Contribution of siderophore systems to growth and urinary tract colonization of asymptomatic bacteriuria Escherichia coli

The molecular mechanisms that define asymptomatic bacteriuria (ABU) E. coli colonization of the human urinary tract remain to be properly elucidated. Here we utilize ABU E. coli strain 83972 as a model to dissect the contribution of siderophores to iron acquisition, growth, fitness and colonization of the urinary tract. We show that E. coli 83972 produces enterobactin, salmochelin, aerobactin and yersiniabactin, and examine the role of these systems using mutants defective in siderophore biosynthesis and uptake. Enterobactin and aerobactin contributed most to total siderophore activity and growth in defined iron-deficient media. No siderophores were detected in an 83972 quadruple mutant deficient in all four siderophore biosynthesis pathways; this mutant did not grow in defined iron-deficient media but grew in iron-limited pooled human urine due to iron uptake via the FecA ferric citrate receptor. In a mixed 1:1 growth assay with 83972 there was no fitness disadvantage of the 83972 quadruple biosynthetic mutant, demonstrating its capacity to act as a ‘cheater’ and utilize siderophores produced by the wild-type strain for iron uptake. An 83972 enterobactin/salmochelin double receptor mutant was outcompeted by 83972 in human urine and the mouse urinary tract, indicating a role for catecholate receptors in urinary tract colonization

Molecular characterization of endocarditis-associated Staphylococcus aureus

Infective endocarditis (IE) is a life-threatening infection of the heart endothelium and valves. Staphylococcus aureus is a predominant cause of severe IE and is frequently associated with infections in health care settings and device-related infections. Multilocus sequence typing (MLST), spa typing, and virulence gene microarrays are frequently used to classify S. aureus clinical isolates. This study examined the utility of these typing tools to investigate S. aureus epidemiology associated with IE. Ninety-seven S. aureus isolates were collected from patients diagnosed with (i) IE, (ii) bloodstream infection related to medical devices, (iii) bloodstream infection not related to medical devices, and (iv) skin or soft-tissue infections. The MLST clonal complex (CC) for each isolate was determined and compared to the CCs of members of the S. aureus population by eBURST analysis. The spa type of all isolates was also determined. A null model was used to determine correlations of IE with CC and spa type. DNA microarray analysis was performed, and a permutational analysis of multivariate variance (PERMANOVA) and principal coordinates analysis were conducted to identify genotypic differences between IE and non-IE strains. CC12, CC20, and spa type t160 were significantly associated with IE S. aureus. A subset of virulence-associated genes and alleles, including genes encoding staphylococcal superantigen-like proteins, fibrinogen-binding protein, and a leukocidin subunit, also significantly correlated with IE isolates. MLST, spa typing, and microarray analysis are promising tools for monitoring S. aureus epidemiology associated with IE. Further research to determine a role for the S. aureus IE-associated virulence genes identified in this study is warranted

Biorecovered precious metals from industrial wastes: Single-step conversion of a mixed metal liquid waste to a bioinorganic catalyst with environmental application

The complete and continuous reduction of 1 mM Cr(VI) to Cr(III) was achieved in a flow-through reactor using a novel bioinorganic catalyst ("MM-bio-Pd(0)"), which was produced by single-step reduction of platinum group metals (PGM) from industrial waste solution onto biomass of Desulfovibrio desulfuricans ATCC 29577. Two flow-through reactor systems were compared using both "MM-bio-Pd(0)" and chemically reduced Pd(0). Reactors containing the latter removed Cr(VI) for 1 week only at the expense of formate as the electron donor, whereas the former gave complete Cr(VI) removal for 3 months of continuous operation. Mass balance analysis showed 100% reduction of Cr(VI) to soluble Cr(III) in the bioreactor exit solution. With the use of electron paramagnetic resonance (EPR) no intermediate Cr(V) species could be detected. Pd(0) was biodeposited similarly using Escherichia coli MC4100 and "bio-Pd(0)". The latter was used to recover Pd(II) from two acidic industrial waste leachates to generate two types of "MM-bio-Pd(0) ": "SI-bio-Pd(0)" and "SII-bio-Pd(0)", respectively. The biomaterial composition was comparable in both cases, and the catalytic activity was related inversely to the amount of chloride in the waste leachate from which it was derived

Induction of membrane permeability in Escherichia coli mediated by lysis protein of the ColE7 operon

