Genetic mapping, cloning and physiological aspects of the glucose kinase gene of Streptomyces coelicolor

Abstract

Glucose kinase in Streptomyces coelicolor has a molecular weight of about 110,000. In crude extracts, the enzyme exhibited apparent Km values of 0.20 mM for ATP, 0.27 mM for glucose, and 2.2 mM for the glucose analogue 2-deoxyglucose. Mutations (glk) to 2-deoxyglucose-resistance, which greatly reduce glucose kinase activity and result in relief of glucose repression of utilisation of various carbon sources, were mapped between proA and hisA in the S. coelicolor linkage map. Glucose kinase activity, 2-deoxyglucose-sensitivity, glucose utilisation and glucose repression, were all restored to glk mutants by a 3.5 kb DNA fragment cloned from S. coelicolor into a phage vector (phi C31 KC515), and by larger (10-30 kb) fragments cloned into a low copy number plasmid vector (pIJ916). The glk gene was further localised to a 2.9 kb BclI fragment of the cloned DNA by sub-cloning. Part or all of this fragment was present in each of five primary plasmid clones tested.

Cytological Evidence for Association of the Ends of the Linear Chromosome in Streptomyces coelicolor

“…S. coelicolor strain J1508 (hisA1 uraA1 strA1 pglNF, SCP2 negative [21]) was used in all FISH experiments because of the low level of fluorescence emitted by this strain (31). Liquid yeast extract-malt extract (YEME) medium (25 ml) supplemented with 50 g of histidine per ml and 7.5 g of uracil per ml (19) was inoculated with 50 l of an S. coelicolor J1508 spore suspension and incubated at 30°C with vigorous shaking for 40 to 45 h (optical density at 600 nm, ϳ0.7).…”

Carbon source regulation of antibiotic production

“…Glucose repression in S. coelicolor operates at the transcriptional level to repress enzymes involved in the use of glycerol, arabinose, fructose and galactose 122 and this effect seems to be due to either intermediates of carbohydrate catabolism, for example, fructose 1,6-diphosphate and glucose 6-phosphate 123,124 or enzymes of the glucose catabolic pathway, such as glucose kinase. [125][126][127][128] In this regard, it has been reported that mutants of S. coelicolor resistant to the nonutilizable glucose analog, 2-deoxyglucose (DOG), appear to be generally deficient in glucose repression. 122 These mutants (Dog R ) can utilize glycerol, arabinose, fructose and galactose in the presence of glucose.…”

“…129 Complementation of these mutants with the Glk gene (glkA) restores not only GlkA activity but also DOG sensitivity and partially restores glucose repression. 125,126 Similarly, when the S. coelicolor glkA gene, is introduced into a S. lividans Dog R mutant (unable to utilize glucose and the chitinase production of which is resistant to glucose repression), sensitivity to DOG and the ability to utilize glucose are restored, but glucose repression of chitinase production is only partially recovered. 128 Interestingly, when S. coelicolor Dog R mutants were complemented with the glk gene of the Gram-negative Zymomonas mobilis, Glk activity and glucose utilization are restored, but not glucose repression.…”

Identification and characterization of phosphoenolpyruvate: fructose phosphotransferase systems in three Streptomyces species

“…Inducible o r constitutive uptake has been reported for several sugars (Sabater et al, 1972;Sabater & Asensio, 1973 ;Hodgson, 1982). Bacteria utilizing glucose, mannose and fructose exhibit activities for ATP-dependent glucokinase, fructokinase and mannokinase (Sabater e t al., 1972;Ikeda e t al., 1984;Angell e t al., 1992) suggesting that transport systems for these sugars are not of the PTS type. At least five different groups have reported evidence leading to the conclusion that the PTS is lacking from the genus Streptomyces (Sabater e t al., 1972;Romano & Margiotta, 1980 ;Novotna & Hostilkk, 1985 ;GarciaDominguez et a/., 1989;L.…”