Type II toxin–antitoxin (TA) systems are generally composed of two genes organized in an operon, encoding a labile antitoxin and a stable toxin. They were first discovered on plasmids where they contribute to plasmid stability by a phenomenon denoted as ‘addiction’, and subsequently in bacterial chromosomes. To discover novel families of antitoxins and toxins, we developed a bioinformatics approach based on the ‘guilt by association’ principle. Extensive experimental validation in Escherichia coli of predicted antitoxins and toxins increased significantly the number of validated systems and defined novel toxin and antitoxin families. Our data suggest that toxin families as well as antitoxin families originate from distinct ancestors that were assembled multiple times during evolution. Toxin and antitoxin families found on plasmids tend to be promiscuous and widespread, indicating that TA systems move through horizontal gene transfer. We propose that due to their addictive properties, TA systems are likely to be maintained in chromosomes even though they do not necessarily confer an advantage to their bacterial hosts. Therefore, addiction might play a major role in the evolutionary success of TA systems both on mobile genetic elements and in bacterial chromosomes.

*Nicotinamide phosphoribosyl transferase (Nampt)/pre-B cell colony-enhancing factor (PBEF)/visfatin is a protein displaying multiple functional properties. Originally described as a cytokine-like protein able to regulate B cell development, apoptosis, and glucose metabolism, this protein also plays an important role in NAD biosynthesis. To gain insight into its physiological role, we have generated a mouse strain expressing a conditional Nampt allele. Lack of Nampt expression strongly affects development of both T and B lymphocytes. Analysis of hemizygous cells and in vitro cell lines expressing distinct levels of Nampt illustrates the critical role of this protein in regulating intracellular NAD levels. Consequently, a clear relationship was found between intracellular Nampt levels and cell death in response to the genotoxic agent MNNG (N-methyl-N-nitro-N-nitrosoguanidine), confirming that this enzyme represents a key regulator of cell sensitivity to NAD-consuming stress secondary to poly(ADP-ribose) polymerases overactivation. By using mutant forms of this protein and a well-characterized pharmacological inhibitor (FK866), we unequivocally demonstrate that the ability of the Nampt to regulate cell viability during genotoxic stress requires its enzymatic activity. Collectively, these data demonstrate that Nampt participates in cellular resistance to genotoxic/oxidative stress, and it may confer to cells of the immune system the ability to survive during stressful situations such as inflammation.

Background Group A Streptococcus (GAS) M protein is an important virulence factor and potential vaccine antigen, and constitutes the basis for strain typing (emm-typing). Although >200 emm-types are characterized, structural data were obtained from only a limited number of emm-types. were aim to evaluate the sequence diversity of near-full-length M proteins from worldwide sources and analyse their structure, sequence conservation and classification. Methods GAS isolates recovered from throughout the world during the last two decades underwent emm-typing and complete emm gene sequencing. Predicted amino acid sequence analyses, secondary structure predictions and vaccine epitope mapping were performed using MUSCLE and Geneious software. Results 1086 isolates from 31 countries were analysed, representing 175 emm-types. emm-type is predictive of the whole protein structure, independent of geographic origin or clinical association. Findings of an emm-type paired with multiple, highly divergent central regions were not observed. M protein sequence length, the presence or absence of sequence repeats, and predicted secondary structure was assessed in the context of the latest vaccine developments. Conclusions Based on these global data, the M6 protein model is updated to a three representative M protein (M5, M80, M77) model, to aid in epidemiological analysis, vaccine development and M protein-related pathogenesis studies.

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Despite increasing interest in coagulase-negative staphylococci (CoNS), little information is available about their bacteriophages. We isolated and sequenced three novel temperate Siphoviridae phages (StB12, StB27, and StB20) from the CoNS Staphylococcus hominis and S. capitis species. The genome sizes are around 40 kb, and open reading frames (ORFs) are arranged in functional modules encoding lysogeny, DNA metabolism, morphology, and cell lysis. Bioinformatics analysis allowed us to assign a potential function to half of the predicted proteins. Structural elements were further identified by proteomic analysis of phage particles, and DNA-packaging mechanisms were determined. Interestingly, the three phages show identical integration sites within their host genomes. In addition to this experimental characterization, we propose a novel classification based on the analysis of 85 phage and prophage genomes, including 15 originating from CoNS. Our analysis established 9 distinct clusters and revealed close relationships between S. aureus and CoNS phages. Genes involved in DNA metabolism and lysis and potentially in phage-host interaction appear to be widespread, while structural genes tend to be cluster specific. Our findings support the notion of a possible reciprocal exchange of genes between phages originating from S. aureus and CoNS, which may be of crucial importance for pathogenesis in staphylococci.

