LexA-independent expression of a mutant mucAB operon.
pKM101 is a naturally occurring plasmid that carries mucAB, an analog of the umuDC operon, the gene products of which are required for the SOS-dependent processing of damaged DNA necessary for most mutagenesis. Genetic studies have indicated that mucAB expression is controlled by the SOS regulatory circuit, with LexA acting as a direct repressor. pGW16 is a pKM101 derivative obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis that was originally identified on the basis of its ability to cause a modest increase in spontaneous mutation rate. In this report, we show that pGW16 differs from pKM101 in being able to enhance methyl methanesulfonate mutagenesis and to confer substantial resistance to UV killing in a lexA3 host. The mutation carried by pGW16 is dominant and was localized to a 2.4-kb region of pGW16 that includes the mucAB coding region and approximately 0.6 kb of the 5'-flanking region. We determined the sequence of a 119-bp fragment containing the region upstream of mucAB and identified a single-base-pair change in that region, a G.C-to-A.T transition that alters a sequence homologous to known LexA-binding sites. DNA gel shift experiments indicate that LexA protein binds poorly to a 125-bp fragment containing this mutation, whereas a fragment containing the wild-type sequence is efficiently bound by LexA. This mutation also alters an overlapping sequence that is homologous to the -10 region of Escherichia coli promoters, moving it closer to the consensus sequence. The observation that the synthesis of pGW16-encoded mucAB proteins in maxicells is increased relative to that of pKM101-encoded mucAB proteins even in the absence of a lexA+ plasmid suggests that this mutation also increases the activity of the mucAB promoter.
McNally
K P
KP
Biology Department, Massachusetts Institute of Technology, Cambridge 02139.
Freitag
N E
NE
Walker
G C
GC
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D015345:Oligonucleotide Probes; D009876:Operon; D010957:Plasmids; D012097:Repressor Proteins / Q000235:genetics*; D012697:Serine Endopeptidases; D014466:Ultraviolet Rays; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D008741:Methyl Methanesulfonate; D015345:Oligonucleotide Probes; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein