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SUMMARY: In Arthrobacter pyridinolis, a respiration-coupled transport system for L-rhamnose caused accumulation of free L-rhamnose, while a phosphoenolpyruvate: L-rhamnose phosphotransferase system caused accumulation of L-rhamnose 1-phosphate (Levinson & Krulwich, 1974). The pathways for subsequent metabolism of L-rhamnose and L-rhamnose 1-phosphate have now been investigated. Arthrobacter pyridinolis contains an inducible L-rhamnose isomerase and L-rhamnulokinase, as well as a constitutive L-rhamnulose 1-phosphate aldolase. Results with mutants which are unable to metabolize L-rhamnose suggest the presence of an L-rhamnose 1-phosphate phosphatase, which forms free L-rhamnose by hydrolysis of L-rhamnose 1-phosphate produced by the phosphotransferase system. Mutants which lack this enzyme exhibited severe inhibition of growth in the presence of L-rhamnose plus any of a variety of carbon sources. There is some evidence that this inhibition was due to accumulation of L-rhamnose 1-phosphate at toxic concentrations within the bacteria. The metabolism of L-rhamnose transported by the phosphotransferase system therefore appears to occur by hydrolysis of L-rhamnose 1-phosphate to free L-rhamnose by a phosphatase. Metabolism of the L-rhamnose thus produced, and of that accumulated by the respiration-coupled transport system, then proceeds by the sequence of reactions: L-rhamnose → L-rhamnulose → L-rhamnulose 1-phosphate → dihydroxyacetone phosphate plus L-lactaldehyde.