Cellobiose transport by mixed ruminal bacteria from a Cow

Area of Science:

• Rumen microbiology
• Carbohydrate metabolism
• Bacterial transport systems

Background:

• Cellobiose is a key disaccharide in ruminant digestion, derived from plant fiber breakdown.
• Understanding its transport is crucial for optimizing energy utilization in livestock.
• Ruminal bacteria possess enzymes for cellobiose hydrolysis, but direct transport mechanisms are less understood.

Purpose of the Study:

• To elucidate the mechanisms of cellobiose transport in mixed ruminal bacteria.
• To quantify the kinetic parameters of cellobiose uptake.
• To determine the contribution of direct transport versus extracellular hydrolysis.

Main Methods:

• Kinetic analysis of cellobiose uptake using radiolabeled substrates.
• Enzyme activity assays for extracellular and cell-associated cellobiases.
• Inhibitor studies using nojirimycin-1-sulfate and assessment of transport under varying pH and glucose concentrations.
• Measurement of phosphoenolpyruvate phosphotransferase (PEP-PT) activity and its role in cellobiose transport.

Main Results:

• Cellobiose uptake exhibited Michaelis-Menten kinetics with Km of 14 μM and Vmax of 10 nmol/min/mg protein.
• Extracellular and cell-associated cellobiases had higher Vmax and lower affinity compared to cellobiose transport.
• Direct cellobiose transport increased significantly at low concentrations (<0.02 mM), exceeding 20%.
• Transport was primarily mediated by the phosphoenolpyruvate phosphotransferase system (PEP-PT), with a secondary ATP-dependent active transport system.

Conclusions:

• Mixed ruminal bacteria actively transport cellobiose, predominantly via PEP-PT.
• Direct cellobiose uptake is a significant pathway, especially at low substrate concentrations.
• Understanding these transport mechanisms can inform strategies for improving ruminal fermentation and nutrient availability.