Field observations on the variation of Streptococcus uberis populations in a pasture-based dairy farm.

Microbiological and molecular tools were used to monitor Streptococcus uberis populations on farm tracks and paddocks on a dairy farm during different seasons of a year to identify and profile potential environmental niches of Strep. uberis in a pasture-based dairying system. Farm tracks of high or low cow traffic were sampled every 2 wk for an entire year and Strep. uberis numbers were enumerated from a selective medium. During each season of the year, paddocks were sampled for the presence of Strep. uberis before and after grazing by dairy cows. Farm tracks of high cow traffic generally had greater concentrations of Strep. uberis isolated compared with tracks with less cow traffic, but there was also significant variation in the concentrations of Strep. uberis contamination among seasons, being highest in winter and lowest in summer. The bacterium was detected in paddocks only after cow grazing had occurred, but the bacteria could still be detected in soil for up to 2 wk following grazing in winter. Multilocus sequence typing showed great heterogeneity, with some commonality between farm track and milk isolates, which may help explain cow-to-environment or environment-to-cow transmission of the bacterium in the dairy setting.

The mouse adrenocorticotropin receptor gene: cloning and characterization of its promoter and evidence for a role for the orphan nuclear receptor steroidogenic factor 1.

To elucidate the mechanism underlying the tissue-specific expression of the ACTH receptor/MC2 receptor (ACTH-R) in the adrenal cortex, we have cloned the mouse ACTH-R promoter. The analysis of the cDNA 5'-end showed an untranslated region of 321 bp, and the ACTH-R gene was demonstrated to be composed of two exons of 113 and 112 bp lying upstream of the single coding exon. S1 nuclease protection assay showed two major transcription start sites separated by 4 bp; 1.8 kb of the 5'-flanking region inserted in a luciferase reporter vector had cell-specific promoter activity because it was functional only in mouse adrenocortical Y1 cells but not in mouse Leydig TM3 cells or fibroblast L cells. There was no dramatic change in the promoter activity in Y1 cells for all the deletions tested up to -113 bp upstream of the transcription start site. In contrast, expression in TM3 cells was switched on from deletion -908 bp, while remaining undetectable in L cells. Site-directed mutagenesis of a steroidogenic factor 1 (SF1)-like site at position -25 bp resulted in a significant reduction in luciferase expression by the promoter in Y1 cells. Gel shift analysis of this site indicated specific binding of a protein in extracts of Y1 and TM3 cells. Moreover, expression of SF1 in L cells induced promoter activity of the construct p(908). In conclusion, cell-specific expression of the mouse ACTH-R appears to be controlled by at least two factors. One of them, most probably SF1, is responsible for steroidogenic cell-specific expression. The other as yet unknown factor binding between position -1236 bp and -908 bp acts as a strong inhibitory factor in nonadrenal steroidogenic cells, resulting in the adrenal-specific expression of ACTH-R.

Functional characterization of naturally occurring mutations of the human adrenocorticotropin receptor: poor correlation of phenotype and genotype.

Several missense mutations of the ACTH receptor (MC2-R) gene have been associated with the autosomal recessive syndrome of familial glucocorticoid deficiency. Attempts to demonstrate the functional role of these mutations have been confounded by difficulties in expression of the cloned receptor in cells lacking endogenous melanocortin receptors. The Y6 cell line, a mutant derived from the Y1 cell line, lacks any endogenous MC2-R and can be used for this purpose. We demonstrate that several MC2-R mutations associated with familial glucocorticoid deficiency result in an impaired maximal cAMP response (S74I, I44M, R146H) or loss of sensitivity for cAMP generation (D103N, R128C, T159K) compared to the wild-type receptor. Considerable variation in clinical phenotype exists even for patients with identical mutations of the MC2-R, and correlation between the estimated severity of the receptor defect in vitro and the age at clinical presentation and degree of clinical severity, as judged by basal and stimulated plasma cortisol concentration, is poor.

Sequence analysis of the potent mitogenic toxin of Pasteurella multocida.

Pasteurella multocida toxin is a potent mitogen for cultured Swiss 3T3 cells where it causes an accumulation of inositol phosphates and activation of protein kinase C. The gene sequence described here coded for a 146 kDa protein. The ORF was preceded by a ribosome binding site and followed by a stem loop. There was no evidence for a signal sequence. The gene had a low G + C base ratio which differs from the rest of the Pasteurella genome. There was no significant homology with other known proteins, although a motif found in certain bacterial toxins which are ADP-ribosyl transferases is present. A recombinant expressing only part of the PMT gene was not mitogenic.

Regulation of spvR, the positive regulatory gene of Salmonella plasmid virulence genes.

