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Applied and Environmental Microbiology, June 2006, p. 4036-4043, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02774-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

A Constitutively Expressed, Truncated umuDC Operon Regulates the recA-Dependent DNA Damage Induction of a Gene in Acinetobacter baylyi Strain ADP1

Janelle M. Hare,1* Sara N. Perkins,1 and Leslie A. Gregg-Jolly2

Department of Biological and Environmental Sciences, Morehead State University, Morehead, Kentucky 40351,1 Department of Biology, Grinnell College, Grinnell, Iowa 501122

Received 23 November 2005/ Accepted 28 February 2006


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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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INTRODUCTION
 
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 UmuD2C complex, which acts as a checkpoint inhibitor of cell division until repair can address the original inducing DNA damage signal (34). However, the subsequent RecA-mediated self-cleavage of the N-terminal 24 amino acids from UmuD within approximately 25 min (34) forms UmuD' (33). UmuD' binds to UmuC to form the (UmuD')2C complex (called DNA polymerase V), which carries out error-prone, translesion replication of damaged DNA (40) in the process called SOS mutagenesis.

Although the E. coli SOS model is the most highly developed model, research with other bacteria has revealed a variety of differences in the ways that cells respond to DNA damage. There are variations in the specific sets of genes induced (7, 8), as well as a lack of a requirement for LexA for regulation of either recA in Geobacter sulfurreducens (25) and Deinococcus radiodurans (5) or gene induction after DNA damage (7, 8). The number of lexA genes present in bacteria is variable, ranging from zero in Rickettsia prowazekii (1), Borrelia burgdorferi, Chlamydia pneumoniae, and Helicobacter pylori (9) to two in, for example, Geobacter sulfurreducens (25) and Xanthomonas axonopodis pv. citri (47). The specific sequences of SOS boxes also vary between and within bacterial classes (7, 8, 9, 15, 18, 25, 46). The SOS box sequences include TACTG(TA)5CAGTA for E. coli (44), TTAG(N6)TACTA for Xylella fastidiosa (9), CGAACRNRYGTTCYC for Bacillus subtilis (11, 46), and GGTT(N2)C(N4)G(N3)ACC for the deltaproteobacterium G. sulfurreducens (25). Finally, in Leptospira interrogans, a LexA binds to an SOS box in the recA promoter but not in its own lexA promoter (13).

Because studying diverse organisms yields a more complete picture of the range of ways in which organisms can respond to DNA damage, the goal of this study was to increase our understanding of DNA damage responses by characterizing the umuDC operon and its regulation and function in the bacterium Acinetobacter baylyi strain ADP1. (The ADP1 strain of Acinetobacter was recently renamed A. baylyi strain ADP1 [42].) ADP1 is a gram-negative, nonpathogenic, naturally transformation-competent soil bacterium belonging to the class Gammaproteobacteria, and its genome has recently been sequenced (2).

ADP1 has some of the characteristics of a typical SOS response. At least one genetic locus (ddrR) that is induced in response to DNA damage in ADP1 requires RecA for induction (G. Whitworth and L. A. Gregg-Jolly, Abstr. 100th Meet. Am. Soc. Microbiol., abstr. H-60, 2000). However, ADP1 also exhibits some unique features in response to DNA damage. First, ADP1 does not respond to DNA damage with SOS mutagenesis (4). Furthermore, although transcription of recA is induced in response to DNA damage in ADP1, as it is in E. coli, this induction does not require the RecA protein (36). Finally, the ADP1 recA promoter does not contain a known SOS box (21). In this study we determined additional unusual features of the SOS response in ADP1, including a constitutively expressed, unusual umuDC operon that is not regulated by DNA damage or recA and does not contain a SOS box in its promoter region but does specifically regulate a DNA damage-inducible gene.


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MATERIALS AND METHODS
 
Cells, plasmids, and growth conditions.
All ADP1 derivatives were grown in minimal medium supplemented with succinate (0.01 M) as a carbon source (MM). E. coli was maintained on Luria broth. Streptomycin was used at a concentration of 10 µg ml–1, ampicillin was used at a concentration of 50 µg ml–1, and kanamycin was used at a concentration of 25 µg ml–1 for E. coli and at a concentration of 10 µg ml–1 for ADP1.

