* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Cloning and expression of chromosomally and plasmid
Human genome wikipedia , lookup
Gene therapy wikipedia , lookup
Transposable element wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Gene nomenclature wikipedia , lookup
Genomic library wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Genetic engineering wikipedia , lookup
Epigenetics in learning and memory wikipedia , lookup
Oncogenomics wikipedia , lookup
Epigenetics of neurodegenerative diseases wikipedia , lookup
Epigenetics of diabetes Type 2 wikipedia , lookup
Long non-coding RNA wikipedia , lookup
X-inactivation wikipedia , lookup
Gene desert wikipedia , lookup
Public health genomics wikipedia , lookup
Quantitative trait locus wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Pathogenomics wikipedia , lookup
Essential gene wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Gene expression programming wikipedia , lookup
History of genetic engineering wikipedia , lookup
Genomic imprinting wikipedia , lookup
Genome evolution wikipedia , lookup
Microevolution wikipedia , lookup
Biology and consumer behaviour wikipedia , lookup
Designer baby wikipedia , lookup
Minimal genome wikipedia , lookup
Genome (book) wikipedia , lookup
Ridge (biology) wikipedia , lookup
Epigenetics of human development wikipedia , lookup
FEMS MicrobiologyLetters 66 (1990) 29-34 29 Pubhshed by Elsevier FEMSLE 03807 Cloning and expression of chromosomally and plasmid-encoded glyceraldehyde-3-phosphate dehydrogenase genes from the chemoautotroph Alcaligenes eutrophus U t e WmdhiSvel a n d Botho Bowlen ]tL¢llllll file MJkeobtologle.Georg-Augusr-Umverstla!Gottmgen.Goltmgen.F R G Received 14 August 1989 Revisionreceived17 AUgUSt1989 Key words" Gene duplication; cfx genes, gap gene; Megaplasmld priG1, lsoenzymes; Calvin cycle; CO 2 fixation 1. SUMMARY Hybndizauons using heterologous glycerald¢hyde-3-phosphate dehydrogenase ( G A P D H ) gene probes suggested the existence of three G A P D H genes in Alcahgenes eutrophus H16. Two of these, located on the chromosome and the megaplasnud priG1 of the orgamsm, respectively, mapped about 2.5 hlobase pairs (kb) downstream of the two duplicated CO 2 ftxauon gene clusters (cfx genes) They were identified as G A P D H genes (cfxGc and cfxGp) by cloning and expression m Escherwhta coh. These genes encode O A P D H tsoenzyrnes functioning in the Calvin cycle. The durd gene ( g a p ) is chromosomaUy encoded but not hnked to the cfx c cluster. Its product Is probably revolved m heterotrophlc carbon metabolism. Correspondenceto n Bowlen, |nstltut fur Mlkroblologte, Georg.August-UmversaatG6ttmgen, Gnsebachstrasse8. D-3400 Gonmgen, F R G 2. I N T R O D U C T I O N The Calvin cycle is the metabohc route of CO: assimilation in the facuhativdy autotrophlc hydrogan-oxtdtzmg bacterium Aleahgenes eutrophus [1]. Except for its key enzymes nbulose-l,5-blsphosphate carhoxylase/oxygenase (RuBisCO) and phosphonbulohnase (PRK) the cycle comprises enzymes catalyzing reactions also involved m heterotropbac carbon metabohsm. Among these enzymes are fructose-l,6-/sedoheptulose-l,7-b~sphosphatase (FSBP) and glyceraldehyde-3-pbosphate dehydrogenase (GAPDH). A general question regarding the regulatory separatmn of the two distinct modes of