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*

Jeganathan Venkatesh {ddagger}, Pradeep Kumar {ddagger}, Pulukuri Sai Murali Krishna, Ramanathapuram Manjunath § and Umesh Varshney 

From the Departments of Microbiology and Cell Biology and §Biochemistry, Indian Institute of Science, Bangalore, 560 012 India

Received for publication, February 28, 2003 , and in revised form, April 3, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {gamma}. These observations suggest Ung as a useful target to control growth of G+C-rich bacteria.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 GxU 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),1 also known as UDG, is the major DNA repair enzyme that initiates uracil excision repair pathway (1). 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 (24).

In herpes simplex virus, UNG gene is necessary for efficient replication and reactivation (5). Furthermore, in human cytomegalovirus, disruption of UNG gene results in delayed DNA synthesis and longer replication cycle (6). On the other hand, although the ung mutants of various bacteria and yeast have shown increased mutator phenotypes (7, 8), they do not show a detectable growth defect. Similarly, knockout mice (ung/ung) presented with no distinct phenotype (9). 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 (10), 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 (11, 12) 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 (13).

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oligodeoxyribonucleotides, Plasmids, Bacterial Strains, Media, and Growth Conditions
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 (14) 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.


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TABLE I
List of plasmids, bacterial strains, and oligodeoxyribonucleotides used in this study

 

Cloning of ugi or ugi-oc36 into Replicative or Integrative Vectors
The open reading frame (ORF) of Ung (UDG) in the pTrcUDG-Ugi and pTrcUDG-Ugi-oc36 (15) 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 (16). 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. 17; 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 (18) 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 (19), 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 (20) 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 (20).

Generation of M. smegmatis ung Knockout Construct
The targeted gene knockout strategy using pPR27 containing a thermosensitive origin of replication of pAL5000 and sacB counter-selective marker was used (21). 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 (22), 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.

Screening of M. smegmatis ung Strains
Cell-free extracts were prepared from the putative ung isolates (23) 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.



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FIG. 4.
Analysis of ung gene disruption in M. smegmatis. A and B, PCR analysis. Chromosomal DNAs from M. smegmatis mc2155 (wild type (WT)) and the two isolates containing disruption in ung gene (ung::aph) were used as template ("Materials and Methods"). Expected sizes are shown in panel A. Agarose gel analysis of the reaction products (panel B) shows the amplification ~1.4 kb (lane 1) or ~2.7 kb (lanes 2 and 3) size DNA from the wild type and the two knockout (ung::aph) isolates. Fragment sizes shown within parentheses are the exact sizes of the expected bands. C and D, genomic blot analysis of the chromosomal DNA from the M. smegmatis strains using a radiolabeled ung gene sequence probe. A diagrammatic representation of the expected sizes of hybridizing bands is shown in panel C, and an autoradiogram with the sizes of the hybridizing bands indicated with arrows in the EcoRV (lanes 1–3) or BclI (lanes 4–6) digests in the wild type (lanes 1 and 4, respectively) or the knockout strains (lanes 2, 3 and 5, 6, respectively) is shown in panel D. Fragment sizes shown within parentheses are the exact sizes of the expected bands.

 

Southern Blot Analysis
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 (24) to Hybond-NX membrane (Amersham Biosciences), and subjected to hybridization (23) with a radiolabeled probe against the ung gene of M. smegmatis. The radiolabeled probe was made in the presence of [{alpha}-32P]dCTP by a PCR-based method using FP2 and RP3 primers (Table I, Fig. 4C).

Preparation of Cell-free Extracts and Ung Assays
Cell-free extracts were prepared from log phase cultures by sonication of M. smegmatis cells (23) or gentle lysis of E. coli and P. aeruginosa cells (25) and quantified (26). Uracil containing synthetic DNA (SSU9, Table I) was 5' end-labeled and used in Ung assays (27).

