Maha Farhat, Megan Murray and colleagues report whole-genome sequencing of 116 Mycobacterium tuberculosis strains selected to be representative of both global diversity and drug resistance. The authors develop a new method to search for resistance markers in microbial genomes based on reconstructing a genome-wide phylogeny and identifying regions showing convergent evolution, and they use this method to identify 39 new candidate drug resistance regions in the M. tuberculosis genome. M. tuberculosis is evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including genetic changes favored by selection in resistant isolates, are incompletely understood. Using 116 newly sequenced and 7 previously sequenced M. tuberculosis whole genomes, we identified genome-wide signatures of positive selection specific to the 47 drug-resistant strains. By searching for convergent evolution—the independent fixation of mutations in the same nucleotide position or gene—we recovered 100% of a set of known resistance markers. We also found evidence of positive selection in an additional 39 genomic regions in resistant isolates. These regions encode components in cell wall biosynthesis, transcriptional regulation and DNA repair pathways. Mutations in these regions could directly confer resistance or compensate for fitness costs associated with resistance. Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifampicin.

Streptococcus pneumoniae is one of the most important causes of microbial diseases in humans. The genomes of 44 diverse strains of S. pneumoniae were analyzed and compared with strains of non-pathogenic streptococci of the Mitis group.

The human intestine is densely populated by a microbial consortium whose metabolic activities are influenced by, among others, bifidobacteria. However, the genetic basis of adaptation of bifidobacteria to the human gut is poorly understood. Analysis of the 2,214,650-bp genome of Bifidobacterium bifidum PRL2010, a strain isolated from infant stool, revealed a nutrient-acquisition strategy that targets host-derived glycans, such as those present in mucin. Proteome and transcriptome profiling revealed a set of chromosomal loci responsible for mucin metabolism that appear to be under common transcriptional control and with predicted functions that allow degradation of various O-linked glycans in mucin. Conservation of the latter gene clusters in various B. bifidum strains supports the notion that host-derived glycan catabolism is an important colonization factor for B. bifidum with concomitant impact on intestinal microbiota ecology.

Streptococcus pneumoniae (the pneumococcus) is the world’s foremost bacterial pathogen in both morbidity and mortality. Switching between phenotypic forms (or ‘phases’) that favour asymptomatic carriage or invasive disease was first reported in 1933. Here, we show that the underlying mechanism for such phase variation consists of genetic rearrangements in a Type I restriction-modification system (SpnD39III). The rearrangements generate six alternative specificities with distinct methylation patterns, as defined by single-molecule, real-time (SMRT) methylomics. The SpnD39III variants have distinct gene expression profiles. We demonstrate distinct virulence in experimental infection and in vivo selection for switching between SpnD39III variants. SpnD39III is ubiquitous in pneumococci, indicating an essential role in its biology. Future studies must recognize the potential for switching between these heretofore undetectable, differentiated pneumococcal subpopulations in vitro and in vivo. Similar systems exist in other bacterial genera, indicating the potential for broad exploitation of epigenetic gene regulation.

Summary: Two main patterns of gene expression of Streptococcus pneumoniae were observed during infection in the host by quantitative real time RT‐PCR; one was characteristic of bacteria in blood and one of bacteria in tissue, such as brain and lung. Gene expression in blood was characterized by increased expression of pneumolysin, pspA and hrcA, while pneumococci in tissue infection showed increased expression of neuraminidases, metalloproteinases, oxidative stress and competence genes. In vitro situations with similar expression patterns were detected in liquid culture and in a newly developed pneumococcal model of biofilm respectively. The biofilm model was dependent on addition of synthetic competence stimulating peptide (CSP) and no biofilm was formed by CSP receptor mutants. As one of the differentially expressed gene sets in vivo were the competence genes, we exploited competence‐specific tools to intervene on pneumococcal virulence during infection. Induction of the competence system by the quorum‐sensing peptide, CSP, not only induced biofilm formation in vitro, but also increased virulence in pneumonia in vivo. In contrast, a mutant for the ComD receptor, which did not form biofilm, also showed reduced virulence in pneumonia. These results were opposite to those found in a bacteraemic sepsis model of infection, where the competence system was downregulated. When pneumococci in the different physiological states were used directly for challenge, sessile cells grown in a biofilm were more effective in inducing meningitis and pneumonia, while planktonic cells from liquid culture were more effective in inducing sepsis. Our data enable us, using in vivo gene expression and in vivo modulation of virulence, to postulate the distinction – from the pneumococcal point of view – between two main types of disease. During bacteraemic sepsis pneumococci resemble planktonic growth, while during tissue infection, such as pneumonia or meningitis, pneumococci are in a biofilm‐like state.

We developed a novel surface display system based on the use of bacterial spores. A protein of theBacillus subtilisspore coat, CotB, was found to be located on the spore surface and used as fusion partner to express the 459-amino-acid C-terminal fragment of the tetanus toxin (TTFC). Western, dot blot and fluorescent-activated cell sorting analyses were used to monitor TTFC surface expression on purified spores. We estimated that more than 1.5 × 10³TTFC molecules were exposed on the surface of each spore and recognized by TTFC-specific antibodies. The efficient surface presentation of the heterologous protein, together with the simple purification procedure and the high stability and safety record ofB. subtilisspores, makes this spore-based display system a potentially powerful approach for surface expression of bioactive molecules.