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 'VPS17' homolog. Complementation of the 'sogA1' and 'sogB1' mutations and identification of nonsense mutations in the 'sogA2' and 'sogB1' alleles confirmed the identification. Vps17p 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 'vps17' mutants. 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.

The Gram-negative anaerobic pathogen 'Dichelobacter nodosus' is the principal causative agent of footrot in sheep. The 'intA', 'intB' and 'intC' elements are mobile genetic elements which integrate into two tRNA genes downstream from 'csrA' (formerly 'glpA') and 'pnpA' in the 'D. nodosus' chromosome. CsrA homologues act as global repressors of virulence in several bacterial pathogens, as does polynucleotide phosphorylase, the product of 'pnpA'. We have proposed a model in which virulence in 'D. nodosus' is controlled in part by the integration of genetic elements downstream from 'csrA' and 'pnpA', altering the expression of these putative global regulators of virulence. We describe here a novel integrated genetic element, the 'intD' element, which is 32 kb in size and contains an integrase gene, 'intD', several genes related to genes on other integrated elements of 'D. nodosus', a type IV secretion system and a putative mobilisation region, suggesting that the 'intD' element has a role in the transfer of other genetic elements. Most of the 'D. nodosus' strains examined which contained the 'intD' gene were benign, with 'intD' integrated next to 'pnpA', supporting our previous observation that virulent strains of 'D. nodosus' have the 'intA' element next to 'pnpA'.

Based on morphological criteria, fungi are often separated into two groups: yeasts and filamentous fungi. Uncellular fungi are known as yeasts. The extraction of nucleic acids from yeasts cells is described in Chapter 9. Filamentous fungi form a mycelia consisting of multinucleate, tubular hyphae, which may be separated into compartments by septa. Some fungi are dimorphic, that is, they can exist in a unicellular or multinucleate form. The switch from filamentous to pathogenic yeast form is temperature dependent in the human dimorphic pathogens 'Coccidioides immitis', 'Blastomyces dermatitidis', 'Histoplasma capsulatum', and 'Paracoccidioides brasiliensis'. In the dimorphic plant pathogen, 'Ustilago maydis', the filamentous form, which is produced by mating, is infectious while the fungus grows in culture as a yeast. Some groups of fungi belonging to the phylum Chytridiomycota do not form a true mycelium. Unlike other fungi, chytrids produce motile spores possessing a flagellum.

In 'Aspergillus nidulans', production of extracellular proteases in response to carbon starvation and to a lesser extent nitrogen starvation is controlled by XprG, a putative transcriptional activator. In this study the role of genes involved in carbon catabolite repression and the role of protein as an inducer of extracellular protease gene expression were examined. The addition of exogenous protein to the growth medium did not increase extracellular protease activity whether or not additional carbon or nitrogen sources were present indicating that induction does not play a major role in the regulation of extracellular proteases. Northern blot analysis confirmed that protein is not an inducer of the major 'A. nidulans' protease, PrtA. Mutations in the 'creA', 'creB' and 'creC' genes increased extracellular protease levels in medium lacking a carbon source suggesting that they may have a role in the response to carbon starvation as well as carbon catabolite repression. Analysis of 'glkA4 frA2' and 'creAΔ4' mutants showed that the loss of glucose signalling or the DNA-binding protein which mediates carbon catabolite repression did not abolish glucose repression but did increase extracellular protease activity. This increase was XprG-dependent indicating that the effect of these genes may be through modulation of XprG activity.

The Aspergillus nidulans xprG gene is involved in the regulation of extracellular proteases. A plasmid which complemented the xprG2 mutation was shown to carry the phoG gene, reported to encode an acid phosphatase. Two phoGDelta mutants were constructed and were identical in phenotype to an xprG2 mutant. Null mutants were unable to use protein as a carbon or nitrogen source, have lost a repressible acid phosphatase and have pale conidial color. XprG shows similarity to the Ndt80 transcriptional activator, which regulates the expression of genes during meiosis in Saccharomyces cerevisiae. The xprG1 gain-of-function mutant contains a missense mutation in the region encoding the putative DNA-binding domain. The response to carbon, nitrogen, sulfur, and phosphate limitation is altered in xprG(-) mutants suggesting that XprG is involved in a general response to starvation. Ndt80 may also be involved in sensing nutritional status and control of commitment to meiosis in S. cerevisiae.

The anaerobic bacterium 'Dichelobacter nodosus' is the principal causative agent of ovine footrot, a mixed bacterial infection of the hoof. Although the bacterium only survives for a few days in soil, this period is crucial for transmission of the disease as sheep are infected by walking through soil or pasture contaminated with infectious bacteria. The 'D. nodosus' genome is only 1.3Mb in size and has a dearth of genes encoding regulatory proteins. A series of genetic elements which integrate into the genome has been identified and we have proposed that these integrated genetic elements control the expression of adjacent genes encoding global regulators of virulence. The intA, intB, intC and intD elements integrate next to csrA or pnpA while the vrl integrates next to ssrA. CsrA, PnpA and the ssrA gene product, a 10SaRNA, have been shown to act as global virulence regulators in other bacteria. We have also identified a bacteriophage, DinoHI, which is integrated into the genome of some 'D. nodosus' strains. Sequence analyses suggest that there are many possible interactions between these integrated genetic elements. The vrl contains a copy of the DinoHI packaging site, indicating that the vrl may be transferred between strains by the bacteriophage. DinoHI and the intB element have a common repressor gene, suggesting that maintenance of the integrated state of these two genetic elements is co-ordinately controlled. Similarly, a DNA segment resembling the bacteriophage P4 immunity region is present on the intA, intC and intD elements and may be responsible for maintaining these three genetic elements in the integrated state. The features of the intD element suggest that it is self-transmissible and also capable of mobilising the intA element. Exchange of sequences between these genetic elements may also occur. We discuss here evidence for a network of interactions between these genetic elements with implications for the control of virulence in 'D. nodosus'.

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'.