Metabolism of carbon monoxide by Rhodopseudomonas gelatinosa: cell growth and properties of the oxidation system

Abstract

Rhodopseudomonas gelatinosa 1 grew as an anaerobic facultative methylotroph with carbon monoxide as the sole carbon and energy source. Carbon from CO was assimilated into cell material via the ribulose 1,5-bisphosphate carboxylase cycle. The CO oxidation system in R. gelatinosa was induced during growth with the gas substrate. Light-grown cells did not oxidize CO. Surprisingly, when strain 1 cells grown in the dark with CO were transferred to growth with both CO and light, they continued to use CO and then photometabolized after the CO gas flow was stopped. This change in the energy-yielding substrate resulted in a diauxic growth response. The use of CO in preference to light energy forms the basis of a system in the cells that controls photosynthetic differentiation. CO oxidation was assayed as CO-methyl viologen oxidoreductase. Methyl viologen reduction only occurred with CO; the dye was not reduced with other C1 compounds. In vitro methyl viologen was reduced best at 24°C and at pH values above 8.5. Whole cells exhibited a Km of 12.5 ,uM for CO and a Vmin of 3,800 nmol of CO oxidized per mg of protein per min. This was a low-potential oxidation reaction that readily reduced the viologen dye triquat (1,1'-trimethylene-2,2'dipyridilium dibromide) (E°' =-548 mV).

Regulation of anaerobic carbon monoxide oxidation activity in Rhodocyclus gelatinosus

“…Cells were collected anaerobically by centrifugation, washed, and resuspended in O2-free 20 mM Tris-HC1 buffer (pH 8.0) with 0.3 mM dithiothreitol [3,6]. Whole cells were solubilized in 0.5% Triton X-100 (v/v) and CO:MV oxidoreductase activity was measured at 578 nm [3,6]. One unit of CO : MV oxidoreductase activity corresponds to 1 nmol CO oxidized/min.…”

“…The fact that CO gas oxidation is a substrateinduced reaction in Rhodocyclus gelatinosus helped explain the diauxic growth response observed when CO was replaced with argon in CO/light cultures [1][2][3]. During diauxie after removal of CO, the gas oxidation reaction decreased to a low level in the culture and finally disappeared as growth resumed photosynthetically.…”

“…This putative interaction would serve to maintain a uniform ATP energy flux during cell growth. [4]) was grown at 30°C under strictly Oz-free conditions in liquid CO medium with 0.1% trypticase [1,3]. Sterile reduced medium was prepared in Hungate culture tubes fitted with gassing vents, or in Blake bottles [3,5].…”

“…[4]) was grown at 30°C under strictly Oz-free conditions in liquid CO medium with 0.1% trypticase [1,3]. Sterile reduced medium was prepared in Hungate culture tubes fitted with gassing vents, or in Blake bottles [3,5]. To maintain a 0.78 mM saturating concentration of CO in cultures during growth up to 0.8 absorbance (A) units at 640 nm, gas flow to Hungate tube cultures was 25-30 ml/min and in Blake bottles, it was about 100 ml/min.…”

“…Medium was inoculated with a 2% (v/v) inoculum and cells were subcultured at least once before use under the appropriate anaerobic lighting condition. Incident light intensity was measured at the front surface of culture vessels [3,5].…”