Screening systems for detecting inhibitors of cell wall transglycosylation in Enterococcus. Cell wall transglycosylation inhibitors in Enterococcus.

We devised two screening systems to detect cell wall transglycosylation inhibitors. One screen utilizes a mutant of Enterococcus faecalis strain A256 that is dependent on vancomycin or moenomycin for growth. In the absence of transglycosylation inhibitors the strain fails to grow, while in the presence of inhibitors, cells are rescued. A second screening organism E. faecalis strain MDD212 utilizes a translational fusion of the lacZ gene to the vanH promoter in a derivative of E. faecalis that contains a vancomycin resistance determinant. Induction of beta-galactosidase occurs when cells are exposed to inhibitors of transglycosylation. Our natural products drug source of fungal fermentations was tested with these screens. Several cultures that produced the same family of compounds, called the thielavins, were detected. Thielavin B inhibited the formation of peptidoglycan in an in vitro assay, suggesting that these screening systems can detect compounds that interfere with cell wall transglycosylation.

Mapping the heterogeneous DNA region that determines the nine A alpha mating-type specificities of Schizophyllum commune.

Classical genetic studies identified nine mating-type specificities at the A alpha locus of the Basidiomycete fungus Schizophyllum commune. We have used Southern blot hybridizations to generate EcoRI restriction maps of the A alpha locus for 18 strains, including all nine specificities. A alpha 1, A alpha 3 and A alpha 4 DNA was subcloned from three cosmids and used as probes. A unique region of DNA was found for each of the three cloned specificities. Hybridization was detected in this region only if the probe(s) and the blotted genomic DNAs were from strains with the same A alpha specificity. DNAs from strains with the same A alpha specificity hybridize regardless of geographic origin, but DNAs from strains with different A alpha specificities do not cross-hybridize. The results demonstrate two size classes of unique A alpha DNA. This unique DNA is about 4.5 kb in A alpha 1 strains and about 7.0-8.5 kb in other strains. Transcription regulators Z and Y, which were deduced previously from the DNA sequence of the A alpha 1, A alpha 3 and A alpha 4 loci, are probably encoded by all non-A alpha 1 loci. The smaller A alpha 1 loci appear to encode only Y and lack sequence for Z. No evidence was found for a locus that encodes only Z. The lack of hybridization detected between A alpha loci with different specificities suggests that the evolution of A alpha has resulted from extensive sequence divergence.

The A alpha mating locus of Schizophyllum commune encodes two dissimilar multiallelic homeodomain proteins.

The A alpha mating locus is one of four multiallelic loci that govern sexual development in the basidiomycete fungus Schizophyllum commune. We have determined the nucleotide sequence encoding three A alpha mating types, A alpha 1, A alpha 3, and A alpha 4. We have found that the locus for A alpha 3 and A alpha 4 consists of two genes: Y and Z. The locus for A alpha 1 encodes only one gene, Y. The Z polypeptides encoded by different alleles exhibit 42% identity. The Y polypeptides exhibit 49-54% identity. The finding that the deduced Z and Y polypeptides have homeodomain motifs suggests that these polypeptides may be DNA-binding regulatory proteins that control the expression of developmental genes. The deduced Z polypeptide also has acidic regions that might be functionally analogous to the acidic regions in yeast GAL4 and GCN4 that activate transcription. The Y polypeptide has a serine-rich region and a basic region that shows some identity to the lysine-rich region of H1 histones.

Functional analysis of the homeodomain-related proteins of the A alpha locus of Schizophyllum commune.

DNA-mediated transformation was used to correlate function with putative genes from three alternative A alpha mating-type loci (A alpha 1, A alpha 3, and A alpha 4) of Schizophyllum commune. Each DNA was tested in at least nine haploid strains, one for each of the nine A alpha mating types found in the world-wide population of S. commune. The Y and Z genes (tentatively identified by sequence analysis elsewhere) individually activate A alpha-regulated development when transformed into any strain with a different A alpha mating type. The only exceptions are when the Y alleles of A alpha 3 or A alpha 4 (i.e., Y3 or Y4, respectively) are introduced into an A alpha 1 strain (the A alpha 1 locus encodes Y1 but lacks a Z gene). These observations indicate that A alpha-regulated development is activated by the interaction (direct or indirect) of products from different genes (e.g., Z3 and Y1) rather than from different alleles of the same gene (e.g., Y1 and Y3). Therefore, the activating interaction is of the form ZiYj where i not equal to j and i and j are the A alpha mating types from which the Z and Y polypeptides, respectively, are derived. Transformations with truncated or mutagenized genes begin to define essential regions of the genes and their products. Activity is in some cases dependent upon the particular A alpha mating type of the recipient. A working hypothesis for the activation of A alpha-regulated development is proposed.