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Importance of Uracil DNA Glycosylase in Pseudomonas aeruginosa and Mycobacterium smegmatis, G+C-rich Bacteria, in Mutation Prevention, Tolerance to Acidified Nitrite, and Endurance in Mouse Macrophages

2003; Elsevier BV; Volume: 278; Issue: 27 Linguagem: Inglês

10.1074/jbc.m302121200

1083-351X

Venkatesh Jeganathan, Pradeep Kumar, Pulukuri Sai Murali Krishna, Ramanathapuram Manjunath, Umesh Varshney,

DNA Repair Mechanisms

Uracil DNA glycosylase (Ung (or UDG)) initiates the excision repair of an unusual base, uracil, in DNA. Ung is a highly conserved protein found in all organisms. Paradoxically, loss of this evolutionarily conserved enzyme has not been seen to result in severe growth phenotypes in the cellular life forms. In this study, we chose G+C-rich genome containing bacteria (Pseudomonas aeruginosa and Mycobacterium smegmatis) as model organisms to investigate the biological significance of ung. Ung deficiency was created either by expression of a highly specific inhibitor protein, Ugi, and/or by targeted disruption of the ung gene. We show that abrogation of Ung activity in P. aeruginosa and M. smegmatis confers upon them an increased mutator phenotype and sensitivity to reactive nitrogen intermediates generated by acidified nitrite. Also, in a mouse macrophage infection model, P. aeruginosa (Ung–) shows a significant decrease in its survival. Infections of the macrophages with M. smegmatis show an initial increase in the bacterial counts that remain for up to 48 h before a decline. Interestingly, abrogation of Ung activity in M. smegmatis results in nearly a total abolition of their multiplication and a much-decreased residency in macrophages stimulated with interferon γ. These observations suggest Ung as a useful target to control growth of G+C-rich bacteria. Uracil DNA glycosylase (Ung (or UDG)) initiates the excision repair of an unusual base, uracil, in DNA. Ung is a highly conserved protein found in all organisms. Paradoxically, loss of this evolutionarily conserved enzyme has not been seen to result in severe growth phenotypes in the cellular life forms. In this study, we chose G+C-rich genome containing bacteria (Pseudomonas aeruginosa and Mycobacterium smegmatis) as model organisms to investigate the biological significance of ung. Ung deficiency was created either by expression of a highly specific inhibitor protein, Ugi, and/or by targeted disruption of the ung gene. We show that abrogation of Ung activity in P. aeruginosa and M. smegmatis confers upon them an increased mutator phenotype and sensitivity to reactive nitrogen intermediates generated by acidified nitrite. Also, in a mouse macrophage infection model, P. aeruginosa (Ung–) shows a significant decrease in its survival. Infections of the macrophages with M. smegmatis show an initial increase in the bacterial counts that remain for up to 48 h before a decline. Interestingly, abrogation of Ung activity in M. smegmatis results in nearly a total abolition of their multiplication and a much-decreased residency in macrophages stimulated with interferon γ. These observations suggest Ung as a useful target to control growth of G+C-rich bacteria. Among all the bases in DNA, cytosine is highly susceptible to deamination of its exocyclic amino group in response to normal physiological reactions or environmental pollutants, resulting in generation of promutagenic G×U mismatches in the genome. If these were to be left unrepaired, an exorbitant increase in accrual of G-C to A-T mutations would occur unabated after each replication cycle and pose a serious threat to the genomic integrity and the very survival of the organism. In cells, uracil-DNA glycosylase (Ung), 1The abbreviations used are: Ung, uracil DNA glycosylase (also UDG); RNI, reactive nitrogen intermediates; LBT, LB supplemented with 0.2% Tween 80; ORF, open reading frame; FCS, fetal calf serum; IFN, interferon; Eco, E. coli; kb, kilobase(s). also known as UDG, is the major DNA repair enzyme that initiates uracil excision repair pathway (1Lindahl T. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 3649-3653Google Scholar). Ung proteins are found in all life forms including many viruses that infect eukaryotic cells. These enzymes are inhibited by free uracil and some of its derivatives as well as by Bacillus subtilis phage PBS-1- and -2-encoded inhibitor Ugi. Ugi is a heat-stable, acidic, low molecular weight protein that forms a tight complex with Ung (2Cone R. Bonura T. Friedberg E.C. J. Biol. Chem. 1980; 255: 10354-10358Google Scholar, 3Krokan H.E. Standal R. Slupphaug G. Biochem. J. 1997; 325: 1-16Google Scholar, 4Mosbaugh D.W. Bennett S.E. Prog. Nucleic Acid Res. Mol. Biol. 1994; 48: 315-370Google Scholar). In herpes simplex virus, UNG gene is necessary for efficient replication and reactivation (5Pyles R.B. Thompson R.L. J. Virol. 1994; 68: 4963-4972Google Scholar). Furthermore, in human cytomegalovirus, disruption of UNG gene results in delayed DNA synthesis and longer replication cycle (6Prichard M.N. Duke G.M. Mocarski E.S. J. Virol. 1996; 70: 3018-3025Google Scholar). On the other hand, although the ung– mutants of various bacteria and yeast have shown increased mutator phenotypes (7Duncan B.K. Miller J.H. Nature. 1980; 287: 560-561Google Scholar, 8Impellizzeri K.J. Anderson B. Burgers P.M. J. Bacteriol. 1991; 21: 6807-6810Google Scholar), they do not show a detectable growth defect. Similarly, knockout mice (ung–/ung–) presented with no distinct phenotype (9Nilsen H. Rosewell I. Robins P. Skielbred C.F. Andersen S. Slupphaug G. Daly G. Krokan H.E. Lindahl T. Barnes D.E. Mol. Cell. 2000; 5: 1059-1065Google Scholar). It is paradoxical that the loss of ung gene, whose phylogenetic distribution is so exceedingly broad and which codes for a highly conserved and functionally relevant protein, does not result in a severe phenotype in the cellular life forms, at least under the conditions investigated. However, it is noteworthy that the G+C-rich organisms, which are naturally at high risk of cytosine deamination, have not yet been used in such studies. The genome-sequencing projects have unfolded greater opportunities to investigate the roles of important genes in various organisms. We chose a fast- and a slow-growing G,C-rich genome-containing bacterium (Pseudomonas aeruginosa and Mycobacterium smegmatis with G+C contents of 67 and 64%, respectively) as model organisms to investigate the biological significance of ung. In addition, M. smegmatis serves as a surrogate model for the pathogenic mycobacteria (10Jacobs Jr., W.R. Kalpana G.V. Cirillo J.V. Pascopella L. Snapper S.B. Udani R.A. Jones W. Barletta R.G. Bloom B.R. Methods Enzymol. 1991; 204: 537-555Google Scholar), which are at an increased risk of cytosine deamination inside host macrophages due to the production of reactive oxygen and nitrogen intermediates. Both reactive nitrogen intermediates (RNI) and reactive oxygen species penetrate through the lipid-rich membranes/cell wall (11Fels A.O. Cohn Z.A. J. Appl. Physiol. 1986; 60: 353-369Google Scholar, 12Lancaster Jr., J.R. Methods Enzymol. 1996; 268: 31-50Google Scholar) of bacteria. One of the types of damage that ensues from the presence of these reactive intermediates is an increased rate of cytosine deamination in DNA (13Wink D.A. Kasprzak K.S. Maragos C.M. Elespuru R.K. Misra M. Dunams T.M. Cebula T.A. Koch W.H. Andrews A.W. Allen J.S. Science. 