Construction of umuD::lacZ reporter, mutant umuD, and mutant recA strains.
pUC19-based plasmids used to construct umuD::lacZ reporter, mutant umuD, and mutant recA strains are described in Table 1. pUC19 does not replicate in ADP1 and so was used as a suicide vector. Allelic exchange of the umuD::lacZ-Kmr reporter gene cassette was performed by transforming naturally competent ADP1 cells (21) with an XbaI-linearized plasmid (either pJH1.1 or pJH1.2) and selecting for growth on kanamycin-containing agar plates.


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  		TABLE 1. Plasmids and strains used in this study

umuD derivatives of both AGC14 and ACN32 were constructed by transforming these strains with the XbaI-linearized plasmid pJH1US and selecting for streptomycin- and kanamycin-resistant colonies (Table 1). PCR experiments confirmed both the absence of the wild-type umuD allele and the presence of the mutant umuD allele in JH1, JH2, ACN32-U, and AGC14-U.

A recA mutant strain of JH1, designated JH3, was constructed by transforming JH1 with the HindIII-linearized plasmid pGW1 and selecting for streptomycin-resistant colonies. PCR experiments confirmed both the absence of the wild-type recA allele and the presence of the mutant recA allele in JH3. The RecA– phenotype of JH3 was confirmed by UV and MMC sensitivity assays (data not shown), which showed that there was increased sensitivity to these conditions.

In all cases, recombinants were screened for ampicillin sensitivity to confirm loss of the donor plasmid.

ß-Galactosidase assays.
Overnight cultures of cells grown in MM at 37°C were diluted 1:10 in MM, and duplicate samples were regrown with shaking at 37°C. After 4 h, the inducing agent, either 2 µg MMC ml–1 for AGC14 and AGC14-U or 3 mM sodium benzoate for ACN32 and ACN32-U, was added to one sample of each strain to induce gene expression. At various times, samples were removed and subjected to a fluorimetric ß-galactosidase assay (FluorAce; Bio-Rad). Fluorescence was measured with a Turner Designs TD-700 fluorometer after fluorometer calibration with 4-methylumbelliferone and was expressed in arbitrary fluorescence units.

umuD phenotype analyses.
Cells were grown overnight in MM before they were diluted 1:50 in MM and regrown with shaking at 37°C to assess growth over time by spectrophotometry. Survival after exposure to UV light was analyzed with overnight cultures grown at 37°C in MM. The cells were diluted in MM and plated onto either L agar or L agar with exposure to 100 or 150 J cm–2 UV-C light in a Stratagene UV Stratalinker 1800 in the dark. Plates were incubated for 18 h at 37°C in the dark before colonies were counted and the numbers of CFU ml–1 were calculated. Survival was calculated by dividing the number of CFU ml–1 in the presence of UV exposure by the number of CFU ml–1 in the absence of UV exposure. Cell filamentation analysis was performed by growing cells for 18 h at 37°C in the presence or absence of 2 µg ml–1 MMC, heat fixing them, staining them with crystal violet, and photographing them under bright-field conditions at a magnification of x1,000.


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RESULTS
 
umuDC operon of ADP1 is interrupted by an insertion sequence fragment.
The ddrR locus in ADP1 encodes an 81-amino-acid (aa) protein that exhibits no homology to any other protein in the database but is induced by DNA damage in a RecA-dependent manner (Whitworth and Gregg-Jolly, Abstr. 100th Meet. Am. Soc. Microbiol.). In our investigation of this locus, DNA regions adjacent to ddrR were sequenced, and an operon with homology to umuDC was identified (Fig. 1A). The sequence that we identified was identical to the sequence that was recently reported by Barbe et al. (2), and the umuD open reading frame (ORF) was annotated as ACAID2729 (umuD) in the study of Barbe et al. (2). However, the features of umuD and the truncation of umuC that we describe here were not identified in the genome annotation for ADP1 (2).


Figure 1
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  		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).

The reading frame of the umuDC genes was oriented away from ddrR (Fig. 2). Typically, a shorter umuD gene precedes a umuC gene (encoding 140 and 423 amino acids, respectively, in E. coli [29]). The umuDC operon of ADP1 that was sequenced had two differences compared with other umuDC operons. The first unusual feature was that the umuD gene product was 1.5 times larger than previously studied UmuD proteins (see below), and the second difference was that there was no homology with UmuC after the first 39 amino acids encoded by umuC. These 39 amino acids exhibit ~75 to 85% similarity (62 to 74% identity) to the amino-terminal 35 amino acids of UmuC proteins from a wide range of bacteria, including Synechococcus elongatus, Porphyromonas gingivalis, Legionella pneumophila, Pseudomonas putida, Salmonella enterica, E. coli, and Vibrio cholerae. Due to the unique characteristics of the ADP1 umuDC operon, below we refer to the UmuD-like protein present in ADP1 as UmuDAb and to the truncated umuC gene product as UmuC*.


