Hexokinase and glucose-phosphoenolpyruvate phosphotransferase synthesis in Klebsiella aerogenes strains growing in continuous culture.
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ABSTRACT:
SUBMITTER: Carter IS
PROVIDER: S-EPMC1165052 | biostudies-other | 1977 Sep
REPOSITORIES: biostudies-other
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Project description:1. Pullulanase synthesis was studied in 16 classified (N.C.I.B.) strains and in an industrial strain (R) of Klebsiella aerogenes grown in chemostats containing maltose as inducer and sole carbon source. 2. Maximum synthesis was associated with carbon-limited growth at a low dilution rate (about 0.2h(-1)). The enzyme remained firmly cell-bound and seemed to be located on the cell surface. 3. Three strains had high activity (R, N.C.I.B. 5938, 8017), twelve were intermediate, and two (N.C.I.B. 8153, 9146) had negligible activity but were inducible with pullulan. 4. Pullulan similarly induced low, but adequate, activity in the other strains in conditions (nutrient limitation other than carbon-limitation) in which pullulanase was otherwise very seriously repressed. Nevertheless, in carbon limitation pullulan induced no more enzyme than did maltose, maltotriose or oligosaccharide mixtures, and ;hyperactivity' never developed on protracted culture. 5. Cyclic AMP relieved the transient repression produced by adding glucose to maltose-limited cultures and a further change to glucose-limited conditions led to constitutive pullulanase synthesis. 6. Amylomaltase and alpha-glucosidase activities were also examined but in less detail. 7. The presence of pullulanase in maltose-limited growth is discussed, but no clear function can be assigned to it at present. The molar growth yields for all the strains were very similar, and no correlation was found between the overgrowth of one strain by another and pullulanase activity. Further, any function as a general branching enzyme in polysaccharide synthesis seems unlikely.
| S-EPMC1168509 | biostudies-other
Project description:1. Phosphatase synthesis was studied in Klebsiella aerogenes grown in a wide range of continuous-culture systems. 2. Maximum acid phosphatase synthesis was associated with nutrient-limited, particularly carbohydrate-limited, growth at a relatively low rate, glucose-limited cells exhibiting the highest activity. Compared with glucose as the carbon-limiting growth material, other sugars not only altered the activity but also changed the pH-activity profile of the enzyme(s). 3. The affinity of the acid phosphatase in glucose-limited cells towards p-nitrophenyl phosphate (K(m) 0.25-0.43mm) was similar to that of staphylococcal acid phosphatase but was ten times greater than that of the Escherichia coli enzyme. 4. PO(4) (3-)-limitation derepressed alkaline phosphatase synthesis but the amounts of activity were largely independent of the carbon source used for growth. 5. The enzymes were further differentiated by the effect of adding inhibitors (F(-), PO(4) (3-)) and sugars to the reaction mixture during the assays. In particular, it was shown that adding glucose, but not other sugars, stimulated the rate of hydrolysis of p-nitrophenyl phosphate by the acid phosphatase in carbohydrate-limited cells at low pH values (<4.6) but inhibited it at high pH values (>4.6). Alkaline phosphatase activity was unaffected. 6. The function of phosphatases in general is discussed and possible mechanisms for the glucose effect are outlined.
| S-EPMC1178562 | biostudies-other
Project description:Dilute cultures of wild-type Escherichia coli K12 and of derivatives impaired in one or other Enzyme-II component of the glucose phosphotransferase system were grown in continuous culture under glucose limitation. Cells harvested from the chemostat took up [U-14C]glucose from 0.1 mM solutions at rates directly related to the rates at which those cells had grown; the activity of the phosphotransferase system in those cells, rendered permeable with optimal accounts of toluene, parallels the ability of the cells to take up glucose. The capacity of these systems was rate-limiting for growth under the negligibly low glucose concentration in the chemostat, but was adequate to account for the stimulation of respiration observed when the cells were presented suddenly with excess glucose.
