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FEMS Microbiology Reviews 28 (2004) 335–352
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A challenge for 21st century molecular biology and biochemistry:
what are the causes of obligate autotrophy and methanotrophy?
Ann P. Wood a, Jukka P. Aurikko a, Donovan P. Kelly
a
b,*
Department of Life Sciences, KingÕs College London, Franklin Wills Building, 150 Stamford Street, London SE1 9NN, UK
b
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
Received 5 August 2003; received in revised form 11 November 2003; accepted 17 December 2003
First published online 30 December 2003
Abstract
We assess the use to which bioinformatics in the form of bacterial genome sequences, functional gene probes and the protein
sequence databases can be applied to hypotheses about obligate autotrophy in eubacteria. Obligate methanotrophy and obligate
autotrophy among the chemo- and photo-lithotrophic bacteria lack satisfactory explanation a century or more after their discovery.
Various causes of these phenomena have been suggested, which we review in the light of the information currently available. Among
these suggestions is the absence in vivo of a functional a-ketoglutarate dehydrogenase. The advent of complete and partial genome
sequences of diverse autotrophs, methylotrophs and methanotrophs makes it possible to probe the reasons for the absence of activity of this enzyme. We review the role and evolutionary origins of the Krebs cycle in relation to autotrophic metabolism and
describe the use of in silico methods to probe the partial and complete genome sequences of a variety of obligate genera for genes
encoding the subunits of the a-ketoglutarate dehydrogenase complex. Nitrosomonas europaea and Methylococcus capsulatus, which
lack the functional enzyme, were found to contain the coding sequences for the E1 and E2 subunits of a-ketoglutarate dehydrogenase. Comparing the predicted physicochemical properties of the polypeptides coded by the genes confirmed the putative gene
products were similar to the active a-ketoglutarate dehydrogenase subunits of heterotrophs. These obligate species are thus genomically competent with respect to this enzyme but are apparently incapable of producing a functional enzyme. Probing of the full
and incomplete genomes of some cyanobacterial and methanogenic genera and Aquifex confirms or suggests the absence of the genes
for at least one of the three components of the a-ketoglutarate dehydrogenase complex in these obligate organisms. It is recognized
that absence of a single functional enzyme may not explain obligate autotrophy in all cases and may indeed be only be one of a
number of controls that impose obligate metabolism. Availability of more genome sequences from obligate genera will enable
assessment of whether obligate autotrophy is due to the absence of genes for a few or many steps in organic compound metabolism.
This problem needs the technologies and mindsets of the present generation of molecular microbiologists to resolve it.
Ó 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Nitrosomonas; Methylococcus; Obligate autotrophy; Methanotrophy; Methanogens; Krebs cycle; Genome probing
Contents
1.
2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Genome sequences and bioinformatic databases as untapped resources. . . . . . . . . . . . . . . .
Background to obligate metabolic modes of life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Bacterial obligate autotrophy and methanotrophy and their relationship to
autotrophy in Archaea. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
*
Corresponding author. Tel.: +44-24-7657-2907;
fax: +44-24-7652-3701.
E-mail address: [email protected] (D.P. Kelly).
0168-6445/$22.00 Ó 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsre.2003.12.001
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A.P. Wood et al. / FEMS Mircobiology Reviews 28 (2004) 335–352
3.2.
3.3.
3.4.
Survival benefits of being an obligate or a facultative autotroph . . . . . . . . . . . . . .
A unitary cause of obligate autotrophy and methanotrophy? . . . . . . . . . . . . . . . .
Origin and evolution of the Krebs cycle: why should it not function in an obligate
autotroph? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5. Testing hypotheses about the incomplete Krebs cycle . . . . . . . . . . . . . . . . . . . . . .
Testing the a-ketoglutarate hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. The a-ketoacid dehydrogenase family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Using the partial and complete bacterial genomes and protein sequences available on
the public databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. In silico detection of the E1 and E2 subunits of a-ketoglutarate dehydrogenase
in target genomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. Evaluation of the validity of the probing technique . . . . . . . . . . . . . . . . . . . . . . .
4.5. Refinement of the identification criteria for the a-ketoglutarate dehydrogenase E2
subunit sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.6. BLAST probing of target genomes for E1 and E2 genes . . . . . . . . . . . . . . . . . . . .
4.7. Positive identification of the E1 and E2 genes in the genomes of N. europaea and
M. capsulatus and computed physicochemical properties of the polypeptides encoded
by the genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.8. A basis for obligate autotrophy and methanotrophy? . . . . . . . . . . . . . . . . . . . . . .
Testing hypotheses about the nature of obligate autotrophy and methanotrophy and the
absence of a-ketoglutarate dehydrogenase activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Detecting the diagnostic coding sequences for a-ketoglutarate dehydrogenase in
obligate autotrophs and methanotrophs: bioinformatic approaches . . . . . . . . . . . .
5.2. Is there a functional transcription product in obligate genera with an E1 coding
sequence: is there an mRNA? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Is there an enzymically inactive translation product from E1 and E2 genes in
obligate genera? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4. Expression of the E1 and E2 genes of obligate genera in heterotrophic hosts
and vice versa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
349
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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4.
5.
6.
1. Introduction
This review is primarily concerned with an ‘‘old
problem’’, namely the attempts to explain the phenomena of ‘‘obligate autotrophy and methanotrophy’’ exhibited by diverse bacteria which otherwise share little
phylogenetic commonality. In the review we concentrate
primarily on the chemolithotrophs, cyanobacteria and
methanotrophs, the ‘‘obligate’’ metabolism of which
was studied in detail up to and during the 1960s and
1970s. Autotrophy among the Archaea and anaerobic
phototrophic bacteria is considered only for comparative purposes as those organisms were not prime subjects
of the ‘‘obligate autotrophy’’ debate at that time. For
the purposes of the review we have used the definition of
autotrophs (and autotrophy) proposed by Whittenbury
and Kelly [137] to encompass all those obligately autotrophic and methanotrophic bacteria which ‘‘synthesize
all their cellular constituents from one or more C1
compounds’’. The organisms central to our discussion
all assimilate carbon by a C1 + C5 condensation and are
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(a) the thiobacilli (deriving energy from inorganic sulfur
compound oxidation), Nitrosomonas (deriving energy
from ammonia oxidation to nitrite) and the phototrophic cyanobacteria, which all use the Calvin ribulose
bisphosphate carboxylase cycle and (b) the obligate
methanotrophs (such as Methanomonas methanica and
Methylococcus capsulatus), which use the Quayle ribulose monophosphate (RuMP) cycle, deriving both energy and carbon from methane. We suggest approaches
to the problem of obligate autotrophy using molecular
biology techniques, which are based on discoveries and
information largely achieved since the time at which
attempts to explain obligate autotrophy virtually ceased
[71,104,112,113,137]. The advent of these techniques
and the consequent explosion of information about
bacterial genomes and the control of gene expression
means that this problem can be looked at from viewpoints that were unthinkable 20 years ago. A holistic
approach, combining historical observations, molecular
methods and new biochemical methods, is needed to
solve this long-standing and intricate problem.
A.P. Wood et al. / FEMS Mircobiology Reviews 28 (2004) 335–352
2. Genome sequences and bioinformatic databases as
untapped resources
The application of sequencing techniques has resulted
in the accumulation of almost inconceivable quantities
of information about the bacterial genome. The public
databases now contain the sequences of billions of nucleotides coding for entire genomes, as well as for individual structural and functional genes, while the protein
databases contain the amino acid sequences of the
polypeptides encoded by many of these genes. The
massively powerful computing systems and search engines, capable of making almost instant comparisons
among these archived sequences and with newly acquired data, have revolutionized bacterial taxonomic
procedures and have helped rewrite taxonomy from
Kingdom to species level. Moreover, evolutionary and
functional relationships among enzymes with the same
catalytic function but from taxonomically diverse organisms can be analyzed in the same way, generating
phylogenetic trees of polypeptides from which much can
be deduced regarding the evolutionary history and
conservation of function of numerous enzymes
[18,50,62,77].
Two applications deriving from the availability of
techniques to identify genes encoding specific functions
have been (1) the ability to identify such functional
genes in the partial or complete genomes of bacteria
and (2) to use molecular probes to detect those genes
in environmental samples, as well as in cultured isolates or uncultured molecular clones [3,5,53,60,91,102].
