Background:The Bordetella polysaccharide (Bps) is involved in Bordetella biofilm formation. Results: BpsB is a periplasmic metal-dependent poly-␤-1,6-N-acetyl-D-glucosamine (PNAG) deacetylase that has unique structural and functional features from known PNAG deacetylases. Conclusion: BpsB-dependent deacetylation of Bps is required for Bordetella bronchiseptica biofilm formation. Significance: Deacetylated Bps is a key component for the structural complexity of Bordetella biofilms.

Transcription of most heat shock genes in Escherichia coli is initiated by the alternative sigma factor sigma(32) (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 factor.

The cellular level of the Escherichia coli heat-shock sigma factor RpoH (s 32 ) is negatively controlled by chaperone-mediated proteolysis through the essential metalloprotease FtsH. Point mutations in the highly conserved region 2.1 stabilize RpoH in vivo. To assess the importance of this turnover element, hybrid proteins were constructed between E. coli RpoH and Bradyrhizobium japonicum RpoH 1 , a stable RpoH protein that differs from region 2.1 of E. coli RpoH at several positions. Nine amino acids forming a putative a-helix were exchanged between the two proteins. Both hybrids were active sigma factors and showed intermediate protein stability.

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, Klebsiellaspp., andEnterococcusspp. 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, Klebsiellaspp., andEnterococcusspp. were each detected with high sensitivity (68%, 78%, 85%), specificity (99%, 99%, 99%), and accuracy (90%, 94%, 96%) at a resolution of ⩾103CFU/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.