Cloning and expression of the carbon monoxide dehydrogenase genes from Pseudomonas thermocarboxydovorans strain C2

FEMS MicrobiologyLetters70 (1990)249-254 Published by E|sevier

249

FEMSLE04078

Cloning and expression of the carbon mono,,dde dehydrogenase genes from Pseudomonas thermocarboxydovorans strain C2 Gary W. Black ~, Catherine M. Lyons 2, Edwin Williams 2, John Colby ~, Michael Kehoe 2 and Catherine O'Reilly School of Biology, Sunderland Polytechnic, Sunderland and 2 Microbiology Department, The Medical School, The Unwersity, Newcastle-upon- Tyne, U.K.

R~ccived2 April 1990 Ac,ccpted 5 April 1990 Key words: Carbon monoxide; Carbon monoxide dehydrogenase; Carboxydotrophic bacterium; Pseudomonas thermocarboxydovorans C2; Immunological screening; Cloning; Restriction analysis;

Western blot analysis; Expression

1. SUMMARY Carbon monoxide dehydrogenase (CODH) from Pseudomonas thermocarboxydovorans strain C2 is composed of three non-identical subunits. A gene library of C2 DNA in lambda vector L,,?.. was generated and screened using anti-CODH serum. Western blotting experiments revealed a protein which co-migrated with and had the same immunological reaction as the large subunit of CODH in some of the clones isolated from the library. The coding region was pinpointed to a 4 kb fragment which was subcloned into plasmid, Western blotting experiments showed that all three subunits of CODH were coded for by the subclone. However, no CODH activity was detected.

Correspondence to: C. O'Reilly, Schoolof Biology,Sunderland

Polytechnic, SunderlandSR1 35D, U.K.

2. INTRODUCTION Bacteria capable of using CO aerobically as sole carbon and energy source are known as carboxydotrophic bacteria or simply as carboxydobacteria [1-4]. Carboxydobacteria are a diverse group of microorganisms and all members are able to: catalyse the oxidation of CO to CO2; transfer the electrons derived from this reaction to the respiratory electron transport chain for the generation of ATP by oxidative phosphorylation; assimilate some of the CO2 formed via the Calvin-Benson cycle; produce NAD(P)H, via reversed electron flow, for CO~ assimilation; and withstand CO inhibition. The key enzyme in the energy metabolism of these bacteria is the molybdenum, FAD and iron-sulfur containing enzyme, CODH (CO oxidoreduetase; CO oxidase). The carboxydobacterium Pseudomonas thermocarboxydovorans C2, isolated from sewage [5], is capable of autotrophic growth at 45-65 ° C on CO or on a mixture of CO and hydrogen, although in

0378.1097/90/$03.50 © 1990 Federation of EuropeanMicrobiologicalSocieties

250 the latter case the hydrogen is not utilized. The organism grows heterotrophically o n a limited range of organic a n d a m i n o acids but not on sugars. O u r work [6] (Halder a n d Colby, u n p u b fished results) indicates that C O D H from C2 is a hexamer of three nonidentical subunits, i.e. an (LMS)2 structure, of molecular masses 87000, 2 9 0 0 0 and 21 000. C O D H from C2 is similar to C O D H enzymes from other c a r b o x y d o b a c t e r i a but a few differences are evident: it has a high thermal stability; a n d it has a n extremely low a p p a r e n t Kr, for CO. These features together with the fact that C2 itself has a m u c h shorter doubling time on C O (3 h) t h a n other c a r b o x y d o b a c t e r i a ( 1 2 - 4 2 h) indicates that C2 is efficient at utilizing C O a n d this bacterium a n d the C O D H from it m a y be particularly useful m the production of biosensors 17,81. C O D H from C2 is maximally induced d u r i n g autotrophic growth o n C O a n d present at approximately one-third o f the m a x i m u m level dur-

ing c a r b o n limited g r o w t h on sodium pyruvate [5]. N o enzyme is present at non-limiting concentrations o f sodium p y r u v a t e [5]. T h u s the p r o d u c t i o n o f C O D H is highly regulated. Analysis o f the genes e n c o d i n g C O D H at the molecular level should help elucidate the m e c h a n i s m of regulation. This p a p e r describt.s the cloning of the genes coding for C O D H from C2 into Escherichia coli a n d the expression of C O D H as indicated b y Western blot analysis.