Current molecular biology is making significant use of
gene probes to establish the presence not only of
specific organism types in environmental samples, but
to draw conclusions about possible metabolic processes that might be occurring in the habitats studied
[56,59,88]. These molecular techniques are useful tools
in indicating what might be possible, but in themselves
cannot prove that any organism types so identified, or
metabolic processes indicated, contribute to the dynamics of a given habitat. As Palleroni [92] has
pointed out ‘‘Modern approaches based on the use of
molecular approaches presumed to circumvent the
need for culturing prokaryotes fail to provide sufficient
and reliable information for estimation of prokaryotic
diversity’’. Similarly, the presumption that the use of
sequence data allows the deduction of the properties
of uncultivated organisms [90] is true only if the genes
detected can actually be expressed in vivo [42]. Thus,
for a full understanding of a natural habitat, molecular and biochemical analysis need to be applied
conjointly. The corollary of this is that molecular
analysis can be applied to known organisms or systems to establish how observable biochemical processes are likely to be underpinned and encoded at the
level of the genome.
337
Our aim is to show how the use of molecular methods
and the public databases can help to understand obligate autotrophy and methanotrophy.
3. Background to obligate metabolic modes of life
3.1. Bacterial obligate autotrophy and methanotrophy and
their relationship to autotrophy in Archaea
Diverse chemolithotrophic, photolithotrophic and
methanotrophic bacteria and some Archaea are characterized by their inability to grow as heterotrophs on
either simple or complex organic media. These are the
obligate autotrophs and type I methanotrophs, whose
one-carbon (C1 )-compound-dependent metabolism requires the fixation of carbon dioxide using energy from
inorganic oxidations or light, or uses methane (or in
some cases methanol) as both energy and carbon substrate [69,71,112,137]. Type I methanotrophs are members of the c-proteobacteria and are characterized by
having bundles of disc-shaped internal membrane vesicles, lacking a complete Krebs cycle, and using the
ribulose monophosphate (Quayle) cycle to assimilate
carbon from C1 -compounds [78]. Some metabolic abilities are common to both obligate autotrophs and type I
methanotrophs. They use similar cyclic sugar–phosphate pathways for the assimilation of carbon dioxide or
reduced C1 -compounds: the ribulose bisphosphate Calvin cycle or the Quayle cycle, respectively [78,108].
Several strains of the obligate methanotroph M. capsulatus also possess the key enzyme of the Calvin cycle,
ribulose bisphosphate carboxylase, and are capable of
autotrophic growth with hydrogen as the energy substrate [6,126]. Similarly, the obligate chemolithoautotroph, Thiobacillus thioparus, can oxidize and assimilate
carbon from methylated sulfides [63,115].
The obligately anaerobic methanogenic Archaea and
some aerobic and facultatively anaerobic sulfur-oxidizing Archaea can also be classified as chemolithoautotrophic. These derive energy from coupling the
oxidation of hydrogen to the reduction of carbon dioxide to methane (or by methane production from
C1 -compounds such as methanol or methylated sulfides), or from the oxidation of elemental sulfur or sulfides to sulfate, and derive cell-carbon from carbon
dioxide or C1 -compounds [33,108,109]. The methanogens and sulfur-oxidizing Archaea employ a variety of
pathways for autotrophic carbon dioxide fixation, none
proved to be like those of the eubacterial autotrophs,
some of which involve acetate or pyruvate as an intermediate, and some of which use a reductive tricarboxylic
acid cycle to fix carbon dioxide [34–36,55]. Central to
the C1 -metabolism of methanogens are carbon monooxide dehydrogenase and the Wood–Ljungdahl pathway, enabling the energy-efficient synthesis of acetyl
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CoA from carbon dioxide and hydrogen as chemolithotrophic growth substrates [30–32,34–36]. A reductive
tricarboxylic acid cycle supports the growth of the obligately anaerobic photolithoautotroph Chlorobium
[34,35], and yet another cyclic carbon dioxide-fixation
pathway, with 3-hydroxypropionate as the key intermediate, is found in the phototroph Chloroflexus
aurantiacus [52,121].
Clearly, obligately methanogenic growth, obligately
anaerobic photolithoautotrophy and the autotrophic
growth by some sulfur- or sulfate-reducing eubacteria
and Archaea, and some sulfur-oxidizing Archaea, are
likely to present problems distinct from those of obligate
chemolithotrophy and methanotrophy in aerobes such
as Nitrosomonas and Methylococcus. Indeed, the diverse
metabolic types represented by these organisms were
little considered from the point of view of obligate
autotrophy at the time of studies in the 1950s–1970s on
obligate methanotrophy and obligate autotrophy in
aerobic chemolithotrophs and cyanobacteria. For this
reason, we have devoted most of this review to a reconsideration of the causes of obligate methanotrophy
and autotrophy in aerobic eubacteria and cyanobacteria, using examples from the Archaea where parallels
can be drawn and when possible biochemical or evolutionary links can be proposed.
3.2. Survival benefits of being an obligate or a facultative
autotroph
The extreme specialization and restricted metabolism
exemplified by obligate chemolithotrophy has been
shown to give selective competitive advantage in the case
of some aerobic sulfur-oxidizing bacteria [75,114]. In
natural or laboratory environments where the ratio of
inorganic substrate to organic nutrients is high, the obligate species tend to dominate over facultatively mixotrophic species [75,114,137]. Several factors can affect
such competition but in general the obligate species
exhibit much greater flexibility in responding to variable
supplies of their inorganic substrate, frequently also
showing higher affinities for the substrate, ensuring their
survival in fluctuating environments [75]. Unlike simple
competition between two heterotrophs for a single
substrate, when one will eventually exclude the other
completely, the evidence indicates that an obligate chemolithotroph will be maintained, albeit sometimes at a
low level, in mixture with a facultative species over long
periods of time, especially under fluctuating conditions
of nutrient supply [45,46,75,114]. Such observations indicate how the obligate mode of metabolism could have
been of selective advantage over geological time. While
the survival benefit of being an obligate species shows
that the ‘‘rigidity’’ of autotrophic metabolism can be
selectively advantageous, it does not help answer the
question of how obligate autotrophy is imposed.
3.3. A unitary cause of obligate autotrophy and methanotrophy?
Obligate autotrophy among the chemo- and photolithotrophic bacteria still lacks satisfactory explanation
over 100 years since its discovery [69,71,99,137,140]. The
cause(s) of obligate autotrophy have never been fully
identified, but the four principal hypotheses proposed
from studies on thiobacilli and nitrifying bacteria up to
the 1970s were: (1) the ‘‘submarine hypothesis’’, which
viewed the bacteria as impermeable to organic nutrients
[132,137,140]. Impermeability alone has, however, been
discounted as various chemolithotrophs, phototrophs
and methanotrophs do incorporate organic compounds
into cell carbon, but only as a supplement to their normal
unique metabolic processes [54,65,69,70,74,80,103,104,
107,112,113,124,137,146]. In some cases the amounts
incorporated supply only a small fraction of the cell
biomass, and in others a compound may be totally excluded (e.g., glucose by some thiobacilli), probably because of the absence of specific transport systems. In
other examples, some amino acids for protein synthesis
can be derived entirely from exogenous sources, and
compounds such as acetate can provide a large part of
the total cell-carbon, for example, to Halothiobacillus
neapolitanus growing chemolithotrophically on thiosulfate [66,69,112]; (2) that organic nutrients were toxic or
inhibited specific metabolic steps [132,137]. Indeed
Winogradsky [140] defined such toxicity as a characteristic of the autotrophic ‘‘Anorgoxydant’’ [71]. Relatively
few organic nutrient compounds have been shown to
exert such toxicity, and growth inhibition by some amino
acids for example is typically due to regulatory effects on
branched biosynthetic pathways, preventing the synthesis of the other product(s) of a branched pathway
[27–29,67,70,137]; (3) that they were unable to derive
energy from NADH oxidation [54,113]. This can be
discounted as NADH oxidation through the cytochrome
system occurs [130] and endogenous ATP levels are in
many cases sustained by the metabolism of organic
storage materials. Several obligate species have been
shown to store polyglucose and other carbohydrates as
carbon and energy reserves, so must be able to derive
energy from their dissimilation [7,8]; (4) in many of these
organisms, a contributory cause of their obligate metabolism has been ascribed to the single enzymatic lesion
of the absence of a-ketoglutarate dehydrogenase activity.
Activity of this enzyme is absent from various thiobacilli,
Nitrosomonas europaea, M. capsulatus and type I methanotrophs, some cyanobacteria and some Archaea
[24,54,69,78,83,96, 111–113,137]. A corollary of the absence of activity of this key enzyme of the Krebs cycle in
some obligate autotrophs is that the reactions of the
Krebs cycle function as a biosynthetic ‘‘horseshoe’’,
channelling carbon from carbon dioxide or methane into
central biosynthetic pathways (Fig. 1).
