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Applied and Environmental Microbiology, February 2008, p. 1273-1275, Vol. 74, No. 4
0099-2240/08/$08.00+0     doi:10.1128/AEM.02347-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Sensitivity of an Acinetobacter baylyi mpl Mutant to DNA Damage{triangledown}

Adityarup Chakravorty,{dagger} Martha Klovstad,{ddagger} Greg Peterson,§ Robin E. Lindeman, and Leslie A. Gregg-Jolly*

Department of Biology, Grinnell College, Grinnell, Iowa 50112

Received 17 October 2007/ Accepted 7 December 2007


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ABSTRACT
 
A mutant derived from Acinetobacter baylyi ADP1 was isolated from a screen for genes involved in the response to DNA damage. This derivative has an insertion in the mpl gene which encodes a peptidoglycan-recycling protein. We demonstrate that the insertion renders cells sensitive to mitomycin C and to UV.


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INTRODUCTION
 
Understanding links between a cell's response to DNA damage and cell division has important implications for the development and application of antimicrobial agents. DNA damage inhibits cell division in most cells. In bacteria, this commonly occurs through derepression of the cell division inhibitor SulA via the LexA-mediated SOS response (4). Since many antibiotics target cell division, it is important to note that cell division inhibitors have been shown to induce the SOS system, which is characteristically activated by DNA damage (12, 15). Unfortunately, SOS induction can increase development of antibiotic resistance (for a review, see reference 9).

Antibiotics that target cell wall synthesis are widely utilized, and identification of agents that interfere with peptidoglycan recycling is a recent focus for drug development (5, 8, 17). For example, efforts are under way to identify Mpl substrate analogues that could target the Mpl recycling enzyme, UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-meso-diaminopimelate ligase (murein peptide ligase), since an mpl-deficient mutant of Acinetobacter baylyi strain ADP1 has increased susceptibility to β-lactam antibiotics (5, 8). Furthermore, Escherichia coli mpl mutants do not show increased susceptibility to β-lactamases, so Mpl is a potential target for species-specific antibacterial compounds (5). Although A. baylyi is not a pathogen, it serves as an important model system since it is especially amenable to genetic studies (18) and is related to the important opportunistic pathogen Acinetobacter baumannii (16).

Efforts to develop antibacterial compounds that compromise cell wall integrity should consider the potential impact of activating the cell's response to DNA damage since an SOS-like response could have the undesirable effect of increasing the rate of evolution of drug resistance. Here we report that in addition to sensitivity to β-lactam antibiotics (5), a mutation in mpl in strain ADP1 also confers sensitivity to DNA damage. The response of ADP1 to DNA damage has been partially characterized (2, 6, 7, 10, 13), and it is known that in strain ADP1 cell division is inhibited in response to DNA damage (2, 7). To better understand the response of ADP1 to DNA damage, we created a mutant library and isolated a clone that was sensitive to the alkylating agent mitomycin C (MMC).

In order to construct the mutant library, chromosomal DNA from strain ADP1 was digested with SalI and ligated with SalI-digested pKOK6 (14). pKOK6 contains a kanamycin resistance gene on a SalI fragment and the pMB1 origin of replication, which does not support replication in ADP1 (11). The ligation mixture was used to transform naturally competent ADP1 cells (6). Kanamycin-resistant (Kmr) recombinants were selected and then screened on plates containing MMC (2 µg/ml). A Kmr derivative that does not form visible colonies on solid media supplemented with MMC was isolated and designated AGC7. For these studies, AGC7 was grown at 37°C and maintained on L agar plates containing 12.5 µg/ml kanamycin.

An XbaI restriction fragment containing the Kmr SalI fragment flanked by 6,019 bp of ADP1 DNA was isolated from strain AGC7. Chromosomal DNA isolated from AGC7 was digested with XbaI and ligated to similarly digested pUC19-derived, ampicillin-resistant vector DNA (14). The ligation mixture was used to transform competent E. coli strain DH5{alpha} cells purchased from Invitrogen Corporation and used according to the manufacturer's directions. Recombinants were selected on medium containing ampicillin (100 µg/ml) and kanamycin (25 µg/ml). Restriction analysis was used to show that a plasmid isolated from a recombinant contained the expected XbaI fragment within the vector. This plasmid was designated pGP7-1. Sequence analysis of pGP7-1 DNA indicated that the pKOK6 SalI fragment is a simple insertion at position 3578437 of the ADP1 genome and resides in the open reading frame (ORF) designated mpl (1) such that the insertion follows the first 114 nucleotides of the predicted ORF and first 38 amino acids of the predicted polypeptide.

