JOURNAL OF BACTERIOLOGY, Mar. 1992, p. 1726-1733

Vol. 174, No. 6

0021-9193/92/061726-08$02.00/0 Copyright X 1992, American Society for Microbiology

Nucleotide Sequence of dcrA, a Desulfovibrio vulganis Hildenborough Chemoreceptor Gene, and Its Expression in Escherichia coli ALAIN DOLLA,t RONGDIAN FU, MICHAEL J. BRUMLIK,4 AND GERRIT VOORDOUW* Division of Biochemistry, Department of Biological Sciences, The University of Calgary, Calgary, Alberta T2N IN4, Canada Received 22 October 1991/Accepted 3 January 1992

The amino acid sequence of DcrA (Mr = 73,000), deduced from the nucleotide sequence of the derA gene from the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, indicates a structure similar to the methyl-accepting chemotaxis proteins from Escherichia coli, including a periplasmic NH2terminal domain (Mr = 20,700) separated from the cytoplasmic COOH-terminal domain (Mr = 50,300) by a hydrophobic, membrane-spanning sequence of 20 amino acid residues. The sequence homology of DcrA and these methyl-accepting chemotaxis proteins is limited to the COOH-terminal domain. Analysis of dcrA-lacZ fusions in E. coli by Western blotting (immunoblotting) and activity measurements indicated a low-level synthesis of a membrane-bound fusion protein of the expected size (Mr = -137,000). Expression of the derA gene under the control of the Desulfovibrio cytochrome c3 gene promoter and ribosome binding site allowed the identification of both full-length DcrA and its NH2-terminal domain in E. coli maxicells.

Bacterial chemotactic signals are mediated through a set of integral membrane proteins (18). The periplasmic NH2terminal domain of these proteins interacts with attracting or repelling ligands, either directly or via periplasmic ligandbinding proteins, while their cytoplasmic COOH-terminal domain is reversibly methylated/demethylated in response to ligand binding. The two domains of these methyl-accepting chemotaxis proteins (MCPs; Mr = 58,000 to 60,000) are connected by a single hydrophobic transmembrane helix of -24 residues. These structural features have been deduced from a limited number of nucleotide sequences of genes for MCPs from members of the family Enterobacteriaceae, primarily the Escherichia coli tap, tar, tsr, and trg genes (4, 5, 14) and the Salmonella typhimurium tar gene (26). There is good experimental evidence that MCPs function more generally in motile prokaryotes in transduction of outside chemical and physical signals to the flagellar motor. Stimulus-dependent reversible methylation of 60- to 70-kDa or higher-molecular-mass integral membrane proteins has been demonstrated in Bacillus subtilis (20), Rhodospirillum rubrum (33), and a number of other eubacteria (see references in reference 21), as well as archaebacteria (1). Structural homology of MCPs of these other bacteria with those from E. coli and S. typhimurium was inferred from immunological cross-reactivity with polyclonal antibodies. However, MCPs from nonenteric bacteria have not been characterized by cloning and sequencing of their genes. The degree and sites of homology between these and the E. coli and S. typhimurium transducers are therefore unknown. In this article, we report the nucleic acid sequence of a gene that is homologous to the E. coli and S. typhimurum family of MCP genes. The gene, isolated from Desulfovibrio vulgaris Hildenbor*

ough, has been named dcrA (DcrA is Desulfovibrio chemoreceptor protein A). Desulfovibrio is a taxonomically diverse genus of motile, gram-negative, dissimilatory sulfate-reducing bacteria (11, 24), which generally require a strictly anaerobic environment, with hydrogen or organic acids (e.g., lactate) as the energy source and sulfate as the respiratory substrate. The chemotactic behavior of sulfatereducing bacteria has so far not been investigated.

MATERIALS AND METHODS Strains, vectors, and medium. The bacterial strains, plasmids, and cloning vectors used are described in Table 1. TY medium has been described before (39). Biochemical reagents. All enzymes used for cloning and dideoxy se,uencing were obtained from Pharmacia. Radiolabeled [a-3 P]dATP (3,000 Ci/mmol, 10 mCi/ml) and a-35SdATP (400 Ci/mmol, 10 mCi/ml) were from Amersham. Tran35S-Label labeling reagent (a mixture of [35S]methionine and [35S]cysteine; 1,000 Ci/mmol; 10 mCi/ml) was purchased from ICN. Immunoblot staining reagents (nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate), anti-pgalactosidase mouse MA6 monoclonal antibody, and antimouse immunoglobulin G (heavy and light chain) alkaline phosphatase-conjugated antibody were from Promega. Prestained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) standards (high range) were from Bio-Rad. All other chemicals and biochemicals were obtained from either BDH or Sigma. Subcloning and dideoxy sequencing of the dcrA gene. The dcrA gene is present immediately upstream from the rbo-rub operon (6). This region had been isolated as a set of overlapping bacteriophage X clones (35). The nucleotide sequence of the dcrA gene was determined as follows (Fig. 1B). (i) The sequence of the 2,036-bp insert of pJK15 was completely determined on both strands by shotgun dideoxy sequencing (2, 28), using M13 vectors (19). (ii) The 340-bp PstI-EcoRI fragment from pJK9 was sequenced in both orientations. (iii) The 1.1-kb insert from pJK33 was se-

Corresponding author.

t Present address: Laboratoire de Chimie Bacterienne, C.N.R.S.,

13277 Marseille Cedex 9, France. : Present address: Department of Microbiology and Infectious Diseases, The University of Calgary, Calgary, Alberta T2N 1N4,

Canada. 1726

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NUCLEOTIDE SEQUENCE OF dcrA

VOL. 174, 1992

TABLE 1. Bacterial strains and DNA vectors used in this study Genotype, comments, and reference

Strain or plasmid

NCIMB no. 8303. Isolated from clay soil near Hildenborough, Kent, United Desulfovibrio vulgaris subsp. vulgaris .......................... Kingdom (24). Source of the dcrA gene. Hildenborougha ....... A(lac-pro) supE thi hsdM hsdR recA (F' traD36 proAB+ lacZAM15I"). From Escherichia coli TG2b .................................... T. J. Gibson. This study Escherichia coli TG108c ................................... 13 Escherichia coli BD1854 ................................... 27 Escherichia coli CSR603 ................................... 34 pUC8, pUC9 ................................... 40 pUC18, pUC19 ................................... 30 pMC1871 ...................................

pJRD215 ....................................8 22 pJ800 ................................... 2.6-kb EcoRI insert in pUC8 (Fig. 1) pJK9 ................................... 2.0-kb EcoRI insert in pUC8 (Fig. 1) pJK15 ................................... 1.1-kb Sall insert in pUC8 (Fig. 1) pJK29 ................................... 1.1-kb Sall insert in pUC8 (Fig. 1) pJK33 ................................... pJ900, pAD1, pFR1, pFR2 pFR3I, pFR5,

pDcr5 ...................................

