Mutations That Confer Resistance to 2-Deoxyglucose Reduce the Specific Activity of Hexokinase from Myxococcus xanthus

The myxobacteria, including their best-characterized representative, Myxococcus xanthus, are social prokaryotes that undergo multicellular development. When a large number (>105) of M. xanthus cells are starved for essential nutrients, individual cells within a starved population coordinate their movements to form a fruiting body that supports the differentiation of a subset of vegetative cells into spores. Spores represent only a small minority (1 to 10%) of developing cells after carbon starvation and are more resistant to heat, UV light, and sonication than vegetative cells. Spores ensure the survival of this organism under extreme environmental conditions (17). Development is a rapid process. Spores mature within fruiting bodies in the short span of 36 h. The cycle leading to spore maturation involves a cascade of gene expression, in which successive subsets of developmental stage-specific promoters are activated (22). Development is accompanied by dramatic changes in the flow of carbon through metabolic pathways. The early stages of development involve the oxidative catabolism of vegetative biopolymers to generate intermediary metabolites. These metabolites fuel a burst of primarily gluconeogenic anabolism during the later stages of development. This gluconeogenic burst begins about 4 to 6 h after starvation, and, in turn, supports the assembly of the spore coat, comprised mainly of polysaccharides (48). Although many studies have focused on the cascade of gene expression that occurs during development, very little is known about the metabolic changes that accompany this adaptive response. Most of the genes known to be required for development function early in this cycle. In contrast, few genes that function late in development have been identified. Thus, little is known about the genes involved in spore maturation, the regulation of gene expression during late development, or the spatial and temporal coordination of polysaccharide biosynthesis with spore coat assembly (17). To understand how polysaccharide biosynthesis and spore maturation are coordinated, we are exploring the regulation of the gluconeogenic pathway during development. Previous studies of intermediary metabolism have shown that M. xanthus makes many of the enzymes that catalyze steps in this pathway. These include phosphoenolpyruvate (PEP) carboxykinase (EC 4.1.1.32), glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), aldolase (EC 7.1.2.7), fructose-1,6-phosphate phosphatase (EC 3.1.3.11), and phosphoglucoisomerase (EC 5.3.1.9), as well as phosphoglucomutase (EC 2.7.5.1) and UDPG pyrophosphorylase (EC 2.7.7.9) (46). Expression of the gluconeogenic enzymes is induced during an alternate developmental pathway, in which cells sporulate in response to high external concentrations of short-chain polyols (10). The gluconeogenic pathway in M. xanthus is unusual in two respects. The M. xanthus PEP carboxykinase, which catalyzes the first committed step in gluconeogenesis, requires GTP or ITP as a phosphoryl donor (46), like eukaryotic PEP carboxykinases and unlike many of their ATP-dependent prokaryotic counterparts. In addition, the M. xanthus glycolytic pathway, which shares many enzymes with the gluconeogenic pathway, initially appeared incomplete. When extracts were prepared from vegetative M. xanthus cells and assayed for glycolytic activities, neither ATP-dependent hexokinase (EC 2.7.1.1) nor pyruvate kinase (EC 2.4.1.40) activities were detected (46). Their inability to detect intracellular ATP-dependent hexokinase, pyruvate kinase, and high-affinity glucose permease activities led Watson and Dworkin (46) to speculate that M. xanthus may exclude glucose for some selective advantage. These results also could suggest that the apparently glycolytic activities produced by M. xanthus may have roles only in the anabolic pathway of gluconeogenesis. However, this notion is not satisfying, because M. xanthus makes an ATP-dependent phosphofructokinase (EC 2.7.1.11), which catalyzes a committed step in glycolysis that must be bypassed in the gluconeogenic pathway (46). During vegetative growth, M. xanthus is predatory and is thought to derive its energy primarily from the oxidative catabolism of proteins released by the lysis of prey organisms by a battery of secreted enzymes. The minimal requirements for the growth of M. xanthus in defined media suggest that proteins comprise the core of its rich diet. M. xanthus is auxotrophic for leucine, isoleucine, and valine, the three branched-chain amino acids, and for methionine or cobalamin. M. xanthus is also a phenylalanine bradytroph, and many nonessential amino acids stimulate its growth (2, 7, 18). Surprisingly, hexose monosaccharides including glucose (Glc), fructose (Fru), galactose, and glucosamine (2-amino-2-deoxyglucose; GlcN), as well as a variety of their disaccharides, do not stimulate the growth of this obligate aerobe on defined medium. In contrast, acetate and pyruvate, as well as the citric acid cycle intermediates malate and succinate, do stimulate growth (2, 18). Again, these results suggest that M. xanthus cannot transport or phosphorylate Glc efficiently or, at the least, utilize Glc as an energy source. Only a small fraction of label added as [14C]glucose is incorporated into biopolymers with high molecular mass during growth on defined media (18, 46), reinforcing the idea that M. xanthus does not require Glc for growth. On the other hand, it is clear that simple sugars can play critical roles during M. xanthus development. One subset of simple sugars, including the reduced triose glycerol (8) and the pentoses ribose and xylose (34), elicit the synchronous and rapid conversion of rod-shaped vegetative cells in liquid culture into spherical spores. These short-chain polyols trigger an abbreviated unicellular developmental pathway leading to the formation of spores that are more heat resistant than vegetative cells but less heat resistant than the more structurally complex spores produced during starvation-induced multicellular development (42). Furthermore, addition of the GlcN, but not the closely related hexose Glc or N-acetylglucosamine (2-deoxy-2-N-acetylamino-d-glucose; GlcNAc), to exponentially growing cells at high concentrations elicits cell lysis (29). Therefore, it is inviting to speculate that M. xanthus uses these sugars or their phosphorylated derivatives as signals to trigger pathways that define distinct cell fates during development. M. xanthus is sensitive to the antibiotic 2-deoxyglucose (2dGlc). In most organisms, the analog 2dGlc is transported by mechanisms that also transport glucose and must be phosphorylated to form 2-deoxyglucose-6-phosphate (2dGlc6P) to exert its toxic effect. Starting with this discovery, we obtained genetic evidence implying that M. xanthus phosphorylates Glc with a soluble hexokinase and have identified this hexokinase activity in vitro.