Plasmid-encoded systems.

The DNA repair and mutagenesis phenotypes conferred by plasmid-encoded mucAB and impCAB opérons are dependent upon a host recAlexA~" genotype (Marsh and Smith, 1969; Mortelmans and Stocker, 1976; Walker, 1977; Waleh and Stocker, 1979; Upton and Pinney, 1983). Sequence analysis has confirmed the position of LexA binding sites, 5' to both pKMlOl muc genes and the TPllO imp operon, overlapping the respective promoter sequences (Perry et d., 1985; reviewed by Strike and Lodwick, 1987).

This highlights the similarity of these plasmid-encoded genes to their chromosomal equivalents (Perry et d., 1985; Kitagawa et 1985).

16.6.12.1. Relationship to chromosomal umuDC genes.

The most significant advance in the understanding of plasmid protection/mutation systems came from the demonstration that plasmids restored mutability to non-mutable umuD and umuC mutants (Kato and Shinoura, 1977; Steinbom, 1978). This was initially demonstrated for plasmids R46 and pKMlOl (Walker, 1979; Walker and Dobson, 1979), and later for TPllO, followed by a range of other plasmids (Dowden and Strike, 1982; Upton and Pinney, 1983). The simplest explanation for these observations is that plasmid- encoded genes are analogues of the chromosomal umuDC genes (section 16.6.7).

Although, the precise role of plasmid gene products remains unclear, these proteins appear to function as well as, if not more efficiently than UmuDC proteins in mediating mutagenesis (Blanco et d ., 1986). Moreover, whereas over-production of mucAB gene products greatly increases UV-induced mutagenesis in bacteria lacking excision repair, overproduction of UmuDC proteins in such cells actually reduced both repair and mutagenesis (Blanco et d., 1986; Marsh and Walker, 1987). McNally et d . (1990) later reported that the physiological effects of the mucAB and umuDC products were strongly influenced by their level of expression.

The gene products of the mucAB operon, but not umuDC operon, can promote mutagenesis in recA430 mutants (Marsh and Walker, 1987); however, mutagenesis is much more effective in recA^ strains (Blanco and Rebollo, 1981; reviewed by Walker, 1984). Since the mucAB operon was originally isolated from a broad-host-range, self- transmissible plasmid, it has been suggested that these proteins might have evolved to become less dependent on specific interactions with RecA and other host-specified proteins than their chromosomal analogues. Consistent with this hypothesis, strains containing a mucAlOUGlu^^) mutation were neither responded to increasing levels of RecA activation, nor required an activated form of RecA to function. However, they did require intact RecA to promote the spontaneous reversion of his-4 (Marsh and Walker, 1987). Such findings led these authors to propose that unactivated RecA might have a role

in MucAB-dependent spontaneous and possibly UV-induced mutagenesis.

Plasmid pKMlOl and TPl 10 gene products are both structurally and functionally related to chromosomal UmuDC proteins. Like UmuD, MucA and ImpA proteins share extensive amino acid homology with the carboxyl terminal of LexA and bacteriophage repressors (Perry et 1985; Eguchi et d., 1988; Battista et 1990). This homology also includes the Ala-25/Gly-26 peptide bond of MucA which, like the Cys-24/Gly-25 peptide bond of UmuD, corresponds to the Ala-84/Gly-85 proteolytic cleavage site of the LexA and bacteriophage repressors (Perry et d., 1985). However, whereas the resulting carboxyl terminal fragment of UmuD, UmuD', was reported by Nohmi et d . (1988) to be both necessary and sufficient for its role in mutagenesis, it was unclear whether the cleavage of MucA was actually required for its function in mutagenesis (Marsh and Walker, 1987; Shiba et d., 1990). However, Tanooka ^ d . (1991) have recently demonstrated that the 17 kDa MucA protein is cleaved into a 14 kDa fragment and an undetectable 3-kDa fragment following mutagenic treatment of a recA'*' E. coli strain. 'Thus, it appears that both chromosomal umuDC and plasmid-encoded mucAB activities are similarly controlled by RecA activation both transcriptionally, via SOS control, and post-transcriptionally (Tanooka et d., 1991).

Despite the significant homology shared by umuDC, mucAB and impAB gene products, this is insufficient to allow hybridisation between opérons (Perry et d ., 1985; reviewed by Strike and Lodwick, 1987; Nohmi e td ., 1991). However, since the molecular weights of the proteins encoded by these opérons are very similar (Perry et d ., 1985; Glazebrook e td ., 1986, Nohmi et d., 1991), and display a remarkable degree of structural and functional homology (Dutreix et d., 1989; Battista et d., 1990; Nohmi et d ., 1991), it is not unreasonable to suggest that the genes have evolved from a common ancestor. Several reports suggest that S. tvphimurium has an SOS regulatory system which closely resembles that of E. coli (MacPhee, 1973; McCann et d ., 1975). Furthermore, Tanooka et d . (1991) demonstrated a RecA-controlled mutagenic pathway in Bacillus subtilis and mucAB genes are expressed in H. influenza (Balganesh and Setlow, 1985). Thus, Sedgwick et d . (1988) proposed that the chromosomal umuDC operon was once carried on a primordial plasmid and has been assimilated by a wide range of plasmids in

exchanges promoted by insertion elements or transposons (reviewed by Strike and Lodwick, 1987; Sedgwick et 1988). Plasmid R46 is known to carry an active insertion element (IS46) capable of promoting such integration (Brown et 1984), while the ‘mutator’ plasmid R391 is permanently integrated within the chromosome (Nugent, 1981). Sedgwick et d . (1988) therefore proposed that plasmids R46 and R391 together with the putative umuDCgx found in Salmonella spp. represented the different stages between autonomous mutator plasmids and their vestigial integrated form. The umuDC operon might alternatively have originally been part of a genuine transposon which has subsequently undergone further internal rearrangement, deletion or double transpositions, and which during recent evolution has subsequently lost the ability to transpose (Sedgwick and Goodwin 1985; Balganesh and Setlow, 1985).

16.6.12.2. Spectrum of activity.

Although pKMlOl restores the mutagenic response of umu mutants to UV light and to many other mutagens (listed in McCann et d ., 1975) it does not confer resistance to the killing effects of all of them. Thus, while R46 is reported (Upton and Pinney, 1983) to increase the levels of UV resistance in a uvrA umuC mutant by more than 1000-fold, it has no effect on the resistance of this strain to mitomycin C or other mutagens that cross-link DNA strands (Attfield and Pinney, 1984).