Detection of Ugi Expression
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 (18). 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 (15). Unlike Ung and most other cellular proteins, Ugi is thermostable and fully refolds to its native structure upon cooling (28), 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.

Analysis of Mutation Frequencies
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.

Effect of Acidified Nitrite on Bacterial Growth
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.

Infection of Macrophages with P. aeruginosa and M. smegmatis
Peritoneal macrophages were isolated from C57BL/6 mice and infected by P. aeruginosa and M. smegmatis as described (29, 30). 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 x 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 {gamma} (IFN{gamma}) 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 x 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.

Estimation of Nitric Oxide in Culture Supernatants
Nitric oxide secretion was determined by measuring the accumulation of nitrite (), 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. 31), incubated for 10 min at room temperature, and spectrophotometrically measured at 550 nm using an enzyme-linked immunosorbent assay reader.

Statistical Treatment
All experiments involving macrophage infections and nitric oxide estimation were statistically analyzed for significance using Student's t test.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (3235). 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.



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FIG. 1.
Sequence alignment of E. coli (Eco)-, P. aeruginosa (Pae)-, M. smegmatis (Msm)-, human (Hu), and herpes simplex virus (RSV) Ung proteins. The numbering scheme is according to EcoUng, and the filled and empty circles above this sequence indicate the side chain and main chain positions, respectively, involved in interaction with Ugi. The identical residues are depicted in a black background, whereas similar residues are shown in gray shade. The sequence alignments were done using PILEUP (GCG package), and BOXSHADE was used to obtain the shaded schematic representation (www.ch.embnet.org).

 

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+ (36). More recently, expression of Ugi in a chicken B cell line, DT40 rendered them Ung (37). 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 (15). 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 (19)) 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).



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FIG. 2.
Analysis of Ugi expression. Ugi expression was detected by its ability to sequester EcoUng (pI 6.6) into EcoUng·Ugi complex (pI 4.9), which can be separated on 15% native PAGE and probed with anti-EcoUng antibodies by immunoblotting. A, detection of Ugi expression in E. coli. Cell-free extracts (100 µg) were electrophoresed on native PAGE and probed by immunoblotting using anti-EcoUng antibodies (lane 1, EcoUng·Ugi marker; lanes 2–4, cell-free extracts from transformants harboring pTrc99c, pTrcUgi-oc36, or pTrcUgi plasmids, respectively. B, detection of Ugi expression in P. aeruginosa. Cellular extracts (200 µg) were thermally denatured at 90 °C for 30 min, and the supernatants were incubated with EcoUng (100 ng), separated on native PAGE, and detected by immunoblotting using anti-EcoUng antibodies. Lane 1, EcoUng·Ugi marker; lanes 2–4, cellular extracts from transformants harboring pBBR, pBBRUgi-oc36, and pBBRUgi, respctively. C, detection of Ugi expression in M. smegmatis. Total cell extract (300 µg) from the strain harboring pDKUgi as a single copy insertion at the L5 att site was heat-treated as above in B, and the supernatant was incubated with either no Ung (lane 2) or with EcoUng (lane 3, 50 ng; lane 4, 100 ng; lane 5, 200 ng), separated on native PAGE, and analyzed by immunoblotting using anti-EcoUng antibodies. Lane M is EcoUng·Ugi marker control.

 

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).



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FIG. 3.
Uracil excision activity in the total cellular extracts of various transformants. Ung assays were performed using the indicated amounts of total cell extracts of E. coli (A), P. aeruginosa (B), and M. smegmatis (C). The presence of various multicopy plasmids in E. coli and P. aeruginosa (panels A and B) or single copy insertion in the chromosome in M. smegmatis (panel C, lanes 3–8) is as indicated. Lanes 9–11 (panel C, ung::aph) represent cell extracts from a strain of M. smegmatis, wherein the ung gene has been knocked out with insertion of a kanamycin cassette (aph). Lanes with – and + symbols indicate controls wherein the uracil-containing substrate DNA was either not supplemented or supplemented with pure EcoUng (100 ng), respectively. The bands corresponding to product and substrate are indicated as P and S, respectively.