This study

pJK9AvaI, pFR3, pJRFR2

This study (Fig. 1) pDcrl, pDcr4 ................................... pAD6, pAD7, pAD8, pAD10, pADll, pAD18 ..................... This study (Fig. 3) a Abbreviated as D. vulgaris Hildenborough. b Constructed from E. coli JM101 by T. J. Gibson and M. D. Biggin at the Laboratory of Molecular Biology, MRC Centre, Cambridge, United Kingdom. c A derivative of E. coli TG2 lacking F'. A

quenced as indicated in Fig. 1 to provide the overlap between sequences i and ii. (iv) The 700-bp PstI fragment from pJK9 was digested with RsaI, and the resulting smallersized fragments were subcloned to provide the entire sequence of both strands. (v) Sequencing of the 400-bp NcoIEcoRI fragment from pJK9 provided an overlap between sequences iii and iv. Construction of dcrA-lacZ fusions and dcrA expression plasmids. Plasmid pJK9AvaI (Fig. IA) was obtained by digestion of pJK9 (5.3 kb) with AvaI, in order to delete the 1.2-kb AvaI fragment, and self-ligation of the 4.1-kb fragment containing the pUC vector. Plasmid pDcrl (Fig. 1A) was constructed by ligating the 2,036-bp EcoRI insert from pJK15 into the EcoRI site of pJK9AvaI. The 696-bp PstI fragment (Fig. 2, nucleotides [nt] 1 to 696) from pJK9AvaI was ligated into the PstI site of pUC9 to give pAD1. The 3.8-kb EcoRI fragment from pMC1871, containing the lacZ coding region, was inserted into the EcoRI site of pAD1 to obtain pAD6 (Fig. 3). The 3.8-kb HindIII-BamHI (partial) fragment from pAD6 was ligated to pUC18 to give pAD7

E

4I

A

pJK9

pJK9AvaI

H,P,S.B

H,P,S,B pDcrl

pDcr4

E

5000 Ab

s

l200 bp

N

pJKl5

E

sIf pJK33

0

E

pJK29

F

r

E EM

B a B

pFR3

E E

E E

(in(inpUC19)

E I._

3

2-

_

(in pUCl9)

(in pJRD215)

pJRFR2

TTGACACGGCATGGCGGACACAATACCATCCCGCTGGGAAATCCTrAACTTACC1TrGTGAAGGAGGTAGTTCGATCCA3O

(Fig. 3).

Plasmid pJ800, containing the D. vulgaris Hildenborough cyc gene (22, 37), was digested with EcoRI. Following end repair with Klenow polymerase and deoxynucleotide triphosphates (dNTPs), BamHI linkers (pCCGGATCCGG) were inserted by ligation. Plasmid pJ900, with an additional BamHI site, was selected by restriction digestion. The 657-bp BamHI insert from pJ900 containing the cyc gene was isolated and digested with Sau3A, generating a 113-bp BamHI-Sau3A fragment containing the cyc promoter and ribosome binding site. Plasmid pFR1 was made by subcloning the 856-bp KpnIEcoRI fragment of pJK9AvaI, containing the 5' end of dcrA (Fig. 2, nt 176 to 1031), in pUC19. Plasmid pFR2 was constructed by digestion of pFR1 with BamHI and KpnI, end repair with T4 DNA polymerase and dNTPs, and religation. This procedure replaced the KjpnI site at nt 176 with a BamHI site. Ligation of the 113-bp BamHI-Sau3A fragment from pJ900 into this BamHI site gave pFR3 (Fig.

B

(i)

(ii)

S

(iii)

(iv)

:'4X

(v)

FIG. 1. (A) Restriction map of a region of the D. vulgaris Hildenborough genome containing the dcrA gene, as well as the rbo and rub genes. Restriction sites forAval (A), BamHI (B), EcoRI (E), KpnI (K), MluI (M), NcoI (N), PstI (P), and Sall (S) are indicated. The leftmost AvaI site (A) of pJK9 is the SmaI site of the pUC8 multiple cloning cassette. The positions of the inserts of plasmids pJK9, pJK15, pJK29, pJK33, pJK9AvaI, and pDcrl (Table 1) are indicated. The expression plasmids pFR3, pDcr4, and pJRFR2 are preceded by 113 bp (hatched box, the sequence of 79 nt is shown) containing the D. vulgaris cyc promoter (L>) and ribosome binding site (underlined in the sequence). (B) Strategy for dideoxy sequencing of the dcrA gene.

1728

DOLLA ET AL.

J. BACTERIOL.

CTGiCAGACTCTTCATGCGGGCACATTGTACGCGRACCCAATCGTAATACACCGGAGATGGTTGATAAGTTATCCGATTGCACATAGGGTCGTTACGGATTCCGGCGG(;CGACCTGGGC 10

50

40

30

20

60

80

70 6 S

T Q R S

90

L

G

M

L

120

110

100

T K V

L

V

S

G

I S

L

GGAATCGCCAACGGCACACCAGTCC2A3CC02GAGGTACCATGTCGACGCAACGCTCCTCGGCACCAAGGTCATGCTCCTTGTGAGCGGCATCTCGCT 0TCCATCCC GG3CAT1CG0 240 220 230 200 210 190 160 170 180 140 150 130 R Q S A L T L I D S G A V R A S E L L L D A I A D P F V Y L I L a V CGGCAATCCGCCCTGACCCTCA:ATCGACAGCGGTGCCGTGCGCGCCTCTGAACTGCTGCTTGATGCCATCGCCGACCC =CACCTCGTACTGGCAGC0 TiTTCGTCTACCTCATCCTCGCCGTAC C 350 360 320 330 340 280 310 300 290 270 260 250 N

