Previous genetic evidence suggested that RecA was required in SOS mutagenesis for its regulatory role and perhaps some other nonregulatory role (Mount, 1977; Blanco et al., 1982). I undertook a genetic study which confirmed the above studies and provided further evidence that RecA protein appeared to have a dual "role in mutagenesis; first, the cleavage of LexA repressor for the derepression of specific SOS genes and second, one or more additional role(s). For these studies a new phage mutagenesis assay was developed which allows rapid scoring of SOS mutagenesis in a large number of host mutants. I next conducted a genetic analysis to determine if the newly defined RecA mutagenesis function was separable by mutation from the numerous other phenotypes which are known to be influenced by RecA protein. From the study of recA mutants it appears that the RecA mutagenesis function(s) is genetically separable from the following RecA phenotypes: LexA cleavage, lambda cI repressor cleavage, UV resistance and homologous recombination. In addition, I discovered that the LexA cleavage function and lambda cI cleavage function is also separable. I also studied in some detail the novel genetic properties that I uncovered for recA432 mutant strains. recA432 was defined as a mutagenesis defective allele (Kato and Shinoura, 1977). LexA cleavage in recA432 cells was more easily induced that in recA⁺ cells, causing lethal filamentation of these mutant cells even at very low UV doses. I concluded that the basis for the Mut⁻ phenotype was this strain's propensity to lethally filament, which complicated the detection of mutant cells. In another set of experiments, I examined the regulatory requirements for SOS mutagenesis and Weigle phage-reactivation; I wanted to determine which SOS operons must be derepressed for this process. lexA(Ind⁻) mutant cells are defective in mutagenesis because they cannot derepress specific SOS genes required in this process. I found that the selective derepression of umuDC was sufficient to restore mutagenesis to these lexA(Ind⁻) mutants; however, derepression of umuDC and recA was required for phage reactivation.

The LexA protein of E. coli is a repressor of at least 20 genes in the SOS regulon, and by this function plays a major role in regulating the SOS response. Two different genetic approaches have been taken to define the DNA binding domain of LexA repressor. First, several mutant repressors which are defective in DNA binding have been isolated. The mutations generating these repressors were dominant to lexA+, indicating that the mutant proteins can act in trans to interfere with binding of normal repressor to an operator sequence. The repressors may be defective due to elimination or disruption of contacts made between side chain(s) within the protein and the DNA helix but dominant because they can still interact with other monomers of LexA protein. In a second experiment to define the DNA binding domain of LexA protein, a novel genetic selection has been used to isolate DNA binding specificity mutants. The recA operator (CTG TATGA.GCATA CAG), a known lexA binding site, has been altered in a symmetric fashion. This choice was based on the assumption that the dyad symmetry of the operator indicates at least two repressor monomers bind to each operator such that each monomer recognizes one half of the operator. A class of mutant repressors which restored binding to this altered operator but had little affinity for the wild-type recA operator was isolated. This type of mutation allowed the identification of amino acids in the repressor which are likely to make specific contacts with base-pair(s) in the DNA binding site. By examining the effects of a series of amino acid substitutions on repressor specificity, it was possible to show that a glutamic acid residue at position 45 (E45) contacts the first and last base-pair of the consensus recA operator (CTG TATGA.GCATA CAG). Both negative dominant and operator recognition mutations were located in a small region that was previously identified to specify a helix-turn-helix motif based on sequence similarity to other repressors. These studies therefore suggest that LexA protein may bind to DNA by a helix-turn-helix motif similar to these repressors.

This report describes the development and use of an expression vector system based on the single-stranded DNA bacteriophage M13. A derivative of M13mp8, designated M13mp8/P, was prepared in which the promoter and N-terminal codons of bacterial genes may be fused to a portion of β-galactosidase, resulting in an easily scorable phenotype. Because transcription from the inserted promoter remains responsive to the host regulatory system, it is simple to screen mutagenized phage for isolates with aberrant regulatory phenotypes, and to determine the mutational changes by dideoxy sequence analysis. The feasibility of this method was demonstrated by identification of a large number of mutations in the regulatory regions of two genes, recA and lexA. Base substitutions that altered the phenotype of recombinant phage were identified both in the single LexA repressor binding site of recA and in the two binding sites of lexA, as well as in other sites that likely affect translational efficiency. My results suggest that this method will be generally useful for mutational analysis of transcriptional and translational regulatory elements. The mutants that were isolated by the above approach were used to investigate the specificity of LexA protein binding by quantifying the repressibility of a several mutant recA and lexA operator/promoter regions fused to the E. coli galactokinase (galK) gene. The results of this analysis indicated that two sets of four nucleotides (terminal nucleotide contacts), one set at each extreme end of the operator, are most critical for repressor binding. In addition, our results indicate that the repressor-operator interaction is symmetric in nature, in that mutations at symmetrically equivalent positions in the recA operator had comparable effects on repressibility. The inferred symmetry of the interaction justified the reevaluation of the consensus sequence by half-site comparison, which yielded the half-site consensus: (5') CTGTATAT. Although the first four positions of this half-site sequence have the greatest effect on LexA repressor binding, the last four are well conserved among binding sites and appear to modulate repressor affinity. The role of the terminal nucleotide contacts and the mechanism by which the internal sequences affect repressor binding is discussed.

The number of foodborne disease outbreaks related to fresh produce has increased in recent years. This has coincided with a growing public demand for minimally processed fruits and vegetables. Effective produce sanitizers are therefore needed that are at least as effective as chlorine, currently the most commonly used sanitizer. Natural antimicrobials from plant extracts and essential oils are a possible alternative. These are highly effective and may also be used in situations in which chlorine is not advantageous; for instance, in situations in which chlorine has limited efficacy or because of concerns over the production of harmful by-products resulting from chlorine use. Plant derived essential oils have been shown to be antibacterial, antiviral, and antifungal. In this study we examined the use of natural antimicrobials from plant extracts and essential oils as possible alternative sanitizers. We examined these antimicrobials for their efficacy against Escherichia coli. In addition, since many of these natural compounds are believed to be membrane active, silver ions were added to some of the tests to assess the potential for synergy between the antimicrobials. Silver ions, although slow-acting on their own, often exhibit a synergistic antimicrobial effect when combined with other membrane active antimicrobials such as oxidizing agents. These studies reveal that plant derived antimicrobials are effective sanitizers with the potential to replace commonly used chlorine

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