In response to the 2011 European health alert caused by a pathogenic Escherichia coli O104:H4 outbreak, the European Academy of Microbiology (EAM), established by the Federation of European Microbiological Societies (FEMS), convened a meeting in Paris on November 30th, 2011 on ‘EHEC infection and control’ attended by world renowned experts in pathogenic E. coli. The major aims of this group were to review the scientific issues raised by the outbreak, to assess the handling of the crisis at the scientific and political levels, and to propose future actions. Several conclusions, which will have impact on future potential E. coli outbreaks, are outlined here.

Although Mycobacterium ulcerans , M. marinum , and M. haemophilum are closely related, their exact taxonomic placements have not been determined. We performed gas chromatography of fatty acids and alcohols, as well as DNA-DNA hybridization and 16S rRNA gene sequence analysis, to clarify their relationships to each other and to M. tuberculosis. M. ulcerans and M. marinum were most closely related to one another, and each displayed very strong genetic affinities to M. tuberculosis ; they are actually the two mycobacterial species outside the M. tuberculosis complex most closely related to M. tuberculosis . M. haemophilum was more distinct from M. ulcerans and M. marinum , and it appeared to be as related to these two species as to M. tuberculosis . These results are important with regard to the development of diagnostic and epidemiological tools such as species-specific DNA probes and PCR assays for M. ulcerans , M. marinum , and M. haemophilum . In addition, the finding that M. ulcerans and M. marinum are more closely related to M. tuberculosis than are other pathogenic mycobacterial species suggests that they may be evaluated as useful models for studying the pathogenesis of M. tuberculosis . M. marinum may be particularly useful in this regard since strains of this species grow much more rapidly than M. tuberculosis and yet can cause systemic disease in immunocompromised hosts.

The bacterial pathogen Neisseria meningitidis expresses long, thin, retractile fibers (called type IV pili) from its cell surface and uses these adhesive structures to mediate primary attachment to epithelial cells during host colonization and invasion. PilQ is an outer membrane protein complex that is essential for the translocation of these pili across the outer membrane. Here, we present the structure of the PilQ complex determined by cryoelectron microscopy to 12 Å resolution. The dominant feature of the structure is a large central cavity, formed by four arm features that spiral upwards from a squared ring base and meet to form a prominent cap region. The cavity, running through the center of the complex, is continuous and is effectively sealed at both the top and bottom. Analysis of the complex using self-orientation and by examination of two-dimensional crystals indicates a strong C4 rotational symmetry, with a much weaker C12 rotational symmetry, consistent with PilQ possessing true C4 symmetry with C12 quasi-symmetry. We therefore suggest that the complex is a homododecamer, formed by association of 12 PilQ polypeptide chains into a tetramer of trimers. The structure of the PilQ complex, with its large and well defined central chamber, suggests that it may not function solely as a passive portal in the outer membrane, but could be actively involved in mediating pilus assembly or modification.

The frequently occurring DNA uptake sequence (DUS), recognized as a 10-bp repeat, is required for efficient genetic transformation in the human pathogens Neisseria meningitidis and Neisseria gonorrhoeae . Genome scanning for DUS occurrences in three different species of Neisseria demonstrated that 76% of the nearly 2,000 neisserial DUS were found to have two semiconserved base pairs extending from the 5′ end of DUS to constitute a 12-mer repeat. Plasmids containing sequential variants of the neisserial DUS were tested for their ability to transform N. meningitidis and N. gonorrhoeae , and the 12-mer was found to outperform the 10-mer DUS in transformation efficiency. Assessment of meningococcal uptake of DNA confirmed the enhanced performance of the 12-mer compared to the 10-mer DUS. An inverted repeat DUS was not more efficient in transformation than DNA species containing a single or direct repeat DUS. Genome-wide analysis revealed that half of the nearly 1,500 12-mer DUS are arranged as inverted repeats predicted to be involved in rho-independent transcriptional termination or attenuation. The distribution of the uptake signal sequence required for transformation in the Pasteurellaceae was also biased towards transcriptional terminators, although to a lesser extent. In addition to assessing the intergenic location of DUS, we propose that the 10-mer identity of DUS should be extended and recognized as a 12-mer DUS. The dual role of DUS in transformation and as a structural component on RNA affecting transcription makes this a relevant model system for assessing significant roles of repeat sequences in biology.

Mycobacterium tuberculosis complex species display relatively static genomes and 99.9% nucleotide sequence identity. Studying the evolutionary history of such monomorphic bacteria is a difficult and challenging task. We found that single-nucleotide polymorphism (SNP) analysis of DNA repair, recombination and replication (3R) genes in a comprehensive selection of M. tuberculosis complex strains from across the world, yielded surprisingly high levels of polymorphisms as compared to house-keeping genes, making it possible to distinguish between 80% of clinical isolates analyzed in this study. Bioinformatics analysis suggests that a large number of these polymorphisms are potentially deleterious. Site frequency spectrum comparison of synonymous and non-synonymous variants and Ka/Ks ratio analysis suggest a general negative/purifying selection acting on these sets of genes that may lead to suboptimal 3R system activity. In turn, the relaxed fidelity of 3R genes may allow the occurrence of adaptive variants, some of which will survive. Furthermore, 3R-based phylogenetic trees are a new tool for distinguishing between M. tuberculosis complex strains. This situation, and the consequent lack of fidelity in genome maintenance, may serve as a starting point for the evolution of antibiotic resistance, fitness for survival and pathogenicity, possibly conferring a selective advantage in certain stressful situations. These findings suggest that 3R genes may play an important role in the evolution of highly clonal bacteria, such as M. tuberculosis. They also facilitate further epidemiological studies of these bacteria, through the development of high-resolution tools. With many more microbial genomes being sequenced, our results open the door to 3R gene-based studies of adaptation and evolution of other, highly clonal bacteria.

Mismatch repair (MMR) is a near ubiquitous pathway, essential for the maintenance of genome stability. Members of the MutS and MutL protein families perform key steps in mismatch correction. Despite the major importance of this repair pathway, MutS–MutL are absent in almost all Actinobacteria and many Archaea. However, these organisms exhibit rates and spectra of spontaneous mutations similar to MMR-bearing species, suggesting the existence of an alternative to the canonical MutS–MutL-based MMR. Here we report that Mycobacterium smegmatis NucS/EndoMS, a putative endonuclease with no structural homology to known MMR factors, is required for mutation avoidance and anti-recombination, hallmarks of the canonical MMR. Furthermore, phenotypic analysis of naturally occurring polymorphic NucS in a M. smegmatis surrogate model, suggests the existence of M. tuberculosis mutator strains. The phylogenetic analysis of NucS indicates a complex evolutionary process leading to a disperse distribution pattern in prokaryotes. Together, these findings indicate that distinct pathways for MMR have evolved at least twice in nature.