1991; 254: 1001-1003Google Scholar). Here, we show that in P. aeruginosa and M. smegmatis abrogation of Ung activity leads not only to an increased mutator phenotype but also to growth inhibition by RNI. Furthermore, in a mouse macrophage infection model, P. aeruginosa (Ung–) shows a significant decrease in survival under the conditions of increased RNI production. Interestingly, in this assay loss of Ung in M. smegmatis results in near abolition of the initial round(s) of bacterial multiplication and in its compromised endurance in macrophage. Various DNA oligomers, plasmids, and bacterial strains used in this study are listed in Table I. Unless specified otherwise, Escherichia coli and P. aeruginosa were grown in Luria Bertani, LB (Difco), and M. smegmatis mc2155 (14Snapper S.B. Melton R.E. Mustafa S. Kieser T. Jacobs Jr., W.R. Mol. Microbiol. 1990; 4: 1911-1919Google Scholar) was grown in LB supplemented with 0.2% Tween 80 (LBT). For growth on a solid surface, 1.5% agar was included in the broth media. Liquid cultures were grown at 37 °C under shaking. When required, the media were supplemented with ampicillin, gentamycin, and kanamycin at 100, 20, and 50 μg/ml, respectively, for E. coli, with chloramphenicol at 200 μg/ml for P. aeruginosa, and with kanamycin and gentamycin at 50 and 5 μg/ml, respectively, for M. smegmatis cultures. Sucrose selection for M. smegmatis was performed on Middlebrook 7H10 (Difco) solid medium with 0.2% glycerol and 10% sucrose.Table IList of plasmids, bacterial strains, and oligodeoxyribonucleotides used in this studyStrain/plasmidRelevant detailsReferencepDK20 (KanR)An integration vector for mycobacteria.20DasGupta S.K. Jain S. Kaushal D. Tyagi A.K. Biochem. Biophys. Res. Commun. 1998; 246: 797-804Google ScholarpDKUgi (KanR)Ugi cloned under M. tuberculosis intiator tRNA gene (metU) promoter at Dral siteThis studypPR27 (GmR)Contains a temperature-sensitive origin of replication and a sacB marker for negative selection in mycobacteria21Pelicic V. Jackson M. Reyrat J.M. Jacobs Jr., W.R. Gicquel B. Guilhot C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10955-10960Google ScholarpPRmsUng::aph (GmR, KanR)pPR27 harboring disrupted M. smegmatis ung gene for allelic exchange of the chromosomal geneThis studypBBRSame as pBBR1MCS (CmR). A shuttle vector for autonomous replication in E. coli and P. aeruginosa17Kovach M.E. Phillips R.W. Elzer P.H. Roop II, R.M. Peterson K.M. Biotechniques. 1994; 16: 800-802Google ScholarpBBRUgi (CmR)Contains Ugi gene in pBBR1MCSThis studypBBRUgi-oc36 (CmR)Contains Ugi-oc36 mutant in pBBR1MCSThis studypTrc99c (AmpR)An E. coli expression vectorAmersham BiosciencespTrcUgi (AmpR)Contains Ugi geneThis studypTrcUgi-oc36 (AmpR)Contains Ugi-oc36 mutant geneThis studyE. coli KL16An E. coli K strainE. coli Genetic stock CenterE. coli KL16 (ung::cat)E. coli KL16 strain wherein ung gene ORF was replaced by chloramphenicol acetyltransferase gene by a protocol described before (56Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Google Scholar).P. Kumar and U. Varshney unpublished data.P. aeruginosa PA01A P. aeruginosa strain57Diver J.M. Bryan L.E. Sokol P.A. Anal. Biochem. 1990; 189: 75-79Google ScholarM. smegmatis mc2 155A high efficiency transformation strain of M. smegmatis14Snapper S.B. Melton R.E. Mustafa S. Kieser T. Jacobs Jr., W.R. Mol. Microbiol. 