 

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 (38) 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) harboring a single copy of ugi gene in the chromosome, expression of Ugi resulted in ~5-fold increase in the appearance of RifR. Importantly, disruption of the ung gene in M. smegmatis in two independent isolates resulted in an increase of mutation frequency by ~8-fold over the control. On the other hand, under the same conditions, both the ung (ung::cat) or Ung (pTrcUgi) strains of E. coli showed an ~4.5-fold increase in appearance of RifR colonies over the vector alone or the pTrcUgi-oc36-harboring controls. These observations lend support to the hypothesis that for G+C-rich genome-containing bacteria, Ung is more crucial in maintaining genomic integrity. Furthermore, a similar increase in the mutation frequencies in both the ung (ung::cat) and Ung (pTrcUgi) strains (4.4 versus 4.5) of E. coli suggest that production of Ugi from multicopy vector resulted in complete sequestration of Ung.


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TABLE II
Effect of depletion of Ung on appearance of rifampicin resistance in P. aeruginosa, M. smegmatis, and E. coli

 

Effect of Acidified Nitrite on the Growth of P. aeruginosa and M. smegmatis—Because nitric oxide is known to enhance the rate of cytosine deamination, we examined the consequences of Ung deficiency in E. coli, P. aeruginosa, and M. smegmatis on tolerance to toxic effects of NaNO2 at acidic pH. Sodium nitrite itself is not toxic to bacteria. However, under acidic conditions, it is converted to nitrous acid, which decomposes to nitric oxide and other RNI (31, 39). The effect of NaNO2 on the growth of P. aeruginosa and M. smegmatis at different initial pH values of the media (7.2, 6.5, 6.0, and 5.5) are shown in Figs. 5, A and B, respectively. As is evident from the longer duration of the lag phases, acidification of culture media was inhibitory to growth of the bacteria. More importantly, as seen in Fig. 5A (+NaNO2), growth of P. aeruginosa (harboring pBBRUgi) at acidic pH of 6.5 and 6.0 was more compromised than that of the control cultures harboring either pBBRUgi-oc36 or the vector (pBBR) alone. The presence of NaNO2 at pH 5.5 was too toxic to P. aeruginosa. However, even at this pH, although the growth of Ung proficient bacteria (pBBR or pBBRUgi-oc36) was detectable, that of Ung (pBBRUgi) was not at all detectable.



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FIG. 5.
Growth curves. Fresh cultures for P. aeruginosa (A), M. smegmatis (B), and E. coli (C) were started with 0.1% inoculum from saturated cultures and grown at 37 °C in the absence (–NaNO2) or the presence (+NaNO2) of 1 mM (P. aeruginosa and E. coli) or 0.2 mM (M. smegmatis) sodium nitrite. The absorbance at 600 nm was monitored at regular intervals for the indicated times. The initial pH values of the LB (P. aeruginosa and E. coli) and LBT media (M. smegmatis) are as shown. The symbols used to represent various strains are as shown.

 

Like P. aeruginosa, at pH 6.5 and lower, the growth of the ung (ung::aph) and the Ung (L5att::pDKUgi) strains of M. smegmatis in the presence of NaNO2 was also less than that of the vector control (L5att::pDK20) (Fig. 5B, +NaNO2). Interestingly, the growth of ung M. smegmatis (ung::aph) strain was more compromised than the Ung strain (L5att::pDKUgi). The difference was more pronounced at lower pH values of 6.0 and 5.5. On the contrary, but consistent with a recent report that E. coli strains lacking Ung showed essentially similar resistance to nitric oxide (40), the growth curves in Fig. 5C reveal that the Ung-deficient strains of E. coli grew as well as the wild type strain in the presence of NaNO2. Not unexpectedly, however, at the low pH of 5.5, compared with the wild type strain, a marginal increase in the lag phase of ung (ung::cat) or Ung (pTrcUgi) strains of E. coli could be detected in the presence of NaNO2. Because all the growth profiles shown in Fig. 5 were consistent in repeat experiments, these data further highlight the importance of Ung in the G+C-rich organisms.

