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. 2006 Jun;72(6):4036-43.
doi: 10.1128/AEM.02774-05.

A constitutively expressed, truncated umuDC operon regulates the recA-dependent DNA damage induction of a gene in Acinetobacter baylyi strain ADP1

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A constitutively expressed, truncated umuDC operon regulates the recA-dependent DNA damage induction of a gene in Acinetobacter baylyi strain ADP1

Janelle M Hare et al. Appl Environ Microbiol. 2006 Jun.

Abstract

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 damage-inducible gene.

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Figures

FIG. 1.
FIG. 1.
(A) Amino acid alignment of the predicted UmuDAb protein from ADP1 and related UmuD and UmuD-like proteins. The first boxed amino acids form the UmuD self-cleavage site observed in E. coli. The serine and lysine residues in boxes are required for this self-cleavage. The dashed box indicates the L-R motif that is required for UmuD self-cleavage in E. coli (38). Alignment was based on the ClustalW algorithm and was performed using the Vector NTI AlignX software. VchoRumA, CfreMucA, SeloUmuD, LpneUmuD, and CvioUmuD are the gene products identified as RumA, MucA, or UmuD from Vibrio cholerae, Citrobacter freundii, Synechococcus elongatus, Legionella pneumophila strain Lens, and Chromobacterium violaceum, respectively. Amino acids present in all sequences in the alignment are indicated by asterisks. (B) Amino acid alignment of the region surrounding the L-R motif (dashed box) that is required for UmuD self-cleavage in E. coli (38).
FIG. 2.
FIG. 2.
Diagram of the umuDC operon and surrounding region in E. coli and ADP1. The beginning of the umuC gene fragment is indicated by the arrow labeled “uC-,” and the end of a umuC gene is indicated by the arrow labeled “-umuC”; the insertion sequence fragment is indicated by the arrow labeled “IS.”
FIG. 3.
FIG. 3.
umuD-ddrR intergenic region of ADP1. Italics indicate potential stem-loop-forming regions, and the directions of the arrows indicate the orientations of the repeats. Potential, predicted matches with −35 and −10 promoter consensus elements, as well as predicted ribosome binding sites (RBS), are underlined. The predicted beginning coding regions of umuD and the DNA-damaged induced ddrR gene are enclosed in boxes.
FIG. 4.
FIG. 4.
Expression of umuD in JH1 and in a RecA background (JH3) compared to expression of a DNA damage-inducible locus (strain AGC14), all in the presence of 2 μg MMC ml−1 for 4.5 h. Strain JH2 showed expression of the lacZ insertion in the orientation opposite the orientation in JH1. The error bars indicate standard deviations of the means for three independent experiments, and β-galactosidase expression is expressed in arbitrary fluorescence units.
FIG. 5.
FIG. 5.
Effects of umuD mutation in JH2 on growth in MM, as measured by spectrophotometry (A), and survival after UV-C exposure of an overnight culture in MM (B). The error bars indicate standard deviations of the means for three (A) or six (B) independent experiments. OD600, optical density at 600 nm.
FIG. 6.
FIG. 6.
Effect of umuD mutation on induction of the DNA damage-inducible gene, ddrR, in the AGC14 strain and of the benzoate-induced gene, benA, in strain ACN32 (12). The inducing conditions for AGC14 and AGC14-U were DNA damage (2 μg ml−1 MMC) for 4 h; the inducing conditions for ACN32 and ACN32-U were 3 mM sodium benzoate for 4 h. The error bars indicate standard deviations of the means for three independent experiments. β-Galactosidase is expressed in arbitrary fluorescence units.

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