1990; 4: 1911-1919Google ScholarM. smegmatis mc2 155 (L5 att::pDK20)Contains pDK20 (vector control) integrated at the L5 att site in genome of M. smegmatis mc2 155This studyM. smegmatis mc2 155 (L5 att::pDKUgi)Contains pDKUgi integrated at the L5 att site in M. smegmatis mc2 155 genomeThis studyM. smegmatis mc2 155 (ung::aph)A M. smegmatis mc2 155 strain wherein the ung gene has been disrupted with kanR cassetteThis studyFP1Forward primer to PCR amplify M. smegmatis ung locus of ≈2.2 kb. (5′-CATGCTGATCAAGCGAAGGCATTTCC-3′)This studyRP1Reverse primer to PCR amplify M. smegmatis ung locus of ≈2.2 kb (5′-GTATCCTGATCAGGTTGTCGGGTC-3′)This studyFP2M. smegmatis ung gene ORF-specific forward primer (5′-CTTTCCGTGGCCGCACGACCGCTGA-3′)27Acharya N. Varshney U. J. Mol. Biol. 2002; 318: 1251-1264Google ScholarRP2M. smegmatis ung gene ORF specific reverse primer (5′-GGAATTCCTACTAGGGCAACTTCCAG-3′)27Acharya N. Varshney U. J. Mol. Biol. 2002; 318: 1251-1264Google ScholarRP3M. smegmatis ung locus reverse primer anneals downstream of RP1 (5′-GTGTTGACGGCCGATTTTGG-3′)This studySSU9Substrate for Ung assays (5′-CTCAAGTGUAGGCATGCAAGAGCT-3′)15Acharya N. Roy S. Varshney U. J. Mol. Biol. 2002; 32: 579-590Google Scholar Open table in a new tab The open reading frame (ORF) of Ung (UDG) in the pTrcUDG-Ugi and pTrcUDG-Ugi-oc36 (15Acharya N. Roy S. Varshney U. J. Mol. Biol. 2002; 32: 579-590Google Scholar) was damaged at its active site by cleaving these plasmids at their unique BamHI sites and end-filling with Klenow DNA polymerase followed by ligation with T4 DNA ligase. The desired recombinants, pTrcUgi and pTrcUgi-oc36 (containing a stop codon, UAA at 36th position in ORF) were selected for expression of Ugi or its mutant Ugi-oc36, respectively, in E. coli KL16 (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). To generate such constructs for introduction into P. aeruginosa, the BamHI to HindIII DNA fragments harboring ugi or ugi-oc36 from pTrcUDG-Ugi and the pTrcUDG-Ugi-oc36 were subcloned into the same sites of pBBR1MCS (Ref. 17Kovach M.E. Phillips R.W. Elzer P.H. Roop II, R.M. Peterson K.M. Biotechniques. 1994; 16: 800-802Google Scholar; kindly provided by Dr. V. Rangaswami, Central Drug Research Institute, Lucknow, India) to create pBBRUgi and pBBRUgi-oc36, respectively. Transcription of ugi gene in these plasmids in P. aeruginosa is driven by a fortuitous promoter. For introduction of ugi gene into the L5 integration site in M. smegmatis, pTZUgi (18Roy S. Purnapatre K. Handa P. Boyanapalli M. Varshney U. Protein Expression Purif. 1998; 13: 155-162Google Scholar) was digested with EcoRI, treated with Klenow DNA polymerase, and subjected to digestion with HindIII after heat inactivation. The DNA fragment containing ugi ORF was then cloned between a blunt-ended BamHI and HindIII sites of pTKmt (19Dastur A. Kumar P. Ramesh S. Vasanthakrishna M. Varshney U. Arch. Microbiol. 2002; 178: 288-296Google Scholar), downstream of metU promoter to generate pTKmtUgi. The pTKmtUgi was digested with EcoRI and HindIII to release a DNA fragment containing ugi along with the metU promoter, end-filled with Klenow DNA polymerase, and cloned at the DraI site of pDK20 (20DasGupta S.K. Jain S. Kaushal D. Tyagi A.K. Biochem. Biophys. Res. Commun. 1998; 246: 797-804Google Scholar) to generate pDKUgi. Integration of ugi into the L5 att Site of M. smegmatis mc2155— M. smegmatis mc2155 was transformed with pDKUgi, a non-replicative vector (in mycobacteria) containing the L5 att sequence, and plated on LBT-agar containing kanamycin. Transformants that arose contained the whole plasmid inserted into the L5 att site in the chromosome (20DasGupta S.K. Jain S. Kaushal D. Tyagi A.K. Biochem. Biophys. Res. Commun. 1998; 246: 797-804Google Scholar). The targeted gene knockout strategy using pPR27 containing a thermosensitive origin of replication of pAL5000 and sacB counter-selective marker was used (21Pelicic V. Jackson M. Reyrat J.M. Jacobs Jr., W.R. Gicquel B. Guilhot C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10955-10960Google Scholar). The nucleotide sequence of M. smegmatis ung gene and its flanking regions was obtained from The Institute of Genomic Research (TIGR) web site (www.tigr.org) to design FP1 and RP1 primers (Table I) for amplification of ∼2.2-kb