S

K G

N

D

T

A

G

I

A

R

R

Y

A

D

I

R A Y

M

T

D

F

R

G

N

I

T

Y

S

T

S K

QiATGTCCAAGGGCAACGACACAGGTA'Al4CGACCGAGAAGTTCGACGGacATCGCCAGACGTTACGcCC ,GACATTCGCGCGTACATGACGGACTTCAGGGGCAACATCACCTACTCCACCTCAAA 360

370

420

410

400

390

430

440

450

460

470

480

R E E Q L A T I D G M A D V L R R D L A V G L Y V G A L L D R F N LCCATCGACGGCATGGC GCCCTGCTCGACAAGTTCAAT4 rG;;CCGCGCTCAAGACCGACAGOC'CGCGhAGAGCAGCTTGCC):AC GACGTGGTGCAGGCACCT7 .IG GCGATGTGCTGCGGCGCGACCTCGCTGr. 560 550 600 590 580 520 570 540 510 530 500 490

PR I L GT L V M L Q D V T P A M S E F Y A T I R S I D Y E V F F H H C H G R S LCOCCTGCCATGAGTGA GTCATGCTGCAGGACGTC AC :AGCOCATCCTCGGTACGCTOCx CACCACTGCCATGGACGCAGC xi GiTrCTACGCCACCATCCGGTCCATACLCCCCAACGCACCAGAATGTI r670 680 700 690 720 710 660 640 630 650 620 610

L

R

L

H

Q

Y

E

G

T

L

L

L

L

V

R

G

A

V

I

S

R V

R

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A

A

GTGGGGCTTTCCGTGGGOC )GGCGTCTGGGC IGI.TGCTGTCGCTCTTCATGCGA(kcCGCGCCGTCATCTCGCGGI ;G,TACAGCGCATCGCCGC .G

G

760

750

740

730

780

770

830

820

810

790

840

DVQ

L A S M V T T I R D Q M Q G F M Q Q G G E H D G N D E ;CTCGCCAGCATGGTCACG ;AC CCATACGCGACCAGAT r-AGCATGACGGCAACGACGAA'kC'TCACACGGCTCAACGCCAAGASC GAiACCAGCGTGGCTTAO x CGTAAGCGGhACAG&TTGAACAGGGO m V

S

G Q

I

E

660

850

900

890

600

070

910

R A V V D A R N Q Y N R S V L E EG V LI PV LP L 'GCCGTGGTCGACGCCAGAAC CC CGIATCATCGCCCGCTT rG 0CAGTACAACCGCTCGGTGCTCGAAU kG 1000 990 1020 1010 980 970

G.CATCGAATTCATCAACCAG 1030

930

920

1040

940

960

950

P M C T I V S T A C H E XCCCATGTGCACCATCGTC:A GCACAGCCTGCCATGA Gc 1080 1070 1050 1060

I S Y R R D D N G V E E D I T A Y S G K D F S A AACCAGCAGGGCAACATC :mGCTACCGCCGCGACGA TTCATGCAACAGGGCGGC DC. GTCGAGGAGGACATCACCGCX CA4RCCCTCCTGAAAGGcGGGAACci AkTACTCGGGCAAGGATTTTTCCGCG I-1 1160 1150 1200 1190 1180 1170 1120 1110 1140 1130 1090 1100 V A T A S F D D R G A V N G A IAV I I D L T Q E E E A R R R I G T V F P L H Y GGCACCGTCTTCCCCCTGCACTACCGAGGTCTCGCCGCTGGCO GATGACAGGGGGGCCGTGAACC ;GCGCCATCGCCGTCATCATOCriACCTGACGCAAGAGGAGC ;AGGCGCGGCGCCGCAT 1280 1270 1320 1300 1230 1310 1290 1260 1250 1210 1220 E T N T V R 1240 V T R M G E L E R D A T T G V A E T L E E Q R I N L L GAG GACAGCGCATCAACCTGTTGCCGTGTCGCCGACGAGGTGIACGGGCGTGGCCGAGACGCTG( 'TGCG;TGCCGCCGAGGCTTTGCXiTCACACGCATGGGCGAAC TCSGAGCGGACGCCAC 1400 1390 1360 1350 1440 1420 1430 1410 1380 1370 1330 1340 A M Q T N L E

A

A

S

E

T

T

E

Q

M

N

V

T

V

S

T

T

A

E

M

A

D

C

Q

W

R

A

GAGGCAGCCTCCGAGACCACGCAGC TGGCTACCGCCATGGAGT IGAGATGAACGTCACCGTCACC ;AGGTGGCACGCAACGCCTCCJAGCACCGCCGAGATGGCC ;ACTGCCAATGGCGAGC 1450

1460

1470

V

A

T

R

1480

E

V

1490

1500

1510

1520

1530

1540

1550

1560

H E L A R R A D N I G R R Q V A Q R T Q S G G T E N A Sx oCATGAACTGGCACGCAGG MGCGGACAACQTCGGGCG C'CAGTCGGGCGGTACGGAGATGGCC :A IACACGTGTGCGGAGACG X CGACAGGTGGCOCAGCGCACC- :G A8GACCTCGCCGRATCGCTG( 1640 1630 1600 1590 1680 1610 1620 1650 1660 1670 1570 1580 C T GTCG

V A

L

SG

A G R G F A L V A D E V L A L N A A I I E V I N E I TTCGACCAGACCAACCTGCXiCTGGCACTCAACGCCGCCATC-Gi;AGGCCGCCCGTGCAGGAGAT(rc!GCCGGACGCGGCTTOGCG X-TGGTGGCCGACGAGGT CMTCATCGAGGTCATCAACGAAATC x 1750 1760 1710 1720 1740 1770 1780 1800 1730 1700 1790 1690 T R I N D L V

V E V M T E T R QQV E E Q A I A A I R K L A E T M GTGGCCACACGCGAAGTG X GTCGAGGTCATGACCGAG NU Ct0CGAAACTGGCCGAGAAGACCATGi ;G kCCCGGCAGCAGGTCGA rGAACAGGCCATOCGGCCATC _C:AGCAGGGCAGCAACGACGCC(N. 1030 1880 1870 1840 1890 1900 1810 1920 1910 1860 1850 1820

