Studies of citrate synthase (CitA) were carried out to investigate its role in morphological development and biosynthesis of antibiotics in Streptomyces coelicolor. Purification of CitA, the major vegetative enzyme activity, allowed characterization of its kinetic properties. The apparent K m values of CitA for acetyl coenzyme A (acetyl-CoA) (32 M) and oxaloacetate (17 M) were similar to those of citrate synthases from other gram-positive bacteria and eukaryotes. CitA was not strongly inhibited by various allosteric feedback inhibitors (NAD ؉ , NADH, ATP, ADP, isocitrate, or ␣-ketoglutarate). The corresponding gene (citA) was cloned and sequenced, allowing construction of a citA mutant (BZ2). BZ2 was a glutamate auxotroph, indicating that citA encoded the major citrate synthase allowing flow of acetyl-CoA into the tricarboxylic acid (TCA) cycle. Interruption of aerobic TCA cycle-based metabolism resulted in acidification of the medium and defects in morphological differentiation and antibiotic biosynthesis. These developmental defects of the citA mutant were in part due to a glucose-dependent medium acidification that was also exhibited by some other bald mutants. Unlike other acidogenic bald strains, citA and bldJ mutants were able to produce aerial mycelia and pigments when the medium was buffered sufficiently to maintain neutrality. Extracellular complementation studies suggested that citA defines a new stage of the Streptomyces developmental cascade.

SummaryBacteria typically undergo intermittent periods of starvation and adaptation, emulated as diauxic growth in the laboratory. In association with growth arrest elicited by metabolic stress, the differentiating eubacterium Streptomyces coelicolor not only adapts its primary metabolism, but can also activate developmental programmes leading to morphogenesis and antibiotic biosynthesis. Here, we report combined proteomic and metabolomic data of S. coelicolor used to analyse global changes in gene expression during diauxic growth in a defined liquid medium. Cultures initially grew on glutamate, providing the nitrogen source and feeding carbon (as 2-oxoglutarate) into the TCA cycle, followed by a diauxic delay allowing reorientation of metabolism and a second round of growth supported by NH 4 + + + + , formed during prediauxic phase, and maltose, a glycolytic substrate. Cultures finally entered stationary phase as a result of nitrogen starvation. These four physiological states had previously been defined statistically by their distinct patterns of protein synthesis and heat shock responses. Together, these data demonstrated that the rates of synthesis of heat shock proteins are determined not only by temperature increase but also by the patterns and rates of metabolic flux in certain pathways. Synthesis profiles for metabolic-and stress-induced proteins can now be interpreted by the identification of 204 spots (SWICZ database presented at http:// proteom.biomed.cas.cz). Cluster analysis showed that the activity of central metabolic enzymes involved in glycolysis, the TCA cycle, starvation or proteolysis each displayed identifiable patterns of synthesis that logically underlie the metabolic state of the culture. Diauxic lag was accompanied by a structured regulatory programme involving the sequential activation of heat-, salt-, cold-and bacteriostatic antibiotic (pristinamycin I, PI)-induced stimulons. Although stress stimulons presumably provide protection during environmental-or starvationinduced stress, their identities did not reveal any coherent adaptive or developmental functions. These studies revealed interactive regulation of metabolic and stress response systems including some proteins known to support developmental programmes in S. coelicolor .

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Large chromosomal DNAfragments containing different parts of the putative rubromycin polyketide synthase "gene cluster were cloned and functionally expressed in S. coelicolor CH999. Expression of these clones yielded 5-10 metabolites that were not detected in S. collinus culture extracts. This paper focusses on one of the new metabolites, termed collinone, that was isolated in large quantities and purified for spectroscopic structure determination and biological screening assays. Collinone is a heavily oxidized angular hexacyclic compound