2249Intracellular concentrations of acetyl-CoA and malonyl-CoA in Escherichia coli K 12 were determined by a malonyl-CoA : acetyl-CoA cycling technique. Under aerobic growth conditions with glucose the acetyl-CoA and malonyl-CoA concentrations varied over a range of 0.05-1.5 nmol (mg dry wt)-' (20-600 WM) and 0-01-0.23 nmol (mg dry wt)-l (4-90 WM), respectively. The intracellular concentration of acetyl-CoA was highest in exponentially growing cells and it fell rapidly to less than 5 % of the maximum level when the organism entered stationary phase after exhaustion of glucose. A linear relationship was observed between the intracellular concentration of total acyl-CoA and the logarithm of the concentration of glucose in the medium. Consequently, the acetyl-CoA/malonyl-CoA ratios also varied drastically, in a range of 0.6-41.7, under different conditions. Of several carbon sources tested, glucose was the most effective for promoting the synthesis of cellular acetyl-CoA. For cells grown on glycerol or acetate the maximum concentrations of total acyl-CoA were significantly lower. In cells incubated with citrate (not used as a carbon source by E. coli), the level was consistent with that in cells starved for exogenous carbon sources.

Thiolactomycin (TLM) and cerulenin are antibiotics that block Escherichia coli growth by inhibiting fatty acid biosynthesis at the 1-ketoacyl-acyl carrier protein synthase I step. Both TLM and cerulenin trigger the accumulation of intracellular malonyl-coenzyme A coincident with growth inhibition, and the overexpression of synthase I protein confers resistance to both antibiotics. Strain CDM5 was derived as a TLM-resistant mutant but remained sensitive to cerulenin. TLM neither induced malonyl-coenzyme A accumulation nor blocked fatty acid production in vivo; however, the fatty acid synthase activity in extracts from strain CDM5 was sensitive to TLM inhibition. The TLM resistance gene in strain CDM5 was mapped to 57.5 min of the chromosome and was an allele of the emrB gene. Disruption of the emrB gene converted strain CDM5 to a TLM-sensitive strain, and the overexpression of the emrAB operon conferred TLM resistance to sensitive strains. Thus, activation of the emr efflux pump is the mechanism for TLM resistance in strain CDM5.The 3-ketoacyl-acyl carrier protein (0-ketoacyl-ACP) synthases are key regulators of dissociated (type II) fatty acid synthase systems typified by Escherichia coli (for reviews, see references 6 and 16). P-Ketoacyl-ACP synthase I is required for a critical step in the elongation of unsaturated acyl-ACP, and fabB mutants lacking synthase I activity synthesize neither palmitoleic nor cis-vaccenic acids and require supplementation with unsaturated fatty acids for growth (29). 3-Ketoacyl-ACP synthase II is responsible for the temperature-dependent regulation of fatty acid composition (for a review, see reference 9). Mutants (fabF) lacking synthase II activity are deficient in cis-vaccenic acid but do not have a growth phenotype (12, 13). O-Ketoacyl-ACP synthase III selectively catalyzes the formation of acetoacetyl-ACP in vitro (19). Synthase III possesses acetyl coenzyme A (acetyl-CoA):ACP transacylase activity (34); however, it is unknown whether synthase III accounts for all of the acetyl transacylase activity. The role of this third condensing enzyme remains to be firmly established, but its position at the beginning of the biosynthetic pathway suggests that it plays a role in governing the rate of fatty acid initiation.Thiolactomycin [(4S)(2E,5E)-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene-4-thiolide] (TLM) is a unique antibiotic structure that inhibits type II (bacterial and plant) but not type I (Saccharomyces cerevisiae and mammalian) fatty acid synthases (14,15,23,24,26,27,31). The antibiotic is not toxic to mice and affords significant protection against urinary tract and intraperitoneal bacterial infections (23). Understanding the mechanism of TLM action is important to the development of more-effective antibiotics that exhibit selective action against type II bacterial fatty acid synthases. synthase suggests that the 3-ketoacyl-ACP synthase and the acetyl-CoA:ACP transacylase activities are the only individual enzymes inhibited by TLM in vitro (24). The findings that malonyl-ACP pr...

Intracellular pools of three CoA molecular species of coenzyme A, CoASH, acetyl-CoA, and malonyl-CoA, in Escherichia coli K-12 cells were studied by acyl-CoA cycling method in replacement culture. The sizes and compositions of CoA pools starved for a carbon source changed within minutes after the addition of one of various carbon sources. A large acetyl-CoA pool formed after the addition of D-glucose, D-fructose, D-mannose, glycerol, or sorbitol, but there was little change when L-glucose, sucrose, maltose, succinate, or acetate was added. The beta-anomer of D-glucose was assimilated 10 times faster than the alpha-anomer. Intracellular CoA pools also changed with stress: in the pH, incubation temperature, or with osmotic stress. The sizes and compositions of CoA pools were not affected by pH changing between 4 and 8, but the breakdown of acetyl-CoA and CoASH was greater at pH 9 than at pH 4 to 8. Production of acetyl-CoA was greatest at 40 degrees C, and at 50 degrees C, an acetyl-CoA pool did not form at all and the size of the CoASH pool declined. When the organism was stressed by the addition of NaCl at concentrations of more than 0.6 M, little acetyl-CoA was produced. The total CoA pool (the sum of the concentrations of CoASH, acetyl-CoA, and malonyl-CoA) remained within the limits of 0.83-1.40 nmol/mg of dry cell weight (0.30-0.52 mM). Whenever acetyl-CoA increased, CoASH decreased. Therefore, the acetyl-CoA/CoASH ratio is an important index of facultative anaerobes that reflects the state of carbon and energy metabolism in vivo.

Solid media on a base of B-12 or CB medium with agarose or agarose of low melting temperature were developed for the cultivation of Microcystis species. The media with 0.4% gel showed the highest number of CFU, and increasing the gel concentration resulted in a reduction of the number of CFU. There was no difference in the numbers of CFU between pour and spread plates made of the solid media. By using the solid media, 31 clones of Microcystis species were isolated from natural blooms in Lake Kasumigaura, and 5 axenic strains (1 of M. wesenbergii and 4 of M. aeruginosa) were established from the clones.

Random amplified polymorphic DNA (RAPD) analysis was used to discriminate genotypes in five species of Microcystis cyanobacteria. Strains of each group with the identical allozyme genotype (T. Kato et al., Algol. Stud., 1991, 129-140; M. Watanabe, in "Toxic Microcystis," ed. by M.F. Watanabe et al., CRC Press, Tokyo, 1966, pp. 13-34) gave similar RAPD patterns characterizing the respective group. On the other hand, no similarities in RAPD patterns were observed among strains of which allozyme genotypes were different. A good accordance between the RAPD analysis and allozyme divergence indicated a high reliability of both methods for discrimination of the affiliated groups of Microcystis. Several amplified DNA fragments, which were expected to be markers for a particular taxon with identical allozyme genotype, were also observed on the RAPD patterns. Genetic homogeneities of M. novacekii, M. viridis, and M. wesenbergii were shown by RAPD analysis as well as the allozyme genotype. However, significant variations were observed in M. aeruginosa and M. ichthyoblabe in the levels of DNA and proteins (allozymes).