A glycogen nonpolyphosphate-accumulating organism (GAO) enrichment culture dominated by the Alphaproteobacteria cluster 1 Defluviicoccus was investigated to determine the metabolic pathways involved in the anaerobic formation of polyhydroxyalkanoates, carbon storage polymers important for the proliferation of microorganisms in enhanced biological phosphorus removal processes. FISH-microautoradiography and post-FISH fluorescent chemical staining confirmed acetate assimilation as polyhydroxyalkanoates in cluster 1 Defluviicoccus under anaerobic conditions. Chemical inhibition of glycolysis using iodoacetate, and of isocitrate lyase by 3-nitropropionate and itaconate, indicated that carbon is likely to be channelled through both glycolysis and the glyoxylate cycle in cluster 1 Defluviicoccus. The effect of metabolic inhibitors of aconitase (monofluoroacetate) and succinate dehydrogenase (malonate) suggested that aconitase, but not succinate dehydrogenase, was active, providing further support for the role of the glyoxylate cycle in these GAOs. Metabolic inhibition of fumarate reductase using oxantel decreased polyhydroxyalkanoate production. This indicated reduction of fumarate to succinate and the operation of the reductive branch of the tricarboxylic acid cycle, which is possibly important in the production of the polyhydroxyvalerate component of polyhydroxyalkanoates observed in cluster 1 Defluviicoccus enrichment cultures. These findings were integrated with previous metabolic models for GAOs and enabled an anaerobic central metabolic pathway model for polyhydroxyalkanoate formation in cluster 1 Defluviicoccus to be proposed

Proton motive force generation from stored polymers for the uptake of acetate under anaerobic conditions

The bacteria facilitating enhanced biological phosphorus removal gain a selective advantage from intracellularly stored polymer-driven substrate uptake under anaerobic conditions during sequential anaerobic : aerobic cycling. Mechanisms for these unusual membrane transport processes were proposed and experimentally validated using selective inhibitors and highly-enriched cultures of a polyphosphate-accumulating organism, Accumulibacter, and a glycogen-accumulating organism, Competibacter. Acetate uptake by both Accumulibacter and Competibacter was driven by a proton motive force (PMF). Stored polymers were used to generate the PMF - Accumulibacter used phosphate efflux through the Pit transporter, while Competibacter generated a PMF by proton efflux through the ATPase and fumarate reductase in the reductive TCA cycle

Investigation of ansB and sspA derived promoters for multi- and single-copy antigen expression in attenuated Salmonella enterica var. typhimurium

Five candidate promoters were examined to determine their utility in directing immunogenic levels of expression of the C fragment from tetanus toxin in attenuated S. enterica used as an oral vaccine in mice. Promoters derived from the genes encoding the stringent starvation protein (sspA) from E. coli and S. enterica, but not ansB derived promoters, expressed immunogenic levels of C fragment from multi-copy plasmids in attenuated S. enterica in vivo and, following oral immunization, induced high titre specific anti-tetanus toxoid serum antibodies. We also demonstrate that not only the choice of promoter, replicon and growth conditions but also how expression constructs are assembled in the chosen plasmid is critical for the successful development of plasmid-based antigen delivery systems using attenuated S. enterica. In addition, the S. enterica sspA promoter is able to elicit anti-tetanus toxoid antibodies in mice when the psspA-tetC expression cassette is integrated in single copy on the S. enterica chromosome

Anaerobic central metabolic pathways active during polyhydroxyalkanoate production in uncultured cluster 1 Defluviicoccus enriched in activated sludge communities

A glycogen nonpolyphosphate-accumulating organism (GAO) enrichment culture dominated by the Alphaproteobacteria cluster 1 Defluviicoccus was investigated to determine the metabolic pathways involved in the anaerobic formation of polyhydroxyalkanoates, carbon storage polymers important for the proliferation of microorganisms in enhanced biological phosphorus removal processes. FISH-microautoradiography and post-FISH fluorescent chemical staining confirmed acetate assimilation as polyhydroxyalkanoates in cluster 1 Defluviicoccus under anaerobic conditions. Chemical inhibition of glycolysis using iodoacetate, and of isocitrate lyase by 3-nitropropionate and itaconate, indicated that carbon is likely to be channelled through both glycolysis and the glyoxylate cycle in cluster 1 Defluviicoccus. The effect of metabolic inhibitors of aconitase (monofluoroacetate) and succinate dehydrogenase (malonate) suggested that aconitase, but not succinate dehydrogenase, was active, providing further support for the role of the glyoxylate cycle in these GAOs. Metabolic inhibition of fumarate reductase using oxantel decreased polyhydroxyalkanoate production. This indicated reduction of fumarate to succinate and the operation of the reductive branch of the tricarboxylic acid cycle, which is possibly important in the production of the polyhydroxyvalerate component of polyhydroxyalkanoates observed in cluster 1 Defluviicoccus enrichment cultures. These findings were integrated with previous metabolic models for GAOs and enabled an anaerobic central metabolic pathway model for polyhydroxyalkanoate formation in cluster 1 Defluviicoccus to be proposed

