The Mycobacterium marinum mel2 locus displays similarity to bacterial bioluminescence systems and plays a role in defense against reactive oxygen and nitrogen species
© Subbian et al; licensee BioMed Central Ltd. 2007
Received: 01 September 2006
Accepted: 19 January 2007
Published: 19 January 2007
Mycobacteria have developed a number of pathways that provide partial protection against both reactive oxygen species (ROS) and reactive nitrogen species (RNS). We recently identified a locus in Mycobacterium marinum, mel2, that plays a role during infection of macrophages. The molecular mechanism of mel2 action is not well understood.
To better understand the role of the M. marinum mel2 locus, we examined these genes for conserved motifs in silico. Striking similarities were observed between the mel2 locus and loci that encode bioluminescence in other bacterial species. Since bioluminescence systems can play a role in resistance to oxidative stress, we postulated that the mel2 locus might be important for mycobacterial resistance to ROS and RNS. We found that an M. marinum mutant in the first gene in this putative operon, melF, confers increased susceptibility to both ROS and RNS. This mutant is more susceptible to ROS and RNS together than either reactive species alone.
These observations support a role for the M. marinum mel2 locus in resistance to oxidative stress and provide additional evidence that bioluminescence systems may have evolved from oxidative defense mechanisms.
Mycobacteria appear to have numerous molecular pathways responsible for their inherent resistance to reactive oxygen species (ROS) [1–3]. In most bacteria, oxidative stress induces a global regulator, OxyR, that induces detoxifying enzymes such as alkyl hydroperoxide reductase (AhpC) and catalase/hydroperoxidase I (KatG) [4, 5]. During normal aerobic metabolism bacteria produce superoxide (O2 •-) that is converted to hydrogen peroxide (H2O2) and oxygen (O2) by superoxide dismutase and H2O2 is converted to water (H2O) and O2 by KatG  or AhpC . The two superoxide dismutase (SOD) genes present in mycobacteria, sodA and sodC, have been suggested to play a role in resistance to ROS. A sodC mutant is more susceptible to ROS, including hydrogen peroxide (H2O2), and displays a defect in growth within activated macrophages [8, 9]. The sodA gene has been down-regulated by antisense methods, resulting in increased sensitivity to H2O2 . Mycobacteria also express a catalase, KatG, that affects resistance to ROS produced by NADPH oxidase activity in activated macrophages . Other pathways must play an important role in resistance of M. tuberculosis to oxidative stress because oxyR is inactive , katG is absent or mutated in numerous human clinical isolates [12–16] and ahpC is expressed at very low levels [17, 18].
Similar to ROS, there are several pathways involved in mycobacterial resistance to reactive nitrogen species (RNS), including noxR1, noxR3 [19, 20], dlaT , msrA [22, 23], cysH , DNA repair, protein degradation in the proteasome and flavin cofactor synthesis . In addition to its role in resistance to ROS, the mycobacterial ahpC is also involved in resistance to the RNS peroxynitrite, but not nitric oxide . Peroxynitrite is produced by SOD in the presence of H2O2 and nitric oxide, linking these two important mechanisms of oxidative stress-mediated cell death . This observation may help to explain the inherent resistance of M. tuberculosis to peroxynitrite as compared to less pathogenic mycobacteria .
Bioluminescence systems can protect cells against ROS [28–32] through a catalase-like reaction between the electron donating ROS and oxidized luciferase-bound flavin mononucleotide, producing water and light . The similarity of luciferases to oxidases  suggests that bioluminescence systems could have evolved from oxygen defense mechanisms . During genetic analysis of factors that affect macrophage infection, we identified the M. marinum mel2 locus, which displays similarity to lux genes involved in bioluminescence . In the current study, more detailed analysis of the genes in the mel2 locus suggests functional similarity between mel2 and bioluminescence systems. Based on this similarity, we asked whether the M. marinum mel2 locus is involved in resistance of mycobacteria to oxidative stress. We constructed an M. marinum mutant that carries a transposon insertion in the first gene in the mel2 locus, melF, by allelic exchange and demonstrated that this mutant displays increased susceptibility to both ROS and RNS. Since this mutation may have polar effects on downstream genes, we complemented this mutant with two constructs, one that carries the melF gene alone and another with the entire mel2 locus. The melF mutant defect is partially complemented by melF alone, but fully complemented by the entire mel2 locus. We recently found that the mel2 mutant displays a defect for growth in activated macrophages that is alleviated by the presence of either ROS scavengers or nitric oxide synthase inhibitors , suggesting that the mel2 mutant is more susceptible to ROS and RNS than wild type bacteria. The data obtained in the current study support and extend these observations through demonstration that the mel2 locus plays a role in susceptibility to several different compounds that produce ROS and RNS in laboratory media. Our results indicate that the M. marinum mel2 locus is the first of a newly identified class of genes with similarity to bioluminescence genes involved in resistance to both ROS and RNS.