Bacterial type II toxin-antitoxin systems are widespread in bacteria. Among them, the RelE toxin family is one of the most abundant. The RelE K-12 toxin of Escherichia coli K-12 represents the paradigm for this family and has been extensively studied, both in vivo and in vitro. RelE K-12 is an endoribonuclease that cleaves mRNAs that are translated by the ribosome machinery as these transcripts enter the A site. Earlier in vivo reports showed that RelE K-12 cleaves preferentially in the 5=-end coding region of the transcripts in a codon-independent manner. To investigate whether the molecular activity as well as the cleavage pattern are conserved within the members of this toxin family, RelE-like sequences were selected in Proteobacteria, Cyanobacteria, Actinobacteria, and Spirochaetes and tested in E. coli. Our results show that these RelE-like sequences are part of toxin-antitoxin gene pairs, and that they inhibit translation in E. coli by cleaving transcripts that are being translated. Primer extension analyses show that these toxins exhibit specific cleavage patterns in vivo, both in terms of frequency and location of cleavage sites. We did not observe codon-dependent cleavage but rather a trend to cleave upstream purines and between the second and third positions of codons, except for the actinobacterial toxin. Our results suggest that RelE-like toxins have evolved to rapidly and efficiently shut down translation in a large spectrum of bacterial species, which correlates with the observation that toxin-antitoxin systems are spreading by horizontal gene transfer.

A mini‐F region 800 bp long, located between the two F origin sites, plays an essential role in the relationship between the F plasmid and its host. This region comprises two sets of overlapping coding sequences: the first set codes for the newly identified H1 and H2 polypeptides; the second set codes for polypeptides G1 and G2. A mini‐F amber mutation (Ham22) causes the virtual disappearance of polypeptides H1 and H2 but only slightly reduces synthesis of polypeptides G1 and G2. This mutation: (i) renders mini‐F hybrids lethal to the host cells (conditional Hos‐ phenotype for host survival) and (ii) causes the induction of a resident prophage in recA+ strains (conditional Map‐ phenotype for maintenance of the prophage). When an additional mutation prevents the synthesis of polypeptides G1 and G2, both the lethal character and the induction of the prophage are abolished. We conclude: (i) that polypeptides G1 and/or G2 are specific mini‐F polypeptides involved in the plasmid‐mediated killing effect and in the recA‐dependent induction of the resident prophage and (ii) that, in normal conditions, polypeptides H1 and/or H2 negatively control (directly or indirectly) the action of polypeptides G1 and/or G2. In relation to the analysis of indirect induction mediated by u.v.‐irradiated lambda mini‐F hybrids, we propose that polypeptides G1 and/or G2 are specific mini‐F products involved in the activation of the bacterial SOS pathway. The H1/H2 and G1/G2 polypeptides could constitute the controlled mini‐F signal enabling the coordination between cell division and F plasmid replication.

BackgroundAsymptomatic nasopharyngeal carriage represents an important biological marker for monitoring pneumococcal serotype distribution and evaluating vaccine effects. Serotype determination by conventional method (Quellung reaction) is technically and financially challenging. On the contrary, PCR-based serotyping represents a simple, economic and promising alternative method.MethodWe designed a novel multiplex PCR assay for specific detection of the 30 classical colonizing S. pneumoniae serogroups/types. This multiplex assay is composed of 7 consecutive PCR reactions and was validated on a large and recent collection of Streptococcus pneumoniae isolated during a prospective study conducted in Belgium at the time of PCV7 adoption.ResultsThe multiplex PCR assay allowed the typing of more than 94% of the isolates of a collection of pneumococci isolated from Belgian preschool attendees (n = 332). Seventy-five percent of the isolates were typed after 3 subsequent PCR reactions. Results were in agreement with the Quellung identification.ConclusionOur novel multiplex assay is an accurate and reliable method which can be used in place of the conventional method for S. pneumoniae carriage studies.