Effect of Abrogation of Ung Activity in P. aeruginosa and M. smegmatis on Survival in C57BL/6 Mouse Peritoneal Macrophages—Mouse macrophage models have been used earlier for infection with P. aeruginosa and M. smegmatis (29, 30). The macrophages respond with production of nitric oxide to encounter the infectious agents. Also, because this nitric oxide response is enhanced upon treatment of the macrophages with IFN{gamma}, we used this model system to investigate the effects of Ung phenotype on the fate of these bacteria in macrophages. Using such ex vivo assays, we repeatedly found that abrogation of Ung activity in P. aeruginosa and M. smegmatis resulted in their poor endurance in the macrophages. As represented by Fig. 6A, at the end of the incubation (90 min) of the infected macrophages, P. aeruginosa harboring pBBRUgi survived poorly (p < 0.05). In contrast, no significant changes were observed in the survival of the bacteria harboring vector alone (pBBR) or the mutant Ugi (pBBRUgi-oc36) under the same conditions (p > 0.05). Furthermore, as shown in Fig. 6B, induction of macrophages with IFN{gamma} resulted in more than a 2.5-fold decrease (p < 0.001) in total viable counts of the Ugi-containing bacteria as opposed to a much smaller decrease (p > 0.05) in the counts of those that harbored the vector alone or the mutant Ugi. Estimation of nitric oxide levels in the culture supernatants (Fig. 6, C and D) showed that the relative levels of nitric oxide production by the macrophages at any one given time point in the three infections was essentially the same for either set of infections (e.g. compare the levels of nitric oxide in culture supernatants of the macrophages infected with bacteria harboring pBBR, pBBRUgi-Oc36, or pBBRUgi at time 0 or 90 in Fig. 6, C or D), suggesting that it is the Ung phenotype of P. aeruginosa that resulted in its poor survival in mouse macrophages.



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FIG. 6.
Infection of mouse (C57BL/6) peritoneal macrophages with P. aeruginosa. A and B, total viable counts of various transformants of P. aeruginosa recovered from C57BL/6 mouse peritoneal macrophages unstimulated (panel A, –IFN{gamma}) or stimulated (panel B, +IFN{gamma}) with 1 unit/ml IFN{gamma} at 0 or 90 min post-infection. C and D, levels of NO in the culture supernatants of C57BL/6 mouse peritoneal macrophages, recovered from panels A and B, respectively. The mean ± S.D. of triplicate values are shown. **, p < 0.001; *, p < 0.05.

 

Similarly, as represented by Fig. 7, experiments with M. smegmatis resulted in differential recoveries of viable counts of the wild type control (L5att::pDK20) versus the ung (ung::aph) or the Ung (L5att::pDKUgi) strains. In the macrophages untreated with IFN{gamma} (Fig. 7A), the numbers of viable counts at 24 h increased by more than 3-fold in the control strain (L5att::pDK20), as opposed to 2-fold for the strains deficient in Ung (L5att::pDKUgi or ung::aph). However, the differences in the recoveries of these bacteria at 48 and 72 h were more significant (p < 0.05). Interestingly, in the IFN{gamma}-stimulated macrophages (Fig. 7B) these differences were further accentuated, whereas the numbers of wild type strain still increased by about 2.5-fold at 24 h and remained at this level until 48 h, the recoveries of Ung-deficient bacteria were significantly lower than the wild type strain (p < 0.001). Taken together, these data show that although the wild type strain of M. smegmatis multiplied 2–3-fold and remained at this level in the macrophages for up to 48 h, the Ung-deficient strains either showed nearly a complete abolition of multiplication (+IFN{gamma}) or multiplied poorly (–IFN{gamma}) and began to decline soon after 24 h.