DNA containing 827 and 663 bp upstream and downstream, respectively, of ung ORF (684 bp). The PCR conditions included an initial denaturation at 94 °C for 4 min, 30 cycles of incubations at 94 °C for 1 min, 55 °C for 30 s, and 72 °C for 4.5 min, and a final extension at 72 °C for 10 min. The PCR product was digested with BclI and cloned into the BamHI site of pPR27 to yield pPRmsUng. Subsequently, a kanR (aph) cassette from pUC4K (Amersham Biosciences) was excised by digestion with BamHI and mobilized into the unique BamHI site present in the active site region of the ung (pPRmsUng) to generate pPRmsUng::aph. The latter construct was introduced into M. smegmatis by electroporation (22Hatfull G.F. Jacobs W.B. Molecular Genetics of Mycobacteria. American Society for Microbiology, Washington, D. C.2002: 313-320Google Scholar), and the transformants were selected at 32 °C on LBT agar plates in the presence of gentamycin and kanamycin. Several transformants were grown at 32 °C in 7H9 medium containing kanamycin and plated on to 7H10 agar containing kanamycin and sucrose at 39 °C. The isolates were further screened for disruption of the chromosomal ung. Cell-free extracts were prepared from the putative ung– isolates (23Vasanthakrishna M. Kumar N.V. Varshney U. Microbiology. 1997; 143: 3591-3598Google Scholar) and assayed for Ung activity. Subsequently, the genomic DNA from the isolates was analyzed by PCR using FP2 and RP2 primers (Table I, Fig. 4A), corresponding to the ung locus, and Taq DNA polymerase. For PCR, DNA samples were heated at 94 °C for 4 min and subjected to 30 cycles of incubations at 94 °C for 45 s, 55 °C for 30 s, and 72 °C for 3 min followed by a final incubation at 72 °C for 10 min. Genomic DNAs (2.5–5 μg) were digested with an excess of restriction enzymes (20 units), separated on a 0.7% agarose gel using Tris-buffered EDTA, transferred (24Reed K.C. Mann D.A. Nucleic Acids Res. 1985; 13: 7207-7221Google Scholar) to Hybond-NX membrane (Amersham Biosciences), and subjected to hybridization (23Vasanthakrishna M. Kumar N.V. Varshney U. Microbiology. 1997; 143: 3591-3598Google Scholar) with a radiolabeled probe against the ung gene of M. smegmatis. The radiolabeled probe was made in the presence of [α-32P]dCTP by a PCR-based method using FP2 and RP3 primers (Table I, Fig. 4C). Cell-free extracts were prepared from log phase cultures by sonication of M. smegmatis cells (23Vasanthakrishna M. Kumar N.V. Varshney U. Microbiology. 1997; 143: 3591-3598Google Scholar) or gentle lysis of E. coli and P. aeruginosa cells (25Varshney U. Lee C.P. RajBhandary U.L. J. Biol. Chem. 1991; 266: 24712-24718Google Scholar) and quantified (26Sedmak J.J. Grossberg S.E. Anal. Biochem. 1977; 79: 544-552Google Scholar). Uracil containing synthetic DNA (SSU9, Table I) was 5′ end-labeled and used in Ung assays (27Acharya N. Varshney U. J. Mol. Biol. 2002; 318: 1251-1264Google Scholar). For detection of Ugi expression in E. coli, total cell extracts (100 μg) were separated on native PAGE (15%) gels, which allow a discrete separation between the free EcoUng (pI 6.6) and the EcoUng·Ugi (pI 4.9) complex. The EcoUng·Ugi complex was then detected by immunoblotting using anti-EcoUng antibodies (18Roy S. Purnapatre K. Handa P. Boyanapalli M. Varshney U. Protein Expression Purif. 1998; 13: 155-162Google Scholar). Detection of Ugi expression in P. aeruginosa and M. smegmatis was also based on the ability of Ugi to sequester EcoUng into a complex. Total cellular proteins (200 or 300 μg for P. aeruginosa or M. smegmatis, respectively, for each treatment) were subjected to thermal denaturation (15Acharya N. Roy S. Varshney U. J. Mol. Biol. 2002; 32: 579-590Google Scholar). Unlike Ung and most other cellular proteins, Ugi is thermostable and fully refolds to its native structure upon cooling (28Reddy G.B. Purnapatre K. Lawrence R. Roy S. Varshney U. Surolia A. Eur. J. Biochem. 