M I A T A S E Q Q S S T G I V G R A E V T A G K A E D AACATCGCCACCGCGTMG NEG;AACAGCAGTCGTCCAC rGGCATCGTGGGCAGGGCCGAA kAkGCATCGCCGACATGGTGCGau ACCGGCAAGGTTCTCTCG(x AGTGACGGCAGGCAAGGCCGAGG&C:A 2000 1990 1960 1950 2010 2020 1970 1980 2030 2040 1940 1930 I R Q V E G M A Q R L E T E A S R D G S D E I N R N V :ACCCXGATCAACGACCTCuACCGAAGCATCCAGAGACGGGGIBrGCGGGAAGCAGGCGATGcCC ciATCCGGChGGTCGAGGGC CAIkTGGCCCAGAGGCTTGA C:AGCGACGAAATACACCGCAACGTC :A 2120 2110 2070 2080 2130 2140 2150 2160 2090 2100 2060 2050 A L V D G F R K CGACGCGTGCCCCTTTCGCA24TCVCACGGGCCCGGFTTTGC0 SCCGGGCGATGAICGGCCA G3CTCGTCGACGGTTTCCGCA hiTAACCGGTCCACAGGxCC PLCCGACAGCACAAGGGGCACGCTKa 2240 2230 2200 2190 2250 2260 2270 2280 2210 2220 2170 2180

GCGAGACATACCATCGCT lkiTGAGAGGGCATACTGTGCCTGGA6AAAGTGAATCACCCCGGACAA(LCATCTTTCCGCTCATGGGNGCCTGGACAAGCCCAGATT ITGGCAACGCCCCCGTGACGTGACACcc 2350 2360 2320 2310 2340 2370 2380 2330 2390 2400 2300 2290 iCGGTCATTGACAGACCOGCCCcGACATGCTGTACACAGGTARZTAAACAGGTATTTCOGGCGGCC(CATTCCCGCCTTTCAGAC,CcCACCGACAACGAACATG AGGATTTACTCGTAAAAAATCACGCcc 2410

2420

2430

2440

2450

2460

2470

2480

2490

2500

2510

2520

GAGGCCCCATG 2530

FIG. 2. Nucleotide sequence of the dcrA gene. The sequence was determined as outlined in Fig. 1B. The dcrA coding region (nt 182 to 2185) has been translated. Restriction sites mentioned in the text are present in the following positions: EcoRI, nt 1031; KpnI, nt 176; MluI, nt 2227; NcoI, nt 652; PstI, nt 1 and 692; Sall, nt 184, 1010, and 2168. A ribosome binding site is found at nt 173 to 177. A possible promoter sequence (nt 61 to 66 and nt 84 to 89) and transcription terminator (nt 2213 to 2239) are indicated. The translational start of the rbo gene is at nt 2529.

1A). Expression of dcrA in pFR3 is from the cyc promoter and ribosome binding site. Plasmid pFR3 was partially digested with PstI and treated with T4 DNA polymerase to delete one of two PstI sites. Self-ligation of the resulting blunt-ended fragment yielded plasmid pFR3I. The 3.1-kb PstI fragment from pAD6 was then ligated into the unique

PstI site of pFR3I to give pAD8 (Fig. 3). The insert of pAD8 was turned around by digestion with both HindIII (complete) and PstI (partial) and ligation of the 3.8-kb HindIII-PstI fragment into pUC18 to give pAD18 (Fig. 3). For construction of pAD11 (Fig. 3), the 5.9-kb Sall fragment of pAD7, which only retains nt 1 to 188 of the dcrA sequence, was end

NUCLEOTIDE SEQUENCE OF dcrA

VOL. 174, 1992

sBSmc XHPJ jPd

1729

p I

Scale * 500 bp

pAD6

FK, S

C

(u9

E

ERV

B,Sm,E,Sm, B,S,PP,

puC18)

E,Ss,K,Sm,B pAD7

laacZcl Ei ERV

E

C

H,Sp,S,

P,S,B,Sm,

Xb,B,E

ELSm,, (pUC 1) 9

I~'

jr'

