Download Genetics of CO2 fixation in the chemoautotroph Alcaligenes eutrophus

FEMS MicrobiologyReviews87 (1990)445-450
Publishedby Elsevier
445
FEMSRE00207
Genetics of
CO 2
fixation in the chemoautotroph
A lcaligenes eutrophus
B. Bowien, U. WindhiSvel, J.-G. Yoo, R. Bednarski and B. Kusian
lnstitut f,;r Mikrobiologie, Georg-August-UniversitiitG~ttingen, GiJttingen,F.R.G.
Key words: Calvin cycle; cfx Genes; Gene duplication; Gene regulation; Hydrogen-oxidizing
bacteria; Isoenzymes
1. SUMMARY
The genome of Alcaligenes eutrophus H16 contains two functional, highly homologous clusters
of genes (cfx genes) for at least six Calvin cycle
enzymes, with one cluster located on the chromosome and the other on megaplasmid priG1.
The genes within each cluster presumably belong
to a single large operon of about 11 kilobase pairs
(kb) in size. Both operons form a cfx regulon that
is controlled by the chromosomally encoded regulatory gen¢ cfxR. The product of cfxR seems to be
a transcriptional activator. Occurrence of isoenzymes within the central carbon metabolism of the
organism is a consequence of this unusual genetic
setup.
several autotrophic bacteria have been initiated in
recent years [1]. They are focussing on those photolithotrophs and chemolithotrophs using the reductive pentose phosphate cycle (Calvin cycle) as
carbon assimilation pathway. Because of the complexity of this cycle--it comprises thirteen reactions catalyzed by ten enzymes--such studies are
a major task. This is rendered even more difficult
by the fact that autotrophic bacteria are not well
amenable to conventional genetic analysis. Nevertheless, substantial progress has been made in the
elucidation of organization and regulation of cfx
genes in some organisms. The present review will
primarily summarize what is known about these
genes and their products in the aerobic hydrogenoxidizing bacterium A. eutrophus which, in this
regard, is the most intensely studied chemoautotroph.
2. INTRODUCTION
Despite the fundamental importance of CO2
assimilation as the primary biosynthetic process in
nature, general knowledge of the genetics of CO2fixing (Cfx) enzyme systems in autotrophs is fragmentary at best. Genetic studies on Cfx systems of
Correspondence to: B. Bowien,Institutf'tirMikrobiologie,Georg-August-Universit~ltG~ttingen,Grisebachstrasse8. D-3400
GBttingen,F.R.G.
3. PHENOTYPE WITH RESPECT TO THE Cfx
ENZYMES
A consideration of central carbou metabolism
of a~totrophic bacteria requires a distinction to be
made between obligate and facultative autotrophs.
Specialist organisms do not need to regulate their
Cfx enzymes, either on the activity or the synthesis level, as efficiently as the nutritionally versatile
ones [2]. The latter are able to grow heterotrophi-
0168-6445/90/$03.50© 1990Federationof EuropeanMicrobiologicalSocieties
446
cally as well as autotrophically or may even prefer
a mixotrophic mode of nutrition at their natural
habitats. Hydrogen-oxidizing bacteria are typical
representatives of this group of organisms [3].
Heterotrophic growth on organic substrates t~sually results in a partial or complete repression of
Cfx enzyme synthesis. The key enzymes of the
Calvin cycle, ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase (PRK) appear to be coordinately regulated in most bacteria (reviewed in [4]). This has
been proved for A. eutrophus H16 and was interpreted to be the manifestation of a possible close
linkage of the respective genes [5,6].
Data obtained for the other Cfx enzymes of A.
