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Journal of Bacteriology, September 2006, p. 6415-6418, Vol. 188, No. 17
0021-9193/06/$08.00+0     doi:10.1128/JB.00557-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Essential Role of Flavohemoglobin in Long-Term Anaerobic Survival of Bacillus subtilis

Michiko M. Nakano^*

Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health and Science University, 20000 NW Walker Road, Beaverton, Oregon 97006

Received 19 April 2006/ Accepted 3 June 2006

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A Bacillus subtilis culture incubated anaerobically in nitrate-containing medium lost viability during the first 3 days but recovered thereafter. A flavohemoglobin mutant showed very poor survival under these conditions unless the cells were prevented from carrying out nitrate respiration.

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Bacillus subtilis responds to nutritional starvation by forming dormant spores, which ensures a long-lasting capability to resume growth upon encountering favorable conditions. B. subtilis strain 168 grows under anaerobic conditions, but it sporulates poorly when oxygen levels are low (8). Therefore, we investigated whether the bacterium is able to survive over prolonged periods under anaerobic conditions.

Flavohemoglobin is essential for long-term anaerobic survival in the presence of nitrate. B. subtilis grows anaerobically, using nitrate as a terminal electron acceptor (4). In the absence of nitrate, it undergoes fermentation to generate ATP through glycolysis. Anaerobic fermentation of B. subtilis requires glucose and pyruvate (13). In the experiments described here, we filled tubes with aliquots of a cell suspension (starting optical density at 600 nm [OD₆₀₀] of 0.02). In these cultures, cells gradually consume the remaining oxygen during incubation, and transcription of anaerobically induced genes is activated after 3 h. All strains used in this study are derivatives of JH642 (Table 1). A B. subtilis strain (LAB2518) was grown anaerobically in 2x yeast extract-tryptone (YT) medium (15) supplemented with 1% glucose, 0.2% nitrate (KNO₃), and 5 µg/ml of chloramphenicol. Cells were plated onto Luria-Bertani (LB) agar at daily intervals, and the plates were incubated under aerobic conditions. Resulting colonies (CFU) were counted to determine the total number of surviving cells. CFU values declined 100- to 1,000-fold during the first 3 days but then recovered and remained relatively stable during the next 7 days (Fig. 1A). The decrease in the number of CFU was not due to a defect in the aerobic recovery of anaerobically grown cells because a similar result was observed when CFU were measured by incubating the plates under anaerobic conditions (data not shown).

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TABLE 1. Bacillus subtilis strains and plasmids used in this studya

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FIG. 1. Survival of B. subtilis after prolonged anaerobic incubation. B. subtilis strains were anaerobically grown in 2x YT medium supplemented with 1% glucose and 0.2% nitrate (A and C) or with 0.5% glucose and 0.5% pyruvate (B). Cells were appropriately diluted at daily intervals and plated onto LB agar to determine CFU. (A and B) Symbols: closed squares, LAB2518 (hmp+ strain); closed circles, ORB3659 (hmp mutant); closed triangles, ORB4960 (hmp mutant with hmp+ at the thrC locus). (C) Symbols: closed circles, ORB3659 (hmp mutant); closed diamonds; ORB3927 (resDE mutant); open squares, ORB5489 (resDE hmp mutant). The experiments were repeated two to five times and showed a similar pattern of survival.

We have previously identified hmp, the gene encoding flavohemoglobin, as a highly induced gene when oxygen becomes limited (9) or nitric oxide (NO) is present (12). Until now, we have not found a phenotype that is conferred by the B. subtilis hmp null mutation. However, in this study, we observed that cells of the hmp mutant are unable to survive prolonged incubation under anaerobic growth conditions. As shown in Fig. 1A, CFU counts of the hmp mutant (ORB3659) were substantially reduced after several days of anaerobic incubation (Fig. 1A). In contrast, the survival curves were similar between hmp⁺ and hmp cells grown in 2x YT medium supplemented with 0.5% glucose, 0.5% pyruvate, and 5 µg/ml chloramphenicol (Fig. 1B), indicating that the hmp mutation may have an adverse effect on long-term anaerobic survival only in nitrate-containing medium.

