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Streptococcus pyogenes, also known as group A Streptococcus or GAS, is a human-restricted pathogen causing a diverse array of infections. The ability to adapt to different niches requires GAS to adjust gene expression in response to environmental cues. We previously identified the abundance of biometals and carbohydrates led to natural induction of the Rgg2/3 cell-cell communication system (quorum sensing, QS). Here we determined the mechanism by which the Rgg2/3 QS system is stimulated exclusively by mannose and repressed by glucose, a phenomenon known as carbon catabolite repression (CCR). Instead of carbon catabolite protein A, the primary mediator of CCR in Gram-positive bacteria; CCR of Rgg2/3 requires the PTS regulatory domain (PRD)-containing transcriptional regulator Mga. Deletion of Mga led to carbohydrate-independent activation of Rgg2/3 by down-regulating rgg3, the QS repressor. Through phosphoablative and phosphomimetic substitutions within Mga PRDs, we demonstrated that selective phosphorylation of PRD1 conferred repression of the Rgg2/3 system. Moreover, given the carbohydrate specificity mediating Mga-dependent governance over Rgg2/3, we tested mannose-specific PTS components and found the EIIA/B subunit ManL was required for Mga-dependent repression. These findings provide newfound connections between PTSMan , Mga, and QS, and further demonstrate that Mga is a central regulatory nexus for integrating nutritional status and virulence.

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By screening a collection of Salmonella mutants deleted for genes encoding small proteins of ≤60 amino acids, we identified three paralogous small genes (ymdF, STM14_1829, and yciG) required for wild-type flagellum-dependent swimming and swarming motility. The ymdF, STM14_1829, and yciG genes encode small proteins of 55, 60, and 60 amino acid residues, respectively. A bioinformatics analysis predicted that these small proteins are intrinsically disordered proteins, and circular dichroism analysis of purified recombinant proteins confirmed that all three proteins are unstructured in solution. A mutant deleted for STM14_1829 showed the most severe motility defect, indicating that among the three paralogs, STM14_1829 is a key protein required for wild-type motility. We determined that relative to the wild type, the expression of the flagellin protein FliC is lower in the ΔSTM14_1829 mutant due to the downregulation of the flhDC operon encoding the FlhDC master regulator. By comparing the gene expression profiles between the wild-type and ΔSTM14_1829 strains via RNA sequencing, we found that the gene encoding the response regulator PhoP is upregulated in the ΔSTM14_1829 mutant, suggesting the indirect repression of the flhDC operon by the activated PhoP. Homologs of STM14_1829 are conserved in a wide range of bacteria, including Escherichia coli and Pseudomonas aeruginosa We showed that the inactivation of STM14_1829 homologs in E. coli and P. aeruginosa also alters motility, suggesting that this family of small intrinsically disordered proteins may play a role in the cellular pathway(s) that affects motility.IMPORTANCE This study reports the identification of a novel family of small intrinsically disordered proteins that are conserved in a wide range of flagellated and nonflagellated bacteria. Although this study identifies the role of these small proteins in the scope of flagellum-dependent motility in Salmonella, they likely play larger roles in a more conserved cellular pathway(s) that indirectly affects flagellum expression in the case of motile bacteria. Small intrinsically disordered proteins have not been well characterized in prokaryotes, and the results of our study provide a basis for their detailed functional characterization.

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Quorum sensing (QS) has been recognized as an important biological phenomenon in which bacterial cells communicate and coordinate their gene expression and cellular processes with respect to population density. Bacillus anthracis is the etiological agent of fatal pulmonary anthrax infections, and the NprR/NprX QS system may be involved in its pathogenesis. NprR, renamed as aqsR for anthrax quorum sensing Regulator, is a transcriptional regulator that may control the expression of genes required for proliferation and survival. Currently, there is no protocol reported to over-express and purify B. anthracis AqsR. In this study, we describe cloning, purification, and confirmation of functional full-length B. anthracis AqsR protein. The AqsR gene was cloned into the pQE-30 vector with an HRV 3C protease recognition site between AqsR and the N-terminal His6-tag in order to yield near native AqsR after the His-tag cleavage, leaving only two additional amino acid residues at the N-terminus.

