Alanine dehydrogenase (ald) is required for normal sporulation in Bacillus subtilis

Abstract

The ski22::Tn917lac insertion mutation in Bacillus subtilis was isolated in a screen for mutations that cause a defect in sporulation but are suppressed by the presence or overexpression of the histidine protein kinase encoded by kinA (spoIIJ). The ski22::Tn9171ac insertion mutation was in ald, the gene encoding alanine dehydrogenase. Alanine dehydrogenase catalyzes the deamination of alanine to pyruvate and ammonia and is needed for growth when alanine is the sole carbon or nitrogen source. The sporulation defect caused by null mutations in ald was partly relieved by the addition of pyruvate at a high concentration, indicating that the normal role of alanine dehydrogenase in sporulation might be to generate pyruvate to provide an energy source for sporulation. The spoVN::Tn9l7 mutation was also found to be an allele of aid. Transcription of ald was induced very early during sporulation and by the addition of exogenous alanine during growth. Expression of ald was normal in all of the regulatory mutants tested, including spoOA, spoOH, spoOK, comA, sigB, and sigD mutants. The only gene in which mutations affected expression of ald was ald itself. This regulation is probably related to the metabolism of alanine.Cells of Bacillus subtilis can differentiate into dormant heat-resistant endospores under appropriate environmental conditions. Regulatory events during the initiation of sporulation lead to the formation of an asymmetric cell division septum, generating two distinct cell types. The smaller cell, or forespore, develops into the mature spore after being engulfed by the larger cell, the mother cell (reviewed in reference 10). Eventually, the mother cell lyses, releasing the mature heatresistant endospore.Dramatic changes in gene expression, physiology, and metabolism underlie the morphological changes associated with spore formation. A variety of regulatory circuits and genes required for sporulation have been characterized. In addition, many genes that are expressed during sporulation are not essential for development (10). While much work has focused on gene expression and regulation, relatively little is known about metabolism and the generation of energy for synthesis of new products required for the morphological development that occurs during sporulation. The tricarboxylic acid cycle seems to play a role in the generation of energy during sporulation, as mutants that are defective in tricarboxylic acid cycle enzymes are defective in sporulation (13,18,56). Protein turnover is known to increase during sporulation (30, 47) and probably plays a role in generating substrates (peptides and amino acids) for further metabolism and new macromolecular synthesis.Previously, we described the isolation and characterization of mutations that caused a defect in sporulation but could be partially suppressed by the presence or overproduction of the histidine protein kinase encoded by kinA (19,20,37). These mutations were called ski (pronounced "sky," for suppressed by kinase) and include spoOK (37) and bofA (20,36). Kin...

Conformational Dynamics of a Mobile Loop in the NAD(H)‐Binding Subunit of Proton‐Translocating Transhydrogenases from Rhodospirillum Rubrum and Escherichia Coli

“…First, peptide bonds within this segment, (a) in the isolated domain I proteins of R. rubrum and E. coli transhydrogenase [40], B. sphaericus [40], B. subtilis [41], Mycobacterium tuberculosis [42]; transhydrogenases (TH), E. coli [S], Bovis [9], R. rubrum [ll], Eimeria tenella [lo]. The numbers in parenthesis refer to the first and last amino acid residues in the sequences shown, as predicted from the nucleotide sequence of the genes or cDNA.…”

Purification, properties and primary structure of alanine dehydrogenase involved in taurine metabolism in the anaerobe Bilophila wadsworthia

“…The B. wadsworthia enzyme was 57%, 56%, 54% and 51% identical to the enzymes from Bacillus subtilis (Siranosian et al 1993), Bacillus sphaericus (Kuroda et al 1990), Mycobacterium tuberculosis (Cole et al 1998) and Phormidium lapideum (Baker et al 1998), respectively. All of these sequences including that of Bilophila wadsworthia contained the conserved residues (KVKEP) of the putative pyruvate binding region (Delforge et al 1997) and of the putative βαβ Rossmann fold responsible for nucleotide binding (Wierenga et al 1986;Bork and Grunwald 1990).…”

“…The enzyme is involved in different metabolic pathways. In Bacillus sp., the main function of alanine dehydrogenase seems to be the generation of pyruvate as an energy source during sporulation (Siranosian et al 1993). When operating in the reverse direction -the reductive amination of pyruvate -the enzyme can be involved in nitrogen assimilation as in, for example, Rhodobacter capsulatus (Caballero et al 1989) 1 Initial steps of the degradation of taurine in Bilophila wadsworthia.…”

Insight into the sporulation phosphorelay: Crystal structure of the sensor domain of Bacillus subtilis histidine kinase, KinD

“…The following rotation angles were observed: 171.6 and 172. 4 in 3E4P and 3E4Q (DctB 29,30 ), 174.4 in KinD-SD (this work), 176.2 in 3C8C, 32 179.2 in 3T4J 34 and 180 in 3LIA, 3LI8, and 3LI9. 31 In 3LI8…”

“…In 1993, a report described that a mutation in the alanine dehydrogenase, the enzyme that converts alanine to pyruvate, resulted in a sporulation defect that could be partially rescued by the addition of pyruvate to the media. 34 We hypothesized that perhaps KinD serves as a phosphatase in the absence of pyruvate and thus drains phosphoryl groups from the system. If this were true, we would have expected that an ald kinD double mutant sporulates better than the ald single mutant.…”