scholarly journals Proton-linked l-fucose transport in Escherichia coli

1987 ◽  
Vol 248 (2) ◽  
pp. 495-500 ◽  
Author(s):  
S A Bradley ◽  
C R Tinsley ◽  
J A R Muiry ◽  
P J F Henderson
Keyword(s):  
Escherichia Coli ◽  
Alkaline Ph ◽  
Electrochemical Gradient ◽  
Ph Change ◽  
Ph Optimum ◽  
Kinetic Measurements ◽  
E Coli ◽  
Steady State Kinetic ◽  
Periplasmic Proteins ◽  
State Kinetic

1. Addition of L-fucose to energy-depleted anaerobic suspensions of Escherichia coli elicited an uncoupler-sensitive alkaline pH change diagnostic of L-fucose/H+ symport activity. 2. L-Galactose or D-arabinose were also substrates, but not inducers, for the L-fucose/H+ symporter. 3. L-Fucose transport into subcellular vesicles was dependent upon respiration, displayed a pH optimum of about 5.5, and was inhibited by protonophores and ionophores. 4. These results showed that L-fucose transport into E. coli was energized by the transmembrane electrochemical gradient of protons. 5. Neither steady state kinetic measurements nor assays of L-fucose binding to periplasmic proteins revealed the existence of a second L-fucose transport system.

scholarly journals Proton-linked l-rhamnose transport, and its comparison with l-fucose transport in Enterobacteriaceae

1993 ◽  
Vol 290 (3) ◽  
pp. 833-842 ◽  
Author(s):  
J A R Muiry ◽  
T C Gunn ◽  
T P McDonald ◽  
S A Bradley ◽  
C G Tate ◽  
...  
Keyword(s):  
Transport System ◽  
Erwinia Carotovora ◽  
Alkaline Ph ◽  
Transport Activity ◽  
Ph Change ◽  
Kinetic Measurements ◽  
E Coli ◽  
Steady State Kinetic ◽  
Competitive Inhibitors ◽  
State Kinetic

1. An alkaline pH change occurred when L-rhamnose, L-mannose or L-lyxose was added to L-rhamnose-grown energy-depleted suspensions of strains of Escherichia coli. This is diagnostic of sugar-H+ symport activity. 2. L-Rhamnose, L-mannose and L-lyxose were inducers of the sugar-H+ symport and of L-[14C]rhamnose transport activity. L-Rhamnose also induced the biochemically and genetically distinct L-fucose-H+ symport activity in strains competent for L-rhamnose metabolism. 3. Steady-state kinetic measurements showed that L-mannose and L-lyxose were competitive inhibitors (alternative substrates) for the L-rhamnose transport system, and that L-galactose and D-arabinose were competitive inhibitors (alternative substrates) for the L-fucose transport system. Additional measurements with other sugars of related structure defined the different substrate specificities of the two transport systems. 4. The relative rates of H+ symport and of sugar metabolism, and the relative values of their kinetic parameters, suggested that the physiological role of the transport activity was primarily for utilization of L-rhamnose, not for L-mannose or L-lyxose. 5. L-Rhamnose transport into subcellular vesicles of E. coli was dependent on respiration, was optimal at pH 7, and was inhibited by protonophores and ionophores. It was insensitive to N-ethylmaleimide or cytochalasin B. 6. L-Rhamnose, L-mannose and L-lyxose each elicited an alkaline pH change when added to energy-depleted suspensions of L-rhamnose-grown Salmonella typhimurium LT2, Klebsiella pneumoniae, Klebsiella aerogenes, Erwinia carotovora carotovora and Erwinia carotovora atroseptica. The relative rates of subsequent acidification varied, depending on both the organism and the sugar. L-Fucose promoted an alkaline pH change in all the L-rhamnose-induced organisms except the Erwinia species. No L-rhamnose-H+ symport occurred in any organism grown on L-fucose. 7. All these results showed that L-rhamnose transport into the micro-organisms occurred by a system different from that for L-fucose transport. Both systems are energized by the trans-membrane electrochemical gradient of protons. 8. Neither steady-state kinetic measurements nor binding-protein assays revealed the existence of a second L-rhamnose transport system in E. coli.

scholarly journals Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase

1999 ◽  
Vol 343 (2) ◽  
pp. 397-402 ◽  
Author(s):  
Daniel M. STOUGHTON ◽  
Gerardo ZAPATA ◽  
Robert PICONE ◽  
Willie F. VANN
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Active Site ◽  
Positive Charge ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
E Coli ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Escherichia Coli K1

Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of CMP-sialic acid from CTP and sialic acid. The active site of the 418 amino acid E. coli enzyme was localized to its N-terminal half. The bacterial CMP-sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, at their N-termini. Several basic residues have been identified at or near the active site of the E. coli enzyme by chemical modification and site-directed mutagenesis. Only one of the lysines in the N-terminal motif, Lys-21, appears to be essential for activity. Mutation of Lys-21 in the N-terminal motif results in an inactive enzyme. Furthermore, Arg-12 of the N-terminal motif appears to be an active-site residue, based on the following evidence. Substituting Arg-12 with glycine or alanine resulted in inactive enzymes, indicating that this residue is required for enzymic activity. The Arg-12 → Lys mutant was partially active, demonstrating that a positive charge is required at this site. Steady-state kinetic analysis reveals changes in kcat, Km and Ks for CTP, which implicates Arg-12 in catalysis and substrate binding.

scholarly journals pH-Dependent Catabolic Protein Expression during Anaerobic Growth of Escherichia coli K-12

2004 ◽  
Vol 186 (1) ◽  
pp. 192-199 ◽  
Author(s):  
Elizabeth Yohannes ◽  
D. Michael Barnhart ◽  
Joan L. Slonczewski
Keyword(s):  
Escherichia Coli ◽  
Ph Regulation ◽  
Metabolic Enzymes ◽  
Anaerobic Growth ◽  
High Ph ◽  
E Coli ◽  
Catabolic Enzymes ◽  
Periplasmic Proteins ◽  
Ph Dependent ◽  
K 12

ABSTRACT During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH. Additional modes of pH regulation were revealed under anaerobiosis. E. coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels. A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures. The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, and HisD), periplasmic proteins (ProX, OppA, DegQ, MalB, and MglB), and stress proteins (DsbA, Tig, and UspA). High-pH induction of the glycolytic enzymes DhaKLM and GapA suggested that there was increased fermentation to acids, which helped neutralize alkalinity. Reporter lac fusion constructs showed base induction of sdaA encoding serine deaminase under anaerobiosis; in addition, the glutamate decarboxylase genes gadA and gadB were induced at the high pH anaerobically but not with aeration. This result is consistent with the hypothesis that there is a connection between the gad system and GabT metabolism of 4-aminobutanoate. On the other hand, 13 other proteins were induced by acid; these proteins included metabolic enzymes (GatY and AckA), periplasmic proteins (TolC, HdeA, and OmpA), and redox enzymes (GuaB, HmpA, and Lpd). The acid induction of NikA (nickel transporter) is of interest because E. coli requires nickel for anaerobic fermentation. The position of the NikA spot coincided with the position of a small unidentified spot whose induction in aerobic cultures was reported previously; thus, NikA appeared to be induced slightly by acid during aeration but showed stronger induction under anaerobic conditions. Overall, anaerobic growth revealed several more pH-regulated proteins; in particular, anaerobiosis enabled induction of several additional catabolic enzymes and sugar transporters at the high pH, at which production of fermentation acids may be advantageous for the cell.

pH-mediated control of anti-aggregation activities of cyanobacterial and E. coli chaperonin GroELs

2020 ◽  
Author(s):  
Tahmina Akter ◽  
Hitoshi Nakamoto
Keyword(s):  
Escherichia Coli ◽  
Lactate Dehydrogenase ◽  
Malate Dehydrogenase ◽  
Citrate Synthase ◽  
Ph Dependence ◽  
Chaperone Activity ◽  
Ph Change ◽  
Aggregation Assay ◽  
E Coli ◽  
Aggregation Activity

Abstract In contrast to Escherichia coli, cyanobacteria have multiple GroELs, the bacterial homologues of chaperonin/Hsp60. We have shown that cyanobacterial GroELs are mutually distinct and different from E. coli GroEL with which the paradigm for chaperonin structure/function has been established. However, little is known about regulation of cyanobacterial GroELs. This study investigated effect of pH (varied from 7.0 to 8.5) on chaperone activity of GroEL1 and GroEL2 from the cyanobacterium Synechococcus elongatus PCC7942 and E. coli GroEL. GroEL1 and GroEL2 showed pH dependency in suppression of aggregation of heat-denatured malate dehydrogenase, lactate dehydrogenase and citrate synthase. They exhibited higher anti-aggregation activity at more alkaline pHs. Escherichia coli GroEL showed a similar pH-dependence in suppressing aggregation of heat-denatured lactate dehydrogenase. No pH dependence was observed in all the GroELs when urea-denatured lactate dehydrogenase was used for anti-aggregation assay, suggesting that the pH-dependence is related to some denatured structures. There was no significant influence of pH on the chaperone activity of all the GroELs to promote refolding of heat-denatured malate dehydrogenase. It is known that pH in cyanobacterial cytoplasm increases by one pH unit following a shift from darkness to light, suggesting that the pH-change modulates chaperone activity of cyanobacterial GroEL1 and GroEL2.

Sign in / Sign up

Export Citation Format

Share Document