Sporulation of Bacillus subtilis

Genetic aspects of bacterial endospore formation.

In many organisms, metabolite interconversion at the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node involves a structurally entangled set of reactions that interconnects the major pathways of carbon metabolism and thus, is responsible for the distribution of the carbon flux among catabolism, anabolism and energy supply of the cell. While sugar catabolism proceeds mainly via oxidative or non-oxidative decarboxylation of pyruvate to acetyl-CoA, anaplerosis and the initial steps of gluconeogenesis are accomplished by C3- (PEP- and/or pyruvate-) carboxylation and C4- (oxaloacetate- and/or malate-) decarboxylation, respectively. In contrast to the relatively uniform central metabolic pathways in bacteria, the set of enzymes at the PEP-pyruvate-oxaloacetate node represents a surprising diversity of reactions. Variable combinations are used in different bacteria and the question of the significance of all these reactions for growth and for biotechnological fermentation processes arises. This review summarizes what is known about the enzymes and the metabolic fluxes at the PEP-pyruvate-oxaloacetate node in bacteria, with a particular focus on the C3-carboxylation and C4-decarboxylation reactions in Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum. We discuss the activities of the enzymes, their regulation and their specific contribution to growth under a given condition or to biotechnological metabolite production. The present knowledge unequivocally reveals the PEP-pyruvate-oxaloacetate nodes of bacteria to be a fascinating target of metabolic engineering in order to achieve optimized metabolite production.

The PEP-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria.

Background: Sediments are massive reservoirs of carbon compounds and host a large fraction of microbial life. Microorganisms within terrestrial aquifer sediments control buried organic carbon turnover, degrade organic contaminants, and impact drinking water quality. Recent 16S rRNA gene profiling indicates that members of the bacterial phylum Chloroflexi are common in sediment. Only the role of the class Dehalococcoidia, which degrade halogenated solvents, is well understood. Genomic sampling is available for only six of the approximate 30 Chloroflexi classes, so little is known about the phylogenetic distribution of reductive dehalogenation or about the broader metabolic characteristics of Chloroflexi in sediment. Results: We used metagenomics to directly evaluate the metabolic potential and diversity of Chloroflexi in aquifer sediments. We sampled genomic sequence from 86 Chloroflexi representing 15 distinct lineages, including members of eight classes previously characterized only by 16S rRNA sequences. Unlike in the Dehalococcoidia, genes for organohalide respiration are rare within the Chloroflexi genomes sampled here. Near-complete genomes were reconstructed for three Chloroflexi. One, a member of an unsequenced lineage in the Anaerolinea, is an aerobe with the potential for respiring diverse carbon compounds. The others represent two genomically unsampled classes sibling to the Dehalococcoidia, and are anaerobes likely involved in sugar and plant-derived-compound degradation to acetate. Both fix CO2 via the Wood-Ljungdahl pathway, a pathway not previously documented in Chloroflexi. The genomes each encode unique traits apparently acquired from Archaea, including mechanisms of motility and ATP synthesis. Conclusions: Chloroflexi in the aquifer sediments are abundant and highly diverse. Genomic analyses provide new evolutionary boundaries for obligate organohalide respiration. We expand the potential roles of Chloroflexi in sediment carbon cycling beyond organohalide respiration to include respiration of sugars, fermentation, CO2 fixation, and acetogenesis with ATP formation by substrate-level phosphorylation.

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Community genomic analyses constrain the distribution of metabolic traits across the Chloroflexi phylum and indicate roles in sediment carbon cycling

Beyond fuelling cellular activities with building blocks and energy, metabolism also integrates environmental conditions into intracellular signals. The underlying regulatory network is complex and multifaceted: it ranges from slow interactions, such as changing gene expression, to rapid ones, such as the modulation of protein activity via post-translational modification or the allosteric binding of small molecules. In this Review, we outline the coordination of common metabolic tasks, including nutrient uptake, central metabolism, the generation of energy, the supply of amino acids and protein synthesis. Increasingly, a set of key metabolites is recognized to control individual regulatory circuits, which carry out specific functions of information input and regulatory output. Such a modular view of microbial metabolism facilitates an intuitive understanding of the molecular mechanisms that underlie cellular decision making.