pAD8 C

S

E

ERV

~~~~~~~B S,Sp,H B,S,P Xb,S,P,Sm,B |Xb,S PS,B| (pUC LII \\\\\\\\\\\F+\dcrA 3acZ I~~~~~~qrIP13 E B Xb,

Sm,E,Sm,

,E,SS,K,SM,B,

pAD 1 8

I

ERV

H,Sp,P,S,Xb,B I

--

C

BS ,S,E

I

kXXXXXXXXXNNXNNXNI lacz KNNNNXNXNXXXIZI (PUC 19)

IL pAD10

[E]

C

ERV

B,Sm,E,

pADI 1

F-*"

18)

I Sm,B,K

E,Ss,K,Sm,B I

8

1puc) pU

IE

'

ERV

C

FIG. 3. Plasmids carrying dcrA-lacZ gene fusions. The dcrA and lacZ coding regions (hatched boxes), the positions of the dcrA, lacZ, and promoters, and the directions of transcription and translation (arrows) as well as the sites for restriction enzymes BamHI (B), ClaI (C), EcoRI (E), EcoRV (ERV), HindlIl (H), KpnI (K), PstI (P), Sall (S), SmaI (Sm), SphI (Sp), SstI (Ss), and XbaI (Xb) are indicated. cyc

repaired and ligated. Plasmid pAD10 (Fig. 3) was obtained by subcloning the 3.1-kb BamHI fragment from pAD6 into pUC19 (Fig. 3). Plasmid pFR5 was made by BamHI digestion of pFR1, end repair with Klenow DNA polymerase and dNTPs, and self-ligation. This procedure fuses the reading frame for the lacZ a-peptide to that of DcrA and adds 17 amino acids to the NH2 terminus of DcrA. Digestion of plasmid pFR5 with EcoRI and ligation with the 2,036-bp EcoRI insert from pJK15 gave pDcr5, which expresses the entire dcrA reading frame (but with a modified NH2 terminus as indicated for pFR5) from the lac promoter and ribosome binding site. Ligation of the 647-bp PstI fragment from pFR3, which expresses dcrA from the cyc promoter and ribosome binding site, using the dcrA start codon at nt 182 (Fig. 2), into the PstI site of pDcr5 gave pDcr4 (Fig. 1A). Finally, plasmid pJRFR2 (Fig. 1A) was obtained by ligating a gel-isolated 2,164-bp BamHI-MluI fragment from pDcr4 into broad-hostrange vector pJRD215. Activity measurements and Western blotting (immunoblotting). Specific activities of p-galactosidase for analysis of dcrA-lacZ fusions were essentially determined and calculated by the method of Silhavy et al. (31), using o-nitrophenyl-o-D-galactopyranoside as the substrate. E. coli TG108, transformed with the appropriate plasmid, was grown in TY medium containing 100 p,g of ampicillin ml-' to an optical density at 600 nm of -1. SDS-PAGE was performed by the method of Laemmli (15), using 6% (wt/vol) polyacrylamide

running gels. Western blots were done as described by Pollock et al. (22) except that anti-p-galactosidase monoclonal antibodies from Promega were used as the primary antibodies. Membrane and soluble fractions were prepared from 200-ml cultures of E. coli TG108(pAD6) as described by Burrows et al. (7). The membrane pellet (50 mg) was resuspended in 2.5 ml of electrophoresis loading buffer containing 8 M urea by the method of Siu et al. (32). Expression of dcrA in E. coli maxicells. Expression of derA in E. coli maxicell strain CSR603 was done essentially as described elsewhere (27, 29), using Tran35S-Label to label plasmid-encoded gene products and cycloserine to kill cells that survived UV irradiation. Samples were electrophoresed on 10% (wt/vol) SDS-PAGE slab gels, dried, and autoradiographed for 2 days. Nucleotide sequence accession number. The nucleotide sequence reported here has been submitted to GenBank and has been given accession number M81168. RESULTS Nucleotide sequence of dcrA. The nucleotide sequence of the dcrA gene, shown in Fig. 2, covers a region of 2,531 nt from the leftmost PstI site of fragment iv (Fig. 1B) to the initiation codon of the rbo gene (6). The suggested dcrA coding region (nt 182 to 2185) encodes a 72,994-Da protein. Codon usage in the 668 derA codons is similar to that in other D. vulgaris genes and reflects the high G+C content of D.

J. BACTrERIOL.

DOLLA ET AL.

1730

(8)

1 ALMFNRIRISTTLFLI ..... LILCGILQIGSNGMSFWAFRDDLQRLNQV 45

1 46 43 94 92

. .MSTQRSLGTKVMLLVSGISLFVYLILAV ... LTSYW ... QRQSALTLI EQSNQQRAALAQTRAVMLQASTALNKAGT..LTALSYPADDIKTLMTTAR D. SGAVRASELLLDAIADPMSKGNDTGTTEKFDAIARRYADIRAYMTDFR ASLTQSTTLFKSFMAMTAGNEHVRGLQKETEKSFARWHNDLEHQATWLES GNITYSTSK . DVLRRDLADVVQAPAL ....... LDKFNAALKTDS ... RE

42 93 91 143 130

144 NQLSDF .... LTAPVQGSQNAFDVN ........ FEAWQLEINHVLEAASA 181 :11..: :I1.:.: .11:.: : ::.1 .1 1.

131 EQLATIDGMAFYATIRSIPNAPECHHCHGRSQPILGTLVMLQDVTPAMSE 180 182 QSQRNYQISALVFISMIIVAAIYISSALWWTRK ...

218

(M)

181 LRLHQYETVGLSVGGLLLLVGVL ... .SLFM. RRAVISRVQRIAAVSGQIE 226 218 . ... 218 227 QGDYSVSFEHDGNDELTRLNAKLASMVITIRDQMQYNRSVLEGIIVPLAV 276 219

.

MIVQPLAIIGS .... HFDS .IAA 232 :

III: . .I .I

.:.::

:

277 VDARNRIEFINQPMCTIVSTACHEYSGKDFSAFMQQGGVEEDITATVLKG 326 233 GNLARPIAVYGRNEITAI ..... FASLKTMQQALRGTVS . DV 268 11 ... I1:1. ..1..1.:..

327 GNNQQGNISYRRDDGTVFPLHYEVSPLRDDRGAVNGAIAVIIDLTQEEEA 376 269 RKGSQEMHI ........

GIAE .

IVAGNNDLSSRTEQQAASL 300

I : : : II:I1 :1I. . :I .. ..:.I.. 377 RRRIEEQRINLLRVADEVTGVAETLVRAAEALVTRMGELERDATEAASET 426

301

(xi)

AQTAASMEQLTATVGQNADNARQASELAKNAATTAQGGGVQVS . 343 11:11.::. .1.1..1::..11. 11. ..1:1 :.. TEVARNASSTAEMA. DCQWRAQSGGTEKANTVRETR 475

427 TQVATAMEEWM 344.

TMTHTMQEIATSSQKIGDIISVIDGIAFQTNILALNAAVEAAR 386

(C)

476 QVAQRTEDLAESLHELARRADNIGRVIEVINEIADQTNLLALNAAIEAAR 525

387 AGEQGRGFAVVAGEVRNLASRSAQAAKEIK .......

526 423 576 473

....... GLIEES

AGDAGRGFALVADEVRKLAEKTMVATREVEQAIAAIQQGSNDAVEVMTET VNRVQQGSKLVNNAAATMIDIVSSVTRVNDIMGEIASASEEQQRGIEQVA RQQVEVTAGKAEDTGKVLSGIVGRAESIADMVRNIATASEQQSSTSDEIN QAVSQMDQVTQQNASLVEEAAVATEQLANQADRLSSRVAVFTLEEHEVAR

422

575 472 625 522

(R1)

626 RNVTRINDLTEASRDGCGKQAMPIRQVEGMAQRLEALVDGFRK ....... 668

523 HESVQLTNCASGILK 668 ...............

FIG. 4. Amino acid sequence comparison of Tap from E. coli (14) (upper sequence) with DcrA (lower sequence). The comparison was made by using the program GAP of the University of Wisconsin genetics computer group (10). Tap sequence elements to which a specific function has been assigned-the signal peptide (S, residues 9 to 34), the membrane-spanning region (M, residues 189 to 211), the methylated regions (Kl, residues 293 to 325; Rl, residues 480 to 505), and the conserved region (C, residues 359 to 405)-have been overlined.

5

