In response to environmentally caused DNA damage, SOS genes are up-regulated due to RecA-mediated relief of LexA repression. In Escherichia coli, the SOS umuDC operon is required for DNA damage checkpoint functions and for replicating damaged DNA in the error-prone process called SOS mutagenesis. In the model soil bacterium Acinetobacter baylyi strain ADP1, however, the content, regulation, and function of the umuDC operon are unusual. The umuC gene is incomplete, and a remnant of an ISEhe3-like transposase has replaced the middle 57% of the umuC coding region. The umuD open reading frame is intact, but it is 1.5 times the size of other umuD genes and has an extra 5 region that lacks homology to known umuD genes. Analysis of a umuD::lacZ fusion showed that umuD was expressed at very high levels in both the absence and presence of mitomycin C and that this expression was not affected in a recA-deficient background. The umuD mutation did not affect the growth rate or survival after UV-induced DNA damage. However, the UmuD-like protein found in ADP1 (UmuDAb) was required for induction of an adjacent DNA damage-inducible gene, ddrR. The umuD mutation specifically reduced the DNA damage induction of the RecA-dependent DNA damage-inducible ddrR locus by 83% (from 12.9-fold to 2.3-fold induction), but it did not affect the 33.9-fold induction of benA, an unrelated benzoate degradation gene. These data suggest that the response of the ADP1 umuDC operon to DNA damage is unusual and that UmuDAb specifically regulates the expression of at least one DNA damageinducible gene.The best-understood model of how bacteria sense and respond to DNA damage, the SOS response, has been developed by studying Escherichia coli (29,43). In the SOS response model of E. coli, when a cell's DNA is damaged (by mitomycin C [MMC] or UV light, for example) between 0.7% (19) and 10% (26) of the genes in the cell are induced. The products of these genes perform DNA repair, replication, and cell cycle control to help the cell recover from the DNA damage (20); these products must be carefully regulated so that cell division does not occur when DNA is damaged but can resume after DNA repair and replication has concluded.The relative amount of SOS gene expression is determined primarily by transcriptional regulation. The key regulatory proteins are LexA and RecA (43). LexA represses gene expression by binding a specific sequence present in the promoters of SOS genes (the SOS box) (32). When a cell's DNA is damaged, RecA undergoes activation, which facilitates the autocleavage of LexA, and this allows the SOS genes to be expressed (6, 29). The strength with which LexA is able to bind to an SOS box also modulates the relative strength of repression and subsequent induction.UmuD and UmuC are important components of the SOS response. They are proteins whose production is induced under DNA damage conditions due to LexA-and RecA-dependent transcriptional up-regulation of the umuDC operon. Immediately after production of UmuD and UmuC, these proteins form a ...
Using the lacZ operon fusion technique, the transcriptional control of the Acinetobacter calcoaceticus r e d gene was studied. A low (approximately twofold) inductive capacity was observed for compounds that damage DNA and/or inhibit DNA replication, e.g. methyl methanesulfonate, mitomycin C, UV light and nalidixic acid. Induction of the r e d gene by DNA damage was independent of functional RecA. The presence of the r e d promoter region on a multicopy plasmid had the same effect on r e d transcription as the presence of DNA-damaging agents. Thus, r e d expression in A. calcoaceticus appears to be regulated in a novel fashion, possibly involving a non-Lea-like repressor. Regulation of the r e d gene in A. calcoaceticus appears not to be part of a regulon responsible for competence for natural transformation : in cells exhibiting extremely low transformation frequencies, the level of transcription of the r e d gene was found to be comparable to the level found in cells in the state of maximal competence.
DNA within Escherichia coli colonies carrying cloned Acinetobacter calcoaceticus genes transforms mutant A. calocaceticus cells with high efficiency. Therefore, E. coli colonies containing such cloned genes can be identified by replica plating onto a lawn of A. calcoaceticus mutant cells. Transformation of A. calcoaceticus also facilitates gap repair and thus allows recovery of specified chromosomal segments in recombinant plasmids. These procedures were used to demonstrate the clustering of A. calcoaceticus genes required for utilization of p-hydroxybenzoate. Chromosomal linkage of the bacterial genes, contained in different operons separated by about 10 kbp of DNA, may have been selected on the basis of their physiological interdependence.
A strain of Acinetobacter calcoaceticus that demonstrates unusually high competence for natural transformation by linear DNA has proven valuable for analysis of genes and gene clusters associated with aromatic catabolism. The transformation system allowed gap repair to be used to recover mutant chromosomal DNA within recombinant plasmids. The sizes of the recovered fragments, 5 and 7 kilobase pairs in length, indicate that gap repair will'be a useful procedure for isolation of wild-type and modified gene clusters from the A. calcoaceticus chromosome.
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