carbon metabohsm m facultatwely autotroph~c bacteria concerns the genetic reformation for these enzymes. Two apparently duphcated clusters of genes (cfx genes) encoding Calvin cycle enzymes have been found In the genome of Alealtgenes eutrophus HI6. They are located on the chromosome and the megaplasmld priG1 [2] that also cartaes the information for hydrogen oxidation [3]. So far, the 0378-1097/89/$03 50 © 1989 Federauon of European Microbiological Soc,eues 30 RuBisCO (cfxL, cfxS), PRK (cfxP) and FSBP (cfxF) genes were identified as constituents of both clusters [2,4]. Simultaneous expression of these genes results m formatmn of Calvin cycle tsoenzymes in the organism. The further analysis of the efx regions lead to the detectmn of a chromosomal GAPDH gene (cfxG¢) and a plasmad-encoded copy (cfxGp) each contained wtttun the respecttve cfx cluster. This paper reports on the locahzatton of the latter genes and their cloning and expressmn an Escher, chla coll. There ,s ctrcumstantial evidence that .4. eutrophu~ possesses a tlurd GAPDH gene (des,gnated as gap) whose product functions m glycolys,s/gluconeogenes~s. 3. MATERIALS AND METHODS Resmctton enzymes, T4 DNA hgase and alkahne phosphatase were obtained from Gthco- BRL (Eggenstem, F.R.G.), Bochnnger (Mannhe,m, F.R.G.) or Pharmacia LKB (Fre,hurg, F.R.G.). The radiochcnucals and a rock-translation kit came from Amersham Buchler (Braunschwetg, F.ILG.). Bacterial strains, phages and plasrmds used m tlus study are listed in Table 1. A. eutrophus strains were grown m Nument Broth (0.8%, w / v ) medmm at 30°C, E. cob XL1-Blue m LB-medmm at 37°C and E coh DF221 also at 37°C either m LB medium supplemented with 0.2% (w/v) glucose or in M9 mineral medmm [9] contaimng succmate (0.4~, w / v ) and glycerol (0.1%, w/v). For gene expression experiments E. coh was grown m LB medium containing 50 /~g a m p i e d h n / m l unUl reaching an optical density of 0.5 measured at 550 nm, then 1 mM isopropyl4~-tluogalactos]de (IPTG) was added and the cultures were incubated for additional 4 h. Cells to be used for the preparauon of extracts Table 1 Bacterial and phage strains and plasmldsused In this study Strata or plasmld Alcahgenes eutrophus Eschertchla coil H16 HF210 b XLI-Blue DF221 Pha~s kAEC2 AEP2 Plasrmds pUC19 pLOI312 pAEC4000 pAEC400I pAEC4010 pAEP4000 pAEP400! pAEp4010 Relevantcharactenstlcs a Cfx, HOX:priG1 Cfx. Hox-, pHOl Source or reference DSM428. ATCC17699 B Fnednch recA - (recAl #ac- endAl ~'rA96zht hsdRl7 supE44relAl {F' proAB laclq tacZAMI5 TnlO }) gap-2 [5l 15 7-kb incompletelydigested EcoRl fragment from A eutrophuschromosomeinserted In ~L47 18-kb Incompletelydigested EcoRI fragment from pHGI of A eutrophtts HI6 inserted i n ~L47 bin lacPOZ" bin lacPOZ', pUC8 with an 1 7-kbrosen carrying sap from ZymomonasmobJhs 3 7-kb EcoRl fragraent from gAEC2inserted m pUCIg, cfxG~oriented m dtrecuon of lacPOZ" 3 7-kb EcoRI fragmentlikein pAEC4000but cfxG~ onented opposite,o dlrecnon of lacPOZ" 2.5-kb Xhol/EcoRl fragmentfrom ?