- Volume 163, Issue 1, 2017

Escherichia coli O157 : H7 is a zoonotic diarrhoeal pathogen of worldwide importance. It belongs to a subset of Shiga toxin-producing E. coli that can form attaching and effacing lesions on intestinal epithelia via the action of a type 3 secretion system that injects bacterial effectors into enterocytes. Infections in humans often arise from contaminated food or direct environmental exposure and can involve life-threatening Shiga toxin-dependent sequelae. In the three decades since E. coli O157 : H7 was first recognized intensive research has helped to unravel the basis of pathogenesis, but few effective options for prevention and treatment of infections exist.

At the end of June, over 120 microbiologists from 18 countries gathered in Dundee, Scotland for the fourth edition of the Young Microbiologists Symposium on ‘Microbe Signalling, Organisation and Pathogenesis’. The aim of the symposium was to give early career microbiologists the opportunity to present their work in a convivial environment and to interact with senior world-renowned scientists in exciting fields of microbiology research. The meeting was supported by the Microbiology Society, the Society of Applied Microbiology and the American Society for Microbiology with further sponsorship from the European Molecular Biology Organisation and the Royal Society of Edinburgh. In this report, we highlight some themes that emerged from the many interesting talks and poster presentations, as well as some of the other activities that were on offer at this energetic meeting.

The important human pathogen Streptococcus pneumoniae is a naturally transformable species. When developing the competent state, it expresses proteins involved in DNA uptake, DNA processing and homologous recombination. In addition to the proteins required for the transformation process, competent pneumococci express proteins involved in a predatory DNA acquisition mechanism termed fratricide. This is a mechanism by which the competent pneumococci secrete a muralytic fratricin termed CbpD, which lyses susceptible sister cells or closely related streptococcal species. The released DNA can then be taken up by the competent pneumococci and integrated into their genomes. To avoid committing suicide, competent pneumococci produce an integral membrane protein, ComM, which protects them against CbpD by an unknown mechanism. In the present study, we show that overexpression of ComM results in growth inhibition and development of severe morphological abnormalities, such as cell elongation, misplacement of the septum and inhibition of septal cross-wall synthesis. The toxic effect of ComM is tolerated during competence because it is not allowed to accumulate in the competent cells. We provide evidence that an intra-membrane protease called RseP is involved in the process of controlling the ComM levels, since △rseP mutants produce higher amounts of ComM compared to wild-type cells. The data presented here indicate that ComM mediates immunity against CbpD by a mechanism that is detrimental to the pneumococcus if exaggerated.

The adaptation of environmental bacteria to laboratory conditions was analysed through the exploration of genomic changes in four strains of Escherichia coli freshly isolated from their natural habitats and belonging to different taxonomic clusters. Up to 25 mutations were present in all cultures of natural isolates within 10 days of transfer in rich media or with a single growth cycle involving an extended stationary phase. Among numerous individual mutations, two genes were affected in parallel in distinct backgrounds. Mutations in rpoS (encoding sigma factor RpoS), altering a multiplication–survival trade-off in E. coli, were present in isolates derived from all four different ancestors. More surprisingly, two different natural isolates acquired mutations in mutL, affecting DNA mismatch repair, and a third also involved higher mutation rates. The elevated mutation rates in these isolates indicate the danger of increased genetic instability arising from laboratory domestication. Neither rpoS nor mutator mutations were detected in the already-acclimatized MG1655 laboratory strain; only one or no new mutations were present in the laboratory strain under the same culture conditions. Our results indicate rapid adaptation to the laboratory environment. Ancestor-specific responses also arise in the laboratory and mutational events are also sensitive to culture conditions such as extended stationary phase. To maintain natural isolates in a stable state, our data suggest that the transition of strains to the laboratory should minimize culture cycles and extended stationary phase.

Neisseria gonorrhoeae is capable of causing gonorrhoea and more complex diseases in the human host. Within the gonococcal genome are over 100 copies of the insertion sequence-like Correia repeat enclosed element (CREE), which has been predicted to be mobile within the neisserial genomes. Although there is evidence of ancestral movement of these elements, no previous study has provided evidence for current mobilization. CREE has the ability to alter gene expression and regulation in many ways: by insertional mutagenesis, by introducing promoter elements, by generating mRNA processing sites and by association with non-coding RNAs. Previous studies have compared the genomic locations of CREEs in the Neisseria spp., demonstrating that otherwise identical regions have either the element or the target TA insertion site. In this study, we report for the first time, to our knowledge, movement of CREEs, through inversion of the element at its chromosomal location. Analysis of Ion Torrent generated genome sequence data from N. gonorrhoeae strain NCCP11945 passaged for 8 weeks in the laboratory under standard conditions and stress conditions revealed a total of 37 inversions: 24 were exclusively seen in the stressed sample, 7 were seen in the control sample and the remaining 3 were seen in both samples. These inversions have the capability to alter gene expression in N. gonorrhoeae through the previously determined activities of the sequence features of these elements, potentially resulting in reversible phase-variable gene expression.