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Generation of genetic diversity is a prerequisite for bacterial evolution and adaptation. Short-term diversification and selection within populations is, however, largely uncharacterised, as existing studies typically focus on fixed substitutions. Here, we use whole-genome deep-sequencing to capture the spectrum of mutations arising during biofilm development for two Pseudomonas aeruginosa strains. This approach identified single nucleotide variants with frequencies from 0.5% to 98.0% and showed that the clinical strain 18A exhibits greater genetic diversification than the type strain PA01, despite its lower per base mutation rate. Mutations were found to be strain specific: the mucoid strain 18A experienced mutations in alginate production genes and a c-di-GMP regulator gene; while PA01 acquired mutations in PilT and PilY1, possibly in response to a rapid expansion of a lytic Pf4 bacteriophage, which may use type IV pili for infection. The Pf4 population diversified with an evolutionary rate of 2.43 × 10(-3) substitutions per site per day, which is comparable to single-stranded RNA viruses. Extensive within-strain parallel evolution, often involving identical nucleotides, was also observed indicating that mutation supply is not limiting, which was contrasted by an almost complete lack of noncoding and synonymous mutations. Taken together, these results suggest that the majority of the P. aeruginosa genome is constrained by negative selection, with strong positive selection acting on an accessory subset of genes that facilitate adaptation to the biofilm lifecycle. Long-term bacterial evolution is known to proceed via few, nonsynonymous, positively selected mutations, and here we show that similar dynamics govern short-term, within-population bacterial diversification.

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Pseudomonas aeruginosa strain 18A is a clinical, nonclonal isolate retrieved from the sputum of a chronically infected cystic fibrosis patient. The genome of 18A was sequenced for comparison with environmental and clinical isolates to identify genes that might facilitate its persistence during infection.

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Persistent lung infection by Pseudomonas aeruginosa is typically associated with the development of biofilms, the appearance of morphotypic variants and reduction in the expression of acute virulence factors. We have characterised and compared functional traits [carbon substrate utilisation, attachment and biofilm formation, protease and elastase activity, quorum-sensing (QS)] of the biofilm dispersal populations of a representative P. aeruginosa isolate from a chronically infected cystic fibrosis individual and P. aeruginosa strain PAO1. The dispersal variants of the clinical strain exhibited significantly greater heterogeneity in all of the phenotypes tested. All morphotypic variants from the dispersal population of the clinical strain showed a significant increase in QS signal and elastase production compared to the parental strain. In contrast, isolates from planktonic cultures were phenotypically identical to the inoculum strain, suggesting that the appearance of these variants was biofilm specific. The clinical strain was shown to have a 3.4-fold higher mutation frequency than PAO1 which corroborated with the increased diversity of dispersal isolates. These data suggest that the development of a chronic infection phenotype can be reversed to recover acute infection isolates and that growth within a biofilm facilitates diversification of P. aeruginosa which is important for ecological adaptation.

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Intractable biofilm infections with Pseudomonas aeruginosa are the major cause of premature death associated with cystic fibrosis (CF). Few studies have explored the biofilm developmental cycle of P. aeruginosa isolates from chronically infected individuals. This study shows that such clinical isolates exhibit biofilm differentiation and dispersal processes similar to those of the better-studied laboratory P. aeruginosa strain PAO1 in the glass flow-cell (continuous-culture) biofilm model, albeit they are initially less adherent and their microcolonies are slower to develop and show heterogeneous, strain-specific variations in architecture. Confocal scanning laser microscopy combined with LIVE/DEAD viability staining revealed that in all CF biofilms bacterial cell death occurred in maturing biofilms, extending from the substratum to the central regions of mature microcolonies to varying degrees, depending on the strain. Bacteriophage activity was detected in the maturing biofilms of all CF strains examined and the amount of phage produced paralleled the degree of cell death seen in the biofilm. Some CF strains exhibited 'seeding dispersal' associated with the above phenomena, producing 'hollowing' as motile cells evacuated from the microcolony interiors as has been described for strain PAO1. Moreover, morphotypic cell variants were seen in the biofilm effluents of all CF strains. For those CF strains where marked cell death and seeding dispersal occurred in the microcolonies, variants were more diverse (up to five morphotypes) compared to those of strain PAO1 (two morphotypes). Given that variants of strain PAO1 have enhanced colonization traits, it seems likely that the similar biofilm dispersal events described here for CF strains contribute to the variability seen in clinical isolates and the overall persistence of the P. aeruginosa in the CF airway.

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