Region C of the Escherichia coli heat shock sigma factor RpoH (σ32) contains a turnover element for proteolysis by the FtsH protease

Transcription of most heat shock genes in Escherichia coli is initiated by the alternative sigma factor σ32 (RpoH). At physiological temperatures, RpoH is rapidly degraded by chaperone-mediated FtsH-dependent proteolysis. Several RpoH residues critical for degradation are located in the highly conserved region 2.1. However, additional residues were predicted to be involved in this process. We introduced mutations in region C of RpoH and found that a double mutation (A131E, K134V) significantly stabilized RpoH against degradation by the FtsH protease. Single-point mutations at these positions only showed a slight effect on RpoH stability. Both double and single amino acid substitutions did not impair sigma factor activity as demonstrated by a groE-lacZ reporter gene fusion, Western blot analysis of heat shock gene expression and increased heat tolerance in the presence of these proteins. Combined mutations in regions 2.1 and C further stabilized RpoH. We also demonstrate that an RpoH fragment composed of residues 37-147 (including regions 2.1 and C) is degraded in an FtsH-dependent manner. We conclude that in addition to the previously described turnover element in region 2.1, a previously postulated second region important for proteolysis of RpoH by FtsH lies in region C of the sigma facto

Engineered reporter phages for detection of Escherichia coli, Enterococcus, and Klebsiella in urine

The rapid detection and species-level differentiation of bacterial pathogens facilitates antibiotic stewardship and improves disease management. Here, we develop a rapid bacteriophage-based diagnostic assay to detect the most prevalent pathogens causing urinary tract infections: Escherichia coli, Enterococcus spp., and Klebsiella spp. For each uropathogen, two virulent phages were genetically engineered to express a nanoluciferase reporter gene upon host infection. Using 206 patient urine samples, reporter phage-induced bioluminescence was quantified to identify bacteriuria and the assay was benchmarked against conventional urinalysis. Overall, E. coli, Enterococcus spp., and Klebsiella spp. were each detected with high sensitivity (68%, 78%, 87%), specificity (99%, 99%, 99%), and accuracy (90%, 94%, 98%) at a resolution of ≥10$^{3}$ CFU/ml within 5 h. We further demonstrate how bioluminescence in urine can be used to predict phage antibacterial activity, demonstrating the future potential of reporter phages as companion diagnostics that guide patient-phage matching prior to therapeutic phage application

Untersuchung der Funktion der putativen Bordetella\it Bordetella Polysaccharid N\it N-deacetylase BpsB in Biofilmbildung und Pathogenese

Bakterien, welche auf Oberflächen in Gemeinschaften leben, werden als Biofilme bezeichnet. Diese sind von einer polymeren Matrix umgeben, welche unter anderem aus Polysacchariden besteht. $\it Bordetellen$ sind Gram-negative Bakterien, die die Atemwege von Säugetieren infizieren. Vor kurzem wurde gezeigt, dass das $\it Bordetella$ Polysaccharid, Bps, unterschiedlicher $\it Bordetellen$ die Biofilmbildung im Nasenrachenraum von Mäusen fördert. Es wird vermutet, dass die Biofilme im Nasenrachenraum für das langfristige Überleben der Bakterien sowie für deren Übertragung verantwortlich sind. Im Rahmen dieser Arbeit wurde die Funktion der putativen Deacetylase, BpsB, in Hinsicht auf Biofilmbildung, Immunabwehr und Pathogenese untersucht. Dabei wurde ersichtlich, dass BpsB eine wichtige Funktion in der Biofilmbildung spielt. Zudem sind $\it bpsB$-Deletionsmutanten empfindlicher gegenüber antimikrobiellen Peptiden und spielen eine wichtige Rolle bei der Besiedlung der Oberflächen der oberen Atemwege der Maus

Region C of the Escherichia coli heat-shock sigma factor RpoH (σ32) contains a turnover element for proteolysis by the FtsH protease

ISSN:0378-1097ISSN:1574-6968ISSN:0168-6496ISSN:0920-8534ISSN:0168-644

Engineered reporter phages for detection of Escherichia coli, Enterococcus, and Klebsiella in urine

Abstract The rapid detection and species-level differentiation of bacterial pathogens facilitates antibiotic stewardship and improves disease management. Here, we develop a rapid bacteriophage-based diagnostic assay to detect the most prevalent pathogens causing urinary tract infections: Escherichia coli, Enterococcus spp., and Klebsiella spp. For each uropathogen, two virulent phages were genetically engineered to express a nanoluciferase reporter gene upon host infection. Using 206 patient urine samples, reporter phage-induced bioluminescence was quantified to identify bacteriuria and the assay was benchmarked against conventional urinalysis. Overall, E. coli, Enterococcus spp., and Klebsiella spp. were each detected with high sensitivity (68%, 78%, 87%), specificity (99%, 99%, 99%), and accuracy (90%, 94%, 98%) at a resolution of ≥103 CFU/ml within 5 h. We further demonstrate how bioluminescence in urine can be used to predict phage antibacterial activity, demonstrating the future potential of reporter phages as companion diagnostics that guide patient-phage matching prior to therapeutic phage application