3. M A T E R I A L S A N D M E T H O D S 3.1. Microbial strains, vectors an~ recombinant DNA These are listed in T a b l e 1. 3.2. Cultivation C2 was g r o w n aut~trophically in bulk as described b y L y o n s et al. [5]. Heterotrophic cultures were g r o w n in a u t o t r o p h i c m e d i u m supplemented

Table l Microbi~ s~.r~n.%v~tors and recombinant I)NA molecules used and constructed Microbial strains Pseudomonos thermocarboxydot'orans C2

Comment Wild type

Source of reference [5]

[9] [10]

HM514

For construction of gene library, lysogenic for P2 For further propagation of recombinant phage and host for pBR322 plasmid and recombinants For further propagation of recombinant phage

JM83

Host for pUC plasmids ana ~ombinants

Escherichiacoil WL95 LE392

Vectors Bacteriophage: klAT.l

Kindly provided by N. Murray [ll K

Recombinants are Gain- and grow on P2 lysogenic strains of E. coli

[9]

~'l~mid: pBR322 pUC9 pUCI 8

Apr, Tc R ApR, lacZ" Apn, lacZ"

[12] [13] [ 14]

Recombinant DNA molecules P22 P40 pCL2 pGB1 pGB2

Purified clone isolated from C2 gene fibrary Purified clone isolated from C2 gene library 7.2kb Sail fragment from P40 subcloned into pBR322. 4kb EcoRI fragment from P22 subcloned into pUC9. 2.7kb Sphl fragment from pCL2 subcloned into pUCI~.

This study This study This study This study This study

251 with 0.2% (w/v) sodium pyru~ate and 0.1% (w/v) yeast extract. Eseherichia coli strains used for the propagation of phage were grown in NZ medium (Gibco) supplemented with yeast extract (5 g/l). Otherwise LB (Luria-Bertani) medium was used for growing E. coli strains. LB medium was solidified with 1.5% (w/v) agar. When used the concentrations of the following supplements were: ampicillin (100 #g/ml), 5-bromo-4-chloro-3-indolyi //-D-gataetopyranoside (70 ,ag/ml) and tetracycline (15/xg/ml). 3.3. Preparation and screening of the C2 library DNA was extracted from a heterotrophic C2 culture grown to an OD600 value of 0.3 using a modification of the technique of Marmur [15]. The C2 DNA was partially digested with Sau3A and the fragments of sizes 4.7 to 19.6 kb purified using the method of Girvitz et al. [16]. The purified C2 DNA was ligated to BamHl cut ~L47.1 DNA. The ligation mixture was packaged in vitro into h particles using the method of Scalenghe et al. [17] and plated out onto E. coli WL95 cells. The resulting recombinant phage were amplified using the plate lys:ne method [10] and about 33,000 recombinant phage from the amplified library were plated out onto E. coli LE392. The chilled plates wcie overlayed with 0.45# nitrocellulose filters for 3 h at room temperature. The filters were removed and transferred to blocking buffer (10 mM Tris, pH 7.5; 2 mM EDTA; 150 mM NaCI; 0.5% bovine serum albumin; 0.05% Triton X-100) for 2 h at room temperature with gentle agitation, then transferred to fresh blocking buffer containing anti-CODH serum fnr "~ b ~' r~oc'.':2.. '.empera*_ure with gentle agitation and then transferred to wash buffer containing 1251-conjugated anti-rabbit lgG for 3 h at room temperature with gentle agitation and subsequently washed for 4 × 15 min (wash buffer with 1 M NaC1). Dried filters were autoradiographed, positive clones isolated, purified and plate lysate stocks made [10]. 3.4. Western blots of recombinant phage and plasmid proteins Recombinant phage proteins were precipitated from cleared phage lysates with chilled trichloroacetic acid (TCA) solution (100% TCA; 0.05

