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

Abstract

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.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 [OD 600 ] 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 2ϫ yeast extract-tryptone (YT) medium (15) supplemented with 1% glucose, 0.2% nitrate (KNO 3 ), and 5 g/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).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 ϩ and hmp cells grown in 2ϫ YT medium supplemented with 0.5% glucose, 0.5% pyruvate, and 5 g/ml chloramphenicol (Fig. 1B), indicating that the hmp mutation may have an adverse effect on long-term anaerobic survival only in nitratecontaining medium.Our previous result showed th...

Functional analysis and subcellular location of two flavohemoglobins from Aspergillus oryzae

“…Although the anaerobic NO reductase activity of FHb remains controversial due to its significantly lower catalytic rate compared to its aerobic NOD activity (Kim et al, 1999), anaerobic protection against NO by FHb has been noted in Salmonella typhimurium (Crawford and Goldberg, 1998), E. coli (Gardner et al, 1998b), and S. cerevisiae (Liu et al, 2000). In addition, FHbs of Staphylococcus aureus and B. subtilis have been shown to protect their cells from NO stress only under O 2 -limiting conditions (Nakano, 2006;Gonçalves et al, 2006). These results emphasize the possible anaerobic roles of FHb and suggest that a possible anaerobic NO metabolism involving FHb may exist.…”

The hmp gene encoding the NO-inducible flavohaemoglobin in Escherichia coli confers a protective advantage in resisting killing within macrophages, but not in vitro: Links with swarming motility

“…Other studies reveal unexpected roles for Hmp. A recent study (Nakano, 2006) has demonstrated that, in Bacillus subtilis, flavohaemoglobin Hmp has a role in long-term anaerobic survival: mutants defective in hmp exhibit poor survival when incubated anaerobically with nitrate unless nitrate respiration is prevented. Although this might be attributed to sensitivity to NO produced under these conditions, addition of NO-releasing NONOate compounds elicited no growth differences in the anaerobic growth of hmp and hmp + strains.…”

ArnR, a Novel Transcriptional Regulator, Represses Expression of the narKGHJI Operon in Corynebacterium glutamicum

“…Flavohemoglobin detoxifies nitric oxide by anaerobically reducing it to N 2 O or by aerobically oxidizing it to nitrate. In B. subtilis, hmp has an essential role in long-term anaerobic survival only in nitratecontaining medium (22). Although C. glutamicum Hmp is not highly similar to either E. coli Hmp (32% identity) or B. subtilis Hmp (31% identity), C. glutamicum Hmp may play some protective role against possible oxidative stress during anaerobic growth with nitrate.…”