The regulation of the spvR promoter from the Salmonella dublin virulence plasmid was monitored using promoter-reporter gene fusion constructs. Activity was dependent upon the presence of the spv region and was affected by the number of copies of the spv region present within the cell. Activity remained constant throughout exponential growth, and increased rapidly with the onset of stationary phase, under both aerobic and anaerobic conditions. Additionally, the level of spvR expression was controlled by the availability of iron, activity being greatest under low iron conditions in stationary phase. The spvA gene product negatively regulated spvR expression in a dose-dependent manner, indicating that SpvA provides a negative feedback mechanism for this operon.

The pathological effect of the Bordetella dermonecrotic toxin in mice.

The effect of dermonecrotic toxin (DNT) expression of Bordetella bronchiseptica was studied in mice by comparing the pathology induced by a wild type strain with that induced by an isogenic DNT- strain in which part of the structural gene has been replaced by an antibiotic resistance cassette. While extracts of strain B58 proved toxic in intravenously inoculated mice, similar extracts from strain B58GP had lost toxic activity. The parent (B58) and the mutant (B58GP) strains of B. bronchiseptica each possessed comparable virulence for mice. These findings confirmed that DNT production was successfully abolished in strain B58GP while other virulence characteristics required for pathogenicity in mice remained intact, at a comparable level to the parent strain. Turbinate atrophy was observed in mice infected with the DNT+ strain, but not in those infected with the DNT- strain. This indicates that DNT is the cause of turbinate atrophy in the mice and not other factors produced by phase I strains of B. bronchiseptica. B. bronchiseptica DNT showed a lienotoxic effect (lymphocyte depletion and a reduction in the intensity of extramedullar haemocytopoieis) that is considered to adversely alter the immune function of the host animal. In mice infected with strain B58GP, catarrhal pneumonia with characteristic lympho-histiocytic peribronchial and perivascular infiltration was noticed. In mice infected with strain B58, large necrotic areas were seen surrounded by an inflammatory reaction. The DNT appears to directly damage lung tissues, at least in mice. DNT production seems to enhance the establishment of B. bronchiseptica in the lungs, presumably by reducing the local resistance and causing severe local damage to the lung tissues.

A Salmonella dublin virulence plasmid locus that affects bacterial growth under nutrient-limited conditions.

This paper reports the characterization of a new locus, vagC/vagD, on the virulence plasmid of Salmonella dublin. Strain G19, harbouring a TnA insertion in vagC, exhibited reduced virulence although vagC was outside the 8 kb essential virulence region. G19 was also unable to grow on minimal-medium containing various sole carbon/energy sources, unlike the wild-type and plasmid-cured strains. Sequencing of the locus revealed the presence of two ORFs (vagC and vagD) which overlapped by one nucleotide. The VagC polypeptide (12 kDa) was observed using minicell expression. Results indicated that vagD was responsible for the phenotypic differences observed between the wild type and G19, and that vagC modulated the activity of vagD. Furthermore, microscopic analysis of G19 cells harvested from minimal-medium plates showed that a high proportion of cells were elongated, which suggested that vagC and vagD might be involved in coordination of plasmid replication with cell division. We propose that vagD, under certain environmental conditions, acts to prevent cell division until plasmid replication is complete, thus aiding plasmid maintenance. vagC and vagD are absent from the related virulence plasmid of Salmonella typhimurium.

Localization of functional domains of the mitogenic toxin of Pasteurella multocida.

The locations of the catalytic and receptor-binding domains of the Pasteurella multocida toxin (PMT) were investigated. N- and C-terminal fragments of PMT were cloned and expressed as fusion proteins with affinity tags. Purified fusion proteins were assessed in suitable assays for catalytic activity and cell-binding ability. A C-terminal fragment (amino acids 681 to 1285) was catalytically active. When microinjected into quiescent Swiss 3T3 cells, it induced changes in cell morphology typical of toxin-treated cells and stimulated DNA synthesis. An N-terminal fragment with a His tag at the C terminus (amino acids 1 to 506) competed with full-length toxin for binding to surface receptors and therefore contains the cell-binding domain. The inactive mutant containing a mutation near the C terminus (C1165S) also bound to cells in this assay. Polyclonal antibodies raised to the N-terminal PMT region bound efficiently to full-length native toxin, suggesting that the N terminus is surface located. Antibodies to the C terminus of PMT were microinjected into cells and inhibited the activity of toxin added subsequently to the medium, confirming that the C terminus contains the active site. Analysis of the PMT sequence predicted a putative transmembrane domain with predicted hydrophobic and amphipathic helices near the N terminus over the region of homology to the cytotoxic necrotizing factors. The C-terminal end of PMT was predicted to be a mixed alpha/beta domain, a structure commonly found in catalytic domains. Homology to proteins of known structure and threading calculations supported these assignments.

Identification of an essential virulence region on Salmonella plasmids.