vulgaris DNA (36). The initiating methionine of the reading frame is separated by only 4 nt from the ribosome binding site (GGAGG, nt 173 to 177), which may not be optimal for efficient translation (9). A possible promoter has been indicated, as well as a potential hairpin-forming structure immediately downstream from the gene (nt 2213 to 2239). Northern (RNA) blotting and Si nuclease protection experiments have previously shown that the rbo-rub operon is transcribed as an mRNA of 680 nt, originating at approximately nt 2420 (6). The sequence in Fig. 2 suggests that the dcrA transcription terminator is located approximately 150 nt upstream from the rbo-rub promoter. Library searching has indicated that DcrA is homologous to the MCPs of E. coli and S. typhimurium. Alignment of the amino acid sequences of DcrA and Tap from E. coli (14) is shown in Fig. 4. The homology is particularly significant in the COOH-terminal domains of both proteins. Only 19% of the residues in the NH2-terminal domains of DcrA (residues 1 to 207) and Tap (residues 1 to 212) are identical, compared with 31.5% of the residues in the COOH-terminal domains (residues 208 to 668 and 213 to 537, respectively). Expression of dcrA-lacZ fusions. Although the homology shown in Fig. 4 provides evidence for a function of the dcrA gene product in Desulfovibnio chemotaxis, expression of the gene remains to be demonstrated. Initial studies in which the minicell strain E. coli BD1854 was transformed with pDcrl (Fig. 1A) did not allow detection of the expected 35S-labeled 73-kDa polypeptide, although expression of the rbo gene (14 kDa) and the bla gene (32 and 30 kDa) present on the same plasmid could be readily demonstrated (data not shown). These results indicated that derA was expressed at a very low level in E. coli BD1854(pDcrl). We therefore resorted to the construction of dcrA-lacZ fusions to characterize the expression level of dcrA in E. coli. Plasmid pAD6 (Fig. 3) was expected to encode a hybrid protein reacting with anti-3-galactosidase antibodies. Analysis of E. coli TG108(pAD6) by SDS-PAGE and Western blotting indicated an additional immunoreactive polypeptide of Mr = 137,000 (Fig. 5A, lane 4) compared with the E. coli TG108 control (Fig. SA, lane 3). This molecular weight was in agreement with that calculated for the dcrA-lacZ gene fusion (Mr = 135,500). The hybrid protein could only be detected by immunostaining and not by Coomassie blue staining. Lower-molecular-weight forms, likely formed by proteolytic degradation of the 137-kDa hybrid protein, were also observed (Fig. 5A, lanes 4 and 6; Fig. 5C, lane 2). The hybrid protein and its derivatives are located in the membrane fraction of E. coli TG108(pAD6) (Fig. 5C, lanes 2 to 3).

6

1

2

3

Hybrid protein _ 8-galactosidase -_

Hybrid protein _* h-galactosidase

A

B

FIG. 5. Detection of DcrA-13-galactosidase hybrid protein expressed in E. coli TG108 by SDS-PAGE and Western blotting. (A) Lanes: 1, SDS-PAGE standards (myosin, 205 kDa; ,B-galactosidase, 116.5 kDa); 2, 0.3 p.g of pure 3-galactosidase; 3, untransformed E. coli TG108; 4 to 6, E. coli TG108 transformed with pAD6, pAD7, and pAD18, respectively. (B) Lanes: 1 to 2, E. coli TG108 transformed with pAD11 and pAD10, respectively (10 times diluted in the case of pAD10); 3, SDS-PAGE standards. (C) Lanes: 1, SDS-PAGE standards; 2, membrane fraction of E. coli TG108(pAD6); 3, soluble fraction of E. coli TG108(pAD6). Positions of both hybrid protein and ,B-galactosidase are indicated.

VOL. 174, 1992

The lower-molecular-weight forms, but not the 137-kDa hybrid protein, could be detected in the soluble fraction when more concentrated samples were loaded (data not shown). Expression of hybrid protein in E. coli TG108(pAD6) results from transcription from either or both the lac and putative derA promoters (Fig. 3). In E. coli TG108(pAD7), the orientation of the insert was inverted relative to the lac promoter. This abolished detectable expression of the hybrid protein (Fig. 5A, lane 5), indicating that the dcrA promoter functions poorly in E. coli. Its substitution by the stronger cyc promoter and ribosome binding site (Fig. 3, pAD18) restored hybrid protein expression (Fig. 5A, lane 6). Only low levels of enzyme activity were observed in transformants expressing the membrane-bound hybrid protein: 140, 72, and 370 nmol min-1 mg of protein-1 for E. coli TG108 transformed with pAD6, pAD7, and pAD18, respectively. Association of the fusion protein with the membrane apparently interferes with the information of a soluble, active, tetrameric 3-galactosidase. Colonies of E. coli(pAD6) and E. coli(pAD18) appeared pale blue on TY plates containing 5-bromo-4-chloro-3-indolyl-4-D-galactopyranoside, isopropyl-p-D-thiogalactoside, and ampicillin, whereas colonies of E. coli(pAD7) were white. To quantitate expression from derA control sequences, we made constructions lacking the signal peptide-containing NH2-terminal domain of DcrA in pAD10 and pAD11 (Fig. 3). Since these fusions express a ,-galactosidase that is only slightly modified at the NH2 terminus, an enzymatically active, non-membranebound product is expected. In E. coli TG108(pAD10), highlevel expression of an Mr-116,500 polypeptide from the lac promoter and ribosome binding site was evident (Fig. SB, lane 2), while in E. coli TG108(pAD11), expression of an Mr-116,500 polypeptide from the dcrA promoter and ribosome binding site could not be detected (Fig. SB, lane 1). Enzyme activity measurements indicated 1.44 x 105 and 71 nmol min-1 mg of protein-1 for these two transformants, respectively. Expression from dcrA control sequences thus appears -2,000-fold less efficient than that from lac control sequences. E. coli maxicell expression of the dcrA gene. Expression of DcrA could be demonstrated in E. coli maxicell strain CSR603 transformed with plasmids pFR3 (expressing an NH2-terminal DcrA domain-containing polypeptide) and pJRFR2 (expressing full-length DcrA). Plasmid pDcr4 also expresses full-length DcrA, but no E. coli CSR603 transformants could be obtained with this plasmid, while E. coli TG2(pDcr4) transformants grew very slowly both on TY plates and in TY liquid medium. It appears that expression of full-length DcrA is toxic to the E. coli host cell. The lower copy number of plasmid pJRFR2 (constructed in pJRD215) compared with pDcr4 (constructed in pUC19) may explain why E. coli CSR603(pJRFR2) grows sufficiently for maxicell analysis. E. coli CSR603(pJRFR2) expresses a polypeptide with an apparent molecular weight of 79,000 (Fig. 6, lane 5) that is absent from the E. coli CSR603(pJRD215) control (Fig. 6, lane 4). E. coli CSR603(pFR3) expresses a polypeptide with apparent molecular weight of 32,500 (Fig. 6, lane 3) that is lacking from the E. coli CSR603(pUC19) control (Fig. 6, lane 2). These values are close to those calculated from a translation of the sequence in Fig. 2, 73 and 32.4 kDa, respectively, and confirm the dcrA reading frame indicated in Fig. 2.

