SummaryCodY, a global regulator of gene expression in low G + C Gram-positive bacteria, was found to repress toxin gene expression in Clostridium difficile. Inactivation of the codY gene resulted in derepression of all five genes of the C. difficile pathogenicity locus during exponential growth and stationary phase. CodY was found to bind with high affinity to a DNA fragment containing the promoter region of the tcdR gene, which encodes a sigma factor that permits RNA polymerase to recognize promoters of the two major toxin genes as well as its own promoter. CodY also bound, but with low affinity, to the toxin gene promoters, suggesting that the regulation of toxin gene expression by CodY occurs primarily through direct control of tcdR gene expression. Binding of CodY to the tcdR promoter region was enhanced in the presence of GTP and branched-chain amino acids, suggesting a link between nutrient limitation and the expression of C. difficile toxin genes.

CodY, a global regulatory protein that monitors the nutrient sufficiency of the environment by responding to the intracellular levels of GTP and the branched-chain amino acids, was previously shown to be a potent repressor of toxin gene expression in Clostridium difficile during growth in rich medium. In the intestinal tract, such derepression of toxin synthesis would lead to destruction of epithelial cells and the liberation of potential nutrients for the bacterium. CodY is likely to play an important role in regulating overall cellular physiology as well. In this study, DNA microarray analysis and affinity purification of CodY-DNA complexes were used to identify and distinguish the direct and indirect effects of CodY on global gene transcription. A codY null mutation resulted in >4-fold overexpression of 146 genes (organized in 82 apparent transcription units) and underexpression of 19 genes. In addition to the toxin genes, genes for amino acid biosynthesis, nutrient transport, fermentation pathways, membrane components, and surface proteins were overexpressed in the codY mutant. Genome-wide analysis identified more than 350 CodY binding regions, many of which are likely to correspond to sites of direct CodY-mediated regulation. About 60% of the CodY-repressed transcription units were associated with binding regions. Several of these genes were confirmed to be direct targets of CodY by gel mobility shift and DNase I footprinting assays.

Clostridium difficile is a Gram‐positive, spore‐forming, anaerobe and is the leading cause of antibiotic‐associated diarrhea, pseudomembranous colitis, and toxic megacolon. Essential to the lifestyle of C. difficile is the ability to form a metabolically dormant spore, germinate, and grow out upon appropriate signals and elicit disease with the secretion of two toxins. To aid in the study of this organism, this unit describes the growth and maintenance of C. difficile. Included are methods to isolate C. difficile from environmental samples, grow in laboratory medium, and produce and purify spores. Curr. Protoc. Microbiol. 12:9A.1.1‐9A.1.10. © 2009 by John Wiley & Sons, Inc.

SummaryConversion of pyruvate to acetate via the phosphotransacetylase-acetate kinase pathway generates ATP and is a major overflow pathway under conditions of carbon and nitrogen excess. In Bacillus subtilis, this pathway is positively regulated by CcpA, a global regulator of carbon metabolism genes. Transcription of the acetate kinase gene (ackA) proved to be activated as well by a second global regulatory protein, CodY. Expression of an ackA-lacZ fusion was reduced in a codY mutant strain. CodY was found to bind in vitro to two sites in the ackA promoter region and to stimulate ackA transcription in a run-off transcription assay. This is the first known case of direct positive regulation by CodY. CodY and CcpA were found to bind to neighbouring sites and their effects were additive both in vivo and in vitro. Surprisingly, positive regulation by CodY, unlike repression, responded primarily to only one type of effector molecule. That is, branched-chain amino acids (BCAAs) served as more potent co-activators of CodY-dependent ackA transcription than did GTP. Given the roles of CcpA and CodY in regulating genes whose products determine the metabolic fate of pyruvate, these two proteins may act together to mediate a hierarchical conversion of pyruvate to its many potential products.