~AEC2inserted In pUCt9, cfxG~ orientedm dffecuonof lacPOZ" 3 9-kb EcoRl fragmentfrom ~AEP2 inserted m pUCIg, cfxGe onemed in d.reclion of lacPOZ' 3 9-kb Et'oRl fragmentlikein pAEP40tl0but cfxG# orientedoppositeto drtectionof lacPOZ" 2 O-kb XhoI/EcoRI fragment from XAEP2inserted m pUC19, cfxUporiented m dtrectlonof lacPOZ' Cfx, ability1o fix CO2, tlox, abilityIn oxidizeH1. b P|asrmd-free mutant derivedfrom A emrophtts H16 [6] [2] [2] [7] 18] This study This study This study This study This study were harvested m the exponentml growth phase, suspensed in 50 mM Tns-HCI, pH 7.5, containing 10 mM MgCI 2, 1 mM EDTA and 1 mM d*thiocrythntol at an optical denmty of about 125 measured at 436 nm and d~srupted by somhcalion. The supernatant resulting from a subsequent centrifugatton of the homogenate at 25000 g at 4 0 C for 1 h was directly used for assays. The actwity of G A P D H was deternuned as described prevmusly [10] but using a pH of 8.5 Sodium dodecyl sulfate-polyacrylamtd¢ gel eloctrophoresls (SDS-PAGE) was performed according to Laemmh [11]. Total bacterial D N A was isolated as detaded m [12]. Estabhshed procedures [9] were employed to isolate phage DNA. Large scale plasmid Isolation was done by an alkahne SDS lysls procedure [13]. Transformants of E. cob XL1-Blue were tested for plasrmd content by the rapid bothng method of Holmes and Qulgley [14]. Digestions of D N A by restricuon endonueleases and incubations in the presence of alkahne phosphatase or T4 D N A hgase were performed under the condtuons recommended by the suppher. Restnettou fragments were isolated from agarose gels by the method of Vogelstem et al. [15] using a kit (Geneclean, BIO 101, La Jolla, Ca., U.S.A.) For cloning experiments digested vector D N A was dephosphorylated. Ligated D N A was transformed into E col* strums according to [16]. Transformants of E col, XL1-Blue were selected on LB plates containing 50/x 8 amplelllm/ml. For complementauon analyXlS of E coh DF221 transformants the plates were addmonally supplemented vath 1 raM IPTG. Digested D N A was separated by agarose gel electrophoresls and transferred to nylon membranes (GeneScreen Plus, Du Pont. Bad Homburg, F . R G . ) following the protocols of the manufacturer. Southern hybndlzauon with 32p. labelled probes were performed at 65 o C. gene(s) m A eutrophus by means of heterolognas hybridization probes appeared to be feasible. A l.l-kb fragcaenl from pS198c encoding the cytosohc G A P D H of mustard (Smap~s alba) [19] and a 1-kb P s t l / B a m H l fragment from pLOt312 carrymg the gap gone of Zymomonas mob~hs [8] were used to probe restriction enzyme-digested D N A from A. eutrophus H16 and its plasmld-eured mutant HF210. The plant probe hybridized with a chromosomal 14-kb EcoRl fragment (Fig. 1A, a, b) that also showed weak homology to the bacterial probe (Fig. ]B, c, d). However, no hybrid formation was observed between the mustard probe and a chromosomal 3.7-kb EcoR.I or a pHGl-denved A B kb ! ! 8 a b c d e "~7 f Fig 1 Lncahzatlon of GAPDH genes m the genome of A 4. RESULTS A N D DISCUSSION 4 1. Locahzaoon of GAPDH genes Because of the strong structural conservation of G A P D H s from orgamsms of different phylogenetic origin [17,18] localization of G A P D H ¢Utrophus HI6 by Southern hybridization using probes from $mapls alba (A) or Z).momonasmobdts (B) labelled wah ~2p DNA isolated from wdd-ty~e strata H16 (lanes a and c). pHGl-cured mutanl HF210 (o and d) or recombinant phages ~AEC2 (¢) and ?