In situ determination of the reduction levels of cytochromes b and c in growing bacteria: a case study with N2-fixing Azorhizobium caulinodans.
The determination of the in situ reduction levels of cytochromes b and c in growing bacteria is achieved by coupling a chemostat with a dual wavelength spectrophotometer. Visible light absorption spectra of cytochromes present in bacterial cells actively growing in a chemostat at a specific growth rate of 0.1 h-1 are recorded. This is accomplished by transporting the emitted light from the spectrophotometer via glass fibers to one side of the chemostat vessel and detecting the transmitted light via a photomultiplier at the other side. The vessel itself is enclosed in a dark box, which contains mirrors on the inside surfaces. The reduction levels of cytochromes b and c during steady state in chemostat cultures are expressed as percentage absorbance of fully reduced cytochromes in the alpha-region of the spectrum. Steady state spectra are recorded in N2-fixing, succinate-limited continuous cultures of Azorhizobium caulinodans at dissolved oxygen tensions in the range between 0.1 and 3.5% O2. Spectra of fully reduced cytochromes are obtained on the basis of spectra recorded after having reached anoxic conditions by sparging pure nitrogen gas through the culture. These spectra of cytochromes b and c reduced by endogenous substrates are corrected as to give the spectrum of fully reduced cytochromes. The respective contributions of cytochromes b and c to spectra in the alpha-region are estimated by deconvolution using best-fit analysis. Using this in situ technique it is observed that at each dissolved oxygen tension the reduction level of the cytochromes b is higher than that of the cytochromes c.(ABSTRACT TRUNCATED AT 250 WORDS)
Pronk
A F
AF
Vrije Universiteit, Department of Microbiology, Amsterdam, The Netherlands.
Boogerd
F C
FC
Stoof
C
C
Oltmann
L F
LF
Stouthamer
A H
AH
van Verseveld
H W
HW
D003573:Cytochrome b Group / Q000032:analysis / Q000378:metabolism*; D003574:Cytochrome c Group / Q000032:analysis / Q000378:metabolism*; D009586:Nitrogen Fixation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000494:pharmacology; D012230:Rhizobiaceae / Q000254:growth & development / Q000378:metabolism*; D013053:Spectrophotometry / Q000295:instrumentation / Q000379:methods
D003573:Cytochrome b Group; D003574:Cytochrome c Group; D010100:Oxygen