eutrophus are more difficult to assess, since these
enzyme activities are not uniquely associated with
the Calvin cycle but also function in glycolysis
a n d / o r ghiconeogenesis. A comparison of the activities, determined with various autotrophically
(lithoautotrophic with H2/CO2; organoautotrophic with formate) or heterotrophically grown
cells, shows that a number of enzymes, including
glyceraldehyde-3-phosphate d e h y d r o g e n a s e
(GAPD), fructose-l,6-bisphosphatase (FBPase)/
sedoheptulose-l,7-bisphosphatase (SBPase), transketolase (TK) and pentosephosphate 3-epimerase
(PPE), follow a similar regulatory pattern like
RuBisCO and PRK (Table 1). Most significant
differences exist between cells grown heterotrophically on the strongly repressing pyruvate and those
cultured lithoautotrophically under limiting supply
of CO 2. Maximal lithoautotrophic derepression
upon carbon limitation, first observed for
RuBisCO [7], therefore also seems to occur with
the Cfx enzymes mentioned, suggesting a common
basis for the regulation of their genes. As genetic
analysis has provided evidence for a close linkage
of these genes (see below in Section 4), another set
of genes must be present in A. eutrophus that
encodes the remaining Cfx enzymes--phosphoglycerate kinase, triosephosphate isomerase, fructose-l,6-bisphosphate/sedoheptulose-l,7-bisphosphate aldolase and pentosephosphate isomerase-and is under different control.
In this context another aspect of carbon
metabolism in the organism deserves attention.
How is the enzymic organization at the interface
of autotrophic and heterotrophic carbon metabolism? It is conceivable that isoenzymes, specifi-
Table 1
Activitiesof Calvincycleenzymesin Alcaligeneseutrophus H16 grownon varioussubstrates
Enz~,me
Ribulose-l,5-bisphosphate
carboxylase/oxygenase
Phosphoribulokinase
Growth substrate
Autotrophica
H2/lim. CO2
H2/CO2
Formate
Heterotrophic
Fructose
Pyruvate
0.475
0.123
0.139
0.026
< 0.001
0.475
0.243
0.257
0.035
< 0.001
1.55
1.78
1.37
1.32
1.07
1.12
1.09
0.083
0.915
0.114
1.25
0.143
0.322
n.d.
0.256
5.59
2.58
0.098
0.090
0.029
3.62
3.66
0.069
n.d.
0.015
3.66
2.57
Phosphoglycerat¢kinase
1.88
1.81
Glyceraldehyde-3-phosphate
2.84
1.72
dehydrogenase
Triosephosphateisomerase
0.633
0.747
Fructose-1,6-bisphosphate
0.338
0.078
aldolase
Fructose-l,6-bisphosphatase
0.674
0.231
Sedoheptulose-l,7-bisphosphatase
n.d.
0.218
Transketolase
0.126
0.152
Pentose-5-phosphate3-epimerase
13.27
9.58
Pentose-5-phosphateisomerase
2.18
3.32
Results are givenas specificactivity(U/mg of p:otcin)
a Lithoautotruphic growth with H2/CO2/O2 (89/1/10% [fimitlngCO2] or
formate (0.2%,w/v) under air.
n.d., not determined.
80/10/10%, v/v); org~.noautotrophicgrowth with
447
cally or preferentiaUy involved either in the Calvin
cycle, i.e. Cfx enzymes, or in glycolysis/
gluconeogenesis, are formed in response to the
different metabolic needs. A separation of the two
modes of metabolism might be required because
the enzymes must have adequate regulatory properties a n d / o r a monoenzyme does not provide
enough catalytic capacity due to insufficient
synthesis. Formation of isoenzymes could occur
for all or only certain enzymes of the reductive
and regenerative parts of the Calvin cycle. Different facultative autotrophs possibly evolved different strategies to solve this metabolic challenge.
Genetically distinct FBPase/SBPa~_ isoenzymes
playing specific physiological roles have been
found in the hydrogen bacterium Nocardia opaca,
one of the first documented cases of Cfx isoenzyme formation in an autotrophic bacterium [8].
Our genetic studies on the Cfx system of A.
eutrophus allow the conclusion that this organism
also forms isocnzymes operating in central carbon
metabolism.
4. ORGANIZATION OF cfx GENES
Wild-type strains of A. eutrophus harbor large
plasmids, some of which are involved in the hydrogen-oxidizing capacity (Hox) of the organism
[9]. The conjugative megaplasmid priG1 of strain
H16 is about 450 kb in size and encodes almost all
Hox-specific functions [9,10]. Early experiments
c~'aracterizing the beterotrophic derepression of
er~,
~x,
RuBisCO and PRK implicated p H G I in this regulation and pointed at a possible extrachromosomal
location of ~fx genes in strain H16 [11]. Subsequent identification of functional chromosomal
and pHGl-encoded RuBisCO (cfxL-cfxS) and
PRK (cfxP) genes indicated a reiteration of these
genes in apparently duplicated clusters of very
high homology on distinct genetic entities [12-14].