Putative Pseudomonas spp. isolated predominantly from raw and processed milk were characterized by automated ribotyping and by biochemical reactions. Isolates were biochemically profiled using the Biolog system and API 20 NE and by determining the production of proteases, lipases, and lecithinases for each isolate. Isolates grouped into five coherent clusters, predominated by the species P. putida (cluster A), P. fluorescens (cluster B), P. fragi (as identified by Biolog) or P. fluorescens (as identified by API 20 NE) (cluster C), P. fragi (as identified by Biolog) or P. putida (as identified by API 20 NE) (cluster D), and P. fluorescens (cluster E). Isolates within each cluster also displayed similar enzyme activities. Isolates in clusters A, C, and D were generally negative for all three enzyme activities; isolates in cluster B were predominantly positive for all three enzyme activities; and isolates in cluster E were negative for lecithinase but predominantly positive for protease and lipase activities. Thus, only isolates from clusters B and E produced enzyme activities associated with dairy product flavor defects. Thirty-eight ribogroups were differentiated among the 70 isolates. Ribotyping was highly discriminatory for dairy Pseudomonas isolates, with a Simpson's index of discrimination of 0.955. Isolates of the same ribotype were never classified into different clusters, and ribotypes within a given cluster generally showed similar ribotype patterns; thus, specific ribotype fragments may be useful markers for tracking the sources of pseudomonads in dairy production systems. Our results suggest that ribogroups are generally homogeneous with respect to nomenspecies and biovars, confirming the identification potential of ribotyping for Pseudomonas spp.Phenotypic microbiological techniques have proven useful for quantifying and describing bacteria causing fluid dairy product spoilage; however, precise location of the sources of these spoilage organisms in the processing environment or on the farm requires reliable, differential strain identification strategies. Currently available phenotypic speciation strategies for the most common dairy product spoilers, i.e., Pseudomonas spp. (26) and Bacillus spp., frequently yield inconclusive results.

The nucleotide sequence of the hemagglutinin ( ha) genes and the transcriptional regulator botR gene were determined in type A Clostridium botulinum strain 62A, and the complete nucleotide sequence of the botulinum neurotoxin (BoNT) gene cluster was determined in strain Hall A- hyper. Comparison of the BoNT/A gene clusters revealed only two nucleotide differences between the two strains. The nucleotide sequences of the regions flanking the BoNT clusters were also determined in strains 62A, Hall A- hyper, and type A(B) strain NCTC 2916. The regions upstream of the BoNT/A clusters in the type A strains shared marked homology with the region upstream of the silent BoNT/B cluster in the A(B) strain, indicating a similar evolutionary origin. The region downstream of the BoNT/A cluster in type A strains encodes putative insertion sequence (IS) elements with multiple internal mutations. These IS elements may have played a role in neurotoxin gene transfer within the host genome and to other Clostridium species.

We examined (i) the persistence of Escherichia coli O157:H7 as a postpasteurization contaminant in fermented dairy products; (ii) the ability of E. coli O157:H7 strains with and without the general stress regulatory protein, RpoS, to compete with commercial starter cultures in fermentation systems; and (iii) the survival of E. coli O157:H7 in the yogurt production process. In commercial products inoculated with 10(3) CFU/ml, E. coli O157:H7 was recovered for up to 12 days in yogurt (pH 4.0), 28 days in sour cream (pH 4.3), and at levels > 10(2) CFU/ml at 35 days in buttermilk (pH 4.1). For the starter culture competition trials, the relative inhibition of E. coli O157:H7 in the experimental fermentation systems was, in decreasing order, thermophilic culture mixture, Lactobacillus delbrueckii subsp. bulgaricus R110 alone, Lactococcus lactis subsp. lactis D280 alone, Lactococcus lactis subsp. cremoris D62 alone, and Streptococcus thermophilus C90 alone showing the least inhibition. Recovery of the rpoS mutant was lower than recovery of its wild-type parent by 72 h or earlier in the presence of individual starter cultures. No E. coli O157:H7 were recovered after the curd formation step in yogurt manufactured with milk inoculated with 10(5) CFU/ml. Our results show that (i) postprocessing entry of E. coli O157:H7 into fermented dairy products represents a potential health hazard; (ii) commercial starter cultures differ in their ability to reduce E. coli O157:H7 CFU numbers in fermentation systems; and (iii) the RpoS protein appears to most effectively contribute to bacterial survival in the presence of conditions that are moderately lethal to the cell.

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