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FIG. 7.
Infection of mouse (C57BL/6) peritoneal macrophages with M. smegmatis. Panels A and B, total viable counts of various strains of M. smegmatis recovered from C57BL/6 mouse peritoneal macrophages unstimulated (panel A, –IFN{gamma}) or stimulated (panel B, +IFN{gamma}) with 1 unit/ml IFN{gamma} at 0, 24, 48, and 72 h post-infection. Panels C and D, levels of NO in the culture supernatants of C57BL/6 mouse peritoneal macrophages, recovered from A and B, respectively. The mean ± S.D. of triplicate values is shown. **, p < 0.001; *, p < 0.05.

 

Also, as shown in Figs. 7, C and D, the nitric oxide levels of the culture supernatants of C57BL/6 mouse peritoneal macrophages after uptake of the bacterial strains at any one given time point in the three infections (L5att::pDK20; L5att::pDKUgi, and ung::aph) were essentially the same within either the IFN{gamma}-treated or untreated sets (e.g. compare the levels of nitric oxide in culture supernatants of the macrophages infected with bacteria harboring L5att::pDK20, L5att::pDKUgi, or ung::aph at time 0 or 24, 48 or 72 h in Fig. 7, C or D), suggesting that the difference in the viable counts is a consequence of abrogation of Ung activity in mycobacteria.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we observed that abrogation of Ung activity in P. aeruginosa and M. smegmatis, G+C-rich bacteria, confers upon them an increase in mutator phenotype by ~7 and 8-fold, respectively, relative to their wild type counterparts. Under similar assay conditions, depletion of Ung activity in both the ung and Ung strains of E. coli showed an increase of ~4.5-fold over the wild type control. These observations lend support to the hypothesis that Ung is more crucial for G+C-rich genome-containing bacteria. Although there are no earlier reports on the effects of depletion of Ung in P. aeruginosa and M. smegmatis or other G+C-rich organisms, several prior studies using E. coli have also reported an increase of ~2.4- to ~5-fold in the mutation rate in ung strains (7, 4143). However, it is important to note that such analyses are susceptible to assay conditions, especially when different reporter assays are used (7, 44). Therefore, to further study the effects of depletion of Ung in P. aeruginosa and M. smegmatis, we analyzed the effects of acidified nitrite, which generates nitric oxide and other RNI, on the growth of these bacteria. The DNA deamination ability of nitric oxide has been elegantly demonstrated both in vitro and in the cellular context (13). It has also been shown that nitric oxide is soluble in lipid and readily penetrates into bacteria to cause multitudes of macromolecular devastation (11, 12). Strikingly, as shown in Figs. 5, A and B, lack of Ung in P. aeruginosa and M. smegmatis resulted in severe growth retardation in the presence of RNI generated by acidification of NaNO2. In contrast, as shown in Fig. 5C, only a marginal effect leading to a slight increase in the lag phase (but not in the overall growth) was recorded for the growth of E. coli upon depletion of Ung (ung or Ung). Taken together, our studies espouse for a significant role for Ung in DNA repair in G+C-rich bacteria.

Ung has already been shown to be important for replication and reactivation of herpes simplex virus and poxvirus as well as in the life cycle of HIV-1 (5, 45, 46). Lack of Ung is also known to result in a strong bias toward transitional mutations in E. coli, Streptococcus pneumoniae, Saccharomyces cerevisiae, and more recently in detouring the hypermutation pathway in the antibody variable region in chicken (7, 8, 37, 47). Thus, uracil excision repair unequivocally represents a crucial DNA repair pathway.