NUCLEOTIDE SEQUENCE OF dcrA 1

94-

2

1731

3 4 5

I- DcrA

6743-

N-DcrA

30-

20 -

FIG. 6. Expression of dcrA in E. coli maxicells. E. coli CSR603 was transformed with no plasmid (lane 1), pUC19 (lane 2), pFR3 (lane 3), pJRD215 (lane 4), or pJRFR2 (lane 5). Bands corresponding to DcrA (79 kDa) and an NH2-terminal domain-containing polypeptide (N-DcrA, 32.5 kDa) are indicated. The positions of molecular mass markers (in kilodaltons) are shown on the left.

DISCUSSION

Analysis and comparison of MCP sequences from E. coli and S. typhimurium led to the definition of five functionally significant regions, indicated for Tap of E. coli in Fig. 4. DcrA shares these five regions. (i) A hydrophobic sequence at the NH2 terminus of DcrA (Fig. 4, residues 7 to 33) corresponds to the signal peptide (S) of Tap. (ii) A second hydrophobic sequence in DcrA (Fig. 4, residues 188 to 207) is similarly positioned as the membrane-spanning sequence (M) of Tap. The presence of both hydrophobic sequences i and ii suggests that the NH2-terminal DcrA domain (residues 34 to 187) is periplasmic, like that of Tap. (iii) A conserved element (C), which is almost invariant in the sequences of the MCPs of the family Enterobacteriaceae, is also conserved in DcrA (Fig. 4, residues 498 to 544 of DcrA and 359 to 405 of Tap share 72% identity). It has been suggested that this conserved sequence interacts with the methylesterase (26). The DcrA sequence allows truly invariant residues to be defined, since the genus Desulfovibnio is evolutionarily distant from the more closely related E. coli and S. typhimunium. The conserved DcrA and Tap sequences are predicted to have a predominantly a-helical secondary structure (not shown). Placing the entire conserved sequence of 47 residues in a helical wheel (Fig. 7) suggests that the helix has a highly conserved, hydrophobic side (92% identity) and a less-conserved, hydrophilic side (52% identity). The strong conservation of the hydrophobic side of this helix may indicate a critical role in signal transduction by internal cooperative interactions between secondary structural elements of the protein. If methylesterase does interact with the less-conserved solvent-exposed hydrophilic side of this helix, then 12 invariant residues that are critical for this interaction are defined in Fig. 7. (iv and v) Two regions, Kl and Ri (17), which have been shown to contain methylation sites (25), show distinct homology. On the basis of the MCP sequences of enteric bacteria, MacNab (17) has defined a consensus sequence of (Glx)-Glx-(Glx)-Xxx-Ala-Pol-HphXxx, where Xxx indicates any, Pol a polar, and Hph a hydrophobic amino acid residue. The corresponding DcrA sequences do not fit this pattern, although a Glu residue and a Gln residue are present in the positions shown to be methylated in the Tap receptor. Other sequences (Glx-Glx-

1732

DOLLA ET AL.

J. BACTERIOL.

into D. desulfuricans G200, carrying genes for cytochrome C3 and a high-molecular-weight cytochrome from D. vulgaris Hildenborough, respectively, led to considerable overexpression of these cytochromes (23, 38). Experiments in which a D. desulfuricans G200(pJRFR2) exconjugant was labeled with L-[methyl-3H]methionine under conditions of protein synthesis inhibition indicated specific labeling of a 79-kDa band that was absent from a D. desulfunicans G200(pJRD215) control (12). These preliminary experiments are strong evidence that DcrA is an MCP. Overexpression of DcrA in D. desulfuricans G200 exconjugants may also help to elucidate the role of the unusual sequence CHHCH in the N-terminal domain of DcrA (Fig. 4, residues 154 to 158), which could serve as a site for covalent binding of heme, as do 18 other sequences CXXCH in the two multiheme c-type cytochromes mentioned above.

FIG. 7. Helical wheel for the conserved sequence of DcrA (Fig. 4, 47 residues, from 498 to 544). The wheel was drawn by using 3.6 residues per helical turn and repeats itself after 18 residues; e.g., position 1 contains in a radially outward direction Ile-1, Ala-19, and Ala-37 of the 47-residue sequence. DcrA residues in boldface are conserved in the Tap sequence (Fig. 4, residues 359 to 405). The diagonal line divides the wheel in a highly conserved hydrophobic side (92%, 22 of 24 residues) and a less-conserved hydrophilic side (52%, 12 of 23 residues).

