TRANSPORT OF METHANOL AND FORMALDEHYDE IN METHYLOMONAS L3 AND METHYLOTROPHIC STRAIN T15 (BACTERIA, ENERGY COUPLING, PROTEINS, PERMEASES)
DIWAN, ANIL RATNAKAR
Doctor of Philosophy
The transport of methanol and formaldehyde in obligate RMP-cycle methylotrophs Methylomonas L3 and strain T15 was studied by using radioactive tracers in anaerobic whole cell suspensions. The corrected accumulation ratios were found to be about 24 to 30 for methanol and about 40 to 75 (respectively) for formaldehyde in Methylomonas L3, depending upon the experimental conditions. In both strains, the transport profiles of methanol and formaldehyde always showed oscillatory efflux and reuptake phases. This was hypothesized to be an effect of the energy limitation imposed by the anaerobic assay conditions coupled with the high permeabilities of the transport substrates. The transport of methanol in M. L3 was not inhibited by protonophores, but was partially inhibited by 10mM arsenate and very strongly inhibited by dicyclohexyl carbodiimide (DCCD). Direct ATP profile monitoring, paralleling the methanol transport experiments, using the highly sensitive luciferase/luciferin assay, showed that ATP was consumed for the transport of methanol. Further, the ATP profiles showed oscillations, in parallel to the oscillations in the methanol profile, indicating that the oscillations in the energy content were responsible for the oscillatory uptake profiles, in support of our hypothesis. In contrast, the formaldehyde transport in M. L3 was inhibited partially by DCCD, and more strongly by protonophores. Thus, perhaps the proton-motive force ((DELTA)pmf) was more closely coupled to the transport of formaldehyde in M. L3, although both ATP and (DELTA)pmf could be used for transport. Similar results were obtained for formaldehyde transport in strain T15, with ca. 40% inhibition being caused by both FCCP and DCCD. Thiol reagents, particularly HgCl(,2) inhibited the transport of both methanol and formaldehyde in M. L3, implying the presence of essential cysteine residues in the transport associated proteins. The formaldehyde transport in M. L3 was also inhibited by diethyl pyrocarbonate (DEPC), indicating an essential histidine residue also. Overall, the results mean that both formaldehyde and methanol M. L3, and at least formaldehyde in strain T15 are transported by (similar) active transport mechanisms.