The phosphotransferase system (PTS) of Streptomyces coelicolor

Abstract

HPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS) controls sugar uptake and carbon utilization in low-GC Gram-positive bacteria and in Gram-negative bacteria. We have purified HPr from Streptomyces coelicolor cell extracts. The N-terminal sequence matched the product of an S. coelicolor orf, designated ptsH, sequenced as part of the S. coelicolor genome sequencing project. The ptsH gene appears to form a monocistronic operon. Determination of the evolutionary relationship revealed that S. coelicolor HPr is equally distant to all known HPr and HPr-like proteins. The presumptive phosphorylation site around histidine 15 is perfectly conserved while a second possible phosphorylation site at serine 47 is not wellconserved. HPr was overproduced in Escherichia coli in its native form and as a histidine-tagged fusion protein.Histidine-tagged HPr was purified to homogeneity. HPr was phosphorylated by its own enzyme I (EI) and heterologously phosphorylated by EI of Bacillus subtilis and Staphylococcus aureus, respectively. This phosphoenolpyruvate-dependent phosphorylation was absent in an HPr mutant in which histidine 15 was replaced by alanine. Reconstitution of the fructose-specific PTS demonstrated that HPr could efficiently phosphorylate enzyme II Fructose . HPr-P could also phosphorylate enzyme II Glucose of B. subtilis, enzyme II Lactose of S. aureus, and IIAMannitol of E. coli. ATP-dependent phosphorylation was detected with HPr kinase/phosphatase of B. subtilis. These results present the first identification of a gene of the PTS complement of S. coelicolor, providing the basis to elucidate the role(s) of HPr and the PTS in this class of bacteria.Keywords: phosphotransferase system; protein phosphorylation; HPr; sugar transport; Streptomyces.Streptomyces are high-GC, Gram-positive soil bacteria which grow vegetatively as a branching mycelial mass using a wide spectrum of carbon sources such as cellulose, chitin, xylan, and many mono-and disaccharides. Upon nutrient depletion this primary metabolic phase may switch to a secondary metabolic phase resulting, for example, in antibiotic production and morphogenesis [1]. Despite the interesting life cycle and the commercial importance, there is limited knowledge on nutrient sensing, carbohydrate transport, and regulation of carbohydrate utilization, and how these may influence secondary metabolic processes.So far, the mechanism of sugar uptake has been described for only a few carbohydrates. It was demonstrated that cellobiose, cellotriose, maltose, and xylobiose are taken up via ABC transport systems, and that fructose is apparently transported via a phosphoenolpyruvate:fructose phosphotransferase system (PTS) [2±5]. The utilization of carbohydrates in many cases is governed by carbon catabolite repression (CCR) [6]. CCR can be exerted by various carbohydrates such as glucose, fructose, mannitol, or gluconate [7]. The signal transduction pathway leading to CCR is not known. Mutations in glkA, encoding glucose kinase, ...

Carbon source regulation of antibiotic production

“…100,101 In S. coelicolor, there are at least four PTS transporters, all of which use the universal PTS energy-coupling enzymes enzyme I (EI, encoded by ptsI) and HPr (encoded by ptsH). [102][103][104] Two of the transporters have been characterized in detail, and are responsible for the transport of D-fructose and N-acetylglucosamine. 102,105 Glucose is not transported by the PTS, but through the specific GlcP permease, which is probably the main reason why PTS is not involved in glucose repression.…”

The phosphotransferase system of Streptomyces coelicolor

“…E. coli DH5a was the host strain for subcloning experiments [19]. E. coli FT1 DptsHIcrr Kan r (pLysS Cm r ) was used to produce native and hexa-histidine (His)-tagged S. coelicolor IIA Crr , Histagged S. coelicolor HPr, and His-tagged E. coli IIA Glc [16]. M15(pREP4, pAG3) was used to produce His-tagged Bacillus subtilis EI [16,20].…”

“…E. coli FT1 DptsHIcrr Kan r (pLysS Cm r ) was used to produce native and hexa-histidine (His)-tagged S. coelicolor IIA Crr , Histagged S. coelicolor HPr, and His-tagged E. coli IIA Glc [16]. M15(pREP4, pAG3) was used to produce His-tagged Bacillus subtilis EI [16,20]. The glucose-negative E. coli crr mutant strain LM1 tonA galT nagE manAI kba ts rpsL xyl metB thi his mglA-C argG crr was used for heterologous complementation experiments [21].…”

“…This could be correlated with the variation of the phosphorylation state of IIA Glc . Recently, another cellular function for IIA Glc has been proposed that suggests that it may be involved in the linkage between carbon metabolism and stress response [14].We have described that the PTS is operative in streptomycetes [15,16]. Analysis of the S. coelicolor genome revealed the presence of nine genes that may encode four sugar-specific permeases, as well as the genes ptsH and ptsI Correspondence to F. Titgemeyer, Friedrich-Alexander-Universita¨t Erlangen-Nu¨rnberg, Lehrstuhl fu¨r Mikrobiologie, Staudtstrasse 5, 91058 Erlangen, Germany.…”

“…We have described that the PTS is operative in streptomycetes [15,16]. Analysis of the S. coelicolor genome revealed the presence of nine genes that may encode four sugar-specific permeases, as well as the genes ptsH and ptsI encoding HPr and EI [17].…”