A.P. Wood et al. / FEMS Mircobiology Reviews 28 (2004) 335–352
CO2
fixation via
the Calvin
Cycle
(autotrophs)
339
CH4
fixation via
the RuMP cycle
(Type I and X
methanotrophs)
Phosphoenolpyruvate
Pyruvate
acetyl CoA
CO2
oxaloacetate
malate
citrate
fumarate
cis-aconitate
succinyl CoA
BLOCKED
isocitrate
α-ketoglutarate
BIOSYNTHESIS
BIOSYNTHESIS
Fig. 1. Simplified schematic to illustrate the biosynthetic ‘‘horseshoe’’ reactions of the incomplete Krebs cycle in obligate autotrophs and methanotrophs [65,66,69,96,112,137].
The summary given above represents the state of
knowledge some 20 years ago and has not subsequently
been substantially advanced at the biochemical level.
Attempts to grow various obligate autotrophs on defined
media, which seek to replace the intermediates of autotrophic metabolism, have all failed, preventing full
analysis of their possible ‘‘heterotrophic’’ control
mechanisms. Recently, the genome of N. europaea has
been shown to contain only limited numbers of genes for
the transport and metabolism of organic molecules [16],
underlining the possibility that its obligate autotrophy is
due to a complex of enzyme ‘‘deficiencies’’ rather than
any single genetic lesion. It remains possible that obligate
autotrophy results from a complex of metabolic controls
and deficiencies: low ability to transport external nutrients in the absence of the normal energy substrate coupled with low rates of energy generation from their
dissimilation and possible repression of essential ‘‘autotrophic’’ processes either by the external nutrients or as a
consequence of the induction of enzymes for the metabolism of those nutrients [75,103]. Obligate autotrophy
could result from the organism being able to vary the
synthesis of enzymes of its central, autotrophically
based, metabolism only within narrow limits, thus preventing it from responding positively to the supply of
exogenous nutrients. There can obviously be unlimited
speculation along these lines, but limited evidence is yet
available to answer the key questions. This topic is in
need of research at the biochemical and molecular levels
using the techniques not available 20 or more years ago,
when these possibilities were considered.
In looking for a primary and unitary cause of obligate autotrophy and methanotrophy, a biosynthetic
or degradative lesion in central metabolism (such as
the lack of a functional a-ketoglutarate dehydrogenase) would seem more probable in evolutionary terms
than organic substrate exclusion or toxicity, and
would be consistent with the occurrence of obligate
autotrophy throughout the phylogenetic tree. This diversity has only recently become apparent with the use
of 16S rRNA sequence analysis to reveal phylogenetic
relationships. Thirty years ago, physiological properties were taken to indicate closer taxonomic relationships than can now be accepted. Extreme examples
are the nitrite-oxidizing Nitrospira and iron-oxidizing
Leptospirillum genera, which are now known to be
members of a phylum distinct from the proteobacteria,
in which physiologically similar bacteria are located
[78].
We explore the viewpoint that obligate autotrophy
results from lesions of central metabolism because it is
only in that area that new information has become
available from studies of the genomes of obligate autotrophs and methanotrophs. For this reason we have
used the absence or repression of a-ketoglutarate dehydrogenase as an example of how a single enzyme of
central metabolism could be the key to understanding
obligate autotrophy. This may be over-simplistic but
does at least provide a starting point from which new
research can begin and might even reveal that as a result
of ‘‘higher-level’’ regulatory phenomena in obligate
organisms, lack of expression of a-ketoglutarate
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dehydrogenase is a consequence rather than a cause of
obligate autotrophy.
It is recognized that the lipoamide-dependent NADþ reducing a-ketoglutarate dehydrogenase typical of most
mitochondria, eukaryotes and aerobic eubacteria is
not the only mechanism for the decarboxylation of
a-ketoglutarate and production of succinyl-CoA. In
various archaeons, including Halobacterium, Haloferax,
Archaeoglobus and a Sulfolobus strain, ferredoxin-dependent 2-oxoacid:ferredoxin oxidoreductases catalyze
the functions associated with eubacterial a-ketoglutarate
and pyruvate dehydrogenases [37,38,58,61,72,73,145].
Interestingly, a mutant of Bradyrhizobium japonicum,
lacking a-ketoglutarate dehydrogenase, ‘‘closes the gap’’
in the Krebs cycle by means of a CoA- and NAD(P)þ independent a-ketoglutarate decarboxylase, producing
succinic semialdehyde [47]. Neither of these types of
enzyme has been sought in obligate autotrophs such as
Nitrosomonas, but their presence in such organisms is
very unlikely, and their absence is indicated by pulselabelling experiments as described below.
The absence of a cyclic Krebs cycle from some of
these obligate genera results in the incorporation of
14
C-labelled acetate into protein only as leucine and
those amino acids derived from a-ketoglutarate: glutamate, proline and arginine [51,65,66,112,124]. In some
cases, including N. europaea, [14 C]acetate is also incorporated into amino acids derived from aspartate, requiring a C4 -precursor for its formation [136]. This was
explained by the formation of C4 -carboxylic acids from
isocitrate by the activity of the glyoxylate bypass enzyme, isocitrate lyase [69,136], which is negligible in
some obligate autotrophs [69,94,136]. Indeed, the facultatively heterotrophic green alga Chlamydomonas
dysosmos was rendered obligately autotrophic with respect to growth on acetate by a mutation which eliminated isocitrate lyase [89].
The facultatively heterotrophic methylotroph, Methylobacterium extorquens, strain AM1 contains a complete Krebs cycle with a functional a-ketoglutarate
dehydrogenase when grown heterotrophically, but
a-ketoglutarate dehydrogenase is repressed during
growth on C1 -compounds and the incomplete Krebs
cycle then acts biosynthetically as in obligate chemolithotrophs and methanotrophs (Fig. 1) [19]. M. extorquens could be rendered obligately methylotrophic by a
mutation that deprived it of a-ketoglutarate dehydrogenase, supporting the view that the basis of naturally
occurring obligate methylotrophy/methanotrophy could
be the absence of this enzyme [80,125]. The latter work
was recently extended by Van Dien et al. [133] to show
that mutants of M. extorquens could be made that
produced neither a-ketoglutarate dehydrogenase nor
pyruvate dehydrogenase and consequently became
greatly impaired in their ability to grow on succinate
and pyruvate. It has also recently been shown that
N. europaea (which does not produce active a-ketoglutarate dehydrogenase) appears to contain the coding
sequences for this enzyme in its genome [16].
Conversely, introducing genes coding for heterotrophic metabolism into obligate autotrophs may enhance
their ability to use exogenous organic materials. Acidithiobacillus thiooxidans is virtually unable to assimilate
glucose during growth with elemental sulfur as energy
substrate, but transconjugants into which the pfkA gene
for phosphofructokinase had been inserted (and expressed) could consume glucose, and showed an increased growth rate and biomass production when
grown chemolithotrophically on sulfur and glucose
compared with the transconjugant grown on sulfur
alone [127]. The growth rate and yield of the transconjugant were, however, considerably lower (by 30–55%)
than those of the wild type A. thiooxidans growing on
sulfur alone [127]. Heterotrophy of the A. thiooxidans
was thus not shown (making the paper somewhat misleading in implying that ‘‘conversion of an obligate autotroph to heterotrophy’’ was shown), and the effect of
the pfkA insert was no more than to enhance the potential for ‘‘mixotrophy’’. Mixotrophy is the wellestablished ability of obligate chemolithoautotrophs
and methanotrophs to obtain carbon simultaneously
from carbon dioxide and organic compounds, but
only at the expense of their normal energy sources
[66,69,70,103,144].
3.4. Origin and evolution of the Krebs cycle: why should it
not function in an obligate autotroph?
The initial steps of the Krebs cycle, from citrate synthase to isocitrate dehydrogenase, are believed to be very
ancient in origin, having entered the textbooks as possibly being ‘‘a remembrance of the early RNA world’’
[122]. The cycle itself probably evolved from a reductive
biosynthetic tricarboxylic acid pathway which had
originated in organisms in the pre-oxic biosphere [134].
One possibility is that a primordial autocatalytic anabolic chemoautotrophic pyrite-generating reductive cycle evolved into the oxidative catabolic Krebs cycle [134].