Similarity of the genes in the mel2 locus to bioluminescence genes
M. marinum mel2 mutant and complementing strains
The mycobacterial mel2 locus affects susceptibility to ROS
The mel2 locus affects susceptibility to RNS
The mel2 locus affects susceptibility to the combination of ROS and RNS
The molecular mechanisms of mycobacterial resistance to ROS and RNS have been an area of intense investigation and suggest that there are multiple pathways involved in resistance . In the current study, we identified a novel set of genes in the mel2 locus that play a role in resistance to both ROS and RNS. As shown in our previous studies, this locus is also important for survival in activated macrophages and virulence in the mouse footpad model of infection . To the best of our knowledge, this is the first description of a mycobacterial pathway that impacts susceptibility to both of these reactive species. Since RNS and ROS are linked through the production of peroxynitrite from nitric oxide and superoxide [1, 46, 47], the mel2 system may be specifically involved in resistance to this reactive species. The presence of the mel2 locus in the tuberculosis complex and M. marinum  and absence in avirulent mycobacteria that are more susceptible to peroxynitrite  supports this concept.
The similarity of the mel2 locus to bioluminescence systems at the amino acid level and the presence of conserved domains between them are intriguing observations. These data are particularly interesting in light of the recent observations that bioluminescent systems can protect cells against oxidative stress [28–32]. In search of a biological role for bioluminescence in bacteria that would explain how such an energy-consuming system could have developed evolutionarily, it has been proposed that these pathways protect against ROS generated in an aerobic atmosphere [35, 54]. Interestingly, it has been observed that ROS play a pivotal role in host-symbiont interactions with bioluminescent bacteria . At present, our model for the biochemical function of mel2 (Figure 2B) is purely hypothetical and is in need of more experimental support, but the large number of conserved functional domain similarities between the lux and mel2 loci suggests that they may have related functions. However, it seems unlikely that this function is bioluminescence, since mycobacteria are not normally bioluminescent and we did not observe any bioluminescence associated with our mutant or complemented strains (data not shown). Our observation that the mel2 locus plays a role in resistance to ROS helps to explain the presence of loci similar to bioluminescence genes in non-luminescent bacterial pathogens.
The inherent resistance of M. marinum to ROS is impacted by a mutation in the mel2 locus. This observation suggests that mel2 has an important role in either directly scavenging oxygen radicals or repairing damage caused by them. Since the mel2 mutant affects susceptibility to H2O2 and the organic peroxides cumene hydroperoxide and t-BOOH, which generate alkoxyl radicals, peroxyl radicals and H2O2 [44, 45], it is unclear whether mel2 is specific to a particular type of ROS. The apparent absence of specificity could be the result of this pathway utilizing an unknown oxidizable substrate that is recycled, similar to luciferin in bioluminescent systems , direct scavenging of H2O2, which all three compounds produce, or repair of damaged DNA, proteins or lipids . Interestingly, luciferase can produce light using H2O2 alone, in the absence of luciferin, suggesting that luciferase can scavenge H2O2, superoxide and hydroxyl radicals . Overall, these data suggest that MelF functions as a FMN-dependent non-heme catalase. The presence of the mel2 locus in pathogenic mycobacteria may at least partially explain why the catalase (katG) gene can be mutated during acquisition of isoniazid resistance , yet katG negative M. tuberculosis are responsible for numerous clinical infections in humans [12–16]. Since oxidative stress increases susceptibility of mycobacteria to isoniazid , it is possible that in some cases there is a relationship between isoniazid susceptibility and the mel2 locus. This possibility can be tested by comparing the effects of a double and single katG and mel2 mutants on virulence and isoniazid resistance.
The role of bioluminescence systems from other bacteria in resistance to RNS has not been examined, but our observations with mel2 suggest that this possibility is worth investigating. Since susceptibility to both SNAP and acidified NaNO2 are impacted by the mel2 mutation, this phenotype is not the result of greater susceptibility to the acidic pH used with NaNO2. The fact that the mel2 mutant displays an obvious defect when exposed to a combination of both ROS and RNS would imply that this locus is important for growth in environments where both of these reactive species are present, such as during infection of mammals. We found that the M. marinum luxA homologue, melF, may play an important role in resistance to both RNS and ROS, since this gene alone can partially complement what may be a polar mutation. Alternatively, this observation could be the result of low levels of expression of the remainder of genes within mel2, as a result of the polar mutation. This polar mutation would allow only low levels of the putative Mel2 protein complex to be formed and provide partial complementation once a functional melF gene is expressed. A better understanding of the biochemical roles of each of the mel2 genes and their importance in susceptibility to ROS and RNS will require analysis of each gene individually as well as in the presence or absence of each of the different Mel2 components.