This seems to be a typical feature of cfx gene
organization in A. e~trophus strains, although the
type strain and strain CH34 only possess chromosomal RuBisCO and PRK genes [4,15]. The
plasmid-encoded tfx genes on priG1 are part of a
larger cluster also containing the box genes [16].
Thus, the megaplasmid confers the essential characters of lithoautotrophy onto the organism.
The two homologous cfx gene clusters of strain
H16 encompass about I kb and contain structural
genes for Cfx enzymes in addition to RuBisCO
and PRK (Fig. 1). Genes for PPE (cfxE),
FBPase/SBPase (cf,:F), TK (cfxT) and GAPD
(cfxG) were identified ([17,18]; Yoo, Kusian and
Bowien, unpublisheci). The functions of the products from two furthe~ genes, cfxX and cfxY, are
still unknown. All genes, except the regulatory
gene cfxR that is loca,.ed within the chromosomal
cluster immediately upstream of cfxLc (see below
in Section 5), have the same relative orientation
and are closely linked. Seq,Jence data will have to
provide information as to whether the intergenic
region of about 0.7 kb between cfxT and cfxG
encodes another open reading frame.
The close linkage of the cfx genes susgests the
c~v~fxE,
dz~
d.P,
dxT,
(= = :J=
boao e ~
cluster
,
I kb
L
Fig. 1. Mapshowing the organization of the chromosomally and plasmid-encoded cfx gene clusters of Alcaligeneseutrophus H16. The
identity of the individual genes, if known, is mentioned in the text (in 4.). Indices c and p refer to chromosome and megaplasmid
priG1, respectively.
448
existence of a common operon within each cluster.
There is genetic evidence in favor of this view. It is
based on the properties of mutants carrying distinct insertional mutations by transposon Tn5
within the chromosomal cfx cluster [19]. Available
sequence information also supports this operon
structure with cfxL as the promoter-proximal and
cfxG as possibly the most distal gene (see Fig. 1).
We have no data to indicate that additional genes
downstream of cfxG would belong to the cfx
region. A pHGl-free mutant deficient in CO 2
fixation (Cfx-) as a result of a Tn5 insertion near
the 5'-end of cfxLc requires a complete intact
gene cluster (cfxL through cfxG, chromosomally
or plasmid-encoded), introduced by means of a
broad-host-range vector, for phenotypic trans
complementation [19].
As both sets of cfx genes in A. eutrophus H16
are function, the organism synthesizes two very
similar isoenzymes for each Cfx enzyme encoded
within the clusters. However, the function of these
enzymes is dispensable in heterotrophically growing cells. Two lines of evidence support this conclusion: (i) the cfx genes, represented by the
RuBisCO and PRK genes, are fully repressed in
pyruvate-grown cells ([5], see Table 1); (ii) a
plasmid-free Cfx- mutant with the cfx genes inactivated by Tn5 insertion grew on all tested
organic substrates with the same rate as the parent
strain [19]. The organism must thus be able to
form 'third' isoenzymes at least for GAPD,
FBPase, TK and PPE operating in gluconeogenesis, giycolysis or pentose phosphate synthesis during heterotrophic growth. The presumed genes gap
(product GAPD), fbp (FBPase), tkt (TK) and rpe
(PPE) remain to be characterized.
Because of the cfx gene cluster duplication, A.
eutrophus H16--and probably other strains of the
species--has a particularly complex inventory of
genes encoding enzymes of the central carbon
metabolism. Partially comparable situations may
prevail in other facultative autotrophs. The h y d r a
gen-oxidizing bacterium Pseudomonasfacilis strain
K harbors megaplasmid pHG22-a that exclusively
carries the cfx and box genes of the organism [20].
Also, in another hydrogen bacterium, Nocardia
opaca, both markers of lithoautotrophy are encoded on a conjugative genetic element [21] which
was recently identified to he a linear plasmid [27].
