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DNA sequences flanking the virulence-associated protein (vap) regions 1,2 and 3 were analysed using the Southern blotting and DNA sequencing. These vap regions of the Dichelobacter nodosus are found in 98% of virulent strains tested which suggest that they may have a role in virulence. They appear to have arisen by the integration of a genetic element into a tRNA gene. A new genetic element integrated into the Dichelobacter nodosus chromosome of vap region 3 was identified as either an integrated bacteriophage or conjugative transposon.

Dichelobacter nodosus is the primary pathogen implicated in ovine footrot. In this paper we have delineated a 27 kb locus, termed the virulence-related locus (vrl), that was essentially specific for virulent D. nodosus isolates. The precise ends of this locus were mapped and the sequences of the junction regions from the virulent strain A198 were compared to corresponding sequences from the benign isolate C305. The left end of the vrl locus was located in a sequence similar to that of the small stable 10Sa RNA molecule of Escherichia coli, next to a phage-'attachment-site-like sequence, which indicated that the vrl locus might have arisen by the integration of a phage. However, no attachment-like sequence could be found at the right end of the vrl locus. In the chromosome of the benign strain the sequences bordering vrl were not contiguous but were separated by about 3 kb. it was concluded that the divergence of the benign and virulent strains at this locus was, a multi-step process. Several potential ORFs were identified at the junction regions but only one ORF, encoding a 126 kDa protein, was expressed in a T7 expression system in E. coli.

The bacterial pathogen Dichelobacter nodosus exhibits variable virulence due to the presence of four homologues of virulence-associated protein genes (vap genes) in the plasmid pJIR318 of the D. nodosus genome. Vap genes have three regions formed by duplication event(s) with insertions and/or deletions and the recombinant plasmid results in the formation of virulent strains.

The extracellular proteases of Aspergillus nidulans are produced in response to limitation of carbon, nitrogen, or sulfur, even in the absence of exogenous protein. Mutations in the A. nidulans xprF and xprG genes have been shown to result in elevated levels of extracellular protease in response to carbon limitation. The xprF gene was isolated and sequence analysis indicates that it encodes a 615-amino-acid protein, which represents a new type of fungal hexokinase or hexokinase-like protein. In addition to their catalytic role, hexokinases are thought to be involved in triggering carbon catabolite repression. Sequence analysis of the xprF1 and xprF2 alleles showed that both alleles contain nonsense mutations. No loss of glucose or fructose phosphorylating activity was detected in xprF1 or xprF2 mutants. There are two possible explanations for this observation: (1) the xprF gene may encode a minor hexokinase or (2) the xprF gene may encode a protein with no hexose phosphorylating activity. Genetic evidence suggests that the xprF and xprG genes are involved in the same regulatory pathway. Support for this hypothesis was provided by the identification of a new class of [xprG.sup.-] mutation that suppresses the xprF1 mutation and results in a protease-deficient phenotype.

The Gram-negative anaerobe Dichelobacter nodosus is the causative agent of footrot in sheep. Different strains of D. nodosus cause disease of differing severities, ranging from benign to virulent. Virulent strains have greater twitching motility and secrete proteases that are more thermostable than those secreted by benign strains. We have identified polynucleotide phosphorylase (PNPase) as a putative virulence regulator and have proposed that PNPase expression is modulated by the adjacent integration of genetic elements. In this study, we compared PNPase activity in three virulent and four benign strains of D. nodosus and found that PNPase activity is lower in virulent strains. We disrupted the pnpA gene in three benign D. nodosus strains and two virulent strains and showed that deletion of the S1 domain of PNPase reduced catalytic activity. In all but one case, deletion of the PNPase S1 domain had no effect on the thermostability of extracellular proteases. However, this deletion resulted in an increase in twitching motility in benign, but not in virulent strains. Reconstruction of the pnpA gene in two mutant benign strains reduced twitching motility to the parental level. These results support the hypothesis that PNPase is a virulence repressor in benign strains of D. nodosus. [ABSTRACT FROM AUTHOR]

XprG, a putative p53-like transcriptional activator, regulates production of extracellular proteases in response to nutrient limitation and may also have a role in programmed cell death. To identify genes that may be involved in the XprG regulatory pathway, xprG2 revertants were isolated and shown to carry mutations in genes which we have named sogA-C (suppressors of xprG). The translocation breakpoint in the sogA1 mutant was localized to a homolog of Saccharomyces cerevisiae VPS5 and mapping data indicated that sogB was tightly linked to a VPS17homolog. Complementation of the sogAl and sogB1 mutations and identification of nonsense mutations in the sogA2and sogB1 alleles confirmed the identification. Vpsi 7p and Vps5p are part of a complex involved in sorting of vacuolar proteins in yeast and regulation of cell-surface receptors in mammals. Protease zymograms indicate that mutations in sogA-C permit secretion of intracellular proteases, as in S. cerevisiae vps5 and vps17mutants. In contrast to S. cerevisiae, the production of intracellular protease was much higher in the mutants. Analysis of serine protease gene expression suggests that an XprG- independent mechanism for regulation of extracellular protease gene expression in response to carbon starvation exists and is activated in the pseudorevertants. [ABSTRACT FROM AUTHOR]