| S-EPMC1186484 | biostudies-other
Project description:In most streptococci, glucose is transported by the phosphoenolpyruvate (PEP):glucose/mannose phosphotransferase system (PTS) via HPr and IIAB(Man), two proteins involved in regulatory mechanisms. While most strains of Streptococcus thermophilus do not or poorly metabolize glucose, compelling evidence suggests that S. thermophilus possesses the genes that encode the glucose/mannose general and specific PTS proteins. The purposes of this study were to determine (i) whether these PTS genes are expressed, (ii) whether the PTS proteins encoded by these genes are able to transfer a phosphate group from PEP to glucose/mannose PTS substrates, and (iii) whether these proteins catalyze sugar transport. The pts operon is made up of the genes encoding HPr (ptsH) and enzyme I (EI) (ptsI), which are transcribed into a 0.6-kb ptsH mRNA and a 2.3-kb ptsHI mRNA. The specific glucose/mannose PTS proteins, IIAB(Man), IIC(Man), IID(Man), and the ManO protein, are encoded by manL, manM, manN, and manO, respectively, which make up the man operon. The man operon is transcribed into a single 3.5-kb mRNA. To assess the phosphotransfer competence of these PTS proteins, in vitro PEP-dependent phosphorylation experiments were conducted with purified HPr, EI, and IIAB(Man) as well as membrane fragments containing IIC(Man) and IID(Man). These PTS components efficiently transferred a phosphate group from PEP to glucose, mannose, 2-deoxyglucose, and (to a lesser extent) fructose, which are common streptococcal glucose/mannose PTS substrates. Whole cells were unable to catalyze the uptake of mannose and 2-deoxyglucose, demonstrating the inability of the S. thermophilus PTS proteins to operate as a proficient transport system. This inability to transport mannose and 2-deoxyglucose may be due to a defective IIC domain. We propose that in S. thermophilus, the general and specific glucose/mannose PTS proteins are not involved in glucose transport but might have regulatory functions associated with the phosphotransfer properties of HPr and IIAB(Man).
| S-EPMC194979 | biostudies-literature
Project description:In Vibrio cholerae, the genes required for chitin utilization and natural competence are governed by the chitin-responsive two-component system (TCS) sensor kinase ChiS. In the classical TCS paradigm, a sensor kinase specifically phosphorylates a cognate response regulator to activate gene expression. However, our previous genetic study suggested that ChiS stimulates the non-TCS transcriptional regulator TfoS by using mechanisms distinct from classical phosphorylation reactions (S. Yamamoto, J. Mitobe, T. Ishikawa, S. N. Wai, M. Ohnishi, H. Watanabe, and H. Izumiya, Mol Microbiol 91:326-347, 2014, https://doi.org/10.1111/mmi.12462). TfoS specifically activates the transcription of tfoR, encoding a small regulatory RNA essential for competence gene expression. Whether ChiS and TfoS interact directly remains unknown. To determine if other factors mediate the communication between ChiS and TfoS, we isolated transposon mutants that turned off tfoR::lacZ expression but possessed intact chiS and tfoS genes. We demonstrated an unexpected association of chitin-induced signaling pathways with the glucose-specific enzyme IIA (EIIAglc) of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) for carbohydrate uptake and catabolite control of gene expression. Genetic and physiological analyses revealed that dephosphorylated EIIAglc inactivated natural competence and tfoR transcription. Chitin-induced expression of the chb operon, which is required for chitin transport and catabolism, was also repressed by dephosphorylated EIIAglc Furthermore, the regulation of tfoR and chb expression by EIIAglc was dependent on ChiS and intracellular levels of ChiS were not affected by disruption of the gene encoding EIIAglc These results define a previously unknown connection between the PTS and chitin signaling pathways in V. cholerae and suggest a strategy whereby this bacterium can physiologically adapt to the existing nutrient status.IMPORTANCE The EIIAglc protein of the PTS coordinates a wide variety of physiological functions with carbon availability. In this report, we describe an unexpected association of chitin-activated signaling pathways in V. cholerae with EIIAglc The signaling pathways are governed by the chitin-responsive TCS sensor kinase ChiS and lead to the induction of chitin utilization and natural competence. We show that dephosphorylated EIIAglc inhibits both signaling pathways in a ChiS-dependent manner. This inhibition is different from classical catabolite repression that is caused by lowered levels of cyclic AMP. This work represents a newly identified connection between the PTS and chitin signaling pathways in V. cholerae and suggests a strategy whereby this bacterium can physiologically adapt to the existing nutrient status.