mg/ml sodium deoxycho!ate) at a concentration of 20% TCA. The proteins were pelleted after 1 h on ice and the pellets washed with 70% acetone; 20% ethanol; 10 mM Tris, pH 7.4; 0,001fg hromophenol blue. The pellets were resuspended in loading buffer (60 mM Tfis, pH 7.4; 5% (w/v) dodecyl sulphate; 1% (w/v) dithiothreol; 20% (v/v) glycerol; 0.005% bromophenol blue~ and boiled for 3 rain prior to loading onto acrylamide gels. Crude cell extracts of C2 were prepared as described by Lyons et al. [5]. E. coli cells harbouring recombinant plasmid were grown up in LB medium containing ampicillin and crude cell extracts were prepared as for C2. Extracts were diluted 1 : 3 with loading buffer prior to loading onto acrylamide gels. TCA precipitated phage proteins and crude cell extracts of C2 proteins and recombinant plasmid proteins were subjected to PAGE using 10% and 12.5% polyacrylamide gels (360 mM Tris, pH 8.8; 0.1% (w/v) ~-ulphate) respectively. The separated proteins were transferred to 0.45tL nitrocellulose filters in accordance with the instructions from the manufacturer (Bio-Rad). The filter was immunostained in the same way as for screening the C2 gene library except that alkaline phosphate conjugated anti-rabbit lgG was used in place of 12Slconjugated anti-rabbit IgG. 3.L Purificatton of CODH from C2 CODH was purified from C2 using the tnethod described by Bell et at. [18]. ¢i Production o( anlisera Anti-CODll serum purified from C2 was raised in rabbits following multiple intraaermal injections (100 #1) of antigen. Injections were repeated fortnightly for two occasions. The antigen (1 mg/ml) was diluted 1 : 2 with phosphate buffered saline. This was hom~,gciiised, by sonicati,~;~, with an equal volume of complete Freund's adjuvant. This mixture was further diluted 1:1 with 2% (v/v) Tween 80 for ease of inoculation. Nine "days after the final vaccinatkm, rabbits were bled (30 ml), Bleeds were repeat,:d after 18-day periods (25 ml). The blood was allowed to clot overnight at 4 ° C and the serum was removed and frozen.

252

3. 7. D/CA manipulation procedures Standard methods were used for Southern blotting, nick translation, phage and plasmid DNA isolations, gel electrophoresis, subcloning, restriction digests and fragment analysis [10]. 3.8. Enzyme assay The C2 crude cell extract, the crude cell extracts of E. coli cells harbouring recombinant plasmids and the recombinant phage lysates were assayed for CODH activity by the method of Lyons et al. [5].

4. RESULTS

4.1. Screening o/clones from the Pseudomonas thermocarboxydooorans C2 gene library Forty-two plaques of the 34000 plaques plated out from the amplified C2 Sau3A partial gene library in MAT.1 cross-reacted with the antiCODH serum. Twenty of the plaques which reacted most strongly with the antiserum were analysed in more detail. Western blots of 15 of the clones (P1, P9, P13, P15, P17, P20, P21, P22, P23,

:7 8 91011121314

t

Fig. 1. Western blot of proteins ©xpres:~cdby some et the clones isolated from the Pseudomonasthermocarboxyd~,,ra~ C2 gen¢library. The proteins were separated on a 105 SDS polyacrylamid¢gel. Lanes 7 and 14: crude cell extract of C2; lanes 1-6: P'22,P'23,P24, P250P28 and P32,respectively;lanes 8-11: P'27, P38, P39 and P40, respectively;lane 12: ~IATA; lane I3: crudec¢11extractof Escherichiaco,[ LE392.

P24, P26, P32, P38, P39 and P40) revealed a large protein which co-migrated with and had the same immunological reaction as the large subunit (LS) of CODH from C2 (Fig. 1). P21 and F37 express a slightly smaller protein which reacted strongly with the antiserum (Fig. 1). The slightly smaller protein is probably a truncated version of the large protein encoded by a clone containing the incomplete coding region for the LS of CODH. The faint bands seen in all the lanes containing proteins expressed by the clones are believed to be due to non-specific interactions with A47.1 and Escheriehia coli LE395. The antiserum reacted with several proteins in the C2 crude cell extract. These proteins are presumably non-specific degradation products of CODH.

4.2. Subcloning the CODH encoding region DNA was isolated from two clones (P22 and P40) picked randomly from the 15 clones shown to be expressing a protein corresponding to the LS of CODH. Preliminary restriction a~.alysis of these clones revealed a region of homology of between 3 kb and 5.9 kb. Since clones P22 and P40 both express the LS of CODH this prote~n must be encoded by the DNA homologous to both clones. To allow further analysis, an internal 7.2kb Sail from P40 was subcloned into pBR322. Western blots of this subclone, designated pCL2, revealed a protein corresponding to the LS of CODH (data not shown). Thus pCL2 was used to pinpoint the DNA encoding the LS of CODH in clone P22. Subclone pCL2 cross-hybridized with a 4 kb EcoRI fragment from P22. This fragment was subcloned into pUC9 to give pGB1. The EcoRl fragment from pGB1 contains only C2 DNA. Subclone pGB1 cross-hybridized with a 2.7 kb Sphl fragment from pCL2. This fragment was subcloned into pUC18 to give pGB2. The Sphl fragment from pGB2 contains 89 bp of pBR322 DNA and the remainder is C2 DNA. A detailed restriction map of P22 and P40 was constructed which shows the region of homology between the two clones and the location of the subcloned fragments (Fig. 2). As expected pGB1 and pGB2 cross-hybridized with an EcoRI digest of C2 DNA to produce a single strong band on autoradiography. However cross-hybridization of an EcoRI

253

digest of C2 DNA with pCL2 produced a multiple banding pattern on autoradiography, suggesting that the DNA subcloned into pCL2 is noncontiguous. This would explain why the region of homology between clones P22 and P40 is confined to between 3 kb and 5.9 kb.