We here report the characterization of two novel proteins encoded by the genes LIC11122 and LIC12287, identified in the genome sequences of Leptospira interrogans, annotated, respectively, as a putative sigma factor and a hypothetical protein. The CDSs LIC11122 and LIC12287 have signal peptide SPII and SPI and are predicted to be located mainly at the cytoplasmic membrane of the bacteria. The genes were cloned and the proteins expressed using Escherichia coli. Proteinase K digestion showed that both proteins are surface exposed. Evaluation of interaction of recombinant proteins with extracellular matrix components revealed that they are laminin binding and they were called Lsa19 (LIC11122) and Lsa14 (LIC12287), for Leptospiral-surface adhesin of 19 and 14 kDa, respectively. The bindings were dose-dependent on protein concentration, reaching saturation, fulfilling the ligand-binding criteria. Reactivity of the recombinant proteins with leptospirosis human sera has shown that Lsa19 and, to a lesser extent, Lsa14, are recognized by antibodies, suggesting that, most probably, Lsa19 is expressed during infection. The proteins interact with plasminogen and generate plasmin in the presence of urokinase-type plasminogen activator. Plasmin generation in Leptospira has been associated with tissue penetration and immune evasion strategies. The presence of a sigma factor on the cell surface playing a secondary role, probably mediating host –pathogen interaction, suggests that LIC11122 is a moonlighting protein candidate. Although the biological significance of these putative adhesins will require the generation of mutants, our data suggest that Lsa19 is a potential candidate for future evaluation of its role in adhesion/colonization activities during L. interrogans infection.

The thioredoxin system plays a central role in the intracellular redox maintenance in the majority of cells. The canonical system consists of an NADPH-dependent thioredoxin reductase (TrxR) and thioredoxin (Trx), a disulfide reductase. Although Trx is encoded in almost all sequenced genomes of methanogens, its incorporation into their unique physiology is not well understood. Methanosarcina acetivorans contains a single TrxR (MaTrxR) and seven Trx (MaTrx1–MaTrx7) homologues. We previously showed that MaTrxR and at least MaTrx7 compose a functional NADPH-dependent thioredoxin system. Here, we report the characterization of all seven recombinant MaTrxs. MaTrx1, MaTrx3, MaTrx4 and MaTrx5 lack appreciable disulfide reductase activity, unlike previously characterized MaTrx2, MaTrx6 and MaTrx7. Enzyme assays demonstrated that, of the MaTrxs, only the reduction of disulfide-containing MaTrx7 is linked to the oxidation of reduced coenzymes. NADPH is shown to be supplied to the MaTrxR–MaTrx7 system through the oxidation of the primary methanogen electron carriers F420H2 and ferredoxin, indicating that it serves as a primary intracellular reducing system in M. acetivorans. Bioinformatic analyses also indicate that the majority of methanogens likely utilize an NADPH-dependent thioredoxin system. The remaining MaTrxs may have specialized functions. MaTrx1 and MaTrx3 exhibited thiol oxidase activity. MaTrx3 and MaTrx6 are targeted to the membrane of M. acetivorans and likely function in the formation and the reduction of disulfides in membrane and/or extracellular proteins, respectively. This work provides insight into the incorporation of Trx into the metabolism of methanogens, and this reveals that methanogens contain Trx homologues with alternative properties and activities.

The endonuclease tRNase Z is responsible for the 3′-end processing of tRNA precursors, which is one of the essential steps in tRNA maturation. The fission yeast Schizosaccharomyces pombe contains two essential tRNase ZL genes (trz1 and trz2) involved in nuclear and mitochondrial tRNA 3′-end processing, respectively. Our previous studies suggest that trz2 is expressed at a very low level. Here we report characterization of the trz2 promoter. Using lacZ as a reporter, we show that the trz2 promoter contains a HomolD box and a very weak diverged TATA element. The HomolD box is usually found in the promoters of S. pombe ribosomal protein genes. lacZ reporter assays suggest that the HomolD box regulates the expression of both trz2 and the ribosomal protein gene rps2501, which are arranged head-to-head on opposite strands. Overexpression of Rrn7, a candidate HomolD box-binding protein, up-regulates expression of lacZ under the control of the trz2 promoter or the rps2501 promoter. Functional complementation studies suggest that the TATA-like element is essential for trz2 expression, whereas the HomolD box may play a nonessential regulatory role. We also demonstrate that a 57 nt negative regulatory element (NRE) located between the HomolD box and the TATA-like element represses the expression of lacZ under the control of the trz2 promoter. Our results suggest that the low-level trz2 expression may arise from a low level of transcription caused by lack of a strong TATA box and the NRE. Our analysis also suggests that trz2 and rps2501 may be coregulated by the HomolD box.