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J Bacteriol. Mar 2011; 193(6): 1327–1333.
Published online Jan 14, 2011. doi:  10.1128/JB.01453-10
PMCID: PMC3067636

Gene Expression Profiling of Corynebacterium glutamicum during Anaerobic Nitrate Respiration: Induction of the SOS Response for Cell Survival [down-pointing small open triangle]

Abstract

The gene expression profile of Corynebacterium glutamicum under anaerobic nitrate respiration revealed marked differences in the expression levels of a number of genes involved in a variety of cellular functions, including carbon metabolism and respiratory electron transport chain, compared to the profile under aerobic conditions using DNA microarrays. Many SOS genes were upregulated by the shift from aerobic to anaerobic nitrate respiration. An elongated cell morphology, similar to that induced by the DivS-mediated suppression of cell division upon cell exposure to the DNA-damaging reagent mitomycin C, was observed in cells subjected to anaerobic nitrate respiration. None of these transcriptional and morphological differences were observed in a recA mutant strain lacking a functional RecA regulator of the SOS response. The recA mutant cells additionally showed significantly reduced viability compared to wild-type cells similarly grown under anaerobic nitrate respiration. These results suggest a role for the RecA-mediated SOS response in the ability of cells to survive any DNA damage that may result from anaerobic nitrate respiration in C. glutamicum.

Corynebacterium glutamicum is a nonpathogenic high-GC Gram-positive soil bacterium that has been widely used for industrial production of various amino acids and nucleic acids (21, 22). In addition, the species is of increasing interest as a model organism for the Corynebacterineae, a suborder of the Actinomycetes, which includes the medically important pathogenic species Corynebacterium diphtheriae and Mycobacterium tuberculosis (5, 41). C. glutamicum was long regarded as an aerobe because there were no reports of its growth by anaerobic respiration or fermentation, despite the fact that most other coryneform bacteria are known facultative anaerobes (35).

It is only recently that C. glutamicum was shown to grow anaerobically, using nitrate as a terminal electron acceptor (48, 57). This ability is attributed to the presence of a narKGHJI operon with high sequence similarity to the combination of the Escherichia coli narK gene and the narGHJI operon encoding a nitrate/nitrite transporter and components of the nitrate reductase complex, respectively (2). C. glutamicum narKGHJI expression is repressed by a novel type of transcriptional regulator, ArnR, under aerobic conditions (47). A shift to anaerobic nitrate respiration relieves the repression, resulting in elevated expression of the nar genes. An hmp gene, encoding a homologue of flavohemoglobin thought to be involved in nitric oxide (NO) detoxification in other bacteria (51), has been identified as an additional target of ArnR-mediated repression (47). This comparatively narrow ArnR function as an aerobic repressor in C. glutamicum is in stark contrast to the global oxygen-sensing E. coli regulator fumarate nitrate reductase regulator (FNR), which activates or represses the expression of many genes involved in anaerobic and aerobic metabolism, including those in nitrate respiration (20). It has been reported that C. glutamicum excretes several organic acids, including lactate, succinate, and acetate, as fermentative end products under oxygen-deprived conditions (12, 23). Transcriptome and metabolome analyses were recently carried out to examine the C. glutamicum response to oxygen deprivation (24, 45); however, it should be noted that cellular growth is arrested under these fermentation conditions (24). C. glutamicum gene regulation in response to anaerobiosis at a genome-wide level remains largely obscure.