4.3. Expres:ion of all three subunits of CODH from C2 by pGBI Western blotting experiments showed that pGB1 expresses 3 proteins, corresponding to the LS, medium subunit (MS) and small subnnit (SS) of CODH (Fig. 3). Unlike CODH produced by C2, the subunits expressed by pGB1 are not pre:~ent in equimolar amounts. In pGBI the I_.S was expressed at a much higher level than the MS and SS of CODH. In pGB2 only the LS and MS were expressed (Fig. 2), and again the LS was expressed at a much higher level than the MS. There is extra insert DNA from pGB1 to the left and right of the insert DNA from pGB2 (Fig. 2). Some of this extra D N A presumably contains coding sequence require.:l for the SS of CODH. The MS and SS were not detected in the TCA precipitations of the proteins expressed by the recombinant phage clones. This may be accounted for by a combination of lower concentrations of nrotein present in TCA precipitations compared to crude cell extracts and the relatively low concentrations of the MS and SS compared to the LS.

~amtrOaONA

insertDNA i

....

;

I~11~ °

,i

P40 R

[3 J

~

I

i~

EH

I

E$ NC ."p Sp Hc

lamb~a DNA

F insertDNA

5 6 7 8

87kD--a"

~--87kD

29kD--~

4 - 29kD

21kD--4,

,1~ " "~-21kD A

B

Fig- 3. ~lJ~S-polyacrylarmde gel arid corre'~l~ .1drag Western blot of proteins expressed by pGBI and pGB2. (A) 12.5% SDS-polyacrylamide gel stained with Coomassie h~illiant blue R: lane I. crude cell extract of P. thermocarboxyak~orans C2; lane 2. purified CODH; lane 3, crude (:el' extract of pGB2; lane 4, crude cell extract of pGBI. (B) Western blot of gel: lane 5, purified CODH; lane 6, crude cell extract of C2: lane 7. crude cell extract of pGB1 ; lane 8. crude cell ext:act of pGB2.

4.4. CODH activity of clones Neither phage nor plasmid clones showed CODH activity when tested using the artificial electron acceptors phenazine ethosulphate and 2,6-dichlorophenolindophenol in a spectrophotometric assay.

~arnbd,1DNA

ES NC

Sc r---

Lanes 1 2 3 4

....

P22

; "--1 ft. : H ,~ L lamb~;t DNA

Fig 2. Restriction map of P22 and P40 showing location of sabcloned fragments ( I , p G B I : ~, pGB2; ~, pCL2). The

minimum re~oo of homologybetween P22 and P40 is indicated by ....... B, BamHl; C, Clal; E. EeoRI; H, Hindlll; He, Hint II; S, Soil; So, Snell; Sp, Sphl; L and R. left aim and right arm respectivelyof lambda 1.47.1 as described by Loenen and llrammar[9]; lines not drawn to t,cale are indicated by . . . . . . .

5. DISCUSSION The structural genes encoding the three subunits of CODH from Pseudomonas thermocarboxydovorans C2 are present on a single 4 kb stretch of DNA. Thus it is possible that the genes encoding the subunits are all translated from a single tranScript, that is they are part of an operon.

In the active CODH enzyme from C2 the 3 non-identical subunits are present in equimolar amounts. However. in pGB1 (a subclone containing 4 kb of C2 DNA and shown to express all 3

