; decreased in stationary phase), Spd-sr37 (80 nt; strongly expressed in all growth phases), and CcnA (93 nt; induced by competence stimulatory peptide). Spd-sr17 and CcnA likely fold into structures containing single-stranded regions between hairpin structures, whereas Spd-sr37 forms a base-paired structure. Primer extension mapping and ectopic expression in deletion/insertion mutants confirmed the independent expression of the three sRNAs. Microarray analyses indicated that insertion/deletion mutants in spd-sr37 and ccnA exerted strong cis-acting effects on the transcription of adjacent genes, indicating that these sRNA regions are also cotranscribed in operons. Deletion or overexpression of the three sRNAs did not cause changes in growth, certain stress responses, global transcription, or virulence. Constitutive ectopic expression of CcnA reversed some phenotypes of D39 ⌬ciaR mutants, but attempts to link CcnA to -E to comC as a target were inconclusive in ciaR ؉ strains. These results show that S. pneumoniae, which lacks known RNA chaperones, expresses numerous sRNAs, but three of these sRNAs do not strongly affect common phenotypes or transcription patterns.A large number of noncoding small RNAs (sRNAs) 50 to 400 nucleotides (nt) in length have been detected and characterized recently in numerous bacterial species (reviewed in references 3, 18, and 78). Some abundant, stable sRNAs, such as RNase P (14), tmRNA (34), and scRNA (4.5S RNA) (23, 33), are highly conserved and play important housekeeping and stress-related functions in RNA metabolism, protein degradation, and secretion. But most regulatory sRNAs are conserved only among closely related species (42). Many sRNAs play key roles in responses to stress conditions, such as iron limitation, osmotic shock, temperature shift, stationary phase, and metabolic imbalance, in different bacterial species (3,15,17,25,26,46,76,78,79). Other sRNAs are expressed during growth or developmental phases that are specific for particular bacterial species (38,64,68,75). In addition, sRNAs have been postulated to mediate virulence gene expression in several pathogenic bacteria and their survival in hosts (3,6,37,55,68,73).Little is known about RNA metabolism in Streptococcus pneumoniae (pneumococcus), which is a major human respiratory pathogen that causes several serious invasive diseases, including pneumonia, otitis media (ear infection), sinusitis, meningitis, and septicemia (49). Pneumococcus exists as a commensal bacterium that inhabits and colonizes the nasopharynx of up to 20 and 50% of healthy adults and children, respectively, at any time (10). The transition from commensal bacterium to opportunistic pathogen often occurs after a respiratory tract infection, and invasive pneumococcal diseases result in over 1.6 million deaths annually worldwide, especially among young, elderly, debilitated, and immunosuppressed individuals (reviewed in references 13 and 30). Clearly, S. pneumoniae has the ability to inhabit numerous niches in the human body (31,32), and responses ...

A biofilm, or a matrix-embedded community of cells, promotes the ability of the bacterium Vibrio fischeri to colonize its symbiotic host, the Hawaiian squid Euprymna scolopes. Biofilm formation and colonization depend on syp, an 18-gene polysaccharide locus. To identify other genes necessary for biofilm formation, we screened for mutants that failed to form wrinkled colonies, a type of biofilm. We obtained several with defects in genes required for cysteine metabolism, including cysH, cysJ, cysK, and cysN. The cysK mutant exhibited the most severe wrinkling defect. It could be complemented with a wild-type copy of the cysK gene, which encodes O-acetylserine sulfhydrolase, or by supplementing the medium with additional cysteine. None of a number of other mutants defective for biosynthetic genes negatively impacted wrinkled colony formation, suggesting a specific role for CysK. CysK did not appear to control activation of Syp regulators or transcription of the syp locus, but it did influence production of the Syp polysaccharide. Under biofilm-inducing conditions, the cysK mutant retained the same ability as that of the parent strain to adhere to the agar surface. The cysK mutant also exhibited a defect in pellicle production that could be complemented by the cysK gene but not by cysteine, suggesting that, under these conditions, CysK is important for more than the production of cysteine. Finally, our data reveal a role for cysK in symbiotic colonization by V. fischeri. Although many questions remain, this work provides insights into additional factors required for biofilm formation and colonization by V. fischeri. The ability of bacteria to grow in biofilms, or communities of cells embedded in a surface-associated, self-produced matrix, permits them to survive environmental assaults and colonize a variety of biotic and abiotic surfaces (1, 2). The biofilm matrix typically contains polysaccharides, proteins, and environmental DNA (eDNA), which together provide a protected environment and permit adherence to surfaces. Production of a biofilm depends on the ability of bacteria to recognize and respond to appropriate environmental cues and produce and export a variety of substances that are assembled to permit a three-dimensional (3D) community architecture from which bacteria can ultimately depart.The complex nature of this process is apparent from the study of numerous bacteria, including the facultative symbiont Vibrio fischeri. This marine microbe forms a transient biofilm on the surface of a specialized organ of its host, the squid Euprymna scolopes (3). V. fischeri cells disperse from this transient biofilm to migrate into pores leading to the internal sites where they multiply to a high cell density and establish a long-term association with the squid (reviewed in reference 4). Mutants defective for the production of this transient biofilm fail to efficiently colonize their squid host (5-8).Some components of the V. fischeri matrix have been identified (Fig. 1). Notably, the 18-gene symbiosis polysaccharide loc...