Figure 2
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  		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."

Examination of the ADP1 genome sequence reported by Barbe et al. (2) indicated that a 348-bp fragment of umuC is located 5.9 kbp downstream of umuDC. This fragment, which is in the same orientation as the umuC fragment present adjacent to umuD, encodes the carboxy-terminal 114 aa of UmuC, but the level of identity to the UmuC sequence of E. coli (28%) is much lower than the level of identity for the amino-terminal 39 aa. The middle ~270 aa of UmuC are not encoded in this region, and no other sequences homologous to umuC are present elsewhere in the genome.

Directly adjacent to the region encoding the amino-terminal 39 amino acids of umuC is the 3' end of a putative transposase gene in the opposite orientation (Fig. 2). The product of this very small, 72-bp region exhibits 87% homology over 24 aa with the product of an unannotated ORF located downstream of the A. baylyi NCIMB9871 cyclohexanol metabolic gene cluster (24). tBLASTx analysis of the unannotated ORF showed that it is the second ORF in a two-ORF insertion sequence that exhibits homology to ISEhe3. Complete or partial sequences of ISEhe3 are present in Erwinia herbicola (22), E. coli (41), Shigella flexneri (45), Wolbachia (37), Shewanella oneidensis (23), and many other gram-negative bacteria. No other significant match with the 72-bp sequence adjacent to the umuC fragment was found at either the nucleotide or amino acid level.

The umuDC operon of ADP1 is not located in an identified bacteriophage (2) or a broad-host-range R plasmid, as some umuDC operons or umuD or umuC genes are (3, 10, 14, 28, 31).

umuD gene product has an extra amino-terminal region.
In contrast to the truncated umuC gene, umuD in ADP1 was about 1.5 times the size of the umuD genes found in other bacteria; the umuD gene encoded 203 aa in ADP1 (23.0 kDa), compared with 140 aa in E. coli (15.0 kDa) (Fig. 1A). The predicted gene product (UmuDAb) exhibited 45% identity (61% similarity) to the entire UmuD protein from E. coli and 37% identity (57% similarity) to 109 aa in the carboxy-terminal domain of E. coli LexA.

Compared to the E. coli umuD product, UmuDAb contains an additional 59 amino-terminal amino acids (Fig. 1A). This region exhibits no similarity to any previously described or studied UmuD protein, nor does it contain any domain that could be identified by an NCBI conserved domain search (30). Predicted gene products identified as UmuD or DNA polymerase V that were similar sizes were encoded in the sequenced genomes of the betaproteobacteria L. pneumophila (43% identity over 192 aa) and Chromobacterium violaceum (35% identity over 197 aa) and the cyanobacterium S. elongatus (38% identity over 190 aa) (Fig. 1A). The levels of identity for the extra amino-terminal region were the same or slightly greater than the levels of identity for the entire protein, and the greatest similarity occurred in aa 9 to 43 (Fig. 1A). This amino-terminal region resembled (51 to 59% identity over 32 aa) 88-aa hypothetical proteins encoded by Shewanella frigidimarina NCIMB 400 and the Shewanella oneidensis MR-1 megaplasmid.

UmuDAb had several amino acids that are required in E. coli for RecA-facilitated UmuD self-cleavage. The cysteine-24/glycine-25 cleavage site (alanine-glycine in most UmuD homologs and RumA, ImpA, and MucA proteins), serine-60, which carries out the nucleophilic attack at the cysteine-glycine site, and lysine-97, which assists in the nucleophilic attack, were all conserved in UmuDAb (Fig. 1A). However, two amino acids required for efficient UmuD self-cleavage in E. coli, leucine-101 and arginine-102 (38), were replaced by isoleucine (at position 163, the equivalent position in ADP1) and aspartic acid (position 164) in UmuDAb. These Ile and Asp residues were more similar to transcriptional repressors belonging to the LexA-like family (Fig. 1B) (38). Furthermore, an additional five amino acids (compared to all other UmuD-like gene products analyzed) were located adjacent to Asp-164.

umuDC promoter lacks an SOS box.
No sequence similar to known SOS boxes was found in the 143-bp umuD-ddrR intergenic region (which presumably contains the promoters for these two genes); however, we did observe a series of repeats in this region (Fig. 3). Sequences 1 and 3 were largely the same, and either one could pair with repeat sequence 2 to form one of two possible inverted repeats. The specific DNA sequences of repeats 1 and 2 formed the inverted repeat AACTTGAA(N11)TTCAAGTT, and repeats 2 and 3 formed the inverted repeat TCAAGTT(N10)AACTTGA. Both of these inverted repeats were different from the SOS boxes of other bacterial species, but the inverted repeat structure and spacing of the palindromic sequences resembled the structure and spacing of a typical SOS box.


