The low-Ca2' response is a multicomponent virulence regulon of the human-pathogenic yersiniae in which 12 known virulence genes are coordinately regulated in response to environmental cues of temperature, Ca2+, and nucleotides such as ATP. Yersinial growth also is regulated, with full growth yield being permitted at 37°C only if Ca2+ or a nucleotide is present. In this study, we constructed and characterized a mutant Yersinia pestis specifically defective in the gene encoding the V antigen, one of the virulence genes of the low-Ca2+ response.An in-frame internal deletion-insertion mutation was made by removing bases 51 through 645 of IcrV and inserting 61 new bases. The altered lcrV was introduced into the low-Ca2' response plasmid in Y. pestis by allelic exchange, and the resulting mutant was characterized for its two-dimensional protein profiles, growth, expression of an operon fusion to another low-Ca2' response virulence operon, and virulence in mice. The mutant had lost its Ca2' and nucleotide requirement for growth, showed diminished expression of Ca2+-and nucleotide-regulated virulence genes, and was avirulent in mice. The mutation could be complemented with respect to the growth property by supplying native V antigen operon sequences in trans in high copy number (on pBR322). Partial complementation of the growth defect and almost complete complementation of the virulence defect were seen with a lower-copy-number complementing replicon (a pACYC184 derivative). The data are consistent with the interpretation that V antigen is bifunctional, with a role in regulating growth and expression of low-Ca2' response virulence genes in addition to its putative role as a secreted virulence protein.The human-pathogenic yersiniae have a multicomponent virulence property called the low-Ca2" response (Lcr phenotype) (8), which contains a set of regulatory genes and 12 identified, coordinately regulated virulence genes. The virulence gene products include a set of 11 surface proteins called Yops, which can be shed into the medium, and a secreted protein called the V antigen (11,35,36,43). The regulatory genes modulate expression of the virulence genes in response to the environmental inputs of temperature, Ca2+, and nucleotides such as ATP (19). The low-Ca2+ response is encoded by a ca. 75-kb plasmid (3, 17, 36), the one in Yersinia pestis KIM being called pCD1 (19).A curious manifestation of the low-Ca2+ response is the requirement of millimolar Ca2+ or ca. 10 to 20 mM nucleotide for the bacteria to show full growth yield at 37°C (20,49

The lcrGVH operon of plasmid pCDl in Yersinia pestis KIM encodes the virulence-associated V antigen, the regulatory protein LcrH, and LcrG, a protein of undefined function. In this study we sequenced krGVH and analyzed it for transcription initiation sites. There were three open reading frames within the sequence, 288, 981, and 507 bases in length, which could encode proteins with molecular weights and isoelectric points corresponding to those of LcrG, LcrV (V antigen), and LcrH, respectively. The predicted LcrV protein lacked an N-terminal signal sequence; however, an internal signallike sequence was present. An Escherichia cofi-like promoter consensus sequence was detected upstream from kIrG. Primer extension analysis showed that (i) the transcriptional start site for krGVH was spaced only three bases upstream from the nearest ATG potential start site, raising the possibility that Y. pestis may use an alternate initiation codon for the V operon; (ii) there was much more primer-extended product in yersiniae grown in the absence of Ca2" than in its presence, showing for the first time that lkiGVH is regulated at the transcriptional level by Ca2m; (iii) no separate krV initiation was detected, indicating that the V antigen is expressed from messages initiating at krG; and (iv) a non-Ca2+-regulated transcriptional start site was found upstream from krH, suggesting that the LcrH protein is expressed constitutively. However, two-dimensional gel analysis showed that net LcrH expression was regulated by Ca2+. We propose that krH lies within two differentially regulated operons, its own and krGVH.Yersinia pestis, the etiologic agent of bubonic plague, contains a 75-kilobase (kb) virulence plasmid called pCD1 in Y. pestis KIM (14,16). pCD1 encodes the low-calciumresponse (Lcr) determinant, a set of temperature-and Ca2+-regulated genes required for virulence (7, 16). The Lcr+ phenotype has two components, a growth requirement for either Ca2e or nucleotides at 37°C (8,17,26,39), and the expression of a set of virulence-associated proteins. These proteins include the V and W antigens (9) and a set of outer membrane proteins called Yops (4,29,33). Maximal expression of the V antigen and the Yops occurs at temperatures above 34°C in the absence of Ca2+ (28,33,39).The association of the V antigen with virulence is based on the ability of partially purified V to protect mice against fully virulent Y. pestis and on the passive protection provided against plague by anti-V antisera (18). Definitive evidence that the V antigen is a classical virulence factor does not exist, because no one has yet studied a mutant of Y. pestis lacking expression of only this protein.A V-mutant of Y. pestis was obtained by insertion mutagenesis, but the transposon insertion also eliminated expression of two other proteins, the ca. 13,000-molecularweight LcrG and the ca. 18,000-molecular-weight LcrH (27). The expression of two genes downstream of lcrGVH was not eliminated by the insertion, and it was concluded that this polar insertion mutation defin...