In this study, we investigate a transcriptional profile of C. glutamicum during anaerobic growth in the presence of nitrate compared with that of aerobically growing cells using DNA microarray analysis. We note differences in the expression of many genes involved in a wide variety of physiological functions, including carbon metabolism and the respiratory chain, between aerobic and anaerobic respiratory conditions. Notably, many SOS genes are markedly upregulated under anaerobic nitrate respiratory conditions. The SOS response, an inducible DNA repair and damage tolerance system, has been intensively studied in E. coli (15). The regulatory mechanism is exerted by the widely conserved regulators RecA and LexA, whose mode of action (7, 8) ensures fine control of the system sensing DNA damage and inducing SOS gene expression (36). Although mutagenic effects caused by nitrosating agents derived from nitrate/nitrite metabolites have been well studied in bacteria (58, 63-65), it has not been reported that anaerobic nitrate/nitrite respiration triggers the SOS response. Here, as in the case of exposure to the DNA-damaging reagent mitomycin C (49), the elongated morphology of C. glutamicum cells was observed during anaerobic growth in the presence of nitrate. None of these transcriptional and morphological changes were observed in a recA mutant strain. Furthermore, the recA mutant exhibited significantly decreased viability under the anaerobic nitrate respiratory conditions. These results suggest that the SOS response is involved in survival of potential DNA damage derived from anaerobic nitrate respiration in C. glutamicum.

MATERIALS AND METHODS

Bacterial strains, media, and culture conditions.

C. glutamicum R wild type (67) and its recA mutant (49) were used in these experiments. C. glutamicum cells were precultured at 33°C overnight in nutrient-rich medium (A medium) (23) supplemented with 1% (wt/vol) glucose. The preculture was harvested by centrifugation, and the resulting cell pellets were washed once with mineral salts medium (BT medium) (23). For aerobic cultivation, the washed cells were subsequently grown at 33°C in 100 ml BT medium supplemented with 0.5% (wt/vol) glucose in a 500-ml flask with vigorous shaking (200 rpm). For anaerobic cultivation, anaerobic BT medium was prepared in advance by bubbling argon for 5 min. Cells from the same batch as those used for aerobic cultivation were subsequently transferred to the anaerobic BT medium in an anaerobic chamber (Coy Laboratory Products, Grass Lake, MI), where the gas composition was kept constant at 95% nitrogen and 5% hydrogen. Cells were grown at 33°C in 50 ml BT medium supplemented with 0.5% (wt/vol) glucose in a 50-ml medium bottle with gentle stirring. Potassium nitrate was added to a concentration of 30 mM wherever necessary. Kanamycin was used at 50 μg/ml for cultivation of the recA mutant strain.

DNA microarray analysis.

C. glutamicum cells were grown aerobically without nitrate or anaerobically with nitrate, and total RNA was extracted from the cells in the logarithmic growth phase with the RNeasy minikit (Qiagen, Germantown, MD) per the manufacturer's instructions. Residual DNA was removed by treating the preparation with RQ1 RNase-free DNase (Promega, Madison, WI). Fluorescent labeling of cDNA from the total RNA, hybridization with the C. glutamicum R whole-genome DNA microarray, and data manipulation were performed as described previously (24). The resultant raw data were expressed as average mRNA ratios of a total of six signals for each gene: there were duplicate spots for each gene on the microarray slide, and three sets of RNA samples from independent experiments were used. Genes that showed significantly altered mRNA levels (P value of <0.02 in a Student t test) by a factor of two or more were determined.

qRT-PCR.

C. glutamicum cells were grown to logarithmic phase aerobically without nitrate or anaerobically with nitrate. Total RNA was prepared as in DNA microarray analysis. Quantitative reverse transcription-PCR (qRT-PCR) analysis was performed using a Power SYBR green PCR Master Mix protocol (Applied Biosystems, Warrington, United Kingdom) according to the manufacturer's instructions with an ABI Prism 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA). Individual target genes were amplified with the primers listed in Table S1 in the supplemental material. A 50-ng total RNA fraction per well was incubated for 30 min at 50°C for reverse transcription, heated for 10 min at 95°C for initial PCR activation, and thereafter amplified for 40 cycles, with each cycle consisting of denaturation at 95°C for 15 s and annealing and extension at 60°C for 30 s. To account for any nucleic acid contamination, negative-control reactions without reverse transcriptase or template RNA were run. RT-PCR for each gene was performed from three independent RNA samples.