An intermediate stage in this evolution could have been a
pathway primarily for amino acid biosynthesis, by means
of a non-cyclic ‘‘horseshoe’’ pathway lacking an enzymatic step to form succinate (see Fig. 1) [82]. Its evolution into an energy-generating system was thus an
opportunistic secondary development, producing the
most biochemically robust system possible from existing
metabolic processes [39,41,82,86,87], dating at least from
the advent of the oxic atmosphere. In that the earliest
self-sustaining metabolism was rooted in chemolithotrophy and that the first autotrophic carbon dioxide
fixation pathway was probably a reductive citric acid
cycle [71,134,135], it is among extant chemoautotrophs
that relicts of these initial processes may be found.
A.P. Wood et al. / FEMS Mircobiology Reviews 28 (2004) 335–352
In its extant form, the Krebs cycle enables the efficient
conservation of biochemical energy from the oxidation
of pyruvate to carbon dioxide. At maximum efficiency,
as in the mitochondrion, the complete oxidation of a
pyruvate molecule by pyruvate dehydrogenase and the
Krebs cycle drives the synthesis of 15 ATP molecules
[108]. In bacteria, the efficiency of electron transport
phosphorylation coupled to NADH oxidation is lower,
typically with a P/O ratio of 2 rather than 3 [108]. Consequently, pyruvate oxidation in an aerobic bacterium
might generate only 10 ATP during pyruvate oxidation.
For an obligate autotroph such as Nitrosomonas to
operate a fully functional Krebs cycle, driven by pyruvate formed from carbon dioxide, would be ‘‘metabolic
suicide’’ and could not occur in nature. The fixation of
three carbon dioxide molecules via the Calvin cycle to
form phosphoglycerate requires 9 ATP and 6 NADH,
with one ATP being regained in the conversion of
phosphoglycerate to pyruvate [100]. NADþ reduction in
the obligate chemolithotrophs (with inorganic sulfur,
iron or nitrogen compounds as substrates) requires energy-dependent ‘‘reverse’’ electron flow from cytochromes. This requires the equivalent of a minimum of
one ATP per NADþ reduced. The synthesis of one
molecule of pyruvate from carbon dioxide thus ‘‘costs’’
at least 14 ATP. For an autotroph to reoxidize this
pyruvate would at best create a futile cycle of no metabolic energy or carbon benefit to the organism.
As outlined above (Fig. 1), the reactions of a
‘‘horseshoe’’ Krebs cycle would be expected primarily
to have a biosynthetic role in obligate autotrophs and
methanotrophs [28,65,66,69,107,112,120,137]. The question arises as to whether this ‘‘horseshoe’’ is a relict of
the evolutionarily ancestral pathway or has resulted
from later deletion of function in the evolution of obligate metabolism from heterotrophic ancestors. In the
case of the anoxygenic autotrophic methanogenic Archaea, it is easy to conclude that these never possessed a
complete Krebs cycle if the archaea are relicts of very
ancient forms of life (>3000 million years (My) before
the present; [79,108,141,142]). If, however, the controversial, minority view of Cavalier-Smith [15] is correct
that the archaebacteria arose only recently (850 My
before present), with methanogenesis as a secondary
character, then they too could have evolved the incomplete, biosynthetic, Krebs cycle by deletion of functions
present in eubacterial ancestors [15]. While the evolutionary timing of archaeal origins is beyond our scope, it
obviously affects deductions about the absence of Krebs
cycle genes. Regardless of the date of their origin, the
modern archaea could be descendants of a line of anaerobic, fermentative, heterotrophic eubacteria which
had never developed a functional oxidative Krebs cycle.
While modern archaea contain citrate synthase and
succinate thiokinase [20,21], they do not seem to produce a functional E1:E2:E3 pyruvate dehydrogenase
341
complex, even though some do contain coding sequences showing high identities to the required genes
[61]. This provides an interesting parallel with the failure
of some autotrophs and methanotrophs to produce
a-ketoglutarate dehydrogenase, although absence of a
functional pyruvate dehydrogenase from archaea is bypassed by the conversion of pyruvate to acetyl CoA by
an oxidoreductase [61].
3.5. Testing hypotheses about the incomplete Krebs cycle
Early studies demonstrated that a-ketoglutarate dehydrogenase activity was absent from diverse obligate
chemo- and photo-lithoautotrophs and some obligate
methanotrophs. It could not at that time necessarily be
concluded that the genes encoding this enzyme were also
absent from the bacteria [54,68,113], although this was
presumed to be a possibility. This presumption was
based on the view that modern obligate autotrophs and
methanotrophs had probably evolved from ancestors in
the pre-oxic biosphere which had never possessed a
functional Krebs cycle, or from an ancestor which had
lost the genes for a-ketoglutarate dehydrogenase. With
the advent of total sequencing of bacterial genomes it
has become feasible to test these assumptions and to
probe more deeply the causes of obligate autotrophy
and methanotrophy.
We have used in silico genome search procedures to
seek the genes encoding the components of a-ketoglutarate dehydrogenase in organisms known, or likely, to
possess the functional enzyme and some which do not
produce detectable activities of it during growth. We
show that two such organisms, N. europaea and
M. capsulatus, appear to be genomically competent with
respect to the genes necessary to encode a-ketoglutarate
dehydrogenase, but are apparently not able to achieve
heterotrophic growth by their expression.
4. Testing the a-ketoglutarate hypothesis
4.1. The a-ketoacid dehydrogenase family
This comprises three cytoplasmic enzymes: a-ketoglutarate dehydrogenase, pyruvate dehydrogenase and
2-oxoisovalerate dehydrogenase, each containing subunits which exhibit evolutionary relationships and
highly conserved amino acid sequences [81,93,101]. The
a-ketoglutarate dehydrogenase complex has been purified from a number of prokaryotes and eukaryotes and
invariably consists of three subunits [106]: these are E1,
E2 and E3, corresponding to a-ketoglutarate dehydrogenase (EC 1.2.4.2), dihydrolipoamide acyltransferase
(EC 2.3.1.61) and dihydrolipoamide dehydrogenase (EC
1.8.1.4), respectively. The overall reaction catalyzed by
the a-ketoglutarate dehydrogenase complex is
342
A.P. Wood et al. / FEMS Mircobiology Reviews 28 (2004) 335–352
a-ketoglutarate þ CoA þ NADþ
! succinyl-CoA þ CO2 þ NADH þ H
þ
The (simplified) component reactions are:
E1 : a-ketoglutarate + lipoamide !
succinyl-lipoamide + CO2
E2 : succinyl-lipoamide + coenzyme A !
succinyl-CoA + dihydrolipoamide
E3 : dihydrolipoamide + NADþ !
lipoamide + NADH + Hþ
The E3 subunit is very highly conserved and is common to all a-ketoacid dehydrogenases [106,118,122] and
it might thus be expected to occur in the genomes even of
obligate autotrophs and methanotrophs. The E1 subunit, however, displays significant interspecies differences
among the a-ketoacid dehydrogenase family members
[81] and might thus be redundant in obligate species.
In a fully functional Krebs cycle, the a-ketoglutarate
dehydrogenase complex converts a-ketoglutarate to
succinyl coenzyme A, which is required for some succinylation reactions and is an essential precursor for
porphyrin synthesis. Succinate formation from succinylCoA is catalyzed by succinyl-CoA synthetase (EC
6.2.1.4), which can also catalyze the reverse reaction.
Succinyl-CoA synthase is also absent from some autotrophically grown bacteria (which lack a-ketoglutarate
dehydrogenase), consistent with its principal role in the
forward direction of the Krebs cycle [82,95,97], indicating alternative CoA-transferase systems for succinylCoA formation in autotrophs [95,96].
The genes encoding the three proteins of the a-ketoglutarate dehydrogenase complex have been identified,
sequenced and characterized from various organisms,
including Escherichia coli (sucA, sucB, lpdA) and Bacillus subtilis (odhA, odhB, pdhD) [22,23,84,117,119]. The
availability of full gene and translated protein sequences
for the E1 and E2 subunits thus provided the possibility
to search for these genes in other genomes. Several
features of the E1 and E2 subunits of the E. coli a-ketoglutarate dehydrogenase complex can serve as biomarkers during in silico-based identification of the
corresponding encoding genes, as was used to show
conservation of these markers between E. coli and
Rhodobacter capsulatus [23]. For E1, the markers are
two phosphorylation sites (residues 391–405 and
440–456) and the cofactor (pyrophosphate thiamine)binding site (residues 362–389). For E2, the markers are
two conserved residues assumed to be located in the active site (His-375 and Asp-379) and the cofactor (a covalently linked lipoic acid moiety)-binding site (Lys-43)
[14,23]. In E. coli, sucA and sucB are part of an operon
sharing the same promoter, transcribed on a common
mRNA, whereas lpdA is part of an unrelated operon,
thus potentially allowing differential regulation of the
E1 + E2 and the E3 components of the a-ketoglutarate
dehydrogenase complex. The odhA and odhB genes for
the E1 and E2 subunits of the B. subtilis a-ketoglutarate
dehydrogenase are similarly contiguous [14].