254 non-identical subunits of C O D H ) the LS is expressed at a higher level than the MS and SS. It may be that in Escherichia coli the LS is expressed constitutively whereas the MS and SS are only expressed at their full level in the presence of CO. To find out if the LS in C2 is expressed constitutively and the MS and SS are inducible, crude cell extracts of C2 cells grown autotrophicaliy and heterotrophically will be analysed As all 3 subunits from pGB1 are expressed to some degree, some C O D H activity would be expected, but our clones showed no C O D H activity. A possible explanation for this may be that the presence of nonequimolar amounts of subunits interferes with the assembly of an active enzyme or it may be that there is so little active enzyme (since the amount of assembled enzyme is determined by the subunit present at the lowest level) that it i~ not detectable, A third possible explanation of failure to detect C O D H activity may be that L. coli cannot supply all the biochemical factors required for C O D H activity since the enzyme is a complex protein requiring molybdenum (present as bactopterin), F A D and iron-sulphur groups [18]. If this is the case, the C O D H activity may be gained by cloning into a more biochemically versatile bacterial species. C2 itself would be an ideal host species since it obviously has the correct biochemical background. It is therefore proposed to reintroduce the cloned genes into C2 on one of the variety of broad host range plasmids already available for use with Pseudomonas species. To do this a strain of C2 carrying a mutation in the C O D H genes will be required. A number of mutants unable to grow on CO as a sole :arbon source ( a u t - mutants) have been isolated by transposon mutagenesis [6] and it is proposed to identify those mutants with inserts in ~he C O D H genes by Southern hybridization of the mutant D N A with p G B I . Once mutants have been identified which carry a mutation in one of the C O D H genes, these strains can then be used for transformation with the cloned C O D H genes.

ACKNOWLEDGEMENT Thanks go to the National Advisory Board for financial support. REFERENCES I1] Colby. J., Williams. E. and Turner, A.P.F. (19851 Trends Biotecbnol. 3, 12-27. [21 Meyer, O., Jacobitz, S. and Kruger, B. ,1986) FEMS Microbiol. Rev. 39, 161-179. [3] Williams, E., Colby. J.. Lyons, C.M. and Bell, J.M. (1986) in Biotechnology and Genetic EngineeringReviews(Russell, G.E., ed.), vol. 4, pp. 169-221. Intercept, Ponteland, Newcastle-upon-Tyne. [4] Williams, E., Colby, J., Logan, G.W. and Lyons, C.M. (1987) in Carbon Substrates in Biotechnology IStowell. J.D., Beardsmore, A.J., Keevil, C.W.. and Woodward. J.R., eds.). SGM Special Publication vol. 21, pp. 185-201. IRL Press, Oxford. 151 Lyons, C.M.. Justin, P., Colby, J. and Williams, E. (1984) J. Gen. Microbiol. 130, 1097-1105. [6] Lyons, C.M. (1987) Ph.D. Thesis, University of Newcastle-upon-Tyne. [7] Tamer, A.P.F.. Aston, W.J., Higgins, I.J., Bell, J.M.. Colby, J.. Davis, G. and Hill, H.A.O. (1984) Anal. Chim. Acta 163, 161-174. [81 Turner, A.P.F., Aston, W.J., Davis. G., Higgins. I.J., Hill, H.A.O. a,~d Colby, J. (19 ;gl i~, t,4icrobial Gas Metabolism. M,:chanistic,Metabolic and BiotechnologicalAspects (Poole, R.K. and Dow, (.S., eds.), SGM Special Publication vol. 14, pp. 161-170, Academic Press, London. [9] Loenen. W.A.M. and Brammar, W.J. (1980) Gene 20, 249-259. [10] Maniatis. T.. Fritscb, E.F. and Sambrook. J. (1982) Molecular Cloning: a Laboratory Manual, Cold Spring Harbour Laboratory, NY. I11] Narrander, J., Kempe. T. and Messing, J. (1983)Gene 26. 101-106. [121 Bolivar, F., Rodriguez, R.L.. Greene, P.J. Betlach, M.C., Heynecker, H.L., Boyer, H.W., Crossa, J.H. and Falcow. S. (1977) Gene 2, 95. [13] Viera, J. and Messing, J. 0982) Gene 19, 259--268. [14] Yanisch-Perron,C., Viera, J. and Messing, J. (1985) Gene 33, 103-119. 115] Marmur, J. (1961) J. dol. Biol. 3, 208-218. [161 Girvitz, S.C.. Bacchetti, A.J., Rainbow, A.J. and Graham, F,L. (1980) Anal. Biochem. 106. 492. [17] Scalenghe. F., Turco. E., Edstrom, J.. Pirotta. V. and Melli, M. (1981) Chromosoma 82, 205. llBI Bell. J.M., Williams, E. and Colby. J. (1985) in Microbial Gas Metabolism: Mechanistic, Metabolic and Biotechnological Aspects (Poole, R.K. and Dow, C.S., eds.). SGM Special Publication vol. 14, pp. 153-159, AcademicPress, London.