Light microscopy.

C. glutamicum cells were grown to logarithmic phase aerobically without nitrate or anaerobically with nitrate. The cells harvested from the cultures were observed under a microscope (AX70; Olympus, Japan).

Evaluation of cell growth and viability.

C. glutamicum cells were cultivated at 33°C in BT minimal medium supplemented with 1% glucose under aerobic conditions or anaerobic conditions with nitrate. Cell growth was monitored by measuring optical density at 610 nm using a spectrophotometer DU640 (Beckman Coulter, Brea, CA). The culture was harvested every other day (1st, 3rd, 5th, and 7th days). A dilution series of the culture was plated onto A medium agar supplemented with 1% glucose. The plates were incubated at 33°C for 24 h, and CFU were counted.

RESULTS

The gene expression profile of C. glutamicum differs between aerobic and anaerobic nitrate respiratory conditions.

A gene expression profile of C. glutamicum wild-type cells grown under anaerobic conditions in the presence of nitrate was compared to that of cells grown under aerobic conditions in the absence of nitrate using a DNA microarray. Transcript levels of 80 genes were comparatively more than 2-fold higher at the significance level of 2% under anaerobic conditions (see Table S2 in the supplemental material), whereas those of 151 genes were more than 2-fold lower (see Table S3). Enhanced expression of the narKGHJI and hmp genes was observed under anaerobic nitrate respiratory conditions compared with aerobic conditions (Table (Table1),1), confirming the expected release of aerobic repression of the two transcriptional units by the ArnR regulator (47). Differential expression of many genes involved in a wide variety of physiological functions was observed.

TABLE 1.
C. glutamicum genes involved in carbon metabolism and respiratory chain showing altered expression under anaerobic nitrate respiratory conditions compared with aerobic conditions

Anaerobic nitrate respiration impacts expression of genes involved in carbon metabolism and the respiratory chain.

Expression of several genes involved in carbon metabolism varied between anaerobic conditions in the presence of nitrate and aerobic conditions as determined from DNA microarray analysis (Table (Table1).1). The transcript levels of some of these genes were additionally evaluated by quantitative RT-PCR (qRT-PCR) analysis. While the expression of the ldhA gene encoding fermentative l-lactate dehydrogenase (LDH) was significantly upregulated during anaerobic growth in the presence of nitrate, several genes encoding tricarboxylic acid (TCA) cycle enzymes, e.g., gltA, icd, odhA, and fum, were downregulated under the same conditions. Similar differential expression of the same genes, to similar magnitudes, was also observed under oxygen-deprived conditions in the absence of nitrate in a previous study (24). However, differences in transcript levels of the gapA-pgk-tpi-ppc gene cluster, which is involved in glycolytic and anaplerotic pathways, between the two conditions were relatively small, in contrast to the markedly induced expression of the same genes previously observed under oxygen-deprived conditions in the absence of nitrate (24).

In addition to the narKGHJI genes for nitrate respiration, several genes involved in the respiratory electron transport chain were sensitive to anaerobic nitrate respiratory conditions (Table (Table1).1). The transcript levels of the cydABDC gene cluster, encoding cytochrome bd oxidase with high oxygen affinity and a putative transporter, were upregulated during anaerobic growth in the presence of nitrate. In contrast, the ndh gene encoding NADH dehydrogenase was expressed at a lower level under the same conditions. Increased expression of the cgR_2818-lldD operon, encoding a putative transporter and respiratory LDH that can use menaquinone as an electron acceptor, was observed under anaerobic nitrate respiratory conditions.

SOS gene expression is enhanced during anaerobic nitrate respiration.