An 8 kilobase pair (kbp) fragment from the Salmonella dublin 2229 plasmid is sufficient to restore virulence for mice to cured strains of both S. dublin and S. typhimurium but only when cloned on a low copy vector. Two regions previously shown to be associated with virulence lie outside the fragment and complementation results suggest that one of these mutated regions affects expression from the 8 kbp fragment. It therefore appears that there are control elements both within and adjacent to the essential virulence fragment.

Identification of proteins expressed by the essential virulence region of the Salmonella dublin plasmid.

An 8 kilobase pair (kb) fragment from the Salmonella dublin 2229 plasmid is sufficient to restore virulence for mice to a cured strain of S. dublin. Deletion analysis of this virulence fragment identified at least one specific region required for virulence expression. Plasmid-directed protein synthesis in minicells has indicated the presence of at least four genes within the essential virulence region of the S. dublin plasmid, encoding proteins of 70, 33, 30 and 26 kDa. Analysis of the proteins expressed by the deletion derivatives suggested that expression of the 33 kDa polypeptide was linked to that of the 30 kDa polypeptide. The proteins expressed by the essential virulence region of the S. dublin plasmid appeared to be similar to the plasmid-encoded virulence proteins recently identified in S. typhimurium.

Escherichia coli cytotoxic necrotizing factor and Pasteurella multocida toxin induce focal adhesion kinase autophosphorylation and Src association.

Cytotoxic necrotizing factor 1 and Pasteurella multocida toxin induced dose- and time-dependent increases in focal adhesion kinase (FAK) Tyr397 phosphorylation in Swiss 3T3 cells. FAK autophosphorylation was sensitive to inhibitors of p160/ROCK and coincided with the formation of stable complexes between FAK and Src family members.

Cytotoxic necrotizing factor 1 from Escherichia coli and dermonecrotic toxin from Bordetella bronchiseptica induce p21(rho)-dependent tyrosine phosphorylation of focal adhesion kinase and paxillin in Swiss 3T3 cells.

Treatment of Swiss 3T3 cells with cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli and dermonecrotic toxin (DNT) from Bordetella bronchiseptica, which directly target and activate p21(rho), stimulated tyrosine phosphorylation of focal adhesion kinase (p125(fak)) and paxillin. Tyrosine phosphorylation induced by CNF1 and DNT occurred after a pronounced lag period (2 h), and was blocked by either lysosomotrophic agents or incubation at 22 degrees C. CNF1 and DNT stimulated tyrosine phosphorylation of p125(fak) and paxillin, actin stress fiber formation, and focal adhesion assembly with similar kinetics. Cytochalasin D and high concentrations of platelet-derived growth factor disrupted the actin cytoskeleton and completely inhibited CNF1 and DNT induced tyrosine phosphorylation. Microinjection of Clostridium botulinum C3 exoenzyme which ADP-ribosylates and inactivates p21(rho) function, prevented tyrosine phosphorylation of focal adhesion proteins in response to either CNF1 or DNT. In addition, our results demonstrated that CNF1 and DNT do not induce protein kinase C activation, inositol phosphate formation, and Ca2+ mobilization. Moreover, CNF1 and DNT stimulated DNA synthesis without activation of p42(mapk) and p44(mapk) providing additional evidence for a novel p21(rho)-dependent signaling pathway that leads to entry into the S phase of the cell cycle in Swiss 3T3.

The virulence plasmid of Salmonella dublin: detailed restriction map and analysis by transposon mutagenesis.

A detailed restriction map of the virulence plasmid of Salmonella dublin has been determined and used for comparison with the virulence plasmid from S. typhimurium. Two regions were identified which appeared to be similar based on blotting and restriction data. One, of about 22 kb, encompassed the virulence region; the other, of about 8 kb, was outside it. The locations of 259 transposon insertions on the S. dublin plasmid were determined and related to their effect on virulence. One gene involved in virulence but outside the essential virulence region was shown to affect citrate metabolism.

Cloning, expression, and molecular characterization of the dermonecrotic toxin gene of Bordetella spp.

A cosmid library of random fragments of Bordetella bronchiseptica genomic DNA was prepared and screened with oligonucleotides designed from the sequence of the B. pertussis dermonecrotic toxin (DNT) gene. Two cosmid clones which apparently contained the complete B. bronchiseptica DNT gene were identified, but they did not express the toxin. A 5-kb fragment containing the DNT gene was subcloned from one of the cosmid clones onto a high-copy-number plasmid, and this resulted in low-level expression of the toxin. The expression level was increased by deletion of a small region upstream of the coding sequence. Assays for biological activity, including the infant mouse dermonecrosis assay, confirmed that the product of the cloned gene was DNT. The complete sequence of the B. bronchiseptica DNT gene was determined and was more than 99% homologous to the DNT gene of B. pertussis. A putative purine nucleotide-binding motif was shown to be important for toxic activity. Extracts containing the recombinant or the native toxin induced DNA synthesis in Swiss 3T3 cells but inhibited cell division leading to binucleation.