1999; 261: 610-617Google Scholar), and it is highly efficient in forming a complex with Ung. The supernatant of the thermally denatured protein extract was used as source of Ugi and incubated with EcoUng (50–200 ng) for 15 min at room temperature followed by 10 min on ice. The samples were then analyzed as above by immunoblotting of the proteins separated on native PAGE. Isolated colonies from plates were inoculated into broth media and grown to early stationary phase (10–12 h for E. coli and P. aeruginosa and 48 h for M. smegmatis). Cells from 1-ml cultures were spread in duplicate on solid media containing rifampicin at a concentration of 50 μg/ml for E. coli and M. smegmatis cultures and 100 μg/ml for P. aeruginosa cultures. Total viable counts in the culture were determined by dilution plating. Mutation frequencies were calculated as the number of colonies (RifR) that appeared on rifampicin-containing media divided by the total viable counts of the bacteria plated. Cultures were started with 0.1% inoculum from freshly prepared saturated cultures. The growth of E. coli and P. aeruginosa in LB was monitored at different initial pH values (7.2, 6.5, 6.0, and 5.5) of the media with or without 1 mm NaNO2 at 37 °C for up to 11 h. The growth curves of M. smegmatis in LBT with or without 0.2 mm NaNO2 were obtained in the same manner except that the growth was monitored for up to 48 h. The NaNO2 was supplemented to the medium from a filter sterilized stock before inoculation. Peritoneal macrophages were isolated from C57BL/6 mice and infected by P. aeruginosa and M. smegmatis as described (29Morissette C. Francoeur C. Darmond-Zwaig C. Gervais F. Infect. Immun. 1996; 64: 4984-4992Google Scholar, 30Rastogi N. Labrousse V. de Sousa J.P. Curr. Microbiol. 1992; 25: 203-213Google Scholar). In brief, the infection assays were as follows. Macrophage Infection Assays with M. smegmatis—Bacteria were opsonized with guinea pig serum, washed with RPMI 1640, and used at a multiplicity of infection of 10. Macrophages (1 × 106) were allowed to adhere for 24 h in a 24-well tissue culture plate in RPMI with 5% fetal calf serum (FCS). The cells were washed with 0.2% FCS-RPMI, incubated with opsonized M. smegmatis for 5 h at 37 °C in 5% CO2 atmosphere in the same medium to allow macrophages to phagocytose and internalize the bacteria, and washed three times with 0.2% FCS-RPMI to remove extracellular bacteria. Cells infected with M. smegmatis were incubated for a further period of 0.5 h in 0.2% FCS-RPMI containing 50 μg/ml gentamycin and washed again three times with 0.2% FCS-RPMI to remove antibiotics. This was considered time 0 of sample collection, and further incubations were for 24, 48, and 72 h in 5% FCS-RPMI. At the end of the incubations, macrophages were subjected to lysis with 0.25 ml of 0.25% SDS supplemented with 0.25 ml RPMI, and the total viable counts of the internalized bacteria were determined by dilution plating. Control experiments wherein bacteria alone or macrophages alone were treated the same were also carried out. No viable counts were detected in these controls. For experiments with stimulated macrophages, cells were treated with 1 unit/ml interferon γ (IFNγ) during adherence to the plates. Macrophage Infection Assays with P. aeruginosa—These were the same as described above except that the wells seeded with 0.5 × 106 macrophages were infected for 0.5 h with P. aeruginosa opsonized with fetal calf serum, washings were done with 0.2% FCS-RPMI without antibiotics, and the post-washing incubations were done either for 0 or 1.5 h in the absence of antibiotics, after which the macrophages were