4.2. Using the partial and complete bacterial genomes and
protein sequences available on the public databases
Molecular probing techniques not available when the
a-ketoglutarate dehydrogenase hypothesis was conceived now enable us to test the possibility that ‘‘absence
of enzyme means absence of gene’’. The following section (Sections 4.3–4.7) are a case study of this approach.
Partial and complete genomes for several well-characterized obligate autotrophs and methanotrophs, some
facultative methylotrophs, phototrophs and Archaea
were probed for the presence of sequences for the E1
and E2 subunits of a-ketoglutarate dehydrogenase
(Table 1).
The amino acid sequences used to seek the presence
of homologous sequences in the target organisms were
those of the E1 and E2 subunits of E. coli strain K12.
Control searches for a-ketoglutarate dehydrogenase
subunits were conducted against the genomes of both
E. coli strain 0157:H7 (EDL933), genome Accession No.
NC_002655 [9], and B. subtilis, genome Accession No.
NC_000964 [76].
4.3. In silico detection of the E1 and E2 subunits of
a-ketoglutarate dehydrogenase in target genomes
The general procedure used to locate the genes encoding the E1 and E2 subunits employed the basic local
alignment search tool (BLAST) as TBLASTX and
TBLASTN (http://www.ncbi.nlm.nih.gov), allowing the
probing of the translated genomes with appropriate
protein sequences. The amino acid sequences selected
were those from the SwissProt database for the E. coli E1
(a-ketoglutarate dehydrogenase) subunit, Accession No.
p07015 (933 amino acids), and E2 (dihydrolipoamide
succinyl transferase) subunit, Accession No. p07016 (404
amino acids) [1,22,23,117]. BLAST results were analyzed
both in silico and manually. Putative identifications were
first based on high scores in terms of ‘‘hits’’ to the sequence being sought. Score bits were computed from the
raw scores using the blosum62 substitution matrix [49].
Entry into further rounds of analysis was restricted to
those hits whose score exceeded 150 bits, where the
‘‘Expect’’ probability value (E-value; [48]) was low, and
which were equivalent to at least half the probe in
physical length. All the putative primary amino acid sequences identified in this way were subjected to manual
analysis involving the identification of the conserved
residues theoretically serving biological functions.
The criteria for the identification of the E2 subunit
were more rigorous and relied on the identification of
the E1 subunit as a prerequisite. Initially criteria analogous to those for the E1 subunit were used, but the
A.P. Wood et al. / FEMS Mircobiology Reviews 28 (2004) 335–352
343
Table 1
Organisms whose genome sequences were used to study genes of the a-ketoglutarate dehydrogenase complex
Organism
Genome status
Source
Escherichia coli K12 strain MG1655
Escherichia coli O157:H7
Bacillus subtilis
Bacillus halodurans strain C-125
Nitrosomonas europaea ATCC25978
Methylococcus capsulatus
Complete
Complete
Complete
Complete
Complete
Incompleteb
Methylobacterium extorquens strain AM1a
Ralstonia metallidurans strain CH34a
Acidithiobacillus ferrooxidans ATCC23270
Prochlorococcus marinus strain MED4
Nostoc punctiforme ATCC29133
Synechocystis sp. strain PCC6803
Synechococcus sp.
Aquifex aeolicus strain VF5
Methanobacterium thermoautotrophicum DeltaH
Methanococcus maripaludis strain LL
Methanococcus jannaschii DSM2661
Methanosarcina barkeri strain Fusaro
Methanosarcina mazei strain GOE1
Incomplete
Incomplete
Incomplete
Incompletec
Incomplete
Complete
Incomplete
Complete
Complete
Incomplete
Complete
Incomplete
Incomplete
Blattner et al. [9]
Perna et al. [98]
Kunst et al. [76]
Takami et al. [123]
Chain et al. [12]
The Institute for Genomic Research and
University of Bergen, Norway
University of Washington
University of Washington
The Institute for Genomic Research
Joint Genome Institute
Joint Genome Institute
Kaneko et al. [64]
Joint Genome Institute
Deckert et al. [25]
Smith et al. [116]
University of Washington
Bult et al. [13]
Joint Genome Institute
G€
ottingen Genomics Laboratory
Completed sequences, sequences in preparation, and experimental and unfinished genomic sequences may be accessed at http://www.tigr.org/tbd/
mdb/complete.html, http://www.tigr.org/tbd/mdb/inprogress.html and at http://pedant.gsf.de.
a
Facultative methylotrophs.
b
Completed subsequent to our analyses [105].
c
Completed subsequent to our analyses, but not yet fully edited (http://www.ncbi.nlm.nih.gov: taxonomy ID 243233).
gene was also required to map adjacent to the E1 gene
within the physical genomic sequence.
Subsequent to identifying the likely E1 and E2 sequences in target organisms, the genomic sequences of
those organisms corresponding to the BLAST hit locations were recovered as open reading frames (ORFs) by
using the NCBI ORF finder (http://www.ncbi.nlm.nih.
gov/gorf.html). This yielded the putative coding sequences corresponding to the E1 and E2 subunits of the
target a-ketoglutarate dehydrogenase genes within the
genomes of the target organisms. These ORFs were
subjected to a translation procedure using the expert
protein analysis system (ExPASy) proteomics server
(http://ca.expasy.org/tools/dna.html) to produce the
amino acid sequences encoded by the ORFs. The Unixbased Genetic Computer Group BESTFIT pairwise
alignment procedure was used to compare these to the
translated sequences from E. coli. The molecular masses
and isoelectric points (pI) of the translated sequences
were calculated for comparison with published data for
a-ketoglutarate dehydrogenase (http://ca.expasy.org/
tools/pi_tool.html).
4.4. Evaluation of the validity of the probing technique
Control searches using the E. coli E1 and E2 amino
acid sequences (p07015 and p07016) were undertaken
against the E. coli 0157:H7 and B. subtilis genomes. For
E. coli the BLAST search provided the expected positive
hits, with p07015 scoring 1896 bits, and p07016 scoring
735 bits (E-values ¼ 0.0). An analogous BLAST search of
the NCBI microbial genome database was conducted
using the E. coli 0157:H7 sucA and sucB gene sequences.
The search with sucA returned hits only to the E1 subunit
of a-ketoglutarate dehydrogenase (e.g., for Pseudomonas
putida, AAC23516, score ¼ 2864 bits). The search with
sucB returned a variety of hits including the entire a-ketoglutarate dehydrogenase family and the 2-oxoisovalerate dehydrogenase of Bacillus halodurans (NP_243627).
When the B. subtilis genome was probed with p07015 and
p07016, the expected odhA (E1) and odhB (E2) genes were
successfully located (with scores of 595 and 341, respectively). The conserved biomarkers were also identified
within the hit sites. Secondary hits were also obtained for
the odhB BLAST (score 237 bits) with B. subtilis, but
these were identified as the corresponding subunit of
2-oxoisovalerate dehydrogenase in each of B. subtilis and
Staphylococcus aureus (NP_372039; score 601) and the
E2 subunit of the pyruvate dehydrogenase of B. halodurans (NP_241081;score 642). The identification criteria of
high score, low E-value and detection of the conserved
biomarkers were thus not necessarily adequate for high
confidence identification of the E1 and E2 subunits of
a-ketoglutarate dehydrogenase across species. BLAST
searches with the B. subtilis odhA (E1) did, however, give
exclusive hits to the E1 sequences in other organisms,
ranging from B. halodurans (NP_243072; score 3293) to
Homo sapiens (BAA01393; score 1345).
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A.P. Wood et al. / FEMS Mircobiology Reviews 28 (2004) 335–352
4.5. Refinement of the identification criteria for the
a-ketoglutarate dehydrogenase E2 subunit sequence
In B. subtilis, the odhA and odhB genes are contiguous
on the genome (genomic locations: 2,108,043–2,110,856
and 2,106,760–2108,013 bp, respectively). In contrast,
the secondary hit by the p07016 (E2) BLAST probing of
B. subtilis (the 2-oxoisovalerate dehydrogenase E2 sequence) was located remotely from the a-ketoglutarate
dehydrogenase genes, at 2,497,143–2,496,061 bp (bfmBaa gene) on the genome. We therefore decided to add a
further criterion to be met in the identification procedure, namely a gene position parameter, requiring close
genomic proximity of the E1 and E2 coding sequences, as
occurs in E. coli and B. subtilis. Inclusion of the positional criterion made high confidence inter-species
E1 + E2 identification feasible.
