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Csr is a conserved global regulatory system, which uses the sequence-specific RNA-binding protein CsrA to activate or repress gene expression by binding to m RNA and altering translation, stability and/or transcript elongation. In E scherichia coli, CsrA activity is regulated by two s RNAs, CsrB and CsrC, which bind to multiple CsrA dimers, thereby sequestering this protein away from its mRNA targets. Turnover of CsrB/ C s RNAs is tightly regulated by a GGDEF-EAL domain protein, CsrD, which targets them for cleavage by RNase E. Here, we show that EIIAGlc of the glucose-specific PTS system is also required for the normal decay of these s RNAs and that it acts by binding to the EAL domain of CsrD. Only the unphosphorylated form of EIIAGlc bound to CsrD in vitro and was capable of activating CsrB/ C turnover in vivo. Genetic studies confirmed that this mechanism couples CsrB/ C s RNA decay to the availability of a preferred carbon source. These findings reveal a new physiological influence on the workings of the Csr system, a novel function for the EAL domain, and an important new way in which EIIAGlc shapes global regulatory circuitry in response to nutritional status. [ABSTRACT FROM AUTHOR]

Indole production by Escherichia coli, discovered in the early 20th century, has been used as a diagnostic marker for distinguishing E. coli from other enteric bacteria. By using transcriptional profiling and competition studies with defined mutants, we show that cyclic AMP (cAMP)-regulated indole formation is a major factor that enables E. coli growth in mixed biofilm and planktonic populations with Pseudomonas aeruginosa. Mutants deficient in cAMP production (cyaA) or the cAMP receptor gene (cip), as well as indole production (tnaA), were not competitive in coculture with P. aeruginosa but could be restored to ~sri1d~type competitiveness by supplementation with a physiologically relevant indole concentration. F. coli sdiA mutants, which lacked the receptor for both indole and N-acyl-homoserine lactones (AHLs), showed no change in competitive fitness, suggesl~ing that indole acted directly on P. aeruginosa. An E. coli tnaA mutant strain regained wild-type competiveness if grown with P. aeruginosa AHL synthase (rhil and rhil las!) mutants. In contrast to the wild type, P. aeruginosa AHL synthase mutants 4ere unable to degrade indole. Indole produced during mixed-culture growth inhibited pyocyanin production and other AHL-re~ulated virulence factors in P. aeruginosa. Mixed-culture growth with P. aeruginosa stimulated indole formation in F. coli cpd)A, which is unable to regulate cAMP levels, suggesting the potential for mixed-culture gene activation via cAMP. These findings illustrate how indole, an early described feature of E. coli central metabolism, can play a significant role in mixed-culture suMval by inhibiting quorum-regulated competition factors in P. aeruginosa. [ABSTRACT FROM AUTHOR]

Although a marked decrease in mortality associated with bacterial infections is attributed to the discovery of antibiotics, antibiotic resistance has become a global health concern due to their misuse. A dynamic in vitro hollow-fiber system was used to study antibiotic resistance in Escherichia coli using ampicillin. An LC-MS/MS assay was validated for quantitative analysis of ampicillin in Luria-Bertani broth. The assay was linear from 0.10-50.00 μg/ml. The assay met acceptance criteria for inter- and intra-assay precisions and accuracies across three quality controls. Stability of ampicillin was confirmed at three different storage conditions. In vitro data were similar to simulated plasma PK data further confirming the appropriateness of the experimental design to quantify antibiotics and study occurrence of antimicrobial resistance in real-time., Competing Interests: Financial & competing interests disclosure The authors would like to thank the Center for Drugs Evaluation and Research for funding this study. All the work presented in this manuscript was supported by intramural grants from the FDA. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.