To further explore the sensitivity of AGC7 to MMC, cells were grown overnight and then diluted 1:200 in fresh L broth containing various concentrations of MMC. The cultures were grown overnight. Then appropriate dilutions were spotted onto L agar plates, and levels of survival were calculated (Fig. 1A). Over the range of MMC concentrations tested, there was a minimal effect on ADP1 survival. AGC7 showed significant sensitivity to MMC, including less than 0.2% survival with 0.5 mmol MMC. It is possible that a deficiency in Mpl could affect cell wall structure, so we examined the sensitivity of AGC7 to UV since mutagenic activity would not be dependent on chemical transport across the cell wall. Overnight cultures of AGC7, ADP1, and the recA-deficient mutant ADP197 (6) were diluted, plated onto L agar, exposed to the indicated doses of UV in a Stratagene UV Stratalinker 1800 in the dark, and then incubated in dark containers. AGC7 showed significantly greater sensitivity to UV than ADP1 at all levels tested, yet it was less sensitive than ADP197 (Fig. 1B). It is not surprising that the mpl-deficient strain AGC7 was less sensitive to UV than the recA-deficient strain ADP197 due to the essential role of RecA in DNA repair and recombination (6).


Figure 1
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  		FIG. 1. Sensitivity of AGC7 to the DNA-damaging agents MMC (A) and UV (B). After exposure to a damaging agent, cells were incubated overnight at 37°C, colonies were counted, and the numbers of CFU ml–1 were calculated. Percent survival was calculated by dividing the number of CFU ml–1 in the presence of the damaging agent by the number of CFU ml–1 in the absence of the agent. Each symbol indicates the mean for at least four independent trials, and the error bars indicate the standard errors of the means.

Since the ADP1 mpl gene is the first of three predicted ORFs (1) and the pKOK6 mutagenic fragment contains a transcriptional stop (11), we examined the possibility that the AGC7 phenotype could be a polar effect. For this experiment, we used ADP1-derived mutants containing the following knockouts of the adjacent ORFs: upstream knockout ACAID3662 and downstream knockouts ACAID3665 and ACAID3666 (2a). Each knockout mutant carries a single deletion in the designated ORF that was created using a PCR-generated DNA fragment with the ORF deleted and replaced by a cassette containing selectable markers. Each of the three predicted ORFs is a homologue of a previously reported gene with an unknown function (1). The levels of survival of the knockout mutants were comparable to each other and to the level of survival of ADP1 after treatment with 100 J/m2 UV, as shown in Fig. 1. Furthermore, we confirmed that the AGC7 phenotype is a direct consequence of the mpl mutation. The ampicillin-resistant plasmid containing the Kmr pKOK6-derived fragment isolated from AGC7, pGP7-1, was digested with XbaI and used to naturally transform ADP1 cells. A Kmr transformant that was also ampicillin sensitive (indicating replacement recombination) was confirmed to be sensitive to UV at a level similar to the AGC7 level (5% survival with 100 J/m2).

While the sensitivity of AGC7 could be due to a physical disturbance of the cell wall that facilitates both the transport of MMC and the penetration of UV, we must also consider the possibility that altering the cell wall composition directly affected the response to DNA damage. Although there is some support for a relationship between cell wall integrity and the SOS system (for a review, see reference 3), other than genes encoding transport proteins, no E. coli genes involved in cell envelope synthesis have been found to be involved in the response to DNA damage. However, Lactococcus lactis cells deficient in the dltD gene involved in cell wall teichoic acid synthesis or the gerC gene involved in cell membrane isoprene synthesis are sensitive to UV and MMC (3). Future studies will examine the effect of the mpl mutation on the expression of recA and ddrR, the only DNA damage-inducible genes identified in ADP1 so far (7, 13).


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ACKNOWLEDGMENTS
 
We thank Veronique de Berardinis from Genoscope in Every, France, for sharing single-gene deletion mutants of ADP1 prior to publication.

This work was funded by Grinnell College. L.G.-J. received support from a Grinnell College Harris Fellowship. Additionally, R.E.L. and A.C. received support from a Howard Hughes Medical Institute Undergraduate Science Education Award to Grinnell College in 2000 and 2004, respectively.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology, Grinnell College, Grinnell, IA 50112. Phone: (641) 269-4978. Fax: (641) 269-4285. E-mail: greggjol{at}grinnell.edu Back

{triangledown} Published ahead of print on 14 December 2007. Back

{dagger} Present address: Department of Biology, University of Rochester. Rochester, NY. Back

{ddagger} Present address: Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, NJ. Back

§ Present address: University of Minnesota Medical School, Minneapolis, MN. Back

Present address: Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI. Back


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Applied and Environmental Microbiology, February 2008, p. 1273-1275, Vol. 74, No. 4
0099-2240/08/$08.00+0     doi:10.1128/AEM.02347-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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