosome dimer, resolution of which generates a chromosome with the TD and an abortive chromosome lacking the duplicated region. We document occurrence of TDs ranging from ∼100 to ∼900 kb in size at various chromosomal locations, including by self-transformation (transformation with recipient chromosomal DNA). We show that self-transformation produces a population containing many different merodiploid cells. Merodiploidy provides opportunities for evolution of new genetic traits via alteration of duplicated genes, unrestricted by functional selective pressure. Transient stimulation of a varied population of merodiploids by transformation, which can be triggered by stresses such as antibiotic treatment in S. pneumoniae, reinforces the plasticity potential of this bacterium and transformable species generally.