Bioenergetic Models for Acetate and Phosphate Transport in Bacteria Important in Enhanced Biological Phosphorus Removal

Most of our understanding of the physiology of microorganisms is the result of investigations in pure culture. However, in order to understand complex environmental processes, there is a need to investigate mixed microbial communities. This is true for enhanced biological phosphorus removal (EBPR), an environmental process that results in the enrichment of the polyphosphate-accumulating organism Accumulibacter spp. and the glycogen non-polyphosphate accumulating organism Defluviicoccus spp. We investigated acetate and inorganic phosphate (P-i) uptake in enrichments of Accumulibacter spp. and acetate uptake in enrichments of Defluviicoccus spp. For both enrichments, anaerobic acetate uptake assays in the presence of the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP) or the membrane potential (Delta psi) uncoupler valinomycin, indicated that acetate is likely to be taken up by a permease-mediated process driven by the Delta psi. Further investigation with the sodium ionophore monensin suggested that anaerobic acetate uptake by Defluviicoccus spp. may in part be dependent on a sodium potential. Results of this study also suggest that Accumulibacter spp. generate a proton motive force (pmf or Delta p) for anaerobic acetate uptake by efflux of protons in symport with P-i through an inorganic phosphate transport (Pit) system. In contrast, we suggest that the anaerobic Delta p in Defluviicoccus spp. is generated by an efflux of protons across the cell membrane by the fumarate respiratory system, or by extrusion of sodium ions via decarboxylation of methylmalonyl-CoA. Aerobic P-i uptake by the Accumulibacter spp. enrichment was strongly inhibited in the presence of an ATPase inhibitor, suggesting that the phosphate-specific transport (Pst) system is important even under relatively high concentrations of P-i. Acetate permease activity in these microorganisms may play an important role in the competition for acetate in the often acetate-limited EBPR process. Activity of a high-velocity Pst system in Accumulibacter spp. may further explain its ability to compete strongly in EBPR

UpaG, a New Member of the Trimeric Autotransporter Family of Adhesins in Uropathogenic Escherichia coli▿ †

The ability of Escherichia coli to colonize both intestinal and extraintestinal sites is driven by the presence of specific virulence factors, among which are the autotransporter (AT) proteins. Members of the trimeric AT adhesin family are important virulence factors for several gram-negative pathogens and mediate adherence to eukaryotic cells and extracellular matrix (ECM) proteins. In this study, we characterized a new trimeric AT adhesin (UpaG) from uropathogenic E. coli (UPEC). Molecular analysis of UpaG revealed that it is translocated to the cell surface and adopts a multimeric conformation. We demonstrated that UpaG is able to promote cell aggregation and biofilm formation on abiotic surfaces in CFT073 and various UPEC strains. In addition, UpaG expression resulted in the adhesion of CFT073 to human bladder epithelial cells, with specific affinity to fibronectin and laminin. Prevalence analysis revealed that upaG is strongly associated with E. coli strains from the B2 and D phylogenetic groups, while deletion of upaG had no significant effect on the ability of CFT073 to colonize the mouse urinary tract. Thus, UpaG is a novel trimeric AT adhesin from E. coli that mediates aggregation, biofilm formation, and adhesion to various ECM proteins

UpaH Is a Newly Identified Autotransporter Protein That Contributes to Biofilm Formation and Bladder Colonization by Uropathogenic Escherichia coli CFT073▿

Escherichia coli is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of uropathogenic E. coli (UPEC) are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. In this study, we identified a new AT-encoding gene, termed upaH, present in a 6.5-kb unannotated intergenic region in the genome of the prototypic UPEC strain CFT073. Cloning and sequencing of the upaH gene from CFT073 revealed an intact 8.535-kb coding region, contrary to the published genome sequence. The upaH gene was widely distributed among a large collection of UPEC isolates as well as the E. coli Reference (ECOR) strain collection. Bioinformatic analyses suggest β-helix as the predominant structure in the large N-terminal passenger (α) domain and a 12-strand β-barrel for the C-terminal β-domain of UpaH. We demonstrated that UpaH is expressed at the cell surface of CFT073 and promotes biofilm formation. In the mouse UTI model, deletion of the upaH gene in CFT073 and in two other UPEC strains did not significantly affect colonization of the bladder in single-challenge experiments. However, in competitive colonization experiments, CFT073 significantly outcompeted its upaH isogenic mutant strain in urine and the bladder