4.6. BLAST probing of target genomes for E1 and E2
genes
Probing the genomes of a diverse range of organisms
with the E. coli sequences for E1 (p07015) and E2
(p07016) gave strong positive as well as negative results
(Table 2). Strong positive results were given for the
heterotrophs E. coli and B. subtilis, the facultative
methylotroph Ralstonia metallidurans, the obligate
methanotroph M. capsulatus and obligate chemolithoautotroph N. europaea (Table 2). Negative results or
very poor scores were found for Prochlorococcus, Synechococcus, Methanobacterium thermoautotrophicum,
Methanococcus jannaschii and Aquifex aeolicus, which
are thus unlikely to contain the E1 gene. The complete
genome sequence of Prochlorococcus marinus SS120 was
also found to lack genes for E1 and E2, and for the
succinyl-CoA genes [26]. Our probing also confirms the
genome analysis by Smith et al. [116], who failed to find
the gene for a-ketoglutarate dehydrogenase in M. thermoautotrophicum, although other Krebs cycle enzyme
genes were identified. The only a-ketoacid dehydrogenase complex component identified in M. jannaschii was
the common E3 subunit, dihydrolipoamide dehydrogenase (EC 1.8.1.4; [13]; located at 567,071–564,878 bp;
http://pedant.gsf.de).
Only a low score for E1 was seen with the facultative
methylotroph Methylobacterium extorquens (Table 2),
which was surprising as this organism does contain a
well-defined E1 gene (NCBI Accession No. AF497852).
This probably indicates a weakness of the technique
which may be due to relative sequence identities of the
genes from different sources (see data in Table 2). It is
also possible that our inclusion of positional criteria,
while giving higher confidence identification, may result
in false negatives if the positional criteria are unjustified
in some cases. The very poor score for the obligate
chemolithoautotroph Acidithiobacillus ferrooxidans
(Table 2) showed that the E1 sequence was absent from
the unfinished genome sequence.
Table 2
BLAST probing for the presence of the a-ketoglutarate dehydrogenase E1 and E2 genes in the genomes of a range of heterotrophic, facultatively
methylotrophic, obligately autotrophic or methanotrophic bacteria, and methanogenic Archaea
Organism
Escherichia coli 0147:H7
Bacillus subtilis
Ralstonia metallidurans
Methylobacterium extorquensa
Acidithiobacillus ferrooxidans
Aquifex aeolicus
Methanobacterium thermoautotrophicum
Methanococcus jannaschii
Methanococcus maripaludis
Methanosarcina barkeri
Methanosarcina mazei
Nostoc punctiforme
Prochlorococcus marinus
Synechococcus sp.
Synechocystis sp.
Nitrosomonas europaeab
Methylococcus capsulatus
E1 gene
E2 gene
Score (bits)
E-value
Score (bits)
E-value
1896
595
450
37
29
0
32
0
28
31
32
31
0
0
56
1011
945
0
e171
e130
0.007
7.8
0
0.099
0
9.8
7.7
0.4
1.1
0
0
5.1
0
0
735
341
430
99
108
69
26
66
nt
31
375
130
129
128
269
401
407
0
7e95
e121
e121
4e24
1.1
2.7
4.5
nt
3
e100
6e31
3e31
3e31
7.3e63
e113
e114
The TBLASTN programme (http://www.ncbi.nlm.nih.gov/blast) used the SwissProt database sequences for E1 (p07015) and E2 (p07016) from
E. coli as probes against the genome listed. nt, not tested.
a
BLAST2 comparison of the gene for KGD from M. extorquens (AF497852) and E. coli (X00661) showed 80.5% identity for 161/200 bases,
corresponding to amino acids 734–800.
b
BLAST2 comparison of the E1 sequence from N. europaea with the E. coli gene for KGD showed a discontinuous identity of 75% for 486/648
bases, corresponding to 214 amino acids with an identity by BLASTX of 54% to that segment of the E. coli E1 polypeptide.
A.P. Wood et al. / FEMS Mircobiology Reviews 28 (2004) 335–352
4.7. Positive identification of the E1 and E2 genes in the
genomes of N. europaea and M. capsulatus and computed
physicochemical properties of the polypeptides encoded by
the genes
The most striking finding was the demonstration of
strong hits for both the E1 and E2 genes for a-ketoglutarate dehydrogenase in both the obligate chemolithoautotroph N. europaea and the obligate methanotroph
M. capsulatus, neither of which has a-ketoglutarate dehydrogenase activity in vivo. Subsequent to our finding
putative E1 and E2 genes in N. europaea, using the then
incomplete genome sequence (http://pedant.gsf.de), the
complete genome sequence was published ([16]; NCBI
nucleotide database sequence AABB02000001). In their
discussion of the genome, Chain and co-authors [16]
noted that genes for all enzymes of the Krebs cycle were
present, including the E1 and E2 of a-ketoglutarate dehydrogenase. We were concerned that putative identifications of genes for specific functions, using BLAST and
other partial alignment methods, are not absolute proof
of identity. Consequently we followed through our
findings by making total sequence comparisons between
the putative E1 and E2 genes of M. capsulatus and
N. europaea and the genes from heterotrophs, and
compared the translated amino acid sequences and predicted physicochemical properties of the polypeptides
encoded by the target genes.
The BLAST hits for the E1 and E2 genes were used to
locate and extract open reading frames (ORFs) from
their ATG start codons (Table 3). Translated amino
acid sequences of the ORFs were obtained by in silico
345
translation of the gene sequences and were aligned
against the corresponding E. coli p07015 (E1) and
p07016 (E2) amino acid sequences to assess sequence
homology and biomarker conservation. The pairwise
alignments using GCG BESTFIT confirmed that the E1
and E2 genes of N. europaea and M. capsulatus had
indeed been detected and identified correctly (Table 4).
The polypeptide sequences obtained from the translated sequences of the E1 and E2 genes in the genomes
of N. europaea and M. capsulatus were used to compute
their molecular masses and isoelectric points (pI; http://
ca.expasy.org/tools/pi_tool.html), and to compare these
features with those of the functional E1 and E2 subunits
of the E. coli a-ketoglutarate dehydrogenase (Table 5).
The polypeptides encoded by the E1 and E2 genes from
all three organisms had very similar properties, showing
that the obligate autotroph and obligate methanotroph
appeared genetically competent to produce a-ketoglutarate dehydrogenase, but do not do so in vivo.
Using the completed genome sequence of N. europaea
ATCC 25,978 (at http://pedant.gsf.de/cgi-bin/wwwfly.pl?Set ¼ Nitrosomonas_europaea_ATCC_25978&
Page ¼ index) enabled us to confirm the findings of our
indirect analyses. The coding sequences for the E1 and
E2 components of a-ketoglutarate dehydrogenase were
positively identified in the genome (orf431 and orf430).
The E1 gene was located at contig positions 420,453–
423,317 bp, comprising 955 amino acids with a molecular mass of 108,487.0 and pI 5.9; while the E2 gene was
located at contig positions 419,154–420,428 bp, comprising 425 amino acids with a molecular mass of
46,188.9 and pI 5.5. These values are comparable to the
Table 3
Locations of the open reading frames for the a-ketoglutarate dehydrogenase E1 and E2 genes in the genomes of M. capsulatus and N. europaea and
the derived amino acid content of the polypeptides they encode
Organism
Subunit
Contig location (bp)
Number of amino acids coded
Contig accession
Methylococcus capsulatus
E1
E2
29841–32655
32620–33766
937
381
gn1jTIGR_414jmcapsul_bmc_79
gn1jTIGR_414jmcapsul_bmc_79
Nitrosomonas europaea
E1
E2
6952–9751
9772–11050
932
425
microbe1_fasta.screen.Contig453
microbe1 fasta screen. Contig453
The open reading frames contain the TGA (stop) codon and the amino acid count excludes this non-coding codon.
Table 4
BESTFIT pairwise alignment statistics for the translated a-ketoglutarate dehydrogenase E1 and E2 open reading frames identified in M. capsulatus
and N. europaea against the a-ketoglutarate dehydrogenase E1 and E2 sequences of E. coli (p07015 and p07016)
Organism
Subunit
Number of nucleotides in deduced orf
Translated sequences
Similarity (%)
Identity (%)
Methylococcus capsulatus
E1
E2
2813
1143
63.0
68.8
54.5
57.5
Nitrosomonas europaea
E1
E2
2796
1275
65.0
65.3
56.5
56.6
For both organisms the biomarkers (E. coli numbering) were well conserved for E1 between amino acids 362 and 405 (75% and 69% identity for
M. capsulatus and N. europaea, respectively), and 440 and 456 (71% and 75% identity, respectively); and for E2 at lys-43, his-375 and asp-79.
