No abstract

Summary The helical cell shape of Helicobacter pylori is highly conserved and contributes to its ability to swim through and colonize the viscous gastric mucus layer. A multi-faceted peptidoglycan (PG) modification program involving four recently characterized peptidases and two accessory proteins is essential for maintaining H. pylori's helicity. To expedite identification of additional shape-determining genes, we employed flow cytometry with fluorescence-activated cell sorting (FACS) to enrich a transposon library for bacterial cells with altered light scattering profiles that correlate with perturbed cell morphology. After a single round of sorting, 15% of our clones exhibited a stable cell shape defect, reflecting 37-fold enrichment. Sorted clones with straight rod morphology contained insertions in known PG peptidases, as well as an insertion in csd6, which we demonstrated has LD-carboxypeptidase activity and cleaves monomeric tetrapeptides in the PG sacculus, yielding tripeptides. Other mutants had only slight changes in helicity due to insertions in genes encoding MviN/MurJ, a protein possibly involved in initiating PG synthesis, and the hypothetical protein HPG27_782. Our findings demonstrate FACS robustly detects perturbations of bacterial cell shape and identify additional PG peptide modifications associated with helical cell shape in H. pylori.

Transcriptional noise is known to be an important cause of cellular heterogeneity and phenotypic variation. The extent to which molecular interaction networks may have evolved to either filter or exploit transcriptional noise is a much debated question. The yeast genetic network regulating galactose metabolism involves two proteins, Gal3p and Gal80p, that feed back positively and negatively, respectively, on GAL gene expression. Using kinetic modeling and experimental validation, we demonstrate that these feedback interactions together are important for (i) controlling the cell-to-cell variability of GAL gene expression and (ii) ensuring that cells rapidly switch to an induced state for galactose uptake.

The innate immune system uses a wide variety of pattern recognition receptors including TLRs, scavenger receptors, and lectins to identify potential pathogens. A carefully regulated balance between activation and inhibition must be kept to avoid detrimental and inappropriate inflammatory responses. In this study, we identify murine-paired Ig-like receptor (PIR)-B, and its human orthologs Ig-like transcript 2 and Ig-like transcript 5 as novel receptors for Staphylococcus aureus. PIR-B contains four ITIM motifs and is thought to be an inhibitory receptor. Expression of these receptors enables NIH3T3 cells to bind S. aureus. In mouse bone marrow-derived macrophages, masking of PIR-B by anti-PIR mAb or genetic deletion of PIR-B shows significantly impaired recognition of S. aureus and enhanced TLR-mediated inflammatory responses to the bacteria. These data suggest a novel mechanism for innate immune regulation by paired Ig-like receptor family members.