Figure 3
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  		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.

umuD is not regulated by DNA damage or RecA.
SOS genes are transcriptionally up-regulated in response to DNA damage. In E. coli, the umuDC operon is induced approximately 15- to 30-fold, depending on temperature (20). Because the umuDC promoter region did not appear to contain a SOS box, we investigated whether expression of the ADP1 umuD gene varied after DNA damage. We inserted a promoterless lacZ reporter gene into umuD to measure its expression. Mitomycin C (2 µg ml–1) was used as a DNA-damaging agent. Cells induced for 4.5 h exhibited uniformly high levels of umuD expression that were even higher (~twofold) than the expression of the ADP1 DNA damage-inducible gene ddrR after DNA damage induction (Fig. 4).


Figure 4
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  		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 ß-galactosidase expression is expressed in arbitrary fluorescence units.

Another characteristic of SOS gene induction is a requirement for RecA. However, unlike the expression in E. coli, the expression of umuD in JH1 was not affected by the absence of RecA, as it was just as high in the otherwise isogenic recA mutant strain JH3 (Fig. 4).

Absence of UmuD in ADP1 does not affect survival in response to DNA damage or the growth rate under normal conditions.
Various aspects of the umuD phenotype were investigated, and no significant morphological or growth-related effects of the umuD mutation were observed. The growth of umuD insertion mutant strains JH1 and JH2 over time in liquid MM was indistinguishable from the growth of ADP1 (Fig. 5A), and the survival of JH2 was the same as that of ADP1 on agar medium after exposure to 100 and 150 J cm–2 of UV light (Fig. 5B) (P = 0.93 and P = 0.73, as determined by t tests assuming equal variances, respectively). Finally, MMC induced cell filamentation in both ADP1 and umuD cells (data not shown). Compared to JH2 and ADP1, JH1 formed smaller colonies on MM agar plates and sometimes appeared to have slightly greater sensitivity to UV (data not shown), possibly due to toxicity resulting from overproduction of ß-galactosidase.


Figure 5
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  		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.

umuD specifically regulates transcriptional induction of a DNA damage-inducible gene.
The Leu-101 and Arg-102 residues required for efficient UmuD self-cleavage (38) were not present in UmuDAb, whereas this region of UmuDAb was more similar to the LexA-like bacteriophage transcriptional repressors (Fig. 1B). We thus hypothesized that UmuDAb was required for regulation of the adjacent gene, ddrR.

This hypothesis was supported by the results of fluorimetric ß-galactosidase experiments (Fig. 6). However, the umuD insertion mutation, instead of derepressing gene expression in a LexA-like manner, reduced the DNA damage-inducible, recA-dependent expression of the ddrR locus by 83% (from 12.9-fold to 2.3-fold). The ~34-fold induction of an unrelated, benzoate-inducible gene (benA) involved in benzoate degradation in strain ACN32 (12) was not affected in a umuD mutant. The effect of the umuD insertion mutation on ddrR induction was statistically significant, as determined by a t test of paired samples for means (P = 0.02). Complementation of AGC14-U with the wild-type umuD allele present in the chromosome resulted in ddrR induction levels comparable to those seen in AGC14.


Figure 6
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  		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. ß-Galactosidase is expressed in arbitrary fluorescence units.


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DISCUSSION
 
Because ADP1 possesses an operon that includes a umuD-like gene preceding two umuC gene fragments, we refer to this operon as umuDC. However, there are significant differences between this operon and the umuDC operons present in E. coli and other bacteria. The two umuC fragments comprise only 43% of the expected length of umuC and are separated by ~6 kbp, perhaps due to a chromosomal rearrangement mediated by the ISEhe3-like fragment located between the two umuC fragments. The absence of a complete umuC gene from the ADP1 genome is the most obvious, and a sufficient, cause for the previously observed lack of SOS mutagenesis in ADP1 (4). Presumably, the UmuD2C-mediated cell cycle checkpoint system, which functions in the short term after DNA damage (up to ~30 min) to regulate DNA synthesis (34), does not function in ADP1 either.

