Respiratory electron transfer in Escherichia coli : components, energetics and regulation

The respiratory chain that is housed in the bacterial cytoplasmic membrane,
generally transfers electrons from NADH to oxygen; in the absence of oxygen
it can use several alternative electron acceptors, such as nitrate and fumarate.
Transfer of electrons through this chain is usually coupled to the translocation
of protons across the membrane. The resulting gradient of protons is then used
for the generation of ATP by the F0F1-ATPase, and for other free energy requiring
processes such as solute transport. Quinones form an essential component
of this electron transfer chain. Generally, for aerobic respiration, Gram-negative
bacteria use ubiquinones and Gram-positive bacteria menaquinones. Many
Gram-negative bacteria, however, also synthesize menaquinones, in order to facilitate
anaerobic respiration. Escherichia coli, the subject central to this study,
even has two different menaquinones: demethyl-menaquinone and menaquinone,
both predominantly with a side chain of 8 isoprenoïd units.
The expression of the components of the respiratory chain is regulated by a
range of regulators, of which the histidine kinase ArcB and the transcriptional
regulator FNR are the best studied representatives. In this thesis I provide new
knowledge with respect to the complex respiratory chain of E. coli, both with
respect to its physiology and with respect to the regulation of its synthesis.
In the General Introduction an overview is given of all the components of this
respiratory chain, including their biochemical and physiological function. Furthermore,
general regulation with respect to oxygen availability is discussed,
with emphasis on the regulation by the histidine kinase ArcB. As histidine kinases
are also heavily involved in the regulation of the expression of virulence
factors, their role as potential targets for (new) antimicrobials is also briefly
touched upon in this chapter.
Chapter 2 describes the development of a method, based on HPLC analysis, for
quantification of the content and redox state of all three (see above) quinone
pools in E. coli. Unresolved questions regarding the species of quinone used
in nitrate respiration, as well as the so far scarcely studied respiratory chain of
Gram-positive bacteria, prompted us to develop a method for analysis of the in
vivo redox state of both ubiquinones and menaquinones, without interference of
quinone auto-oxidation during sampling and analysis. The data obtained indicate
that indeed significant auto-oxidation does not take place in the methods
developed. Application of this methodology has led to the surprising observation
that even under anaerobic conditions in E. coli, the menaquinone pool is
fully oxidized. Identification of the process that causes this anaerobic oxidation
of the menaquinones is a topic of ongoing research within the Molecular Microbial
Physiology group.
Chapter 3 describes the use of the newly developed method of quinone quantification
to assess in vivo the consequences of perturbing an aerobic culture
with a sudden anaerobiosis. In this experiment the redox state and content of the
ubiquinone-pool and the content of the two menaquinone pools were analyzed.
We demonstrate that biosynthesis of the two menaquinones proceeds faster than
can be explained by regulation of the expression of menaquinone biosynthetic
enzymes. Therefore, we conclude that menaquinone biosynthesis is regulated at
the biochemical level, although the exact detail of this regulation has not been
resolved. In batch cultures of E. coli a correlation between the redox state of the
ubiquinone pool and the dissolved oxygen tension in the medium was demonstrated.
This is surprising because the affinity of all the quinol:oxygen oxidoreductases
is far lower (<7 μM) than the highest oxygen concentrations at which
this correlation is observed.
The proton translocation efficiency and kinetic parameters of the three quinol
oxidases of the respiratory chain of E. coli were studied in chapter 4. Clear
differences with respect to the affinity for two of their substrates, i.e. ubiquinol
and oxygen, were observed among them. These data strengthen our hypothesis
that both cytochrome bd-I and bd-II are used under micro-aerobic conditions,
and that cytochrome bo mainly functions under fully aerobic conditions. Nevertheless,
we could also show that, in contrast to what is generally assumed with
respect to the physiology of E. coli, cytochrome bd-II significantly contributes
to the respiratory flux during aerobic glucose-limited growth. Interestingly, we
were able to deduce, by calculations on the growth yield of E. coli, that electron
transfer to oxygen via the cytochrome bd-II complex does not contribute to
proton translocation across the cytoplasmic membrane. This, in contrast to the
cytochrome bd-I complex, for which this reaction does result in translocation of
1 proton per electron transferred to oxygen.
The respiratory flux, and thus the rate of proton pumping, is partially regulated
via the expression of selected respiratory enzymes, under control of the ArcB/A
two-component system. In chapter 5 we show that the activity of ArcB, the histidine
kinase of this system, can not be directly correlated to the redox state of
the ubiquinone pool only, nor to the cellular content of the oxidized form of this
quinone, as was thought previously. Therefore, contributions by other regulators
were investigated and it was found that under anaerobic conditions a menaquinone-
biosynthetic enzyme is essential for activation of ArcB kinase activity.
This strongly suggests that menaquinone itself is essential for this activation.
An extended model (adapted from the Georgellis model, discussed in chapter 5)
has been proposed in which under low micro-aerobic conditions menaquinone
functions as the main regulator for ArcB kinase activity (also due to low cellular
ubiquinone contents), whilst in the high aerobiosis range ubiquinone takes over
this role.
Chapter 6 shows that the HPLC-based analysis for quantification of the redox
state and content of quinone pools can straightforwardly be applied to a variety
of organisms, particularly to those that have quinones with isoprenoid chains
longer than 7 units.
In the General Discussion several questions are addressed with respect to the
complexity of the respiratory chain and the use of uncoupled enzymes. The
presence of no less than four different uncoupled NADH dehydrogenases in
E. coli may be necessary for the variety of quinone species that the organism
can synthesize and for the successful survival in the redox environments it may
encounter. As for the presence of uncoupling-, and thus energy-wasting, enzymatic
complexes: Some organisms use these as oxygen scavenging enzymes
and/or as a way to produce heat locally in order to volatilize insect attractants.
As for E. coli, the exact role of uncoupling enzymes remains elusive, although
it is speculated that these enzymes could assist the cell in coping with a change
from a strongly oxidizing- to a reducing environment, e.g. upon addition of a
glucose-pulse to a glucose-limited culture.
Uncoupled enzymes may also be of use to the bio-fuel industry, to reach optimal
substrate to product conversion ratios. This topic is currently studied with
respect to its usefulness in the synthesis of a variety of industrially relevant
chemicals.