Alternative pathways of glucose transport in Prevotella bryantii B14

Abstract

Prevotella bryantii B14 grew faster on glucose than mannose (0.70 versus 0.45 h−1), but these sugars were used simultaneously rather than diauxically. 2‐deoxy‐glucose (2DG) decreased the growth rate of cells that were provided with either glucose or mannose, but 2DG did not completely prevent growth. Cells grown on glucose or mannose transported both 14C‐glucose and 14C‐mannose, but cells grown on glucose had over three‐fold higher rates of 14C‐glucose transport than cells grown on mannose. The 14C‐mannose transport rates of glucose‐ and mannose‐grown cells were similar. Woolf‐Augustinsson‐Hofstee plots were not linear, and it appeared that the glucose/mannose/2DG carrier acted as a facilitated diffusion system at high substrate concentrations. When cultures were grown on nitrogen‐deficient (excess sugar) medium, isolates had three‐fold lower 14C‐glucose transport, but the 14C‐mannose transport did not change significantly. 14C‐glucose and 14C‐mannose transport rates could be inhibited by 2DG and either mannose or glucose, respectively. The 14C‐glucose transport of mannose‐grown cells was inhibited more strongly by mannose and 2DG than those grown on glucose. Cells grown on glucose or mannose had similar ATP‐dependent glucokinase activity, and 2DG was a competitive inhibitor (Ki=0.75 mM). Thin layer chromatography indicated that cell extracts also had ATP‐dependent mannose phosphorylation, but only a small amount of phosphorylated 2DG was detected. Glucose, mannose or 2DG were not phosphorylated in the presence of PEP. Based on these results, it appeared that P. bryantii B14 had: (1) two mechanisms of glucose transport, a constitutive glucose/mannose/2DG carrier and an alternative glucose carrier that was regulated by glucose availability, (2) an ATP‐dependent glucokinase that was competitively inhibited by 2DG but was unable to phosphorylate 2DG at a rapid rate, and (3) virtually no PEP‐dependent glucose, mannose or 2DG phosphorylation activities.

The glucomannokinase of Prevotella bryantii B14 and its potential role in regulating β-glucanase expression The SWISS-PROT accession number for the sequence of P. bryantii B14 glucomannokinase reported in this paper is P82680.

“…Glucose and mannose were, however, utilized simultaneously and P. bryantii B " 4 had a glucose\mannose carrier (Fields & Russell, 2000). P. bryantii B " 4 also had an alternative glucose carrier that was induced by high concentrations of glucose, but mutants deficient in the alternative glucose carrier still repressed β-glucanase expression when glucose was available (Fields & Russell, 2000).…”

“…Glucose and mannose were, however, utilized simultaneously and P. bryantii B " 4 had a glucose\mannose carrier (Fields & Russell, 2000). P. bryantii B " 4 also had an alternative glucose carrier that was induced by high concentrations of glucose, but mutants deficient in the alternative glucose carrier still repressed β-glucanase expression when glucose was available (Fields & Russell, 2000). Because P. bryantii B " 4 does not have phosphotransferase system activity (Martin & Russell, 1986) and has very low concentrations of cAMP (Cotta et al, 1994), it appeared that previously defined catabolite regulatory mechanisms could not explain the regulation of β-glucanase in P. bryantii B " 4.…”

“…The ["%C]glucose phosphorylation was not significantly inhibited by a 10-fold excess of unlabelled glucose 6-phosphate, but was inhibited by unlabelled mannose (Fields & Russell, 2000). ["%C]Mannose phosphorylation could be strongly inhibited by unlabelled glucose and a 1 : 1 ratio of unlabelled glucose to ["%C]mannose caused more than a 50 % reduction in the mannose phosphorylation rate (Fig.…”

“…" 4 transports glucose and mannose rapidly, and the carriers appear to operate as facilitated diffusion systems at high substrate concentrations (Fields & Russell, 2000). P. bryantii B " 4 lacks phosphotransferase activity (Martin & Russell, 1986), but enzyme assays indicated that cells had ATP-dependent glucokinase activity (Fields & Russell, 2000).…”

“…P. bryantii B " 4 lacks phosphotransferase activity (Martin & Russell, 1986), but enzyme assays indicated that cells had ATP-dependent glucokinase activity (Fields & Russell, 2000). The glucokinase was inhibited by mannose, but it was unclear whether the glucokinase was catalysing mannose phosphorylation.…”