Typically, a short umuD gene is located just before a long umuC gene (29). In ADP1, however, the umuD gene itself is longer than other bacterial umuD genes, with an extra region at its 5' end. All five additional species of Acinetobacter for which a umuD homolog has been sequenced also contain a nearly identical, extralong, umuD-like gene, suggesting that this umuD allele is highly conserved in Acinetobacter (C. Lin and J. Hare, unpublished data). Three other bacterial species also encode UmuDAb-like proteins: L. pneumophila (strains Paris, Philadelphia 1, and Lens), S. elongatus, and C. violaceum (Fig. 1). In L. pneumophila and S. elongatus, the umuD homolog appears to be in an operon with an intact umuC homolog and is annotated as encoding an "SOS response transcriptional regulator" or the "putative SOS mutagenesis protein UmuD." In C. violaceum, however, the umuD homolog is ~300 kbp from a monocistronic umuC homolog. The presence of both umuD and umuC in these species suggests that in these organisms, the UmuDAb homologs have a canonical UmuD role, unlike the role in ADP1, which has no intact umuC. Although these UmuD homologs lack the E. coli L-R motif, the conservation of all other amino acid residues required for cleavage suggests that the L-R motif, while required in E. coli, may not be required in other classes or phyla.

It is interesting that the only bacteria encoding UmuDAb-like proteins are not close relatives of the gammaproteobacterium ADP1; L. pneumophila and C. violaceum are betaproteobacteria, while S. elongatus belongs to a completely different phylum (Cyanobacteria). The umuDC region in ADP1 was not found to be in a bacteriophage or other horizontally acquired region of DNA (2).

A unique feature of UmuDAb is the five additional amino acids that are present immediately after the typical location of the L-R motif. The effect of these extra amino acids at this location may be minimal, as this region is located in a solvent-exposed loop according to the crystal structure of the E. coli UmuD' homodimer (17, 35).

The umuD gene of ADP1 is expressed very highly (the expression is twofold higher than the induced expression of the DNA damage-inducible locus ddrR) (Fig. 4), although this expression, unlike that in other species, is not induced in response to DNA damage, nor is it reduced in a recA-deficient background. These data, together with the high level of conservation of UmuC* and the N terminus of E. coli UmuC, could imply that there is continuing selection for UmuDAb and UmuC* and their constitutive expression. It is possible that UmuC* interacts with UmuDAb to contribute to the regulatory phenotype observed in ADP1.

Alternately, the loss of umuC and the subsequent loss of SOS mutagenesis function may simply have released the umuDC operon from selection for a functional SOS box to mediate appropriate up-regulation upon DNA damage, resulting in the constitutive expression and lack of dependence on recA, and presumably lexA, that we observed. Our data indicate that UmuDAb is not essential, as there was no reduction in the growth of knockout strains (Fig. 5A).

Instead of playing the expected role in SOS mutagenesis and as a cell cycle checkpoint, UmuDAb is required in ADP1 for full induction of ddrR, an adjacent DNA damage-inducible locus (Fig. 6). Loss of UmuDAb does not seem to affect global gene expression or transcriptional regulation since expression and regulation of benA were not affected in a umuDC background (Fig. 6). This was manifested not as derepression of ddrR expression in the AGC14-U umuD strain but as a reduction in DNA damage induction to a level that was approximately 20% of the level seen in the AGC14 umuD+ strain. The persistence of a small amount of ddrR induction in the absence of UmuDAb suggests that other mechanisms may also contribute to ddrR regulation. Because the umuD insertion mutation may be polar on the umuC fragment, we cannot conclude that the effects on ddrR regulation are solely due to a lack of UmuDAb.

It is thus tempting to speculate that UmuDAb plays a role that is similar to but opposite from the role of LexA, with SOS gene induction requiring UmuDAb for activation, as the action of UmuDAb is more consistent with a transcriptional activator than with a repressor. The conservation in UmuDAb of amino acids needed for cleavage suggests a model in which (UmuDAb)2 is cleaved after DNA damage, yielding (UmuD'Ab)2. The activated (UmuD'Ab)2 could positively affect ddrR transcription, working either alone or with UmuC*. Considering the UmuD2 structure proposed by Sutton et al. (39), the extra N-terminal residues of UmuDAb may not interfere with cleavage. The "extra" amino-terminal part of UmuDAb could be required for this regulatory function. However, while UmuDAb possesses the typical UmuD protein motifs, it lacks any obvious motif for DNA binding, such as the helix-turn-helix motif used by LexA. The significance of the DNA damage-sensitive regulatory role of UmuDAb is further highlighted by the absence of a LexA homologue in ADP1.