~AEP2 (f) was digested with EcoR[ and elcelrophoretlcally separated (10 [=$)m a 08% (w/v) agarose gel Numbers indicate the sizes On kb) of hybridizing fragments 32 Table 2 Expressionof cfxG¢ and cfxGp from .4 euzroph~ measuredas GAPDH actlvsty m transformants of E coh XLI-Bluecontmrung different plasrmds B ~ ~'~' ~? ~ ~ ~ ~' F~g 2 Physicaland geneuc maps of the chromosomall~* (A) and pHGl-encoded(B) cfx gene clusters of A eutrophusH16 The arrowsshow the ]ocauons and transcnptlonalorientations of the genes Cleavagestiesfor resmctlonenzymes EcoRl (E), 8amHl (B) and Xhol (X) are 81ven Indices c and p refer to chromosome and pHGI, respecevely 3 9-kb EcoRI fragment, both of wluch hybridized strongly wtth the Z mobths probe (Fig. 1B, c, d). Hybndtzatton of the latter probe with recombinant phages from a A. eutrophus gene bank (Ftg. 1B e, f) tdenttfied the two shorter EcoRI fragments as those located lmmedtately downstream of the chromosomally and plasrmd-encoded cfxP genes, respectively (Fig. 2). Probmg wtth the gapB gene from the purple bacterium Rhodobacter sphaeroldes [20] confirmed these hybndtzation patterns (not shown) This su~ested the existence of three GAPDH genes m A. eufrophus wtth two of them, cfxG¢ and cfxGe, belongmg to the dupheared cfx clusters. The tlurd gene, gap, encoded on the chromosovae (dtscussed in 4 3 ) is not hnked to the cfx genes Plasnud Specificactwltyof GAPDH (U/rag extract protein) 3 92 40 36 4 02 42 20 23 62 3 86 8 90 pUC19 pAEC4000 pAEC400] pAEC4010 pAEP4000 pAEP4001 pAEP4010 same transcripUonai orientation as the other cfx genes. These findings were substanttated and extended by expression experiments designed to determine the activity of the recombinant G A P D H isoenzymes and to detect the products of their genes. The expt~esslons wece performed wtth transfor- I,- I 4 2 Subclonmg and expresston of the efxG genes m E colt The 3.7-kb and 3.9-kb EcoRI fragment and their 2.5-kb and 2.0-kb XhoI/EcoRl subfragments, respecttvely (see Fig. 2), were cloned m E colt using the expression vector pUC19 to prove functmnality and identity of the cfxG genes. The resuhmg hybrid plasmlds (see Table 1) were transformed into the gap mutant E. coh DF221 for testing of phenotyptc trans complementation Growth of transformants on LB agar would indicate expression of a GAPDH gene encoded on the respective plasmid. All plasmids except pAECd00] and pAEP4001 complemented the mutant, showing that the cfxG genes were mdeed funcUonal and expressed under the control of the lae promoter of the vector. The genes apparently had the -14 a b c d e f g Fig. 3. SDS-PAGEof cell extracts (10 ~tgprotein) from transformants of E cob XLl-Blueharbonng differentple.smlds (a) pUCI9, (b) pAEC4000, (c) pAEC400I, (d) pAEC4010, (e) pAEP40~0, (I0 pAEP400I and (8) pAEP4010 The arrow head points at the band of the putative recombinant GAPDH subumt The electrophoresss was performed in a gel polymer. Bed from 147o (w/v) acrylarmde Numbers mdeatc the sazes 0n kllodalton[kDa]) of molecularmass markers. 33 m a n t s of E. cob XL1-Blue smce m u t a n t D F 2 2 1 tended to loose the hybrid plasrmds. Extracts of cells contaimng plasnuds with the cfxG genes under control of the lac p r o m o t e r exhibtted twoto tenfold higher G A P D H acuvities t h a n those of the reference strain h a r b o n n g p U C 1 9 ( T a b l e 2), O p p o s i t e orientation of the genes did not cause an increase o f enzyme activity. S D S - P A G E of these extracts revealed a strong synthesis In the clones with high G A P D H acuvity of a 35-kdodalton polypeptide (Fig. 3). T h i s molecular mass correlates closely with that of subumts of other G A P D H s [17], m a k i n g formatton of fuston proteins during heterologous expresston of cfxG unlikely. 