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Capsule Shields the Function of Short Bacterial Adhesins. Mark A. Schembri, 2004.Bacterial surface structures such as capsules and adhesins are generally regarded as important virulence factors . Here we demonstrate that capsules block the function of the self-recognizing protein antigen 43 through physical shielding . The phenomenon is not restricted to Escherichia coli but can occur in other gram-negative bacteria . Likewise, we show that other short adhesins exemplified by the AIDA-I protein are blocked by the presence of a capsule. The results support the notion that capsule polysaccharides sterically prevent receptor-target recognition of short bacterial adhesins . This negative interference has important biological consequences, such as affecting the ability of bacteria to form biofilms. The Vibrio cholerae FlgM Homologue Is an Anti-  28
Factor That Is Secreted through the Sheathed Polar Flagellum.Nidia E. Correa, 2004.Vibrio cholerae has a single polar sheathed flagellum that propels the cells of this bacterium . Flagellar synthesis, motility, and chemotaxis have all been linked to virulence in this human pathogen. V . cholerae expresses flagellar genes in a hierarchy consisting of 54-
and
28-dependent
transcription . In other bacteria,
28
transcriptional activity is controlled by an anti-28
factor, FlgM . We demonstrate that the V . cholerae FlgM
homologue (i) physically interacts with
28,
(ii) has a repressive effect on some V . cholerae
28-dependent
flagellar promoters, and (iii) is secreted through the polar sheathed
flagellum, consistent with anti-28
activity . Interestingly, FlgM does not have a uniform repressive
effect on all
28-dependent
promoters, as determined by measurement of
28-dependent
transcription in cells either lacking FlgM ( flgM)
or incapable of secretion (fliF) .
Further analysis of a
fliF
strain revealed that this flagellar assembly block causes a decrease
in class III (FlrC- and
54-dependent)
and class IV (28-dependent),
but not class II (FlrA- and
54-dependent),
flagellar transcription . V . cholerae flgM and fliA (encodes
28)
mutants were only modestly affected in their ability to colonize the
infant mouse intestine, a measure of virulence . Our results
demonstrate that V . cholerae FlgM functions as an anti-28
factor and that the sheathed flagellum is competent for secretion of
nonstructural proteins .
Biochemical and Mutational Characterization of the Heme Chaperone CcmE Reveals a Heme Binding Site. Elisabeth Enggist, 2003.CcmE is a heme chaperone that binds heme transiently in the periplasm of Escherichia coli and delivers it to newly synthesized and exported c-type cytochromes . The chemical nature of the covalent bond between heme and H130 is not known . We have purified soluble histidine-tagged CcmE and present its spectroscopic characteristics in the visible range . Alanine scanning mutagenesis of conserved amino acids revealed that H130 is the only residue found to be strictly required for heme binding and delivery . Mutation of the hydrophobic amino acids F37, F103, L127, and Y134 to alanine affected CcmE more than mutation of charged and polar residues . Our data are in agreement with the recently solved nuclear magnetic resonance structure of apo-CcmE (PDB code 1LIZ) and suggest that heme is bound to a hydrophobic platform at the surface of the protein and then attached to H130 by a covalent bond . Replacement of H130 with cysteine led to the formation of a covalent bond between heme and C130 at a low level . However, the H130C mutant CcmE was not active in cytochrome c maturation . Isolation and characterization of the heme-binding peptides obtained after a tryptic digest of wild-type and H130C CcmE support the hypothesis that heme is bound covalently at a vinyl group . Genome Sequences of Two Closely Related Vibrio parahaemolyticus Phages, VP16T and VP16C. Victor Seguritan, 2003.Two bacteriophages of an environmental isolate of Vibrio parahaemolyticus were isolated and sequenced . The VP16T and VP16C phages were separated from a mixed lysate based on plaque morphology and exhibit 73 to 88% sequence identity over about 80% of their genomes . Only about 25% of their predicted open reading frames are similar to genes with known functions in the GenBank database . Both phages have cos sites and open reading frames encoding proteins closely related to coliphage lambda's terminase protein (the large subunit) . Like in coliphage lambda and other siphophages, a large operon in each phage appears to encode proteins involved in DNA packaging and capsid assembly and presumably in host lysis; we refer to this as the structural operon . In addition, both phages have open reading frames closely related to genes encoding DNA polymerase