lysed with 0.5 ml of 0.01% BSA in distilled water. Nitric oxide secretion was determined by measuring the accumulation of nitrite ( NO2−), a stable metabolite of the reaction of nitric oxide with oxygen. Briefly, macrophage culture supernatants (100 μl) were added to a 96-well flat-bottomed enzyme-linked immunosorbent assay plate in duplicate, supplemented with an equal volume of the Griess reagent (0.5% sulfanilamide and 0.05% n-1-naphthylethylenediamine hydrochloride in 2.5% phosphoric acid, Ref. 31Stuehr D.J. Gross S.S. Sakuma I. Levi R. Nathan C.F. J. Exp. Med. 1989; 169: 1011-1020Google Scholar), incubated for 10 min at room temperature, and spectrophotometrically measured at 550 nm using an enzyme-linked immunosorbent assay reader. All experiments involving macrophage infections and nitric oxide estimation were statistically analyzed for significance using Student's t test. Comparison of Primary Sequences of Ung Proteins—Primary sequences of Ung proteins from P. aeruginosa (Pae) and M. smegmatis (Msm), E. coli (Eco), human (Hu), and herpes simplex virus (HSV) are shown in Fig. 1. The comparison shows a high degree of similarity and identity of 63.5 and 55.9% for the Eco-/Pae-Ung pair and 50.4 and 43.8% for the Eco-/Msm-Ung pair, respectively. Both proteins possess all the conserved motifs such as the water-activating loop, 62-GQDPY-66; the Pro-Ser loop, 84-AIPPS-88; the uracil specificity pocket, 120-LLLN-123; and the DNA intercalation loop, 187-HPSPLS-192 (numbering according to EcoUng). These motifs are characteristic of Ung proteins and lie at the interface with Ugi in the Ung-Ugi complexes (32Savva R. Pearl L.H. Nat. Struct. Biol. 1995; 2: 752-755Google Scholar, 33Mol C.D. Arvai A.S. Sanderson R.J. Slupphaug G. Kavli B. Krokan H.E. Mosbaugh D.W. Tainer J.A. Cell. 1995; 82: 701-708Google Scholar, 34Ravishankar R. Bidya Sagar M. Roy S. Purnapatre K. Handa P. Varshney U. Vijayan M. Nucleic Acids Res. 1998; 26: 4880-4887Google Scholar, 35Putnam C.D. Shroyer M.J.N. Lundquist A.J. Mol C.D. Arvai A.S. Mosbaugh D.W. Tainer J.A. J. Mol. Biol. 1999; 287: 331-346Google Scholar). Furthermore, a closer analysis (Fig. 1) shows that in P. aeruginosa and M. smegmatis-Ung proteins, the majority of the functional equivalents of the crucial amino acids that interact with Ugi are also conserved. Expression of Ugi in E. coli, P. aeruginosa, and M. smegmatis and Inactivation of Ung Activity—Expression of Ugi in B. subtilis by phage PBS-1 and -2 as an early gene product allows these uracil containing DNA phages to survive and replicate in the host that is genotypically ung+ (36Friedberg E.C. Ganesan A.K. Minton K. J. Virol. 1975; 16: 315-321Google Scholar). More recently, expression of Ugi in a chicken B cell line, DT40 rendered them Ung– (37Noia J.D. Neuberger M.S. Nature. 2002; 419: 43-48Google Scholar). Hence, in one of the approaches to obtain Ung– phenotype, we expressed Ugi in these bacteria. The indirect immunoblot analysis (Fig. 2, wherein Ugi is detected as EcoUng·Ugi complex, see “Materials and Methods”) shows that introduction of multicopy ugi expression vectors in E. coli (pTrcUgi) and P. aeruginosa (pBBRUgi) resulted in production of Ugi (lanes 4, Figs. 2, A and B, respectively). The band corresponding to EcoUng·Ugi was not present in the transformants harboring vector alone (lanes 2) or the Ugi-oc36 mutant (lanes 3) that does not form a complex with Ung (15Acharya N. Roy S. Varshney U. J. Mol. Biol. 2002; 32: 579-590Google Scholar). However, a similar approach of expression of Ugi in M. smegmatis from a mycobacterial plasmid (pTKUgi), most likely because of the vector instability effects, resulted in inconsistent observations (data not shown). Therefore, to abrogate Ung activity in this organism, we cloned the ugi ORF downstream of a mycobacterial promoter (metU (19Dastur A. Kumar P. Ramesh S. Vasanthakrishna M. Varshney U. Arch. Microbiol. 