Interestingly, expression of many SOS genes was higher under anaerobic conditions in the presence of nitrate than under aerobic conditions. The DNA microarray data showed that out of 48 genes belonging to the C. glutamicum SOS regulon (28), the transcript levels of 17 were greater under anaerobic nitrate respiratory conditions than under aerobic conditions (Table (Table2),2), although the induction level of most of these genes was lower than that previously observed both with a lexA mutant and in the presence of mitomycin C (28). Both lexA and recA genes encode regulatory proteins that play a central role in the SOS response, and both are members of the SOS regulon themselves (15, 37). Other notable upregulated genes include ruvC, recN, and uvrA genes, involved in nucleotide excision and recombination repair (62), and divS, which encodes a novel cell division suppressor (49) and forms an operon with nrdR, which in turn codes for a regulator of deoxyribonucleotide biosynthesis (5). dnaE2 encodes a putative DNA polymerase subunit, a homologue of which is known to be regulated by LexA in M. tuberculosis (10). Notably, the induction levels of the recN gene and the divS-nrdR operon under anaerobic nitrate respiratory conditions were particularly high. A similar situation was observed in the previous lexA disruptant and mitomycin C study (28). In contrast, the transcript levels of four SOS genes, cgR_0465, cgR_0466, cgR_0846, and cgR_1245 (putP), were downregulated under anaerobic nitrate respiratory conditions to less than half their levels under aerobic conditions. Among these genes, expression of cgR_0846 and putP, encoding a proline/sodium symporter (50), is reported to be activated by LexA (28). To confirm the DNA microarray data, the expression levels of 7 representative SOS genes, i.e., lexA, recA, uvrA, ruvC, recN, and the divS-nrdR operon, were evaluated by qRT-PCR analyses (Fig. (Fig.1).1). As expected, all 7 in wild-type cells were more strongly induced under anaerobic nitrate respiratory conditions than under aerobic conditions.

FIG. 1.
Relative transcript levels of the SOS genes determined by qRT-PCR. The mRNA levels of the SOS genes (uvrA, recN, ruvC, nrdR, divS, lexA, and recA) from the C. glutamicum wild-type (white and dark gray bars) and the recA mutant (light gray and black bars) ...
TABLE 2.
C. glutamicum SOS genes showing altered expression under anaerobic nitrate respiratory conditions compared with that under aerobic conditions

To examine the involvement of RecA in the increased expression of these SOS genes during anaerobic growth in the presence of nitrate, the expression levels of 6 of the 7 genes above (besides recA) in a recA mutant strain were analyzed by qRT-PCR (Fig. (Fig.1).1). In the recA mutant strain, expression of all 6 genes under anaerobic nitrate respiratory conditions was reduced to a level equal to or less than that in the wild-type strain under aerobic conditions. This observation clearly suggests that the RecA-dependent SOS response occurs when the cells are transferred from aerobic conditions to anaerobic nitrate respiratory conditions.

Anaerobic growth in the presence of nitrate induces abnormal elongation of C. glutamicum cells.

During their SOS response, E. coli and Bacillus subtilis cells elongate without septation and exhibit a filamentous morphology (19, 39). C. glutamicum cells show a different, RecA-dependent elongated morphology following DNA damage induced by mitomycin C treatment (30, 49). To further examine the SOS response during anaerobic growth in the presence of nitrate, C. glutamicum wild-type and recA mutant cells grown under aerobic or anaerobic nitrate respiratory conditions were observed under a microscope. Wild-type cells were approximately 2 to 3 times longer under anaerobic nitrate respiratory conditions than were aerobically grown cells (Fig. (Fig.2).2). This elongated morphology was significantly suppressed in a recA mutant, in agreement with previous observations in mitomycin C-treated cells (49). Cells over 3 μm in length, rarely observed in aerobically grown wild-type cells (3% of more than 250 cells measured), comprised 76% of cells grown under anaerobic nitrate respiratory conditions. In contrast, the frequency of elongated cells in the recA mutant was 23% under anaerobic nitrate respiratory conditions, although it is unclear why this frequency is still higher than that of aerobically grown elongated wild-type cells. These observations in combination with the transcriptional data strongly suggest that the RecA-dependent SOS response is induced during anaerobic nitrate respiration in C. glutamicum.