Biofilm formation by Vibrio fischeri is a complex process that requires multiple regulators. One such regulator, the NtrClike response regulator SypG, controls biofilm formation and host colonization by V. fischeri via its impact on transcription of the symbiosis polysaccharide (syp) locus. SypG is predicted to activate syp transcription by binding to the syp enhancer (SE), a conserved sequence located upstream of four syp promoters. In this study, we performed an in-depth analysis of the sequences necessary for SypG to promote syp transcription and biofilm formation. We found that the SE sequence is necessary for SypG-mediated syp transcription, identified individual bases necessary for efficient activation, and determined that SypG is able to bind to syp promoter regions. We also identified SE sequences outside the syp locus and established that SypG recognizes these sequences as well. Finally, deletion of the SE sequence upstream of sypA led to defects in both biofilm formation and host colonization that could be restored by reintroducing the SE sequence into its native location in the chromosome. This work thus fills in critical gaps in knowledge of the Syp regulatory circuit by demonstrating a role for the SE sequence in SypG-dependent control of biofilm formation and host colonization and by identifying new putative regulon members. It may also provide useful insights into other bacteria, such as Vibrio vulnificus and Vibrio parahaemolyticus, that have syp-like loci and conserved SE sequences.

Summary Biofilm formation by Vibrio fischeri is a complex process involving multiple regulators, including the sensor kinase (SK) RscS and the response regulator (RR) SypG, which control the symbiosis polysaccharide (syp) locus. To identify other regulators of biofilm formation in V. fischeri, we screened a transposon library for mutants defective in wrinkled colony formation. We identified LuxQ as a positive regulator of syp-dependent biofilm formation. LuxQ is a member of the Lux phosphorelay and is predicted to control bioluminescence in concert with the SK AinR, the phosphotransferase LuxU, and the RR LuxO. Of these, LuxU was the only other regulator that exerted a substantial impact on biofilm formation. We propose a model in which the Lux pathway branches at LuxU to control both bioluminescence and biofilm formation. Furthermore, our evidence suggests that LuxU functions to regulate syp transcription, likely by controlling SypG activity. Finally, we found that, in contrast to its predicted function, the SK AinR has little impact on bioluminescence under our conditions. Thus, this study reveals a novel connection between the Lux and Syp pathways in V. fischeri, and furthers our understanding of how the Lux pathway regulates bioluminescence in this organism.

Bacterial biofilms are recalcitrant to antibiotic therapy and a major cause of persistent and recurrent infections. New antibody-based therapies may offer potential to target biofilm specific components for host-cell mediated bacterial clearance. For Pseudomonas aeruginosa, human monoclonal antibodies (mAbs) targeting the Psl biofilm exopolysaccharide exhibit protective activity against planktonic bacteria in acute infection models. However, anti-Psl mAb activity against P. aeruginosa biofilms is unknown. Here, we demonstrate that anti-Psl mAbs targeting three distinct Psl epitopes exhibit stratified binding in mature in vitro biofilms and bind Psl within the context of a chronic biofilm infection. These mAbs also exhibit differential abilities to inhibit early biofilm events and reduce biomass from mature biofilms in the presence of neutrophils. Importantly, a mAb mixture with neutrophils exhibited the greatest biomass reduction, which was further enhanced when combined with meropenem, a common anti-Pseudomonal carbapenem antibiotic. Moreover, neutrophil-mediated killing of biofilm bacteria correlated with the evident mAb epitope stratification within the biofilm. Overall, our results suggest that anti-Psl mAbs might be promising candidates for adjunctive use with antibiotics to inhibit/disrupt P. aeruginosa biofilms as a result of chronic infection.