| S-EPMC5573088 | biostudies-literature
Project description:PurposeThis study aimed to evaluate the molecular characteristics and prevalence of clinical carbapenem-resistant Klebsiella aerogenes (CRKA), collected during an outbreak in a Chinese tertiary hospital.MethodsAntimicrobial susceptibility test, using 17 antibiotics, was performed on 14 CRKA isolates. The strains were examined for the presence of β-lactamase genes by PCR, and efflux pump phenotype was determined by efflux pump inhibition test. Presence of outer-membrane porins was examined. Clonal relatedness among the isolates was investigated by pulsed-field gel electrophoresis (PFGE). S1 nuclease-PFGE and plasmid incompatibility group analysis were performed to determine plasmids, and the genetic environment of bla KPC-2 was analyzed. Epidemiological data were collected via chart review.ResultsThe 14 CRKA isolates were all resistant to carbapenems; five distinct groups (PFGE types A-E) were observed. All 14 isolates carried the bla KPC-2 gene. S1 nuclease-PFGE indicated the size of bla KPC-2-carrying plasmids to range from 20 kb to 200 kb, and the 14 plasmids belonged to various incompatibility groups. The most frequent genetic environment of bla KPC-2 was Tn1721- bla KPC-2-ΔTn3-IS26. PFGE type A group, including 11 KPC-2-producing clinical isolates, was primarily responsible for dissemination.ConclusionOur findings suggest both transposons and vertical transmission to contribute to the transformation of bla KPC-2. The results strongly suggest strict implementation of infection control of CRKA, in healthcare facilities.
| S-EPMC6814356 | biostudies-literature
Project description:1. A rapid-sampling technique was used to obtain perchloric acid extracts of cells growing in a chemostat culture, so that meaningful values for ATP content could be obtained in spite of the fact that the turnover time for the total ATP content was about 1sec. 2. For steady-state growth, it was found that, in a glucose-limited chemostat culture, the ATP/ADP concentration ratio was approximately constant with changes in dissolved-oxygen tensions above the critical value, but fell when the culture was grown under oxygen-limited conditions and was at a minimum in anaerobically grown cultures. The steady-state ATP content was lower in cells growing under nitrogen-limited conditions with glucose in excess than in glucose-limited cells. The steady-state ATP content was independent of growth rate at growth rates over 0.1hr.(-1). 3. When the respiration rate of the cells was stimulated by lowering the oxygen tension the ATP content did not increase, indicating either an increased turnover rate of ATP or a fall in the P/O ratio. The sudden addition of extra glucose or succinate to a glucose-limited culture increased the respiration rate of the cells, but the ATP content quickly returned to the steady-state value after initial perturbations. This control over ATP content is explained in terms of regulation by adenine nucleotides of the catabolism and anabolism of glucose. An exception to this control over ATP content was found when the respiration rate was stimulated by addition of an antifoam.
| S-EPMC1187768 | biostudies-other
Project description:In Escherichia coli, the transport and phosphorylation of glucose is mainly accomplished by the phosphoenolpyruvate-dependent glucose-specific phosphotransferase system (PTSGlc), which is, therefore, frequently selected as a target for engineering to increase the intracellular level of phosphoenolpyruvate. Here we characterized the effects of a low glucose concentration on the growth, glucose consumption, and acetate secretion of individual strains with a single PTSGlc mutation. We found that most mutants accumulated similar amounts of biomass, consumed glucose at lower rates, and secreted less acetate compared with the wild-type parental strain. The exception was the growth-impaired strain MG1655I harboring a ptsI deletion. In summary, the fermentation performance of mutant strains under 5 g/L glucose was obviously different with those strains under 20 g/L glucose. This study is a good complement to the knowledge of PTSGlc in E. coli and indicates that engineering the components of PTSGlc should be carefully optimized, particularly during fermentation in the presence of low concentrations of glucose.