There are three types of UmuD-like proteins: the "true" UmuD proteins that function in concert with UmuC to regulate the cell cycle and carry out translesion DNA synthesis; the UmuD-like transcriptional repressors, such as LexA and bacteriophage-encoded repressors; and the signal peptidases (38). UmuDAb shares features with the "true" UmuD proteins, as well as with the LexA-like repressors, as demonstrated by its unusual, LexA-like size and function. These features, together with other previously observed features of the DNA damage response of ADP1, indicate that an atypical suite of DNA damage response mechanisms operate in ADP1. Previous work indicated that in ADP1, although recA is induced by DNA damage, this induction is not RecA dependent, nor is there a typical SOS box in its promoter (21).

This work also revealed the lack of any known SOS box in the promoters of either umuDC or the DNA damage-inducible gene ddrR. The lack of a known SOS box in these promoters is not without precedent, as many bacteria possess their own specific SOS box sequences, and it could simply mean that an alternate SOS box, such as the inverted repeats in the umuD-ddrR intergenic region (Fig. 3), is present in ADP1. Proposal of a functional SOS box, however, invokes an alternate LexA-like repressor protein, as no LexA homolog has been identified in the genome of ADP1. The gene product most similar to LexA in ADP1 is UmuDAb described in this paper; querying the ADP1 genome with E. coli LexA using the BLAST algorithm yielded UmuDAb (data not shown). Second, although a possible SOS box is present in the promoter of ddrR, no similar sequence is in the promoter region of ADP1 recA. However, the ddrR locus requires RecA for induction upon DNA damage (due to either MMC or UV light) (Whitworth and Gregg-Jolly, Abstr. 100th Meet. Am. Soc. Microbiol.), whereas recA does not require RecA for induction under the same conditions (36). Thus, in ADP1 there may be two separate mechanisms of gene induction after DNA damage: one for RecA-dependent genes (exemplified by ddrR) and one for RecA-independent genes (e.g., recA). The capacity of ADP1 and its derivatives to undergo natural transformation, coupled with the availability of constructs (pJH1.1, pJH1.2, and pJH1US) as a result of this study, should facilitate work to determine whether UmuDAb plays a role in the regulation of other DNA damage-inducible genes in ADP1.

The position of short repetitive sequences near both of the putative umuD and ddrR promoter consensus elements suggests an alternative to LexA-mediated SOS gene expression: a posttranscriptional mechanism in which secondary structure changes (e.g., a bent DNA stem-loop structure) affect the translation of either (or both) mRNA transcripts. UmuDAb could exert its effects posttranscriptionally, by binding to the 5' end of ddrR mRNA.

Future work will focus on understanding the range of mechanisms by which this metabolically versatile, industrially important microbe deals with DNA damage. The unexpected constitutive expression of UmuDAb in ADP1 and the lack of a RecA requirement for recA induction (36) distinguish this organism from E. coli and other bacteria, yet the specific regulation of an SOS-like (RecA-regulated induction in response to DNA damage) gene in this bacterium suggests that some SOS functions have been conserved. Determining what aspects of E. coli UmuD function are present in ADP1 will involve examining whether UmuDAb is cleaved upon DNA damage. Understanding how a constitutively expressed gene can be coupled to the induction of a gene responding to an environmental signal makes the ADP1 DNA damage response system important to understand.


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ACKNOWLEDGMENTS
 
Support for this work was provided by Grinnell College, by grants from the Morehead State University Research and Creative Productions Committee to J.M.H., by a Morehead State University undergraduate research fellowship to S.N.P., and by grants from the National Science Foundation (grant DBI-0070310) and the Howard Hughes Medical Institute (grant 71100503702) to Grinnell College.

We thank the lab of Ellen Neidle for providing the ACN32 strain and Graham Walker for helpful discussions.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biological & Environmental Sciences, 327-G Lappin Hall, Morehead State University, Morehead, KY 40351. Phone: (606) 783-2951. Fax: (606) 783-5002. E-mail: jm.hare{at}morehead-st.edu. Back


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Applied and Environmental Microbiology, June 2006, p. 4036-4043, Vol. 72, No. 6
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