The RNA Chaperone Hfq Is Essential for Virulence and Modulates the Expression of Four Adhesins in Yersinia enterocolitica

In Enterobacteriaceae, the RNA chaperone Hfq mediates the interaction of small RNAs with target mRNAs, thereby modulating transcript stability and translation. This post-transcriptional control helps bacteria adapt quickly to changing environmental conditions. Our previous mutational analysis showed that Hfq is involved in metabolism and stress survival in the enteropathogen Yersinia enterocolitica. In this study we demonstrate that Hfq is essential for virulence in mice and influences production of surface pathogenicity factors, in particular lipopolysaccharide and adhesins mediating interaction with host tissue. Hfq inhibited the production of Ail, the Ail-like protein OmpX and the MyfA pilin post-transcriptionally. In contrast Hfq promoted production of two major autotransporter adhesins YadA and InvA. While protein secretion in vitro was not affected, hfq mutants exhibited decreased protein translocation by the type III secretion system into host cells, consistent with decreased production of YadA and InvA. The influence of Hfq on YadA resulted from a complex interplay of transcriptional, post-transcriptional and likely post-translational effects. Hfq regulated invA by modulating the expression of the transcriptional regulators rovA, phoP and ompR. Therefore, Hfq is a global coordinator of surface virulence determinants in Y. enterocolitica suggesting that it constitutes an attractive target for developing new antimicrobial strategies

HpaC Controls Substrate Specificity of the Xanthomonas Type III Secretion System

The Gram-negative bacterial plant pathogen Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to inject bacterial effector proteins into the host cell cytoplasm. One essential pathogenicity factor is HrpB2, which is secreted by the T3S system. We show that secretion of HrpB2 is suppressed by HpaC, which was previously identified as a T3S control protein. Since HpaC promotes secretion of translocon and effector proteins but inhibits secretion of HrpB2, HpaC presumably acts as a T3S substrate specificity switch protein. Protein–protein interaction studies revealed that HpaC interacts with HrpB2 and the C-terminal domain of HrcU, a conserved inner membrane component of the T3S system. However, no interaction was observed between HpaC and the full-length HrcU protein. Analysis of HpaC deletion derivatives revealed that the binding site for the C-terminal domain of HrcU is essential for HpaC function. This suggests that HpaC binding to the HrcU C terminus is key for the control of T3S. The C terminus of HrcU also provides a binding site for HrpB2; however, no interaction was observed with other T3S substrates including pilus, translocon and effector proteins. This is in contrast to HrcU homologs from animal pathogenic bacteria suggesting evolution of distinct mechanisms in plant and animal pathogenic bacteria for T3S substrate recognition

Models of classroom assessment for course-based research experiences

Course-based research pedagogy involves positioning students as contributors to authentic research projects as part of an engaging educational experience that promotes their learning and persistence in science. To develop a model for assessing and grading students engaged in this type of learning experience, the assessment aims and practices of a community of experienced course-based research instructors were collected and analyzed. This approach defines four aims of course-based research assessment—(1) Assessing Laboratory Work and Scientific Thinking; (2) Evaluating Mastery of Concepts, Quantitative Thinking and Skills; (3) Appraising Forms of Scientific Communication; and (4) Metacognition of Learning—along with a set of practices for each aim. These aims and practices of assessment were then integrated with previously developed models of course-based research instruction to reveal an assessment program in which instructors provide extensive feedback to support productive student engagement in research while grading those aspects of research that are necessary for the student to succeed. Assessment conducted in this way delicately balances the need to facilitate students’ ongoing research with the requirement of a final grade without undercutting the important aims of a CRE education

Mutations in the Regulatory Gene hrpG of Xanthomonas campestris pv. vesicatoria Result in Constitutive Expression of All hrp Genes

hrpG is a key regulatory gene for transcriptional activation of pathogenicity genes (hrp) of Xanthomonas campestris pv. vesicatoria. We identified three mutations in hrpG which render hrp gene expression constitutive in normally suppressing medium. The mutations in hrpG result in novel amino acid substitutions compared to mutations in related proteins, such as OmpR. In addition, mutated hrpG enhances the timing and intensity of plant reactions in infection assays

The Legionella pneumophila tatB Gene Facilitates Secretion of Phospholipase C, Growth under Iron-Limiting Conditions, and Intracellular Infection

Our previous mutational analysis of Legionella pneumophila demonstrated a role for type II protein (Lsp) secretion and iron acquisition in intracellular infection and virulence. In gram-negative bacteria, the twin-arginine translocation (Tat) pathway is involved in secretion of proteins, including components of respiratory complexes, across the inner membrane to the periplasm. To assess the significance of Tat for L. pneumophila, tatB mutants were characterized. The mutants exhibited normal growth in standard media but grew slowly under low-iron conditions. They were also impaired in the Nadi assay, indicating that the function of cytochrome c oxidase is influenced by tatB. Consistent with this observation, a subunit of the cytochrome c reductase was shown to be a Tat substrate. Supernatants of the tatB mutants showed a 30% reduction in phospholipase C activity while maintaining normal levels of other Lsp secreted activities. When tested for infection of U937 macrophages, the tatB mutants showed a 10-fold reduction in growth. Double mutants lacking tatB and Lsp secretion were even more defective, suggesting tatB has an intracellular role that is independent of Lsp. tatB mutants were also impaired 20-fold in Hartmannella vermiformis amoebae cultured in the presence of an iron chelator. All mutant phenotypes were complemented by reintroduction of an intact tatB. Thus, L. pneumophila tatB plays a role in the formation of a respiratory complex, growth under low-iron conditions, the secretion of a phospholipase C activity, and intracellular infection

Strategies for Bacteriophage T5 Mutagenesis: Expanding the Toolbox for Phage Genome Engineering

International audiencePhage genome editing is crucial to uncover the molecular mechanisms of virus infection and to engineer bacteriophages with enhanced antibacterial properties. Phage genetic engineering relies mostly on homologous recombination (HR) assisted by the targeted elimination of wild-type phages by CRISPR-Cas nucleases. These strategies are often less effective in virulent bacteriophages with large genomes. T5 is a virulent phage that infects Escherichia coli . We found that CRISPR-Cas9 system (type II-A) had ununiform efficacies against T5, which impairs a reliable use of CRISPR-Cas-assisted counterselection in the gene editing of T5. Here, we present alternative strategies for the construction of mutants in T5. Bacterial retroelements (retrons) proved to be efficient for T5 gene editing by introducing point mutations in the essential gene A1 . We set up a protocol based on dilution-amplification-screening (DAS) of phage pools for mutant enrichment that was used to introduce a conditional mutation in another essential gene ( A2 ), insert a new gene ( lacZ α), and construct a translational fusion of a late phage gene with a fluorescent protein coding gene ( pb10-mCherry ). The method should be applicable to other virulent phages that are naturally resistant to CRISPR/Cas nucleases