FIG. 2.
Elongated morphology of C. glutamicum cells during anaerobic growth in the presence of nitrate. Cells were cultured under aerobic conditions (+O2) and anaerobic nitrate respiratory conditions (−O2 +NO3). Differential interference ...

RecA influences long-term viability of C. glutamicum under anaerobic nitrate respiratory conditions.

Mutants with defective SOS induction show ultrasensitivity to DNA-damaging agents or UV irradiation (16, 55). recA mutant cells of C. glutamicum exhibit considerable reduction of viability upon mitomycin C treatment (49). Here, effects of recA mutation on growth and viability of C. glutamicum during cultivation under anaerobic nitrate respiratory conditions were examined. The recA mutant growth followed almost a normal growth curve during 24-h cultivation (data not shown). CFU of the wild-type strain and the recA mutant strain were counted after entry into stationary phase during cultivation under aerobic conditions as well as under anaerobic nitrate respiratory conditions (Fig. (Fig.3).3). Under aerobic conditions, the two strains showed similar viabilities over the course of the cultivation, with an approximately 2-log decrease in CFU count from day 1 to day 7. Under anaerobic nitrate respiratory conditions, recA mutant strain CFU count decreased much more rapidly than that of the wild-type strain, probably due to accumulation of DNA damage caused by toxic nitrate/nitrite metabolites: from day 1 to day 7, the recA mutant strain exhibited approximately a 4-log decrease in CFU count whereas the wild-type strain exhibited only a 1- to 2-log decrease in CFU count. These results are evidence that RecA is involved in the long-term viability of C. glutamicum under anaerobic nitrate respiratory conditions.

FIG. 3.
Viability of C. glutamicum cells cultured under anaerobic nitrate respiratory conditions. CFU of the wild-type (closed symbols) and the recA mutant (open symbols) strains were counted under aerobic (circles) or anaerobic (triangles) nitrate respiratory ...

DISCUSSION

Properties of shift between aerobic and anaerobic metabolic pathways in C. glutamicum.

This study revealed that in addition to the predictable upregulation of the narKGHJI operon essential for nitrate respiration, cydABDC and ldhA are also markedly upregulated in C. glutamicum cells grown under anaerobic nitrate respiratory conditions compared to aerobically grown cells. cydAB encodes cytochrome bd oxidase, which exhibits higher oxygen affinity than does the other cytochrome bc1-aa3 oxidase supercomplex (4). While the latter terminal oxidase complex is of major importance in the aerobic respiratory chain in C. glutamicum, the former alternative menaquinol oxidase of the bd type supposedly plays an important role under low oxygen tension. l-Lactate dehydrogenase encoded by ldhA is a key enzyme that couples production of l-lactate, a major fermentation product, to reoxidation of NADH formed during glycolysis under oxygen limitation (23). In contrast, expression of a respiratory NADH dehydrogenase gene, ndh (4), and the TCA cycle genes gltA and odhA (3, 13) was markedly repressed under anaerobic nitrate respiratory conditions. This aerobic-anaerobic shift in the gene expression profile is similar to the situations observed in other bacteria (20, 66). It is noteworthy that, in E. coli and B. subtilis, nitrate is the preferred electron acceptor under anaerobic conditions and acts to repress synthesis of other anaerobic pathways (9, 26, 29, 33, 34). E. coli cydAB exhibits higher expression under microaerobic conditions than under anaerobic conditions (6). It is likely that the fine regulation in response to anaerobiosis contributes to efficient energy generation along with balancing of the cellular redox state in these bacteria. In C. glutamicum, upregulation of several key enzymes of glycolysis due to low oxygen tension, which we reported in a previous study (24), seems to be curtailed to some extent under the anaerobic nitrate respiratory conditions in this study, although it should be noted that unique conditions prevailing in the previous study include arrested cellular growth and absence of nitrate (24).