Our previous result showed that the ykjA gene is transcribed at least partly from the hmp promoter by readthrough past a putative factor-independent terminator present between hmp and ykjA (9). Therefore, the effect of the hmp mutation on long-term anaerobic survival could be caused by a polar effect of the hmp null mutation on ykjA expression. We examined this possibility by complementation analysis. A fragment encompassing the 3' part of ykhA, an intact hmp, and the 5' part of ykjA was isolated from pML32 (9) and subcloned into pDG795 (5) to generate pMMN576. Transformation of B. subtilis with pMMN576 resulted in the integration of hmp⁺, including its promoter, into the thrC locus. As shown in Fig. 1A, hmp⁺ in thrC was able to complement the hmp null mutation with respect to long-term survival. These results clearly demonstrated that Hmp is essential for long-term anaerobic survival.

We have shown that the ResD-ResE two-component signal transduction system is required for nitrate respiration in B. subtilis (17). Since our previous results showed that hmp expression is largely dependent on ResDE (9), we expected that the resDE mutant would not survive during long-term anaerobic incubation. The resDE mutant is unable to activate genes involved in nitrate respiration and grows at a much slower rate than the wild type (19). However, after overnight incubation, the resDE mutant (ORB3927) showed CFU values close to those of resDE⁺ hmp⁺ cells and survived much better than the hmp mutant (Fig. 1C). In order to eliminate the possibility that a secondary-site mutation arose after long-term cultures, which was responsible for the survival of the resDE mutant, six colonies isolated from cultures grown overnight and from 4-day-old cultures were examined for phenotypic characteristics of the resDE mutation. All 12 clones tested showed retarded short-term anaerobic growth similar to that of the resDE mutant (Fig. 2). In addition, hmp expression was much lower in these strains than in the resDE⁺ cells, and the level was similar to that observed in the resDE mutant (Fig. 2). The result indicated that the survival ability of the resDE mutant is intrinsic to the mutant and is not due to the emergence of a suppressor mutation. Taken together, this result suggested that Hmp is required for anaerobic survival only when cells undergo nitrate respiration, consistent with the result that the hmp mutant survived well under fermentation conditions (Fig. 1B). Hence, Hmp is not needed for anaerobic survival of the resDE mutant that is unable to reduce nitrate. To examine this possibility, we tested anaerobic survival of ORB5489 (resDE hmp). Figure 1C shows that the survival curves of ORB3927 (resDE hmp⁺) and ORB5489 were similar, demonstrating that Hmp is required only for prolonged anaerobic survival when cells can undergo nitrate respiration. Therefore, whereas ResDE accelerates short-term anaerobic growth, its activity results in diminished long-term survival.

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FIG. 2. Growth and hmp expression in cells grown anaerobically in 2x YT medium supplemented with 1% glucose and 0.2% nitrate. Symbols: squares, LAB2518 (resDE+ strain); circles, ORB3927 (resDE mutant); triangles, a colony of ORB3927 that survived after a 4-day anaerobic incubation. Closed symbols represent growth, and open symbols represent hmp-lacZ expression. Time zero indicates the end of exponential growth. Six independent colonies isolated from ORB3927 cultures grown overnight and six colonies isolated from the 4-day-old cultures showed similar patterns of growth and hmp expression.