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A.P. Wood et al. / FEMS Mircobiology Reviews 28 (2004) 335–352
Table 5
Computed physicochemical properties of the a-ketoglutarate dehydrogenase E1 and E2 polypeptides encoded by the respective genes in E. coli,
M. capsulatus and N. europaea located in this study
Organism
Subunit
Molecular mass of encoded polypeptide
(Dalton)
pI of encoded polypeptide
(pH units)
Escherichia coli
E1
E2
105061.7
43880.2
6.04
5.58
Methylococcus capsulatus
E1
E2
105388.4
41553.8
6.06
5.98
Nitrosomonas europaea
E1
E2
105923.3
44987.5
5.96
5.60
data obtained by our independent methods (Tables 3
and 5). The closest homologues to the Nitrosomonas E1
database sequence by BLASTP and TBLASTN were the
odhA gene (E1) of Ralstonia solanacearum and Ralstonia
eutropha, with scores of 1145 and 1138 (E-value ¼ 0.0).
By TBLASTN these showed 566/955 (59%) and 519/952
(58%) amino acid sequence identity, respectively, to the
N. europaea sequence. Identity to E. coli E1 (X00661)
was 517/948 (54%), which was comparable to the identity of the E. coli E1 sequence to those of Xanthomonas
(NP_641868.1) and Xylella (NP_778980.1) at 55% (526/
941 and 521/937, respectively). TBLASTN amino acid
sequence identity of the N. europaea E1 sequence to that
of Methylobacterium extorquens was lower, at 429/990
(43%). The closest homologue of the Nitrosomonas E2
sequence was the dihydrolipoamide succinyltransferase
of R. eutropha.
It is also noteworthy that the genes encoding both the
a- and b-chains of succinyl-CoA synthetase occur in the
N. europaea genome (Scaffold_1 contig: orf128 and
orf129; http://pedant.gsf.de). The presence of this enzyme, which normally functions to produce succinate
from succinyl-CoA (the product of a-ketoglutarate dehydrogenase activity), has not been reported in Nitrosomonas, but clearly merits further study.
4.8. A basis for obligate autotrophy and methanotrophy?
The reason for the failure of N. europaea and
M. capsulatus to synthesize functional a-ketoglutarate
dehydrogenase and the significance of this to their obligate physiologies remain to be established. Among the
possibilities are: (i) failure to transcribe one or both of
the genes into mRNA, (ii) deletion of regulatory parts of
the operons, (iii) that the obligate nature of their central
metabolism results from regulatory controls causing
permanent repression of the genes, or (iv) some kind of
post-transcriptional gene silencing, possibly by endogenous small interfering RNAs [17,131].
A parallel is seen in the facultative autotroph and
methylotroph, P. versutus strain A2, when autotrophic
and heterotrophic growth are compared. Both a-ketoglutarate dehydrogenase and succinyl-CoA synthetase
are completely repressed during chemolithotrophic
growth with thiosulfate, whereas high activities occur in
heterotrophically grown bacteria [97]. During autotrophic growth of P. versutus on thiosulfate or formate,
14
C-acetate is incorporated heavily into leucine, glutamate, proline and arginine, but when grown heterotrophically, increased labelling of aspartate, alanine and
other amino acids occurs in cultures ‘‘spiked’’ with
[14 C]acetate [137].
Transcriptional repression of the expression of genes
for a-ketoglutarate dehydrogenase in response to anaerobiosis is well known in enterobacteria [2,10,93], and
a permanently ‘‘switched off’’ state may exist in obligate
autotrophs. Alternatively, expression may occur but is
accompanied by deficiencies at the translational or
polypeptide assembly levels, resulting in failure to produce enzymatically active a-ketoglutarate dehydrogenase protein. Such a situation appears to occur in the
archaeon Haloferax volcanii where an insert in the E2
gene of the pyruvate dehydrogenase complex appeared
to be the cause of failure of the complex to assemble to
produce a functional enzyme [61]. Another possible situation is represented by the ushB gene of Salmonella
(encoding a UDP–sugar-hydrolase), which has an orthologue, ushB(c) in E. coli, which does not produce this
enzyme activity. In fact the ushB(c) gene shares 100%
identity with the E. coli gene for CDP-diglyceride hydrolase (cdh) but the ushB(c) gene does not produce any
detectable protein in vivo despite being transcribed
normally [110]. The possibility thus remains that the sequences found in obligate autotrophs and methanotrophs, seemingly encoding the E1 of a-ketoglutarate
dehydrogenase may in fact not even be capable of that
function if expressed. The corollary of course is that in
some species in which active a-ketoglutarate dehydrogenase is found, the protein catalyzing the reactions may
be structurally unrelated to the E. coli enzyme, and encoded by genes of analogous rather than homologous
function, as has been extensively demonstrated by
Galperin et al. [40].
Identifying which, or what combination, of these
possibilities is responsible may require an extensive and
multi-targeted approach.
A.P. Wood et al. / FEMS Mircobiology Reviews 28 (2004) 335–352
5. Testing hypotheses about the nature of obligate
autotrophy and methanotrophy and the absence of
a-ketoglutarate dehydrogenase activity
5.1. Detecting the diagnostic coding sequences for
a-ketoglutarate dehydrogenase in obligate autotrophs
and methanotrophs: bioinformatic approaches
To date, the number of obligate species (including
methanogens) to which it has been possible to apply in
silico methods to seek the E1 and E2 sequences is very
limited. As more complete genome sequences become
available it will be possible to screen these to see whether
other genera also possess genes which do not translate in
vivo into active enzyme. When such sequences are
found, the same diagnostic procedures described above
for Nitrosomonas and Methylococcus can be applied to
detect key amino acid sequences in the translated polypeptides and to predict their molecular masses and pI
values. In the short term the genomes of A. ferrooxidans,
A. aeolicus, Synechocystis PCC6803, Synechococcus,
Nostoc, Prochlorococcus, Methanoccous spp. and Methanosarcina spp., which all appear to lack the E1 gene
(Table 2), will be of particular interest. Some of the
genomes which are unfinished (Table 1) may eventually
be shown to contain the E1 coding sequence. The unfinished genome of A. ferrooxidans appears to have the
E2 coding sequence (Table 2) but apparently not that
for E1, although these two genes would be expected to
be contiguous on the genome (see Section 4.5). The E1
sequence is probably absent from A. ferrooxidans, as we
have used BLASTN to probe the essentially complete
genome ([128]; 3164654 bp) using the E1 nucleotide sequence (bases 2,580,584–2,583,451 of the complete genome of N. europaea), and found no significant sequence
similarity. The search for E1 and E2 coding sequences,
using specific gene probes, needs to be extended to
genera such as Thiobacillus, Thermithiobacillus, Halothiobacillus, Methylomonas and Methylobacter as well as
facultative genera and organisms such as Nitrobacter
and Methylosinus, which can produce a functional
a-ketoglutarate dehydrogenase enzyme.
5.2. Is there a functional transcription product in obligate
genera with an E1 coding sequence: is there an mRNA?
Having located the E1 and E2 genes within the genome, sequencing of the contiguous regions of DNA
may give information about the regulatory elements associated with the genes. Failure to transcribe the genes
could result from: (1) a missing promoter or one that is
shared with and overridden by another gene or (2) the
gene being in some way ‘‘faulty’’, possibly resulting in an
untranslatable or nonsense polypeptide or (3) the absence or a transcriptional failure of an essential chaperone gene. If there is a regulatory failure such as deletion
347
of part of an operon, then no mRNA would be produced. This could be tested using probes from the known
Escherichia and Nitrosomonas sequences and standard
techniques (Northern blots, labelled probe hybridization) used to seek transcriptional mRNA products. If
mRNA were to be produced, amplification of the cDNA
using revere transcriptase (RT)-PCR would be a powerful means of identifying and analyzing the product.
5.3. Is there an enzymically inactive translation product
from E1 and E2 genes in obligate genera?