In contrast to the above-noted differences in the expression of glycolytic genes, the induction levels of ldhA and cydABDC in this study are comparable to those under oxygen-deprived conditions in the absence of nitrate in the previous study (24). It has recently been found that transcription of the C. glutamicum ldhA gene is directly regulated by a sugar-responsive regulator, SugR (11, 14, 60), and an l-lactate-responsive regulator, LldR (59). However, for gene regulation mechanisms in response to anaerobiosis, no oxygen and/or redox-sensing global regulators similar to ResDE, Fnr, ArfM, Rex, and ArcAB, which coordinately control anaerobic respiration and fermentation genes in E. coli and B. subtilis (9, 20, 27, 33, 52), have been identified in C. glutamicum. Instead, our previous DNA microarray data revealed that in C. glutamicum, aerobic repression by a novel transcriptional regulator, ArnR, is limited to the narKGHJI operon and a homologue of the hmp gene involved in detoxifying nitric oxide in other bacteria (47). It is interesting that GlxR, a Crp/Fnr-type global transcriptional regulator, binds to the promoter regions of the narKGHJI operon and the ldhA gene in a cyclic AMP (cAMP)-dependent manner (31), but GlxR was recently shown to activate expression of the narKGHJI operon in not only anaerobically growing cells but also aerobically growing cells (46). Further studies are needed to elucidate the mechanism of genome-wide regulation of fermentative and respiratory pathways in C. glutamicum.

A characteristic feature observed in this study is marked induction of expression of lldD under anaerobic nitrate respiratory conditions. lldD encodes a membrane-bound, menaquinone-dependent lactate dehydrogenase essential for growth on l-lactate (4, 53), while ldhA encodes a cytoplasmic NAD-dependent lactate dehydrogenase responsible for l-lactate formation (23). The induction of lldD along with ldhA and narKGHJI may imply that coupling of LdhA and LldD reactions is involved in electron transfer from NADH to menaquinone via l-lactate, and the reduced menaquinone is utilized as an electron donor to reduce nitrate to nitrite by nitrate reductase. Similarly, electron transfer from NADH to menaquinone via coupling of malate dehydrogenase and malate:quinone oxidoreductase reactions has been proposed in a strain carrying a mutation in the ndh gene encoding NADH dehydrogenase (42, 44). It is noted that ndh is downregulated under the anaerobic nitrate respiratory conditions, as described earlier. Expression of C. glutamicum lldD and ldhA is repressed by a common regulator, LldR (17, 18, 59), which may contribute to efficient electron transfer for energy generation under the conditions. However, the induction of lldD was not observed under oxygen-deprived conditions in the absence of nitrate in our previous study (24). Therefore, it is possible that another unidentified regulator is also involved in lldD expression in response to the presence of nitrate under anaerobic conditions. In contrast, E. coli lldD expression is strongly repressed by a redox-sensing two-component regulatory system, ArcAB, under anaerobic conditions in the presence and absence of nitrate (25).

Induction of the SOS response under anaerobic nitrate respiratory conditions.

We here particularly focused on the finding that induction of the SOS response was detected under anaerobic nitrate respiration. Several representative genes belonging to the SOS regulon (28) were significantly upregulated during anaerobic growth in the presence of nitrate, and the induction was fully dependent on RecA. It is likely that the RecA-dependent cell elongation observed under anaerobic nitrate respiration is attributed to the most inducible cell division suppressor, DivS, as previously shown with mitomycin C-treated cells (49). The RecA-LexA-dependent induction of the SOS genes has been extensively studied in E. coli treated with UV irradiation or DNA-damaging reagents such as mitomycin C (15). It has been established that the system is induced in response to various environmental conditions, such as oxidative stress, starvation, inhibition of cell wall synthesis, and high-pressure stress (15). Therefore, the SOS response is thought to function as an important bacterial system in adaptation, mutation, and thus diversification under a broad range of environmental conditions.