Morphological change associated with long-term anaerobic cultures. Next, we examined cell viability of the hmp⁺ and hmp strains using the fluorescent LIVE/DEAD BacLight bacterial viability kit (Molecular Probes). The BacLight stain consists of two nucleic acid stains, namely, Syto-9, which stains all cells, and propidium iodide, which penetrates only bacteria with damaged membranes. Live bacteria with intact membranes fluoresce green, while dead bacteria with damaged membranes fluoresce red. As shown in Fig. 3, cellular morphology changes drastically during anaerobic incubation. In cultures grown overnight, both the hmp⁺ and hmp strains showed normal rod-shaped cells (data not shown). In the following few days, when CFU counts declined, cells became smaller than exponentially growing cells, and the majority of the cells were almost coccoid (3-day culture). These cultures also contained mixtures of live and dead cells. When hmp⁺ cells had partially recovered their colony-forming ability (6-day-old culture), small aggregates of rod-shaped cells started to reappear (Fig. 3), and these rod-shaped cells sometimes formed multicellular filaments (data not shown). Most of the rod-shaped cells were alive and coccoid cells were dead. In contrast, the hmp mutant cells remained coccoid during prolonged incubation and were apparently dead.

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FIG. 3. LIVE/DEAD BacLight staining of the hmp+ (LAB2518) and hmp mutant (ORB3659) strains after anaerobic incubation in 2x YT medium supplemented with 1% glucose and 0.2% nitrate for 3 and 6 days. Cells in red are dead, and those in green are alive.

It was shown that mutants that have a growth advantage in stationary phase appear after prolonged stationary-phase incubation of Escherichia coli (20). To test this possibility, the wild-type cells from 8-day-old cultures were plated onto LB agar, and four clones were chosen for reinoculation under the same anaerobic conditions. All strains showed survival curves similar to that of the parent strain, indicating that these strains did not acquire mutations that confer a growth advantage in stationary phase.

Hmp is likely required for protection against prolonged nitrosative stress. What are the damaging effects associated with nitrate respiration and how does Hmp protect cells from that damage? Flavohemoglobin (Hmp), which is composed of an N-terminal globin module and a C-terminal reductase domain, has been found in prokaryotes and lower eukaryotes (reviewed in reference 18). Hmp is an NO dioxygenase that converts NO to nitrate under aerobic conditions (2, 3, 6, 7), and hmp mutants of several species were previously shown to be sensitive to NO (1, 2, 7, 10, 11).

Since B. subtilis hmp is the most highly induced gene in the NO stimulon (12) and B. subtilis endogenously produces NO during nitrate respiration (14), we asked whether the B. subtilis hmp mutant is sensitive to NO under anaerobic conditions. hmp⁺ and hmp cells were cultured in 2x YT medium supplemented with 0.5% glucose and 0.5% pyruvate, and when the OD₆₀₀ of cultures reached 0.08 to 0.1, spermine NONOate (Cayman Chemical) was added at a final concentration of 1 mM. Spermine NONOate generates 2 mol of NO per mole at a neutral pH (the half-life at 37°C is 39 min). The growth of both strains was retarded in the presence of NO, but the growth curve was almost indistinguishable between the hmp⁺ and the hmp strains (Fig. 4). An alternative explanation for the hmp mutant phenotype is that oxygen present in the medium at the beginning in our culture conditions is sufficient to oxidize NO to generate N₂O₃, a strong mutagen that causes DNA deamination and alkylation.

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FIG. 4. Effect of NO on B. subtilis growth. B. subtilis cells were cultured anaerobically in 2x YT medium supplemented with 0.5% glucose and 0.5% pyruvate. When the OD600 reached around 0.08 to 0.1, 1 mM spermine NONOate was added (time zero). The experiments were repeated three times and showed similar results. Symbols: circles, LAB2518 (hmp+ strain); rectangles, ORB3659 (hmp mutant). Open and closed symbols indicate cultures without and with spermine NONOate, respectively.

 
I thank Anda Cornea for her assistance in fluorescence microscopy. I also thank Peter Zuber for critical reading of the manuscript.

This study was supported by grant MCB0110513 from the National Science Foundation.

 
* Mailing address: Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health and Science University, 20000 NW Walker Road, Beaverton, OR 97006. Phone: (503) 748-4078. Fax: (503) 748-1464. E-mail: mnakano{at}ebs.ogi.edu.

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Journal of Bacteriology, September 2006, p. 6415-6418, Vol. 188, No. 17
0021-9193/06/$08.00+0     doi:10.1128/JB.00557-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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