The gene encoding the toxin A protein of Clostridium difficile (strain VPI 10463) was cloned and sequenced. The coding region of 8,133 base pairs has a mol% G+C of 26.9 and encodes 2,710 amino acids. The deduced polypeptide has a molecular mass of ca. 308 kilodaltons. Nearly a third of the gene, at the 3' end, consists of 38 repeating sequences. The repeating units were grouped into two classes, I and II, on the basis of length and the low levels of DNA sequence similarities between them. There were seven class I repeating units, each containing 90 nucleotides, and 31 class II units, which, with two exceptions, were either 60 or 63 nucleotides in length. On the basis of DNA sequence similarities, the class II repeating units were further segregated into subclasses: 7 class IIA, 13 class IIB, 5 class IIC, and 6 class IID. The dipeptide tyrosine-phenylalanine was found in all 38 repeating units, and other amino acid sequences were unique to a specific class or subclass. This region of the protein has epitopes for the monoclonal antibody PCG-4 and includes the binding region for the Galoa1-3Gal01-4GlcNAc carbohydrate receptor. Located 1,350 base pairs upstream from the toxin A translation start site is the 3' end of the toxin B gene. Between the two toxin genes is a small open reading frame, which encodes a deduced polypeptide of ca. 16 or 19 kilodaltons. The role of this open reading frame is unknown.

Poultry and livestock are the most important reservoirs for pathogenic Escherichia coli and use of antimicrobials in animal farming is considered the most important factor promoting the emergence, selection and dissemination of antimicrobial-resistant microorganisms. The aim of our study was to investigate antimicrobial resistance in E. coli isolated from food animals in Jiangsu, China. The disc diffusion method was used to determine susceptibility to 18 antimicrobial agents in 862 clinical isolates collected from chickens, ducks, pigs, and cows between 2004 and 2012. Overall, 94% of the isolates showed resistance to at least one drug with 83% being resistance to at least three different classes of antimicrobials. The isolates from the different species were most commonly resistant to tetracycline, nalidixic acid, sulfamethoxazole, trimethoprim/sulfamethoxazole and ampicillin, and showed increasing resistance to amikacin, aztreonam, ceftazidime, cefotaxime, chloramphenicol, ciprofloxacin. They were least resistant to amoxicillin/clavulanic acid (3.4%) and ertapenem (0.2%). MDR was most common in isolates from ducks (44/44, 100%), followed by chickens (568/644, 88.2%), pigs (93/113, 82.3%) and cows (13/61, 21.3%). Our finding that clinical E. coli isolates from poultry and livestock are commonly resistant to multiple antibiotics should alert public health and veterinary authorities to limit and rationalize antimicrobial use in China.

Escherichia coli O157:H7 is a highly acid-resistant food-borne pathogen that survives in the bovine and human gastrointestinal tracts and in acidic foods such as apple cider. This property is thought to contribute to the low infectious dose of the organism. Three acid resistance (AR) systems are expressed in stationaryphase cells. AR system 1 is S dependent, while AR systems 2 and 3 are glutamate and arginine dependent, respectively. In this study, we sought to determine which AR systems are important for survival in acidic foods and which are required for survival in the bovine intestinal tract. Wild-type and mutant E. coli O157:H7 strains deficient in AR system 1, 2, or 3 were challenged with apple cider and inoculated into calves. Wild-type cells, adapted at pH 5.5 in the absence of glucose (AR system 1 induced), survived well in apple cider. Conversely, the mutant deficient in AR system 1, shown previously to survive poorly in calves, was susceptible to apple cider (pH 3.5), and this sensitivity was shown to be caused by low pH. Interestingly, the AR system 2-deficient mutant survived in apple cider at high levels, but its shedding from calves was significantly decreased compared to that of wild-type cells. AR system 3-deficient cells survived well in both apple cider and calves. Taken together, these results indicate that E. coli O157:H7 utilizes different acid resistance systems based on the type of acidic environment encountered.