The phosphoenolpyruvate-dependent carbohydrate: Phosphotransferase system enzymes II as chemoreceptors in chemotaxis of Escherichia coli K12

Abstract

In Escherichia coli K12, eight substrate-specific, membrane-bound enzymes II of the PEP-dependent carbohydrate: phosphotransferase system (PTS), specific for hexoses, hexosamines and hexitols, have been characterised in a series of isogenic and constitutive strains. In such mutants, lacking all but one enzyme II, the transport and vectorial phosphorylation activities as well as the chemotactical response in capillary tube assays have been compared. According to the data obtained, all enzymes II not only are directly involved in the transport and vectorial phosphorylation of their substrates, but they have also a primary role as the chemoreceptors for these substrates: (1) Metabolism of the attractant beyond the phosphorylation step is not a pre-requisite to eliciting positive chemotaxis. (2) Mutants, having only one enzyme II react in the capillary tube assay only to substrates of this enzyme II, but not to substrates of the missing enzymes II. This holds for enzymes II consisting of one membrane-bound protein as well as for systems containing a soluble factor III (FIII). (3) The substrate specificities or affinities, whether tested by transport and chemotaxis assays in vivo or by phosphorylation tests in vitro, are in correspondence. (4) The activities of enzymes II, regulated in a complex way at the level of enzyme synthesis and activity and tested as above, are also in agreement, (5) Mutants lacking the soluble proteins enzyme I or HPr of the PTS no longer respond chemotactically to any substrate taken up and phosphorylated by enzymes II. It is concluded that in PTS enzymes II some functions required for transport and chemotaxis are identical. It is suggested furthermore, that the alternation of intrinsic membrane-bound proteins between a phosphorylated and a dephosphorylated state, rather than binding of the substrate to the enzyme II, is the decisive stimulus in the chemotaxis toward carbohydrates taken up by these transport systems.

Analysis of Sucrose Catabolism in Klebsiella pneumoniae and in Scr+ Derivatives of Escherichia coli K12

“…Interestingly, the phosphorylation depended on the presence of the soluble EIII of the PTS, product of the gene crr. This dependence of a membrane-bound and sucrose-specific EIIScr upon EIII has also been observed in the pUR400-encoded sucrose system (Lengeler et al, 1982). To isolate mutants with defects in sucrose transport and metabolism, strain KAY2026 was mutagenized with ethyl methanesulphonate and treated with the selective agent streptozotocine $ EIISCr activity was tested using 0.56 pM-[ 14C]sucrose, purified membrane vesicles of the strains and cultures indicated and membrane-free cytoplasmic extracts of KAY2026 or PRL-R3 from cells pregrown on D-glUCit01.…”

“…Metabolism of the disaccharide sucrose (D-glucopyranosyl-~l,2-~-fructofuranoside) by bacteria has been studied in detail in Bacillus subtilis (Lepesant et al, 1976;Aymerich et al, 1986;Amory et al, 1987), in the cariogenic oral streptococci (St Martin & Wittenberger, 1979;Mimura et al, 1984) and in a clinical isolate of a sucrose-positive strain of Salmonella carrying the sucrose plasmid pUR400 (alias pSCR53) (Wohlhieter et al, 1975;Schmid et al, 1982;Lengeler et al, 1982). In these organisms, sucrose is taken up and phosphorylated by a specific enzymeIIScr (EIISr) of the phosphoenolpyruvate (PEP) dependent carbohydrate phosphotransferase system (PTS) to yield, in combination with a soluble enzyme111 (EIII), sucrose 6-phosphate.…”

The enigmatic lack of glucose utilization in Streptomyces clavuligerus is due to inefficient expression of the glucose permease gene

“…Two glucose transport-negative mutants, E. coli LM1 and E. coli LR2-175 (Aulkemeyer et al, 1991;Lengeler et al, 1981), were transformed with plasmid pglcP, in which S. clavuligerus glcP is expressed from the lacZ promoter, and with plasmids pFT76 (containing S. coelicolor glcP1) and pBRel, as positive and negative controls, respectively. The strains were streaked on MacConkey agar and on MacConkey agar supplemented with 50 mM glucose.…”

The phosphotransferase system of Streptomyces coelicolor

“…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].…”

“…IIA Glc becomes phosphorylated by the general PTS proteins, which are histidine-containing phosphoryl carrier protein (HPr) and enzyme I (EI). In turn, it phosphorylates the sugar-specific PTS permeases that catalyse the uptake of glucose, trehalose, and sucrose [8,10,11]. Mutations in the respective gene crr exhibit a pleiotropic catabolite repression resistant phenotype [12].…”