In this study, we confirmed that the mel2 locus plays a role in the susceptibility of M. marinum to ROS and RNS. Although this locus displays similarity to bioluminescent systems in other bacterial species, further biochemical studies are necessary to demonstrate the functional significance of the conserved domains that are present. These observations suggest that mel2 represents a previously unrecognized pathway for resistance of bacterial pathogens to ROS and RNS and support the concept that bioluminescence systems may have evolved from oxidative stress defense mechanisms.
Strains and growth conditions
M. marinum strain M, a clinical isolate obtained from the skin of a patient , was used in these studies. M. marinum strains were grown at 33°C in 7H9 broth (Difco, Detroit, Mich.) supplemented with 0.5% glycerol, 10% albumin-dextrose complex (ADC) and 0.25% Tween 80 (M-ADC-TW) for 5 days. M. smegmatis strain mc2155  cultures were grown in M-ADC-TW for 3 days at 37°C and M. tuberculosis strain Erdman (ATCC35801) cultures were grown in M-ADC-TW for 10 days at 37°C. The number of viable bacteria was determined for each assay using the LIVE/DEAD assay (Molecular Probes, Eugene, OR.) and by plating dilutions for colony forming units (cfu) on 7H9 (M-ADC) agar (Difco, Detroit, Mich.). All inocula used were > 99% viable. E. coli strains were grown in Luria-Bertani (LB, Difco) media at 37°C. Where appropriate, kanamycin was added at a concentration of 25 μg/ml (E. coli) or 10 μg/ml (M. marinum).
Construction of M. marinum mel2 mutant and complementing strains
The M. marinum mel2 mutant carries a mini-Mu transposon insertion near the amino terminus of the melF gene as described previously . Our previous studies have found no functional differences between the M. tuberculosis and M. marinum mel2 loci, both confer wild type host cell infection and growth in macrophages to the M. marinum melF insertion mutant [36, 37], so either can be used for complementation studies. The M. marinum melF::pJDC79 strain is the melF mutant that carries the plasmid pMV262  expressing the melF gene from M. tuberculosis that has been previously shown to complement the macrophage infection defect of the M. marinum mel2 mutant . The M. marinum melF::pJDC75 strain is the melF mutant that carries the single-copy integrating plasmid pYUB178  with the entire M. tuberculosis mel2 locus cloned into its single NheI site. Construction of all strains was confirmed by Southern analyses and PCR as described previously .
Sequence (5' -> 3')
In silico analysis of the melF-melK genes
Detailed analysis of the amino acid sequence of MelF-MelK was carried out initially using protein-protein National Center for Biotechnology Information (NCBI) BLAST  and Conserved Domain Search  as described previously . Once motifs of interest were identified, they were compared to the appropriate bioluminescence genes and the mel2 gene and homologues were aligned and dendograms constructed using MegAlign (DNASTAR). Domain scores were considered significant if greater than 150 and the expectation values were less than 1 × 10-10.
Susceptibility to reactive oxygen species
Mycobacterial strains were exposed to ROS generated by H2O2, cumene hydroperoxide and tert-butyl hydroperoxide (t-BOOH). The susceptibility of mycobacteria to these compounds was determined by treatment for various periods of time at the appropriate growth temperature for the mycobacterial strain used and plating dilutions on M-ADC agar to determine CFU at each time point as compared to the original inoculum (To), i.e. percent survival = (CFU Tx/CFU To) × 100. Dimethyl sulfoxide (DMSO) was used as a solvent for t-BOOH and was tested for effects on viability of all mycobacterial strains and no solvent affected mycobacterial viability during the time periods examined or at the final concentrations used.
Susceptibility to reactive nitrogen species
Mycobacterial strains were exposed to RNS generated by S-nitroso-N-acetyl penicillamine (SNAP) and acidification of sodium nitrite (NaNO2) to pH 5.2 for various periods of time. Susceptibility was determined in the same manner as that described for ROS. DMSO was used as a solvent for SNAP and had no effects on viability of mycobacteria at the concentrations and time periods used.
All experiments were carried out in triplicate and repeated at least three times. The significance of the results was determined using the Student t-test. P values of < 0.05 were considered significant.
This work was supported by grant AI47866 from the National Institutes of Health. We thank Drs. David McMurray and James Samuel for critical review of this manuscript.
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