Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth

Abstract

Bacillus subtilis can grow anaerobically by respiration with nitrate as a terminal electron acceptor. In the absence of external electron acceptors, it grows by fermentation. Identification of fermentation products by using in vivo nuclear magnetic resonance scans of whole cultures indicated that B. subtilis grows by mixed acid-butanediol fermentation but that no formate is produced. An ace mutant that lacks pyruvate dehydrogenase (PDH) activity was unable to grow anaerobically and produced hardly any fermentation product. These results suggest that PDH is involved in most or all acetyl coenzyme A production in B. subtilis under anaerobic conditions, unlike Escherichia coli, which uses pyruvate formate lyase. Nitrate respiration was previously shown to require the ResDE two-component signal transduction system and an anaerobic gene regulator, FNR. Also required are respiratory nitrate reductase, encoded by the narGHJI operon, and moaA, involved in biosynthesis of a molybdopterin cofactor of nitrate reductase. The resD and resDE mutations were shown to moderately affect fermentation, but nitrate reductase activity and fnr are dispensable for fermentative growth. A search for genes involved in fermentation indicated that ftsH is required, and is also needed to a lesser extent for nitrate respiration. These results show that nitrate respiration and fermentation of B. subtilis are governed by divergent regulatory pathways.Recent studies have shown that Bacillus subtilis, which had been widely believed to be a strict aerobe, can grow anaerobically in the presence of nitrate (3,8,13,15,20,25,27,29,34). Respiratory nitrate reductase encoded by the narGHJI operon (3) was shown to be responsible for nitrate respiration (13, 15). Mutations in moaA (formerly narA), the product of which shows homology to the Escherichia coli moaA gene product (26), impair nitrate respiration, probably by conferring a defect in the biosynthesis of the nitrate reductase cofactor (8). Transcription of narGHJI and narK (required for nitrite extrusion [3]) is induced by oxygen limitation, and the induction is completely abolished by mutations in fnr, the second gene of the narK-fnr operon (3,15). FNR is known to be a global anaerobic gene regulator in E. coli and has amino acid sequence similarity to the catabolite activator protein (30). In E. coli, the activity of FNR, but not the expression of fnr, was shown to be stimulated by anaerobiosis. This is believed to be due to the cluster of cysteine residues in the amino terminus of the protein that may play a role in modulating FNR activity by a mechanism involving bound iron (9, 10, 37). Unlike E. coli, in which fnr expression is weakly repressed by anaerobiosis, fnr expression in B. subtilis is strongly induced by oxygen limitation (3,20). Anaerobic induction of fnr transcription is controlled at two levels. First, fnr transcription at an intergenic fnr-specific promoter is activated by oxygen limitation and requires phosphorylated ResD, the production of which depends on a cognate histidine se...

Bacillus stamsii sp. nov., a facultatively anaerobic sugar degrader that is numerically dominant in freshwater lake sediment

“…It was concluded that pyruvate might serve as a stimulating agent that triggers expression of genes required for mixed acid fermentation, as the intracellular pyruvate pool might be too small to initiate expression of genes required for fermentation. Also, pyruvate was discussed as a precursor of certain amino acids that cannot be synthesized by B. subtilis [19]. Similar conclusions were drawn before from observations made with the methanogenic archaeon Methanosarcina barkeri [17].…”

“…Limited growth was possible also if no supplements but only glucose and pyruvate were added. Also Bacillus subtilis can grow by fermentation of glucose in anoxic, non-reduced Spizizen's medium amended with amino acid mixtures, however, addition of pyruvate to cultures with glucose augmented growth significantly [19]. It was concluded that pyruvate might serve as a stimulating agent that triggers expression of genes required for mixed acid fermentation, as the intracellular pyruvate pool might be too small to initiate expression of genes required for fermentation.…”

“…Several Bacillus species can also grow anaerobically by a fermentative metabolism, e.g., B. subtilis [19], B. cereus, B. thuringiensis, B. licheniformis, B. coagulans, B. polymyxa, B. macerans, B. alvei, B. laterosporus, B. larvae, B. popilliae, and B. lentimorbus [35]. Nonetheless, syntrophic cooperation with methanogenic partners has not been described so far for any Bacillus species.…”

Growth Inhibition of Pathogenic Bacteria by Sulfonylurea Herbicides

“…For experiments with B. pseudomallei, Burkholderia thailandensis, or Pseudomonas aeruginosa, cultures were grown in M9 medium (17) that contained glucose (0.2%) and thiamine (0.5 g/ml). For experiments with Bacillus subtilis, cultures were grown in Spizizen's minimal medium (18) containing glucose (1%) and tryptophan (50 g/ml) along with glutamate (2 mg/ml), KNO 3 (0.2%), and trace elements as described by Nakano et al (19). The MIC values reported for each strain are the median values from three to six independent experiments.…”

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

“…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).…”