2002; 178: 288-296Google Scholar)) in pDKUgi and integrated it as a single copy gene into the L5 attachment (att) site of M. smegmatis genome. As detected by the indirect immunoblot analysis, this strain resulted in consistent expression of Ugi (Fig. 2C, lanes 3–5). To ascertain that expression of Ugi in these organisms resulted in abolition of Ung activity, assays were performed in the cell-free extracts of the various strains, using a 5′ 32P-labeled uracil containing synthetic DNA oligomer (SSU9) as substrate (S). Uracil excision by Ung generates an apyrimidinic site in the DNA oligomer, which is sensitive to alkaline conditions and results in two fragments, one of which (32P-labeled) is detected as faster migrating product band (P) upon gel electrophoresis. As seen in Fig. 3, expression of Ugi in E. coli (panel A, lanes 9–11), P. aeruginosa (panel B, lanes 9–11), and M. smegmatis (panel C, lanes 6–8) resulted in undetectable Ung activities as opposed to the extracts of bacteria harboring vector alone (lanes 3–5 in all panels) or the Ugi-oc36 mutant (lanes 6–8 in panels A and B). ung Gene Knockout in M. smegmatis and Ung Assays—Because in M. smegmatis expression of Ugi was achieved from a single copy of the chromosomally inserted gene of ugi (L5att::pDKUgi), we employed a yet another approach to deplete Ung activity in this organism. This approach involved disruption of the ung gene by insertion of a 1.264-kb kanamycin resistance cassette (aph) into the active site region. Amplification of a 1.384-kb DNA (Fig. 4B, lane 1) from the wild type strain and a 2.648-kb DNA from the knockout strains (Fig. 4B, lanes 2 and 3) upon PCR with the primers flanking the ung gene are exactly as per the expectations (Fig. 4A) and show that the ung gene was disrupted by the kanamycin (aph) marker. Furthermore, the genomic blot analyses (Fig. 4, C and D) also show the bands of expected sizes of 1.822 kb (lane 1) versus 3.086 kb (lanes 2 and 3) in the EcoRV and 3.049 kb (lane 4) versus 4.313 kb (lanes 5 and 6) in the BclI digests of the DNA from wild type and the knockout strains, respectively. The presence of single bands corresponding to the ung plus aph cassette size in the knockout strains confirms the disrupted nature of the ung gene in the isolates. And as shown in Fig. 3C, lanes 9–11, the M. smegmatis strain with disrupted gene (ung::aph) did not contain any detectable Ung activity. It may also be mentioned that susceptibility of M. smegmatis DNA to restriction by BclI (Fig. 4D, lanes 5 and 6) further confirms an earlier report (38Hemavathy K.C. Nagaraja V. FEMS Immunol. Med. Microbiol. 1995; 11: 291-296Google Scholar) of the absence of methylation at GATC (Dam) sequences in this bacterium. Effect of Depletion of Ung Activity in P. aeruginosa and M. smegmatis on the Appearance of Rifampicin Resistance— One of the consequences of the loss of a DNA repair enzyme is an increase in the rate of mutations in the organism. Hence, to investigate the consequence of the Ung– phenotype in P. aeruginosa and M. smegmatis, we plated bacterial cultures on rifampicin-containing media and scored for rifampicin resistant (RifR) colonies. As shown in Table II, expression of Ugi in P. aeruginosa (pBBRUgi) resulted in an increase in the appearance of RifR colonies by ∼7-fold when compared with the vector alone (pBBR) control. We noticed that in P. aeruginosa but not in E. coli the Ugi mutant (Ugi-oc36) offered a detectable protection over the vector alone control. Hence, the actual increase in the appearance of RifR colonies in P. aeruginosa upon Ugi expression may actually be more than 7-fold. In M. smegmatis (L5att::pDKUgi) ha