and helicase proteins . Both phages also encode several putative transcription regulators, an apparent polypeptide deformylase, and a protein related to a virulence-associated protein, VapE, of Dichelobacter nodosus . Despite the similarity of the proteins and genome organization, each of the phages also encodes a few proteins not encoded by the other . We did not identify genes closely related to genes encoding integrase proteins belonging to either the tyrosine or serine recombinase family, and we have no evidence so far that these phages can lysogenize the V . parahaemolyticus strain 16 host . Surprisingly for active lytic viruses, the two phages have a codon usage that is very different than that of the host, suggesting the possibility that they may be relative newcomers to growth in V . parahaemolyticus . The DNA sequences should allow us to characterize the lifestyles of VP16T and VP16C and the interactions between these phages and their host at the molecular level, as well as their relationships to other marine and nonmarine phages . Signal Transduction Cascade between EvgA/EvgS and PhoP/PhoQ Two-Component Systems of Escherichia coli. Yoko Eguchi, 2004.Transcriptional analysis of a constitutively active mutant of the EvgA/EvgS two-component system of Escherichia coli resulted in enhanced expression of 13 PhoP/PhoQ-regulated genes, crcA, hemL, mgtA, ompT, phoP, phoQ, proP, rstA, rstB, slyB, ybjG, yrbL, and mgrB . This regulatory network between the two systems also occurred as a result of overproduction of the EvgA regulator; however, enhanced transcription of the phoPQ genes did not further activate expression of the PhoP/PhoQ-regulated genes . These results demonstrated signal transduction from the EvgA/EvgS system to the PhoP/PhoQ system in E . coli and also identified the genes that required the two systems for enhanced expression . This is one example of the intricate signal transduction networks that are posited to exist in E . coli . Concentration and Prevalence of Escherichia coli O157 in Cattle Feces at Slaughter. F. Omisakin, 2003.The concentration and prevalence of Escherichia coli O157 in cattle feces at the time of slaughter was studied over a 9-week period from May to July 2002 . Fecal samples (n = 589) were collected from the rectums of slaughtered cattle, and the animal-level prevalence rate was estimated to be 7.5% (95% confidence interval [CI], 5.4 to 9.6%) while the group prevalence was 40.4% (95% CI, 27.7 to 53.2%) . Of the 44 infected animals detected, 9% were high shedders that contained E . coli O157 at concentrations of >104 CFU g-1 . These 9% represented >96% of the total E . coli O157 produced by all animals tested . All isolates possessed the vt2 gene, 39 had the eaeA gene, and a further five had the vt1 gene also . The presence of high-shedding animals at the abattoir increases the potential risk of meat contamination during the slaughtering process and stresses the need for correctly implemented hazard analysis and critical control point procedures . Prevalence of Escherichia coli O157 in Cattle Feeds in Midwestern Feedlots. Charles C. Dodd, 2003.Comparisons of enrichment methods (with or without antibiotics and with or without a preenrichment step) using gram-negative (GN) broth or tryptic soy broth (TSB) were conducted with feeds inoculated with Escherichia coli O157:H7 . TSB was more sensitive than GN broth, and TSB with a preenrichment step followed by TSB with antibiotics was more sensitive than plain TSB enrichment, in detecting E . coli O157 in inoculated feeds . Feed samples were collected from feed bunks from 54 feedlots to determine the prevalence of E . coli O157 in cattle feeds . TSB preenrichment followed by TSB with antibiotics and the standard GN broth enrichment were used for each feed sample . All samples underwent immunomagnetic separation and were plated onto sorbitol MacConkey agar with cefixime and potassium tellurite . Identification of E . coli O157 was based on indole production, positive latex agglutination for O157 antigen, API 20E test strip results, PCR for the eaeA gene, and the presence of at least one Shiga toxin . E . coli O157 was detected in 52 of 504 feed samples (10.3%) by using GN broth enrichment and in 46 of 504 feed samples (9.1%) by using TSB followed by TSB supplemented with cefixime and vancomycin . E . coli O157 was detected in 75 of 504 feed bunk samples (14.9%) by one or both methods . There was no correlation between E . coli O157 prevalence and generic coliform counts in feeds . The prevalence of E . coli O157 in cattle feed warrants further studies to increase our knowledge of the on-farm ecology of E . coli O157 in order to develop strategies to prevent food-borne disease in humans .
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