Coordination of microbial metabolism

Role of Pyruvate Carboxylase, Phosphoenolpyruvate Carboxykinase, and Malic Enzyme during Growth and Sporulation of Bacillus subtilis

Enzyme activities of glycolysis and glyconeogenesis are present in spores of Bacillus subtilis, the rate-limiting step of glucose (GLC) metabolism being its phosphorylation. GLC allows initiation of germination in the presence of fructose (FRU) and asparagine (ASN), not because it is used via the Embden-Meyerhof path, but because it is oxidized in the nonphosphorylated form via the spore-specific GLC dehydrogenase. Spores of mutants lacking GLC-phosphoenolpyruvate transferase, FRU-6-P-kinase, or phosphoglucoisomerase activity can still be initiated by the above substrate combination. Furthermore, GLC can be replaced by 2-deoxy-GLC, which is also oxidized by GLC-dehydrogenase, but not by α- or β-methylglucoside, which are not substrates of this enzyme. GLC probably acts by reducing nicotinamide adenine dinucleotide (or nicotinamide adenine dinucleotide phosphate), which is used for some metabolic reaction other than the cytochrome-linked electron transport system, since inhibitors of this system do not inhibit initiation. Spores of a mutant lacking FRU-1-P-kinase activity can no longer be initiated by GLC+FRU+ASN, but they do respond to the combination of GLC+mannose+ASN. Since spores of a FRU-6-P-kinase (or phosphoglucoisomerase) mutant can still respond to either FRU or mannose, FRU-6-P (or some derivative) apparently is needed for initiation (in addition to reduced nicotinamide adenine dinucleotide and an amino donor). Alanine can initiate germination in spores of all of the above mutants, indicating that it can form all required compounds. However, in a mutant lacking P-glycerate kinase activity, alanine initiates only after a long lag and at a slow rate, indicating that some compound in the upper metabolic subdivision is required for initiation, in agreement with the above findings. All initiating agents of B. subtilis probably produce the same required compound(s) by different metabolic routes.

Initiation of spore germination in glycolytic mutants of Bacillus subtilis.

Most strains of Bacillus subtilis, dervied from the 168 (Marburg) strain, grow slowly on aspartate as sole carbon source. We isolated a mutant (aspH) that grows rapidly on aspartate because it produces aspartase constitutively. Thus, aspartase is needed for rapid growth on aspartate, whereas aspartate-alpha-ketoglutarate aminotransferase is not needed, as was demonstrated by a mutant lacking that enzyme activity. By two--and three-factor crosses using PBSl transduction, the aspH mutation was located between the aroD and the lys markers of the genetic map. Although sodium ions do not affect growth on glucose or L-malate, they specifically stimulate growth on aspartate in both the parent and the aspH mutant strains. Enzyme activities of crude aspartase and fumarase and of purified aspartase do not increase in the presence of sodium. These results show that stimulation by sodium involves some reaction other than the enzymes catabolizing aspartate. The ease of purification from the aspH strain and the stability of aspartase suggest that the B. subtilis enzyme is particularly useful for aspartate determinations.

Sodium effect of growth on aspartate and genetic analysis of a Bacillus subtilis mutant with high aspartase activity.

Pyruvate kinase of bacillus subtilis.

Mutants (aspT) of Bacillus subtilis which lack the high affinity transport of L-aspartate and L-glutamate have been isolated by their resistance to DL-threo-β-hydroxyaspartate. They transport low concentrations (100 μM) of all three amino acids at a greatly reduced rate but they can still grow at the normal rate on high concentrations of aspartate (25 mM) as sole carbon source. The aspT mutation has been mapped between argC and glpK, glpD. In glucose/citrate medium, an aspB mutant, auxotrophic for aspartate, requires much lower aspartate concentrations for growth than an aspB aspT double mutant. These results demonstrate that a low affinity aspartate transport system is still present in aspT mutant strains. This was also shown by the fact that both aspH and aspH aspT mutants, which carry the gene (aspH) for high aspartase production, grew at the same rate in media containing high concentrations (25 mM) of L-aspartate as sole carbon source. The high affinity aspartate transport system (K
m = 67 μM) alone can satisfy the growth requirements of aspartate auxotrophs in media containing glucose or some other carbon source. However, the maximum rate (V
max) of this transport system is too low to allow rapid growth on aspartate as sole carbon source. For such rapid uptake the low affinity aspartate transport system is needed. Membrane vesicles, energized by glycerophosphate, exhibit only the active high affinity transport which is saturated at about 100 μM-L-aspartate (K
m = 66 μM). Apparently, the proton motive force, which energizes the high affinity aspartate (and glutamate) transport, is not (directly) required for the low affinity aspartate transport.

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Evidence for a Low Affinity but High Velocity Aspartate Transport System Needed for Rapid Growth of Bacillus subtilis on Aspartate as Sole Carbon Source

Martin D. Diesterhaft

Papers

Role of Pyruvate Carboxylase, Phosphoenolpyruvate Carboxykinase, and Malic Enzyme during Growth and Sporulation of Bacillus subtilis

Initiation of spore germination in glycolytic mutants of Bacillus subtilis.

Sodium effect of growth on aspartate and genetic analysis of a Bacillus subtilis mutant with high aspartase activity.

Pyruvate kinase of bacillus subtilis.

Evidence for a Low Affinity but High Velocity Aspartate Transport System Needed for Rapid Growth of Bacillus subtilis on Aspartate as Sole Carbon Source