Glx) are present in the COOH-terminal domain of DcrA which could function as methylation sites. The similarities in these five functionally significant regions confirm DcrA as a member of the MCP transducer family. Expression studies of dcrA-lacZ fusions in E. coli TG108 and of dcrA in the maxicell strain E. coli CSR603 confirm the reading frame indicated in Fig. 2. The membrane-bound nature of the 137-kDa fusion protein (Fig. SC) is in agreement with the structural features discussed above. The signal peptide of DcrA (residues 7 to 33) initiates secretion which is blocked by the ,-galactosidase moiety of the fusion protein (16). Partial secretion of a malE-lacZ fusion product, initiated through the signal peptide of the maltose-binding protein, severely impaired the growth of the E. coli host (3). We found, similarly, that expression of dcrA-lacZ fusions and overexpression of derA, e.g., from plasmid pDcr4, caused very slow growth of the E. coli host. The low level of expression of dcrA-lacZ fusions from derA control sequences may be caused by inefficient transcription and/or inefficient translation. The strong decrease in fusion protein expression upon insert turnaround (Fig. SA, lanes 4 and 5) indicates a low transcription efficiency as the likely cause, since the ribosome binding site is not affected in this process. The number of copies of DcrA present in a D. vulgaris Hildenborough cell could thus be too low to allow identification of DcrA in D. vulgaris Hildenborough by labeling with L-[methyl-3H]methionine in the absence of protein synthesis, a technique that is very commonly used for detection of MCPs (1, 20, 21, 33). However, the demonstration (Fig. 6, lane 5) of overexpression of derA from cyc control sequences in E. coli CSR603(pJRFR2) offers a solution to this problem. The pJRD215 vector used in the construction of pJRFR2 has been successfully transferred by conjugation from E. coli to Desulfovibno desulfuricans G200 (38). Introduction of plasmids pJRDC800-1 and pBPHMC-1

ACKNOWLEDGMENTS This work was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada to G.V. A.D. was the recipient of an EMBO postdoctoral fellowship. Harm Deckers is thanked for constructing plasmid pJ900. REFERENCES 1. Alam, M., and G. L. Hazelbauer. 1991. Structural features of methyl-accepting taxis proteins conserved between archaebacteria and eubacteria revealed by antigenic cross-reaction. J. Bacteriol. 173:5837-5842. 2. Bankier, A. T., and B. G. Barrell. 1983. Shotgun DNA sequencing, p. 1-34. In R. A. Flavell (ed.), Techniques in the life sciences, B5. Nucleic acid biochemistry, B508. Elsevier Scientific Publishers Ireland, Ltd., Shannon, Ireland. 3. Bassford, P. J., T. J. Silhavy, and J. R. Beckwith. 1979. Use of gene fusion to study secretion of maltose-binding protein into Eschenichia coli. J. Bacteriol. 139:19-31. 4. Bollinger, J., C. Park, S. Harayama, and G. L. Hazelbauer. 1984. Structure of the Trg protein: homologies with and differences from other sensory transducers of Escherichia coli. Proc. Natl. Acad. Sci. USA 81:3287-3291. 5. Boyd, A., K. Kendall, and M. I. Simon. 1983. Sensory transducers of E. coli are encoded by homologous genes. Nature (London) 301:623-625. 6. Brumlik, M. J., and G. Voordouw. 1989. Analysis of the transcriptional unit encoding the genes for rubredoxin (rub) and a putative rubredoxin oxidoreductase (rbo) in Desulfovibrio vulgaris Hildenborough. J. Bacteriol. 171:4996-5004. 7. Burrows, G. G., M. E. Newcomer, and L. Hazelbauer. 1989. Purification of receptor protein Trg by exploiting a property common to chemotactic transducers of Escherichia coli. J. Biol. Chem. 264:17309-17315. 8. Davison, J., M. Heusterpreute, N. Chevalier, V. Ha-Thi, and F. Brunal. 1987. Vectors with restriction site banks. V. pJRD215, a wide-host-range cosmid vector with multiple cloning sites. Gene 51:275-280. 9. De Boer, H. A., and A. S. Hui. 1990. Sequences within ribosome binding site affecting messenger RNA translatability and method to direct ribosomes to single messenger RNA species. Methods Enzymol. 185:103-114. 10. Devereux, J., P. Haeberli, and 0. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12:387-395. 11. Devereux, R., M. Delaney, F. Widdel, and D. A. Stahl. 1989. Natural relationships among sulfate-reducing eubacteria. J. Bacteriol. 171:6689-6695. 12. Fu, R., J. Rapp-Giles, J. D. Wall, and G. Voordouw. Unpublished data. 13. Jensen, K. F., J. N. Larsen, L. Schack, and A. Sivertsen. 1984. Studies on the structure and expression of Escherichia coli pyrC, pyrD and pyrF using the cloned genes. Eur. J. Biochem. 140:343-352.

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NUCLEOTIDE SEQUENCE OF dcrA

28. 29.

30.

31.

32.

33.

34. 35. 36. 37.

38.

39.

40.

1733

for identification of plasmid-coded proteins. J. Bacteriol. 173: 692-693. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. Sankar, P., and K. T. Shanmugam. 1988. Biochemical and genetic analysis of hydrogen metabolism in Escherichia coli: the hydB gene. J. Bacteriol. 170:5433-5439. Shapira, S. K., J. Chou, F. V. Richaud, and M. J. Casadaban. 1983. New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxy-terminal portion of P-galactosidase. Gene 25:71-82. Silhavy, T. J., M. L. Berman, and L. W. Enquist. 1984. Experiments with gene fusions, p. 266-282. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Siu, C. H., R. A. Lerner, and W. F. Loomis. 1977. Rapid accumulation and disappearance of plasma membrane proteins during development of wild-type and mutant strains of Dictyostelium discoideum. J. Mol. Biol. 116:469-488. Sockett, R. E., J. P. Armitage, and M. C. W. Evans. 1987. Methylation-independent and methylation-dependent chemotaxis in Rhodobacter sphaeroides and Rhodospirillum rubrum. J. Bacteriol. 169:5808-5814. Vieira, J., and J. Messing. 1982. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259-268. Voordouw, G. 1988. Cloning of genes encoding redox proteins of known amino acid sequence from a library of the Desulfovibrio vulgaris (Hildenborough) genome. Gene 69:75-83. Voordouw, G., and S. Brenner. 1985. Nucleotide sequence of the gene encoding the hydrogenase from Desulfovibnio vulgaris (Hildenborough). Eur. J. Biochem. 148:515-520. Voordouw, G., and S. Brenner. 1986. Cloning and sequencing of the gene encoding cytochrome C3 from Desulfovibrio vulgaris (Hildenborough). Eur. J. Biochem. 159:347-351. Voordouw, G., W. B. R. Pollock, M. Bruschi, F. Guerlesquin, B. J. Rapp-Giles, and J. D. Wall. 1990. Functional expression of Desulfovibrio vulgaris Hildenborough cytochrome C3 in Desulfovibrio desulfuricans G200 after conjugational gene transfer from Escherichia coli. J. Bacteriol. 172:6122-6126. Voordouw, G., J. D. Strang, and F. R. Wilson. 1989. Organization of the genes encoding [Fe] hydrogenase in Desulfovibrio vulgaris subsp. oxamicus Monticello. J. Bacteriol. 171:38813889. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33:103-119.