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Glucose toxicity in Prevotella ruminicola: methylglyoxal accumulation and its effect on membrane physiology
1Agricultural Research Service, Cornell University, Ithaca, New York 14853-8101.
Abstract:
When the ruminal bacterium prevotella ruminicola B(1)4-M was grown in a defined medium with an excess of glucose (3.6 mM ammonia and 50 mM glucose), the cells accumulated large amounts of cellular polysaccharide and the viable cell number decreased at least 1,000-fold. This decrease in viability was correlated with an accumulation of methylglyoxal in the supernatant (3 to 4 mM). Other genetically distinct strains of P. ruminicola produced methylglyoxal, but methylglyoxal production was not ubiquitous among the strains. When P. ruminicola B(1)4-M was grown in continuous culture (dilution rate, 0.1 h-1) with an excess of glucose, there was an oscillating pattern of growth and cell death which was correlated with the accumulation and washout of methylglyoxal from the culture vessel. Mutants which resisted an excess of glucose took up glucose at a slower rate and produced less methylglyoxal than the wild type. These mutants were, however, not stable. There was always a long lag time, and the mutants could only be maintained with a daily transfer schedule. When the mutants were transferred less frequently, methylglyoxal eventually accumulated and the cultures died. The mutants transported glucose at a threefold-slower rate than the wild type, and in each case the carrier had more than one binding site for glucose. Because glucose transport could not be driven by phosphoenolpyruvate or ATP, the glucose carrier of P. ruminicola is probably a proton symport system. When P. ruminicola B(1)4-M cultures were treated with 4 mM methylglyoxal, the delta psi decreased even though intracellular ATP concentrations were high.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
High glucose levels in Prevotella ruminicola cultures caused cell death due to methylglyoxal accumulation. Mutants showed slower glucose uptake and reduced methylglyoxal production, but were unstable.
Area of Science:
- Microbiology
- Bacterial Physiology
- Rumen Microbiology
Background:
- Prevotella ruminicola is a key ruminal bacterium.
- High glucose concentrations can negatively impact bacterial growth and viability.
- Methylglyoxal is a reactive byproduct of glycolysis.
Purpose of the Study:
- Investigate the effects of excess glucose on Prevotella ruminicola B(1)4-M.
- Determine the role of methylglyoxal in glucose-induced cell death.
- Characterize glucose transport mechanisms and their regulation.
Main Methods:
- Culturing Prevotella ruminicola B(1)4-M in defined media with excess glucose.
- Continuous culture experiments to observe growth and death patterns.
- Isolation and characterization of glucose-resistant mutants.
- Measurement of methylglyoxal accumulation and glucose uptake rates.
- Analysis of membrane potential (delta psi) and intracellular ATP levels.
Main Results:
- Excess glucose led to polysaccharide accumulation, significant cell death, and methylglyoxal production in P. ruminicola B(1)4-M.
- Methylglyoxal accumulation oscillated with growth and death in continuous culture.
- Mutants resistant to excess glucose exhibited slower glucose uptake and lower methylglyoxal production but were unstable.
- The glucose transporter in P. ruminicola appears to be a proton symport system.
- Methylglyoxal treatment reduced membrane potential (delta psi) despite high intracellular ATP.
Conclusions:
- Methylglyoxal is a key factor in glucose-induced cell death in Prevotella ruminicola.
- The bacterium's glucose transport system and its regulation are linked to its susceptibility to high glucose conditions.
- Further research is needed to understand the stability of resistant mutants and the precise mechanisms of methylglyoxal toxicity.
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