P. aeruginosa and the mycobacterial species are important human pathogens, and the phagocytic cells of the host immune system such as macrophages play a crucial role in killing these pathogens. The reactive oxygen intermediates and RNI generated by the NADPH oxidase and the nitric oxide synthase pathways, respectively, play a major role in defense against bacteria and other pathogens (4850). The nitric oxide production by macrophages is stimulated upon treatment with cytokines such as IFN{gamma}. And even though M. smegmatis used in this study is a nonpathogenic saprophyte, it is widely used as a model organism for the pathogenic mycobacteria (10). Although neither of these bacteria survives in macrophages for long, the macrophage infection models are useful in studying gene functions in bacteria (51). Using such ex vivo assays, we reproducibly found that abrogation of Ung activity in P. aeruginosa and M. smegmatis results in their poor survival in macrophages (Figs. 6B and 7B). Furthermore, the studies using M. smegmatis presented us with an interesting observation. Although the wild type strain multiplied in the macrophages within 24 h, the strains abrogated for Ung activity failed to multiply to any significant extent. It is unclear if this feature of ung M. smegmatis physiologically resembles the observation made with human cytomegalovirus wherein disruption of UNG gene resulted in delayed DNA synthesis and longer replication cycle (6). More recently, in E. coli it was shown that DNA damage results in a replication checkpoint regulated by the uncleaved form of umuD (together with umuC). This allows the cell sufficient time for accurate repair before the error prone repair fostered by the cleaved form of umuD' and umuC as a part of SOS response (52). Hence, whether the absence of cell duplication in the Ung-deficient M. smegmatis in macrophages that we have observed is mechanistically similar and/or fostered by some other proteins related to E. coli umuC and umuD remains to be investigated.

Because a biochemical study (53) suggests weaker interaction between Ugi and M. smegmatis Ung (i.e. as compared with EcoUng), milder effects (Fig. 5, Table II) of Ung depletion seen in the strain (L5att::pDKUgi) expressing Ugi from a single copy gene as compared with the strain wherein ung gene was disrupted (ung::aph) are most likely because of some residual Ung activity in the L5att::pDKUgi strain. On the other hand, in the macrophage infection assays depletion of Ung activity either by expression of Ugi or by the disruption of ung gene resulted in identical phenotypes (Fig. 7). This observation suggests that Ugi (or a shorter peptide derived from it) when present in stoichiometrically larger amounts could actually be used to target mycobacterial Ung. Together with the observation that ung/ung mice showed no overt histopathological symptoms (9) targeting Ung may provide a new approach to controlling bacterial infections, particularly those where the causative agents are susceptible to RNI or other such host responses.

Interestingly, genome sequence analysis (54) of Mycobacterium tuberculosis, a pathogen that continues to cause a large number of casualties worldwide, has revealed that this organism may contain only three (base excision repair, nucleotide excision repair, and recombinational repair) of the major DNA repair pathways. The recA strain of M. bovis has been isolated and shown to be susceptible to DNA-damaging agents (55). Because our studies show Ung as a crucial DNA repair enzyme for G+C-rich organisms, it will be interesting to carry out macrophage infection studies using M. bovis and M. tuberculosis mutants deficient in Ung alone or in combination with recA and/or the uvr (uvrA, uvrB, or uvrC) genes. If such strains remain viable in vitro but fail to replicate in macrophages, these may allow development of novel strains useful in vaccine development for the dreaded disease caused by M. tuberculosis.


    FOOTNOTES
 
* This work was supported by research grants from Council of Scientific and Industrial Research, Department of Biotechnology, and Indian Council of Medical Research, New Delhi, India. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Joint first authors. Back

To whom correspondence should be addressed: Dept. of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560 012, India. Tel.: 91-80-293-2686; Fax: 91-80-360-2697; 91-80-360-0683; E-mail: varshney{at}mcbl.iisc.ernet.in.

1 The 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). Back


    ACKNOWLEDGMENTS
 
We thank Drs. C. Guilhot, Institut Pasteur, Paris, and A. K. Tyagi, University of Delhi, South Campus, New Delhi, for providing pPR27 and pDK20 vectors and our laboratory colleagues for suggestions on the manuscript.



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