Xanthobacter sp. strain H4-14, a hydrogenmethanol autotroph, apparently contains only
chromosomal cfx genes clustered similarly like in
A. eutrophus ([22]; W. Meijer and L. Dijkhuizen,
personal communication). Two chromosomaHy
located cfx gene clusters designated the form I
and form II regions were detected in the phototrophic non-sulfur purple bacterium Rhodobacter
sphaeroides. They are similar but not identical,
and their gene arrangement is reminiscent of that
within the A. eutrophus clusters [1,23].
5. REGULATION OF cfx GENES
Expression of the cfx genes in A. eutrophus
does not only depend on the substrates used as
carbon and energy sources but is also influenced
by the presence/absence of the megaplasmid.
Partial derepression of RuBisCO and PRK in
strain H16 during growth on fructose (see Table 1)
is completely abolished in a plasmid-cured mutant
[11]. This regulatory influence of the megaplasmid
on cfx gene expression was observed for other
strains as well, except for the type strain which
lacks the plasmid-encoded cfx gene copies [4].
Recent experiments with Tn5 insertional mutants
inactivated in the chromosomal copies showed
that both sets of genes must be functional for the
heterotrophic derepression to occur (WindhSvel
and Bowien, unpublished). The basis for this regulatory interrelation of the duplicate cfx operons is
still unclear.
Derepression/repression rather than induction/repression might generally control expression of the cfx genes [5,7], although formate was
considered as potential inducer [24]. Expression
control appears to be primarily exerted by transcriptional regulation [14]. Signal metabolites, such
as the Calvin cycle intermediate 3-phosphoglycerate or the indirect cycle product phosphoenolpyruvate, that vary in intracellular concentration depending on the metabolic status may
function as effectors [4,24,25]. The. effector could
interact with a regulatory protein, the liganded or
unliganded form of which would be involved in
transcriptional control of the genes. In fact, the
449
properties of a class of C f x - mutants derived
from A. eutrophus H16 were compatible with this
hypothesis. These mutants carried a chromosomal
mutation within the regulatory locus cfxR located
immediately upstream of cfxL c (WindhiJvel and
Bowien, unpublished; see Fig. 1). Inactivation of
cfxR abolished expression of both the chromosomal and plasmid cfx operons. Thus, cfxR
encodes a product, CfxR, acting m cis and in
trans as a transcriptional activator. The duplicated
operons form a cfx regulon. Sequence analysis of
the upstream region of cfxL c indicated the presence of an open reading frame that is divergently
oriented from the cfx operon. The N-terminal
portion of the deduced protein has significant
similarity to bacterial regulatory proteins of the
LysR family (WindhiSvel and Bowien, unpublished). Like CfxR, most members of this family
are activators [26]. This suggests that the expression of cfx genes in A. eutrophus is governed by a
very general mechanism of bacterial gene regulation.
6. C O N C L U S I O N S
The organization of cfx genes represents an
unusual genetic s¢.tup: two highly homologous
functionaJ gene clusters that are located on the
chromosome and on megaplasmid priG1 of A.
eutrophus H16 and are under common control.
Expression of the genes results in the formation of
Cfx isoenzymes. The metabolic advantage of this
arrangement is not evident, since the type strain of
A. eutrophus lacks the plasmid copies of cfx genes
without being affected in its autotrophic capability. It is of interest to note that only six out of ten
Cfx enzymes are encoded within the cfx operons.
The products of the cfxX and cfxY genes are
unlikely to be Calvin cycle enzymes (Kusian and
Bowien, unpublished).
The remaining four Cfx enzymes have to be
encoded by genes that might not be linked to the
known clusters and are differently regulated. These
genes might form (an)other cfx cluster(s) or be
dispersed throughout the genome of the organism.
Even reiteration on chromosome and megaplasmid is conceivable. However, their products do
not necessarily have to be specific Cfx enzymes
like those coded for within the cfx operons already identified, but could also operate in the
heterotrophic carbon metabolism of this facultarive autotroph. The elucidation of the genetic and
enzymic integration of the two modes of carbon
metabolism in A. eutrophus will certainly be a
fascinating aim for future investigations.
ACKNOWLEDGEMENT
This work was supported by grants from the
Deutsche Forschungsgemeinschaft.
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