Enterobacterial animal pathogens exhibit aggregative multicellular behavior, which is manifested as pellicles on the culture surface and biofilms at the surface-liquid-air interface. Pellicle formation behavior requires production of extracellular polysaccharide, cellulose, and protein filaments, known as curli. Protein filaments analogous to curli are formed by many protein secretion systems, including the type III secretion system (TTSS). Here, we demonstrate that Erwinia chrysanthemi, which does not carry curli genes, requires the TTSS for pellicle formation. These data support a model where cellulose and generic protein filaments, which consist of either curli or TTSS-secreted proteins, are required for enterobacterial aggregative multicellular behavior. Using this assay, we found that hrpY, which encodes a two-component system response regulator homolog, is required for activity of hrpS, which encodes a 54 -dependent enhancer-binding protein homolog. In turn, hrpS is required for activity of the sigma factor homolog hrpL, which activates genes encoding TTSS structural and secreted proteins. Pellicle formation was temperature dependent and pellicles did not form at 36°C, even though TTSS genes were expressed at this temperature. We found that cellulose is a component of the E. chrysanthemi pellicle but that pellicle formation still occurs in a strain with an insertion in a cellulose synthase subunit homolog. Since the TTSS, but not the cellulose synthase subunit, is required for E. chrysanthemi pellicle formation, this inexpensive assay can be used as a high throughput screen for TTSS mutants or inhibitors.Erwinia chrysanthemi is an economically important enterobacterial plant pathogen that causes soft rot and wilt diseases on numerous species of plants. Aggregative multicellular behavior, which results in the formation of large cell aggregates on the culture surface known as pellicles, was demonstrated in E. chrysanthemi over 40 years ago (32). In related species, pellicle formation requires cellulose and protein filaments, known as aggregative fimbriae or curli (37,45,49,52). The recently sequenced E. chrysanthemi 3937 (Ech 3937) genome revealed homologs of genes required for cellulose production in related enterobacteria, but no homologs of genes required for curli synthesis (N. Perna, personal communication). Ech 3937 also does not encode csgD, a regulatory protein that controls curli and cellulose production in related species (18). Thus, the regulatory proteins controlling pellicle formation and whether or not there are protein filaments that play an analogous role to curli in formation of E. chrysanthemi pellicles were unknown.In plant-pathogenic bacteria, the type III secretion system (TTSS) is encoded by hrp (hypersensitive response and pathogenicity) and hrc (hypersensitive response conserved) genes. The TTSS functions as a molecular syringe, injecting virulence proteins into host cells; some of these proteins may interfere with host defense machinery (9,17,23). The hrc genes, many of which are hom...

The prevailing "plug-in-the-bottle" model suggests that macrolide antibiotics inhibit translation by binding inside the ribosome tunnel and indiscriminately arresting the elongation of every nascent polypeptide after the synthesis of six to eight amino acids. To test this model, we performed a genome-wide analysis of translation in azithromycin-treated Staphylococcus aureus. In contrast to earlier predictions, we found that the macrolide does not preferentially induce ribosome stalling near the 5′ end of mRNAs, but rather acts at specific stalling sites that are scattered throughout the entire coding region. These sites are highly enriched in prolines and charged residues and are strikingly similar to other ligandindependent ribosome stalling motifs. Interestingly, the addition of structurally related macrolides had dramatically different effects on stalling efficiency. Our data suggest that ribosome stalling can occur at a surprisingly large number of low-complexity motifs in a fashion that depends only on a few arrest-inducing residues and the presence of a small molecule inducer.antibiotic | ribosome stalling | Staphylococcus aureus

The recognition of a C-terminal motif in E. coli SecM (150FXXXXWIXXXXGIRAGP166) inside the ribosome tunnel causes translation arrest, but the mechanism of recognition is unknown. While single mutations in this motif impair recognition, we demonstrate that new arrest-inducing peptides can be created through remodeling of the SecM C-terminus. We found that R163 is indispensable, but that flanking residues that vary in number and position play an important secondary role in translation arrest. The observation that individual SecM variants showed a distinct pattern of crosslinking to ribosomal proteins suggests that each peptide adopts a unique conformation inside the tunnel. Based on the results, we propose that translation arrest occurs when the peptide conformation specified by flanking residues moves R163 into a precise intra-tunnel location. Our data indicate that translation arrest results from extensive communication between SecM and the tunnel and help explain the striking diversity of arrest-inducing peptides found throughout nature.

In opportunistic Gram-positive Staphylococcus aureus, a small protein called hibernation-promoting factor (HPFSa) is sufficient to dimerize 2.5-MDa 70S ribosomes into a translationally inactive 100S complex. Although the 100S dimer is observed in only the stationary phase in Gram-negative gammaproteobacteria, it is ubiquitous throughout all growth phases in S. aureus. The biological significance of the 100S ribosome is poorly understood. Here, we reveal an important role of HPFSa in preserving ribosome integrity and poising cells for translational restart, a process that has significant clinical implications for relapsed staphylococcal infections. We found that the hpf null strain is severely impaired in long-term viability concomitant with a dramatic loss of intact ribosomes. Genome-wide ribosome profiling shows that eliminating HPFSa drastically increased ribosome occupancy at the 5′ end of specific mRNAs under nutrient-limited conditions, suggesting that HPFSa may suppress translation initiation. The protective function of HPFSa on ribosomes resides at the N-terminal conserved basic residues and the extended C-terminal segment, which are critical for dimerization and ribosome binding, respectively. These data provide significant insight into the functional consequences of 100S ribosome loss for protein synthesis and stress adaptation.