Regulation of both gene expression and protein stability provides genetically assisted target evaluation (GATE) for microbial target validation.

The attempt to develop novel antibiotics, active against organisms resistant to current therapies, has led researchers to seek and explore new drug targets. The rapid sequencing and analysis of entire microbial genomes has identified large numbers of genes that may be sufficiently different from their human counterparts to be exploited as targets for antimicrobial treatment. As a first step, the importance of the various putative targets for microbial growth and survival must be assessed. Emerging validation technologies are becoming increasingly sophisticated and, in certain cases, allow prioritisation of the best targets. In this paper, genetically assisted target evaluation (GATE) is introduced as a versatile target validation technology. GATE concomitantly manipulates both synthesis and stability of the targeted protein using copper ions as an effector. This technology allows rapid quantitation of the lethal consequences of inactivation of targeted gene products in Saccharomyces cerevisiae. Additional tools can then be applied to extend these results into pathogenic organisms, such as Candida albicans.

Human natural killer cells account for non-MHC class I-restricted cytolysis of porcine cells.

Despite similarities in the cellular response to allografts and xenografts, some aspects of the xenogeneic immune response are unique. We find that both freshly isolated and primed human peripheral blood lymphocytes manifest MHC unrestricted cytolysis of porcine cells. While natural antibody-mediated mechanisms account for variable levels of cytotoxicity, reproducible killing in the absence of human serum is attributable to natural killer (NK) cells. This was shown by cold target inhibition with K562 cells, increased antiporcine cytotoxicity after enrichment for CD56+ cells, and significantly reduced lytic activity after depletion of CD56+ cells. Increased anti-porcine cytotoxicity after mixed culture of human and porcine cells was due to differentiation of NK cells to lymphokine-activated killer (LAK) cells and was IL-2 dependent. After depletion of NK cells, T-cell-mediated anti-porcine cytotoxicity could also be demonstrated. We conclude that the human anti-porcine cellular cytotoxic response is due to multiple cell types that include T cells in addition to NK and LAK cells.

The groE gene products of Escherichia coli are dispensable for mucA+B(+)-dependent UV mutagenesis.

UV mutagenesis in Escherichia coli requires the groES+EL+ chaperonins as well as the umuD+C+ SOS-regulated genes. GroES and GroEL appear to be required to stabilize UmuC. The mucA+B+ genes, which are encoded on a broad host range plasmid, are functionally analogous and structurally similar to the umuD+C+ genes of E. coli. While these gene pairs are quite similar, differences have been reported in the functioning of these gene products. We tested whether mucA+B+ function requires the groE+ gene products as well. We show that mucA+B(+)-induced UV mutagenesis, UV resistance, phage reactivation and cold sensitivity do not require the groE+ heat shock genes. These findings suggest that the requirement of UmuC for groES+EL+ function is not shared by its analog, MucB.

Coexpression of UmuD' with UmuC suppresses the UV mutagenesis deficiency of groE mutants.

The GroE proteins of Escherichia coli are heat shock proteins which have also been shown to be molecular chaperone proteins. Our previous work has shown that the GroE proteins of E. coli are required for UV mutagenesis. This process requires the umuDC genes which are regulated by the SOS regulon. As part of the UV mutagenesis pathway, the product of the umuD gene, UmuD, is posttranslationally cleaved to yield the active form, UmuD'. In order to investigate what role the groE gene products play in UV mutagenesis, we measured UV mutagenesis in groE+ and groE strains which were expressing either the umuDC or umuD'C genes. We found that expression of umuD' instead of umuD will suppress the nonmutability conferred by the groE mutations. However, cleavage of UmuD to UmuD' is unaffected by mutations at the groE locus. Instead we found that the presence of UmuD' increased the stability of UmuC in groE strains. In addition, we obtained evidence which indicates that GroEL interacts directly with UmuC.

Genetic analyses of cellular functions required for UV mutagenesis in Escherichia coli.

In Escherichia coli, most UV and chemical mutagenesis is not a passive process and requires the participation of the umuD and umuC gene products. However, the molecular mechanism of UV mutagenesis is not yet understood and the roles of the UmuD and UmuC proteins have not been elucidated. The umuDC operon is induced by UV irradiation and regulated as part of the SOS response. Genetic evidence now indicates that RecA-mediated cleavage activates UmuD for its role in mutagenesis. The COOH-terminal fragment of UmuD is both necessary and sufficient for this role. The RecA protein appears to have a third role in UV mutagenesis besides mediating the cleavage of LexA and UmuD at the time of SOS induction. In addition, we have obtained evidence which indicates that the GroEL and GroES proteins also play a role in UV mutagenesis. Similarities of the amino acid sequence of UmuD to the sequence of gene 45 protein of bacteriophage T4 and of the sequence of UmuC to those of the gene 44 and gene 62 proteins suggest possible roles for UmuD and UmuC in mutagenesis that are supported by preliminary evidence.

groE mutants of Escherichia coli are defective in umuDC-dependent UV mutagenesis.

Overexpression of the SOS-inducible umuDC operon of Escherichia coli results in the inability of these cells to grow at 30 degrees C. Mutations in several heat shock genes suppress this cold sensitivity. Suppression of umuD+C+-dependent cold sensitivity appears to occur by two different mechanisms. We show that mutations in lon and dnaK heat shock genes suppress cold sensitivity in a lexA-dependent manner. In contrast, mutations in groES, groEL, and rpoH heat shock genes suppress cold sensitivity regardless of the transcriptional regulation of the umuDC genes. We have also found that mutations in groES and groEL genes are defective in umuDC-dependent UV mutagenesis. This defect can be suppressed by increased expression of the umuDC operon. The mechanism by which groE mutations affect umuDC gene product function may be related to the stability of the UmuC protein, since the half-life of this protein is shortened because of mutations at the groE locus.

Amino acid similarities to other proteins offer insights into roles of UmuD and UmuC in mutagenesis.

The products of the umuD and umuC genes are required for most uv and chemical mutagenesis in Escherichia coli. The genes are organized in an operon that is repressed by LexA and regulated as part of the SOS response. The umuD protein shares homology with the carboxyl-terminal domain of LexA. Genetic evidence now indicates that RecA-mediated cleavage activates UmuD for its role in mutagenesis. The COOH-terminal fragment of UmuD is both necessary and sufficient for this role. Similarities of UmuD to gene 45 protein of bacteriophage T4 and of UmuC to gene 44 protein and gene 62 protein suggest possible roles for UmuD and UmuC in mutagenesis that are supported by preliminary evidence.

Mutations in the lac P2 promoter.

We used site-directed mutagenesis to generate mutations in the -10 region of the lac P2 promoter. The mutations were crossed onto lambda bacteriophage carrying the lac regulatory elements and an intact lacZ gene, and the effects of the various mutations were determined in vivo and in vitro. Two of four mutations had effects on the start point of the P2-directed transcript and had very little effect on lac expression. Another mutation, which abolishes P2 promoter activity in vitro, also had very little effect on lac expression in vivo. We suggest that the P2 promoter plays little or no role in the activation of the P1 promoter by catabolite activator protein in complex with cyclic AMP.

Promoter-probe plasmid for Bacillus subtilis.

We have constructed a promoter-probe expression vector for Bacillus subtilis. This plasmid, pCED6, can be used to fuse various DNA sequences to the structural gene of Escherichia coli beta-galactosidase, permitting analysis of the promoter activity of such sequences. pCED6 replicates and confers drug resistances in both E. coli and B. subtilis.