If the genes appeared to be transcribed into mRNA,
standard methods could be applied to seek polypeptide
products based on the predicted properties of these
products, and by using antibodies raised against homologous polypeptides. Fractionation of cell-free extracts to concentrate polypeptides of the expected
molecular mass followed by two-dimensional electrophoresis [138,139] using bacteria such as Methylococcus
and Nitrosomonas (with E. coli or Ralstonia as controls)
might reveal polypeptides of the predicted size and
charge, which could be subjected to N-terminal sequencing and to fragmentation pattern analysis and
internal amino acid sequencing by mass spectrometric
methods. A combination of in silico prediction from the
gene sequences of the likely secondary and tertiary
folding of the polypeptides and direct analysis of the
actual translated product and its active site(s) might
reveal that the gene product could not function in vivo
as part of an active a-ketoglutarate dehydrogenase.
5.4. Expression of the E1 and E2 genes of obligate genera
in heterotrophic hosts and vice versa
Using a recipient such as E. coli (which is capable of
fermentative growth without use of the Krebs cycle) from
which the E1 and E2 genes of a-ketoglutarate dehydrogenase had been deleted, the corresponding genes from an
obligate genus could be inserted and their expression
sought. This should restore the ability of the mutant
E. coli to grow aerobically with a functional Krebs cycle.
Alternative recipients could be other gram-negative
bacteria capable of facultative fermentative growth. If
this approach worked, it would enable subsequent overexpression of the autotroph genes and production of
quantities of protein for further analysis. It would also
give leads into the regulatory factors missing or controlling expression in autotrophs. In this context, the citrate
synthase gene from Sulfolobus solfataricus was expressed
at high levels when introduced into E. coli [20].
A parallel approach could be to introduce the E1 and
E2 genes of E. coli or another heterotroph (such as
Paracoccus, Methylobacterium or Ralstonia) into an
obligate species such as Methylococcus or Halothiobacillus to see whether they could be transcribed and
348
A.P. Wood et al. / FEMS Mircobiology Reviews 28 (2004) 335–352
translated into functional a-ketoglutarate dehydrogenase, and whether the presence of active enzyme allowed
heterotrophic growth on any organic substrates. Such
an approach would be essential in establishing whether
an overriding regulatory control existed that prevented
the expression of the introduced genes. That such an
approach might have success is exemplified by the succesful introduction of the gene for phosphofructokinase
into A. thiooxidans [127].
The phenotype of such recombinants would be of
considerable interest, such as in assessing stimulatory or
deleterious effects of the inserted genes on methanotrophic or chemolithotrophic growth, both in the presence and absence of added nutrients such as Krebs cycle
intermediates.
6. Concluding remarks
The dual purposes of this review have been (1) to
emphasize how bioinformatics can be exploited to help
solve and provide new approaches to unresolved metabolic problems and (2) to stimulate the interest of
‘‘modern molecular microbiologists’’ in a neglected area
of microbiology: the causes of obligate autotrophy and
methanotrophy.
We have stressed studies on a-ketoglutarate dehydrogenase but acknowledge that absence of this functional enzyme may not be the only, or even most
important, factor in bestowing obligate metabolism.
This was recognized by Whittenbury and Kelly [137] in
1977, when they noted that the ‘‘absence of 2-oxoglutarate dehydrogenase in itself does not impose obligate
autotrophy, but obligate autotrophy can result if the
Ômetabolic environmentÕ into which a lesion such as lack
of this enzyme in introduced is such that no alternative
metabolic process can be growth-supporting’’. It may
transpire that a number of genes for central metabolic
pathways essential for heterotrophic growth and some
transport processes are commonly absent from, or are
present and not expressed in, obligate genera, and that
obligate autotrophy is imposed by the ‘‘concerted absence’’ of these functions. In the present state of
knowledge we have concentrated on the historical candidate, a-ketoglutarate dehydrogenase, as a model for
the investigation of some obligate species. a-Ketoglutarate dehydrogenase is a strong contender for a role in
obligate autotrophy as its apparent absence is widespread among chemolithoautotrophs and methanotrophs throughout the phylogenetic tree. Most such
a-ketoglutarate dehydrogenase-negative species do not
seem to lack other enzymes of the Krebs cycle or of
other central metabolic pathways. A parallel line of
enquiry needs to be undertaken with the facultative
autotrophs and methylotrophs such as Paracoccus and
Methylobacterium in which a-ketoglutarate dehydroge-
nase activity is repressible during growth on C1 -compounds, but in which little is known about the
mechanism of repression. Understanding this could
throw light on the obligate autotrophy phenomenon. It
may be that the regulation of autotrophic metabolism in
obligate genera is much more complex than the simple
regulation of one or two genes, and involves cross-regulatory processes from their central carbon pathways
(the Calvin or Quayle cycles). This idea requires an
answer to the question ‘‘can genomics and proteomics
ever tell us that an obligate species is so because a gene
or gene product is non-functional, or that it is obligate
because there is a higher level of genomic organization
that prevents expression of genes such as a-ketoglutarate dehydrogenase?’’. That is to say, is obligate autotrophy determined or serendipitous?
Once the complete genome sequences are known for a
wider range of obligate chemo- and photo-lithoautotrophs and methanotrophs, it is likely that they will fall
into two categories: those with genes encoding a-ketoglutarate dehydrogenase and other central metabolic
enzymes, and those without. The explanation of obligate
metabolism in the latter group will require survey of their
overall metabolism to see if lack of specific functional
enzymes is the prime cause. Absence of the genes raises
the question of why they are lacking: does the organism
come from an ancestral line which never possessed them
or were they lost by a process of evolutionary reductionism, akin to that proposed for mycoplasmas? It may
be that at least some obligate autotrophs may have
evolved by the progressive loss of non-essential or potentially deleterious genes. A trend seems to be emerging
to indicate that obligate chemolithoautotrophs do indeed possess small to medium-sized genomes. Examples
are A. ferrooxidans, A. thiooxidans and Leptospirillum
ferrooxidans with chromosomes that are 2.29–3.33 (eight
strains), 3.14 and 1.90 Mb in size [4,57,128]. A. thiooxidans also contains five plasmids totalling 475 kb, indicating a maximum genome size of 3.62 Mb. Similarly the
chromosome of M. capsulatus comprises about 3.31 Mb
[129]. While these genome sizes are far greater than those
of metabolically fastidious symbionts and pathogens
such as Buchnera (0.45 Mb) and Mycoplasma (0.58 Mb),
they are significantly smaller than those of heterotrophs
including E. coli (4.64 Mb), B. subtilis (4.21 Mb) and
Streptomyces (8.66 Mb) [43,78,85]. The genomes of
strains of the cyanobacterium Prochlorococcus (the
smallest oxygen-evolving phototroph known) are also
very small at 1.66–2.4 Mb [26,105]. The genome of
N. europaea is also compact (2.8 Mb) and Chain et al.
[16] proposed that is indicative that a small genome is
sufficient to support an obligate autotroph. They also
suggest that N. europaea may be continuing to evolve a
‘‘more compact genome’’ by loss of functional genes [16].
Gene inactivation and subsequent erosion and deletion
may be the major force that maintains relatively small
A.P. Wood et al. / FEMS Mircobiology Reviews 28 (2004) 335–352
genome size in bacteria and selects against insertion of
laterally transferred genes unless these are selectively
advantageous [85]. In the case of a-ketoglutarate dehydrogenase in obligate genera, its permanent repression or
deletion would be selectively advantageous in avoiding
lethal or futile carbon cycling during autotrophic
growth. An intriguing question is how the selective
pressure has been exerted to prevent retention or expression of functional a-ketoglutarate dehydrogenase
genes, possibly acquired repeatedly by lateral transfer,
during evolutionary time, given that horizontal DNA
transfer appears to have been (and continues to be) a
major means for the acquisition of genes by bacteria
[143]. Thus, in that it is estimated that 17.6% of the genes
of E. coli have been acquired by horizontal transfer [11],
a comparable proportion could have arisen in gram
negative obligate autotrophs and methanotrophs such as
Nitrosomonas and Methylococcus. This may be indicated
by a BLAST search using the N. europaea genome which
showed 12% and 31% of its predicted open reading
frames also to be present, respectively, in Pseudomonas
aeruginosa and R. solanacearum [16]. Equally significant
is the likelihood of gene acquisition from archaea: as
many as 24% of the genes of Thermotoga are reportedly
archaeal [12]. While modern autotrophs and methanotrophs may be subject to a constant flux of gain and loss
of environmental DNA, their fundamental genetic makeup may have been established early in their evolutionary history, when horizontal gene transfer might also
have played a significant role [44].
Acknowledgements
Some of this work used preliminary sequence data
obtained from The Institute of Genomic Research
website at http://tigr.org. We thank Phil Cunningham
for advice on Unix-based computer systems. We thank
Noel Carr, Colin Murrell and Ian McDonald (Warwick)
for valuable critical comment on drafts of the review,
and anonymous reviewers for helpful criticism.
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