Fermentative metabolism of Bacillus subtilis: physiology and regulation of gene expression

Abstract

Bacillus subtilis grows in the absence of oxygen using nitrate ammonification and various fermentation processes. Lactate, acetate, and 2,3-butanediol were identified in the growth medium as the major anaerobic fermentation products by using high-performance liquid chromatography. Lactate formation was found to be dependent on the lctEP locus, encoding lactate dehydrogenase and a putative lactate permease. Mutation of lctE results in drastically reduced anaerobic growth independent of the presence of alternative electron acceptors, indicating the importance of NADH reoxidation by lactate dehydrogenase for the overall anaerobic energy metabolism. Anaerobic formation of 2,3-butanediol via acetoin involves acetolactate synthase and decarboxylase encoded by the alsSD operon. Mutation of alsSD has no significant effect on anaerobic growth. Anaerobic acetate synthesis from acetyl coenzyme A requires phosphotransacetylase encoded by pta. Similar to the case for lctEP, mutation of pta significantly reduces anaerobic fermentative and respiratory growth. The expression of both lctEP and alsSD is strongly induced under anaerobic conditions. Anaerobic lctEP and alsSD induction was found to be partially dependent on the gene encoding the redox regulator Fnr. The observed fnr dependence might be the result of Fnr-induced arfM (ywiD) transcription and subsequent lctEP and alsSD activation by the regulator ArfM (YwiD). The two-component regulatory system encoded by resDE is also involved in anaerobic lctEP induction. No direct resDE influence on the redox regulation of alsSD was observed. The alternative electron acceptor nitrate represses anaerobic lctEP and alsSD transcription. Nitrate repression requires resDE- and fnr-dependent expression of narGHJI, encoding respiratory nitrate reductase. The gene alsR, encoding a regulator potentially responding to changes of the intracellular pH and to acetate, is essential for anaerobic lctEP and alsSD expression. In agreement with its known aerobic function, no obvious oxygen- or nitrate-dependent pta regulation was observed. A model for the regulation of the anaerobic fermentation genes in B. subtilis is proposed.

FIG. 1

Proposed pathways for anaerobic fermentation and related catabolism in B. subtilis (modified from references and 19). Enzymes with known coding genes are as follows: LctE, lactate dehydrogenase; AlsS, acetolactate synthase; AlsD, acetolactate decarboxylase; Pta, phosphotransacetylase; Ack, acetate kinase; AcoABC, acetoin dehydrogenase; Pdh, pyruvate dehydrogenase; PycA, pyruvate carboxylase; AcsA, acetyl-CoA synthetase. TCA, tricarboxylic acid.

FIG. 2

Growth of wild-type B. subtilis (■); mutants with mutations in the alsS (●), lctE (▴), and pta (▾) genes; and a pta alsS double mutant (⧫) under fermentative conditions (A) and nitrite (B) and nitrate (C) respiratory conditions. Growth was monitored by determination of the optical density at 578 nm (OD₅₇₈ nm) at the indicated time points. Values reported are the averages from at least five independent experiments performed in triplicate.

FIG. 3

mRNA analysis of the lctEP operon. (A) Northern blot analysis. The lctE- and lctP-specific probes were generated and the blotting was performed as outlined in Materials and Methods. (B) Primer extension mapping. The location of the 5′ end of the lctEP mRNA was deduced from the lengths of the cDNA bands. The length was obtained by comparison with the sequencing reaction products (in the order A, C, G, and T) performed with the primer used for the extension reaction. For both panels, the RNA used in each experiment was extracted from B. subtilis grown under the following conditions: lane 1, exponential phase in complex medium in the presence of oxygen; lane 2, without oxygen and with 10 mM nitrate; 3, without oxygen or further additions; 4, without oxygen and with 5 mM fumarate; 5, control without RNA.

FIG. 4

DNA sequences of the B. subtilis lctEP and alsSD promoter regions. The mapped mRNA ends are indicated by arrows. Predicted −10 and −35 regions are boxed. Predicted Fnr binding sites are also boxed. The 3′ ends of the lctE and alsS promoter regions in the Plct-lacZΔFnr and Pals-lacZΔFnr fusions are shown. The alsSR start point was described by Renna et al. (29).

FIG. 5

Regulatory model for the anaerobic expression of the lctEP, alsSD, and pta loci.