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Methionine transport in Yersinia pestis
Abstract:
Yersinia pestis TJW, an avirulent wild-type strain, requires phenylalanine and methionine for growth. It was of interest to examine and define the methionine transport system because of this requirement. The methionine system showed saturation kinetics with a Km for transport of approximately 9 times 10(-7) M. After 8 s of methionine transport, essentially all of the methionine label appeared in S-adenosyl-L-methionine (SAM) as detected in ethanol extracts. Small amounts of free methionine was detected intracellularly after 1 min of transport. Addition of glucose increased significantly the amount of intracellular methionine at 1 min. A series of SAM metabolic products was detected after 90 s to 5 min of transport including: 5'-thiomethyladenosine, homoserine lactone, S-adenosyl homoserine, and a fluorescent methyl receptor compound. Results from assays for SAM synthetase in spheroplast fractions showed a small (16%) but significant portion of synthetase associated with the membrane. However, most of the enzyme activity was associated with the cytoplasmic fraction. Methionine transport was characterized by a high degree of stereospecificity. No competition occurred from structurally unrelated amino acids. Although uptake was inhibited by uncoupling and sulfhydryl reagents, no efflux was observed. Results using energy inhibitors on unstarved and starved cells showed that respiratory inhibitors such as potassium cyanide (KCN) and amytal were most effective, and that arsenate was least effective. KCN plus arsenate completely blocked utilization of energy derived from glucose, and KCN completely blocked utilization of energy deived from D-lactate. The data indicate that methionine transport in Y. pestis is linked to the trapping of methionine in SAM. The results further suggest that this transport system can be classified as a permease-bound system where transport is coupled to an energized membrane state and to respiration.
Insights
Yersinia pestis TJW utilizes a stereospecific methionine transport system. This system is linked to S-adenosyl-L-methionine synthesis and requires an energized membrane state for function.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Yersinia pestis TJW, an avirulent strain, has specific growth requirements, including phenylalanine and methionine.
- Understanding methionine transport is crucial due to its essential role in Y. pestis TJW growth.
Purpose of the Study:
- To define the methionine transport system in Yersinia pestis TJW.
- To elucidate the biochemical mechanisms and energy requirements of methionine uptake.
Main Methods:
- Kinetic analysis of methionine transport.
- Metabolite analysis using ethanol extracts to identify intracellular products.
- Enzyme assays for S-adenosyl-L-methionine synthetase activity in spheroplast fractions.
- Stereospecificity and competition assays with various amino acids.
- Inhibition studies using uncoupling, sulfhydryl, and energy inhibitors (KCN, amytal, arsenate).
Main Results:
- Methionine transport exhibited saturation kinetics with a high affinity (Km ~ 9 x 10(-7) M).
- Intracellular methionine was rapidly converted to S-adenosyl-L-methionine (SAM) and its metabolic products.
- Methionine transport is stereospecific and not inhibited by unrelated amino acids.
- Transport is dependent on an energized membrane state, primarily linked to respiration, and is inhibited by respiratory inhibitors.
Conclusions:
- Methionine transport in Y. pestis TJW is coupled to SAM synthesis, suggesting a 'trapping' mechanism.
- The system functions as a permease-bound system, requiring energy from respiration.
- The findings provide insights into nutrient transport mechanisms in Yersinia species.