4.3. Conclustens T h e two cfxG genes of A. eutrophus H 1 6 do not have promoters that are active m E colt. However, their ribosome-bmding sites seem to be recogmzed by the foreign host as in the case of the o t h e r cfx genes [2]. T h e i r location relative to cfxP resembles that of prkB and gapB m P~ sphaerotdes [20]. It is possible that the cfxG genes of A eutrophus lack own promoters and are p a r t of large cfx operons whtch may comprise all genes within each of the duplicated cfx clusters T r a n s poson T n S - m d u c e d mutants defective in C O 2 assimilation requtred a complete cluster including cfxG for phenotyplc trans complementation (unpublished results). Thus, the phystologoeal role of the cfxG-eueaded G A P D H lsoonzymes ts m the C a l v i n cycle. Since these mutants with inactive cfxG ,¢ere able to grow helerotrophically another gene coding for a third G A P D H isoenzyme opera t m g in glycolysis and ghiconengenests must exast m strata H ) 6 . T h i s gap cane is presumably located on a chromosomal 14-kb EcoRl fragment (see Fig. 1). ACKNOWLEDGEMENTS W e are indebted to B. Bachmann, R. Cerff, T Conway, D . G . Fraenkel, J.L. Gtbson, L.O. Ingrain and F R T a h i t a for providing plasmads, probes or strmns. Thts w o r k was supported by a g r a n t of the Deutsche Forschangsgememschaft. REFERENCES [I] Bowlen, B and Schlcg¢l,H G (1981) Annu Re.v Mlcrobwl 35, 405-452 12] Husemann, M, Khntwonh. R, Buttcher. V, Salmkow, J, We~ssenborn, C and Bovaen, B (19881 Mol Gen Genet 214,112-120 [3] Kordhke, C, Hogrefe, C. Ebcrz. G , Puhler, A and Fnednch, B (19871 Mol Gen Genet 210.122-128 [4] Kogmann. J, Khmworth. R and Bo'men, B (19891Gene, In press [5] Bullock, W O, Fernandez, J M and Short. J M (19871 BioTechmques 5. 376-378 [6] Hdlman. J D and FraenkeL D G {19751J Bactgnol 122, 1175-179 [71 Yamsch-Perron, C, Vmwa, J and Messing. J (1985) Gen¢ 33.103-119 iS] Conway. T , SewelL G W and Ingrain. LO 0987) J Bactenol 169, 5653-5662 [9] Mamatls. T . Fmsch. E F and Sambrook, J {1982) Molecular Cloning Cold gpnng Harbor Laboralory, Cold spnng Harbor, NY [101 Fnednch, C G . Fnednch, B and Bowzen, B (1981)J Gen Mtcrobiol 122, 69-78 [11} Laemmh. U K (1970) Nature 227, 680-685 [121 Ausubel, F M. BrenL R, Kingston, R E, Moore, D D, Seldmam J G . Srmth, J A and StruhL K (19871 Currenl Protocols m Molecular Biology Wiley. New York [13] Blmbolm. HC and Doly, J (19791 Nucl Acids Res 7, 1513-1523 [14] Holmes. DS and Qmgley. M {1981) Anal Biochem 114, 193-197 115] Vogelstcm. B and Gillesple, D (19791 Proc Natl Aead Set U S A 76.615-619 [16] Mandel, M and Hlga. A (1970) J Mol lhol 53.159-162 [17] Hams, J 1 and Waters, M (19761 in The Enzymes (Boyer. P D. ed ). Vol 13. pp 1-43. Acaderrac Press, New York [18] SkarzynskL T . Moody. P C E and Wonacott, A J 098"/) J Mol BIol 1';3.171-187 [19] Martin. W and Cerff. R (1986) Eur J 8mchem 15o 323-331 [20] Gibson, J L and Tablta. F R (19881 J Bact©nol 170, 2153-2158