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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. REFERENCES [1] Tabita, F.R. (1988) Molecular and cellular regulation of autotrophic carbon dioxide fixation in microorganisms. Microbiol. Rev. 52,155-189. [2] Smith, A.J. and Hoare, D.S. (1977) Specialistphototrophs, lithotrophs, and methylotrophs: a unity among a diversity of procaryotes? Bacteriol. Rev. 41, 419-448. [3] Bowien, B. and Schlegel, H.G. (1981) Physiology and biochemistry of aerobic hydrogen-oxidizing bacteria. Anna. Rev. Microbiol.35, 405-452. [4] Bowien, B. (1989) Molecular biology of carbon dioxide assimilation in aerobic chemolithotrophs, in Autotrophic Bacteria (Schlegel, H.G. and l~wien, B., Eds.), pp. 437460. ScienceTech, Madison. [5] Friedrich, C.G., Friedrich, B. and Bowien,B. (1981) Formation of enzymesof autotrophic metabofismduring beterotrophic growth of Alcaligeneseutrophus.J. Gen. Microbiol. 122, 69-78. [6] Leadbeater, L.. SieberL K., Schobert, P. and Bowien, B. (1982) Relationship between activities and protein levels of ribulosebisphosphate carboxylas¢ and phosphoribulokinase in Alcaligeneseutrophus. FEMS Microbiol. Lett. 14, 263-266. [7] Friedrich, C.G. (1982) Derepression of hydrogenase during limitation of electron donors and derepression of ribuiosebisphosphate carboxylase during carbon limitation of Alcaligeneseutrophus.J. Bactcriol.149, 203-210. [8] Amachi,T. and Bowien,B. (1979) Characterizationof two fructose bisphosphatase isoenzymes from the hydrogen bacterium Nocardia opaca lb. J. Gen. Microbiol. 113, 347-356. [9] Friedrich, B. (1989) Geneticsof energyconvertingsystems in aerobic chemolidiotrophs, in Autotrophic Bacteria (Schlcgel, H.G. and Bowicn, B., Eds.), pp. 415-436. Science Tech, Madison. [10] Friedrich, B., Hogrefe,C. and Schlegel,H.G. (1981) Naturally occurringgenetic transfer of hydrogen-oxidizingabib 450 ity between strains of Alcaligenes eutrophus. J. Bacteriol. 147,198-205. [11] Bowien, B., Friedrich, B. and Friedrich, C.G. (1984) Involvement of megaplasmids in heterotrophic derepression of the carbon-dioxide assimilating enzyme system in AIcaligenes spp. Arch. Mierobiol. 139, 305-310. 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Gen. Genet. 210,122-128. [17l Kossmann, J., Klintworth, R. and Bowien, B. (1989) Sequence analysis of the chromosomal and plasmid genes encoding phosphoribulokinase from AIcaligenes eutrophus. Gene 85, 247-252. [18] Windh~vel, U. and Bowien, B. (1990) Cloning and expression of chromosornally and plasmid-encoded glyceraidehyde-3-phosphate dehydrogenase genes from the chemo- autotroph Alcaligenes eutrophus. FEMS Microbiol. Left. 66, 29-34. [19] WindhSvel, U. and Bowien, B. (1990) On the operon structure of the cfx gene clusters in Alcaligenes eutrophus. Arch. Microbiol., 154, 85-91. [20] Warrelmann, J. and Friedrich, B. (1989) Genetic transfer of llthoautotrophy mediated by a plasmid.cointegrate from Pseudomonasfacilis. Arch. Micrubiol. 151, 359-364. [21] ,Sensfuss,C., Reh, M. and Schlegel, H.G. (1986) 14o correlation exists between the conjugative transfer of the autotrophic character and that of plasmids in Nocardia opaca strains. J. Gen. Microbiol. 132, 997-1007. [22] Lehmicke, L.G. and Lidstsom, M.E. (1985) Organization of genes necessary for growth of the hydrogen-methanol autotroph Xanthobacter sp. strain H4-14 on hydrogen and carbon dioxide. J. Bacteriol. 162,1244-1249. [23] Gibson, J.L. and Tabita, F.R. (1988) Localization and mapping of CO2 fixation genes within two gene dusters in Rhodobacter sphaeroides. J. Bacteriol. 170, 2153-2158. [24] Ira, D.-S. and Friedrich, C.G. (1983) Fluoride, hydrogen, and formate activate ribuiosebisphosphate carboxylase formation in Alcaligenes eutrophus. J. Bacteriol. 154, 803808. [25] Dijkhuizen, L. and Harder, W. (1984) Current views on the regulation of autotrophic carbon dioxide fixation via the Calvin cycle in bacteria. Antonie van Lceuwanhoek 50, 473-487. [26] Henikoff, S., Haughn, G.W., Cairo, J.M. and Wallace, J.C. (1988) A large family of bacterial activator proteins. Proc. Natl. Acad. Sci. USA 85, 6602-6606. [27] Kalkus, J., Reh, M. and Schlegel, H.G. (1990) Hydrogen autotrophy of Nocardia opaca strains is encoded by linear megaplasmids. J. Gen. Microbiol. 136,1145-1151.