| S-EPMC6346689 | biostudies-literature
Project description:Urease from Klebsiella aerogenes is composed of three subunits (UreA-UreB-UreC) that assemble into a (UreABC)(3) quaternary structure. UreC harbors the dinuclear nickel active site, whereas the functions of UreA and UreB remain unknown. UreD and UreF accessory proteins previously were suggested to reposition UreB and increase the level of exposure of the nascent urease active site, thus facilitating metallocenter assembly. In this study, cells were engineered to separately produce (UreAC)(3) or UreB, and the purified proteins were characterized. Monomeric UreB spontaneously binds to the trimeric heterodimer of UreA and UreC to form (UreABC*)(3) apoprotein, as shown by gel filtration chromatography, integration of electrophoretic gel band intensities, and mass spectrometry. Similar to the authentic urease apoprotein, the active enzyme is produced by incubation of (UreABC*)(3) with Ni(2+) and bicarbonate. Conversely, UreB?1-19, lacking the 19-residue potential hinge and tether to UreC, does not form a complex with (UreAC)(3) and yields negligible levels of the active enzyme when incubated under activation conditions with (UreAC)(3). Comparison of activities and nickel contents for (UreAC)(3), (UreABC*)(3), and (UreABC)(3) samples treated with Ni(2+) and bicarbonate and then desalted indicates that UreB facilitates efficient incorporation of the metal into the active site and protects the bound metal from chelation. Amylose resin pull-down studies reveal that MBP-UreD (a fusion of maltose binding protein with UreD) forms complexes with (UreABC)(3), (UreAC)(3), and UreB in vivo, but not in vitro. By contrast, MBP-UreD does not form an in vivo complex with UreB?1-19. The soluble MBP-UreD-UreF-UreG complex binds in vitro to (UreABC)(3), but not to (UreAC)(3) or UreB. Together, these data demonstrate that UreB facilitates the interaction of urease with accessory proteins during metallocenter assembly, with the N-terminal hinge and tether region being specifically required for this process. In addition to its role in urease activation, UreB enhances the stability of UreC against proteolytic cleavage.
| S-EPMC3202645 | biostudies-literature
Project description:Efficient cofermentation of D-glucose, D-xylose, and L-arabinose, three major sugars present in lignocellulose, is a fundamental requirement for cost-effective utilization of lignocellulosic biomass. The Gram-positive anaerobic bacterium Clostridium acetobutylicum, known for its excellent capability of producing ABE (acetone, butanol, and ethanol) solvent, is limited in using lignocellulose because of inefficient pentose consumption when fermenting sugar mixtures. To overcome this substrate utilization defect, a predicted glcG gene, encoding enzyme II of the D-glucose phosphoenolpyruvate-dependent phosphotransferase system (PTS), was first disrupted in the ABE-producing model strain Clostridium acetobutylicum ATCC 824, resulting in greatly improved D-xylose and L-arabinose consumption in the presence of D-glucose. Interestingly, despite the loss of GlcG, the resulting mutant strain 824glcG fermented D-glucose as efficiently as did the parent strain. This could be attributed to residual glucose PTS activity, although an increased activity of glucose kinase suggested that non-PTS glucose uptake might also be elevated as a result of glcG disruption. Furthermore, the inherent rate-limiting steps of the D-xylose metabolic pathway were observed prior to the pentose phosphate pathway (PPP) in strain ATCC 824 and then overcome by co-overexpression of the D-xylose proton-symporter (cac1345), D-xylose isomerase (cac2610), and xylulokinase (cac2612). As a result, an engineered strain (824glcG-TBA), obtained by integrating glcG disruption and genetic overexpression of the xylose pathway, was able to efficiently coferment mixtures of D-glucose, D-xylose, and L-arabinose, reaching a 24% higher ABE solvent titer (16.06 g/liter) and a 5% higher yield (0.28 g/g) compared to those of the wild-type strain. This strain will be a promising platform host toward commercial exploitation of lignocellulose to produce solvents and biofuels.
| S-EPMC3208996 | biostudies-literature