Download Phototropic bacteria - useful organisms for class experiments

Biochemical Society Transactions
Phototropic bacteria
- useful organisms for class experiments
J. B. Jackson
School of Biochemistry, University of Birmingham, Edgbaston, Birmingham B I 5 2TT, U.K.
976
The phototrophic bacteria, though interesting in
their own right (they have a remarkably versatile
physiology), have been used extensively in research
to provide ‘model systems’ in the study of photosynthetic reaction centres, electron transport, ion
translocation, ATP synthesis, pigment biosynthesis,
membrane-protein assembly and cell mobility. In
undergraduate class experiments they provide useful material for the demonstration of several aspects
of bioenergetics. Organisms such as Rhodobacter
capsulatus are easy to grow on liquid or solid minimal medium, either under aerobic conditions in the
dark or anaerobically in the light.
Three types of experiment will be described
that are appropriate for small groups of students
(up to about 20). They will make use of readily available or easily constructed inexpensive apparatus: (i)
Intact cells of Rh. capsulatus respire in the dark.
Upon illumination, respiration is inhibited. Addi-
tions of specific inhibitors and uncoupling agents
show that light-inhibition of respiration results from
the ‘back pressure’ exerted by an elevated proton
motive force (pmf). (ii) The pigments from membranes of Rh. capsulatus (including pigment-biosynthesis mutants) can be extracted with organic
solvents, and carotenoid- and bacteriochlorophyllrich fractions separated. Absorbance spectra of the
membranes can be reconstructed from the spectra
of the pigment fractions. (iii) There is a highly
active, pmf-driven transhydrogenase (NADP +
NADH NADPH + NAD) in membranes of Rh.
capsulatus. The reaction is easily measured and provides a good demonstration of the effects of
electron-transport inhibitors, uncouplers and ionophores.
Further details of Rh. capsulatus and of these
experiments, as presented in the Colloquium, can
be obtained by writing to Dr Jackson.
Abbreviation used: pmf, proton motive force.
Received 24 July 1001
-.
Using the bacterium, Paracoccus denitrificans and other ‘runaway mitochondria’
as classroom models for respiratory electron transport studies
Barbara Bolgiano,*§ Helen C. Daviest and Robert K. PooleS
*Glycoconjugates Section, M.R.C. Clinical Research Centre, Watford Road, Harrow, Middlesex HA I 3UJ.U.K.,
tDepartment of Microbiology, University of Pennsylvania, School of Medicine, Philadelphia, PA I 9 104-6076, U.S.A.
and +Division of Biosphere Sciences, King’s College London, Campden Hill Road, London W8 7AH, U.K.
Introduction
Electron transport reactions in the inner mitochondrial membrane are of fundamental importance in
bioenergetics. The sequence of the components that
accept and donate electrons and protons, and participate in the ultimate utilization of oxygen and the
mechanism of synthesis of ATP are covered in all
undergraduate biochemistry courses and in many
microbiology courses. In laboratory practicals, however, it is not easy to demonstrate, for example, how
an estimated 60 000-70 000 molecules of cytochrome oxidase per heart mitochondrion [ 11 contribute towards the production of the necessary 200
Abbreviations used: DMPL), N,N,N’,N’-dimethyl-pphenylenediamine; TMPD. N.N,N’.N’-tetramethyl-pphenylenediamine.
§To whom correspondence should be addressed.
Volume 19
kg or so of ATP needed each day as an energy
source for a person weighing 70 kg [21.
Mitochondria1 preparations are often too
laborious and expensive for laboratory courses, but
a cheap, simple and suitable alternative for mammalian tissue could be found by using any one of a
large range of bacteria grown in the presence of
oxygen. The bacterial respiratory complexes,
located in the cytoplasmic membrane, are much
simpler in that they contain fewer components [3].
Since their functions have been conserved through
evolution, essential features of their structure and
function are often very similar to mitochondria1 systems. Haem ligands and the protein folding around
the redox centres are often almost identical for distantly related species [4]. Research over the past 30
years emphasizes the validity of comparative
studies owing to this structural conservation
between the bacterial and higher eukaryote proteins