In situ determination of the reduction levels of cytochromes b and c in growing bacteria: a case study with N2-fixing Azorhizobium caulinodans

A.F. Pronk, F C Boogerd, C. Stoof, L F Oltmann, A.H. Stouthamer, H W van Verseveld

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

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)

Original languageEnglish
Pages (from-to)149-55
Number of pages7
JournalAnalytical Biochemistry
Volume214
Issue number1
DOIs
Publication statusPublished - Oct 1993

Keywords

  • Cytochrome b Group
  • Cytochrome c Group
  • Nitrogen Fixation
  • Oxidation-Reduction
  • Oxygen
  • Rhizobiaceae
  • Spectrophotometry
  • Journal Article
  • Research Support, Non-U.S. Gov't

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