Nitrate respiration is an important physiological process that allows bacteria to generate sufficient energy to permit anaerobic growth. Two dissimilation pathways of nitrate respiration, ammonification and denitrification, involve formation of a common intermediate, nitrite, but end in different products, ammonia and dinitrogen, respectively (43, 68). During nitrate or nitrite metabolism, mutagenic reactive nitrogen species (RNS) such as nitric oxide (NO) and its derivatives are formed endogenously (51). Therefore, optimization of the relevant metabolic pathways along with RNS detoxification systems, various types of which are identified in bacteria (51), is important to avoid the damaging effects. Nitrosative alkylation or deamination on DNA is known to occur during nitrate or nitrite metabolism in the E. coli mutants defective in the repair system (58, 63). Expression of the E. coli sulA gene, which is involved in the inhibition of cell division in the SOS response, is induced in nonaerated cells upon treatment with alkylating agents (61). The SOS response is also induced by adding physiological NO donors like dinitrosyl iron complexes (DNIC) with thiol and S-nitrosothiols (RSNO) in E. coli (38, 54). However, to our knowledge, induction of the SOS response triggered by an endogenous inducer during anaerobic nitrate respiration in bacteria has not yet been reported.

It is noteworthy that C. glutamicum lacks the ability of nitrite reduction and excretes nitrite at an almost equimolar ratio to nitrate consumed (48), in contrast to many other nitrate-reducing bacteria possessing an ammonification or denitrification pathway (43, 68). Actually, nitrate cannot be utilized as the sole nitrogen source for either aerobic or anaerobic growth of C. glutamicum (48, 57). These traits may lead to accumulation of DNA-damaging RNS during nitrate respiration and induce the SOS response, although this bacterium possesses the putative NO-detoxifying flavohemoglobin Hmp. Induction of lactate production pathways during nitrate respiration, as described earlier, may also be involved in the induction of the SOS response because acidification is known to enhance the generation of toxic reactants from nitrite (32). Here, we showed that inactivation of the SOS response by the recA mutation results in a significant reduction of prolonged anaerobic survival in the presence of nitrate, suggesting that the SOS response plays an important role in tolerance and survival of potential nitrosating agents generated from anaerobic nitrate respiration in C. glutamicum.

It is interesting that the signal transduction of the induction of the SOS response seems to be more diverse depending on the situation than previously thought (15). It was recently shown that the SOS system is induced in response to β-lactam-induced cell wall stress through the DpiBA two-component signal transduction system (40). Induction of the SOS response dependent on the intracellular cAMP level was reported in starved E. coli cells (56). It was also found that high pressure induced the SOS response by triggering activation of the cryptic type IV restriction endonuclease Mrr, resulting in DNA double-strand breaks (1). The transcriptome analyses of this study showed that the SOS genes upregulated during anaerobic nitrate respiration constitute part of a large number of the genes in the SOS regulon. We also showed that exposure to NO-donating agents under anaerobic conditions markedly induces expression of the hmp gene but not that of the SOS genes (data not shown). Various RNSs and their derivatives along with some other exogenous and/or endogenous factors may be cooperatively involved in the RecA-LexA-dependent induction of the SOS genes during nitrate respiration. Further study is needed to understand the homeostasis of RNS and its relation to the induction mechanism of the SOS response during anaerobic nitrate respiration in C. glutamicum.

Supplementary Material

[Supplemental material]

Acknowledgments

We thank Crispinus A. Omumasaba (Research Institute of Innovative Technology for the Earth) for critical reading of the manuscript.

This study was partially supported by a grant from the New Energy and Industrial Technology Development Organization (NEDO), Japan.

Footnotes

[down-pointing small open triangle]Published ahead of print on 14 January 2011.

Supplemental material for this article may be found at http://jb.asm.org/.

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