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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.

Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States. Uropathogenic Escherichia coli (UPEC), the most common cause of CAUTI, can form biofilms on indwelling catheters. Here, we identify and characterize novel factors that affect biofilm formation by UPEC strains that cause CAUTI. Sixty-five CAUTI UPEC isolates were characterized for phenotypic markers of urovirulence, including agglutination and biofilm formation. One isolate, E. coli MS2027, was uniquely proficient at biofilm growth despite the absence of adhesins known to promote this phenotype. Mini-Tn5 mutagenesis of E. coli MS2027 identified several mutants with altered biofilm growth. Mutants containing insertions in genes involved in O antigen synthesis (rmlC and manB) and capsule synthesis (kpsM) possessed enhanced biofilm phenotypes. Three independent mutants deficient in biofilm growth contained an insertion in a gene locus homologous to the type 3 chaperone-usher class fimbrial genes of Klebsiella pneumoniae. These type 3 fimbrial genes (mrkABCDF), which were located on a conjugative plasmid, were cloned from E. coli MS2027 and could complement the biofilm-deficient transconjugants when reintroduced on a plasmid. Primers targeting the mrkB chaperone-encoding gene revealed its presence in CAUTI strains of Citrobacter koseri, Citrobacter freundii, Klebsiella pneumoniae, and Klebsiella oxytoca. All of these mrkB-positive strains caused type 3 fimbria-specific agglutination of tannic acid-treated red blood cells. This is the first description of type 3 fimbriae in E. coli, C. koseri, and C. freundii. Our data suggest that type 3 fimbriae may contribute to biofilm formation by different gram-negative nosocomial pathogens.

Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of UPEC are fimbrial adhesins, which mediate attachment to specific receptors, enhance persistence and trigger innate host responses. UPEC produce a range of fimbrial adhesins, with type 1 and P fimbriae of the chaperone-usher subclass being the best characterized. The prototype UPEC strain CFT073 contains ten gene clusters that contain genes characteristic of this class of fimbriae. However, only five of these gene clusters have been characterized in detail. In this study the F9 fimbrial gene cluster (c1931-c1936) from CFT073 has been characterized. The F9 fimbriae-encoding genes were PCR amplified, cloned and expressed in a K-12 background devoid of type 1 fimbriae. While F9 fimbrial expression was not associated with any haemagglutination or cellular adherence properties, a role in biofilm formation was observed. E. coli K-12 cells expressing F9 fimbriae produced a dense and uniform biofilm in both microtitre plate and continuous-flow biofilm model systems. In wild-type UPEC CFT073, expression of the F9 major subunit-encoding gene was detected during exponential growth in M9 minimal medium. F9 expression could also be detected following selection and enrichment for pellicle growth in a CFT073fim foc double mutant. The F9 genes appear to be common in UPEC and other types of pathogenic E. coli. However, their precise contribution to disease remains to be determined.

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. [ABSTRACT FROM AUTHOR]

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 coliMC4100 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(O)": "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. [ABSTRACT FROM AUTHOR]

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.