Organization of the hydrogenase gene cluster from Bradyrhizobium japonicum: sequences and analysis of five more hydrogenase-related genes

Gene, 145 (1994) 91-96 Q 1994 Elsevier Science B.V. AR rights reserved. 0378-l 119/94~SO7.~

91

GENE 07982

Organization of the hydrogenase gene cluster from Bradyrhizobium japonicum: sequences and analysis of five more hydrogenase-related genes (Hydrogen oxidation; nickel enzyme; nitrogen fixation; soybeans)

Changlin Fu and Robert J. Maier De~ffrtment qf Biology, The Johns Hopkins Uniuersity, Baltimore, MD 21218, USA

Received by A.M. Campbell: 24 November 1993; Revised/Accepted: 3 February/23 February 1994; Received at publishers: 24 March 1994

SUMMARY

Previously, the deletion of a 2.9-kb chromosomal EcoRI fragment of DNA located 2.2 kb downstream from the end of the ~r~~yr~izo~iu~ j~po~ic~~ hydrogenase structural genes caused lack of normal-sized hydrogenase (Hup) subunits and complete loss of Hup activity. It was suggested that this region encodes one or more genes required for Hup processing. Sequencing of a 3322-bp XcmI fragment of DNA covering this 2.9-kb EcoRI fragment within the hup gene cluster revealed the presence of five open reading frames (ORFs) designated hupG, hupH, hupl, hupJ and hupK, encoding polypeptides with calculated molecular masses of 15.8, 30.7, 7.6, 18.1 and 38 kDa, respectively. Based on deduced amino acid (aa) sequences, all five products of the hupGHlJK genes showed significant homology with other genes’ products in several HZ-utilizing bacteria. Of particular interest are HupG and HupI. HupG showed 70% similarity (28% identity) to the HyaE of the Escherichia coli hydrogenase-1 operon which was demonstrated to be involved in the processing of hydrogenase-1. HupI showed strong identity to rubredoxin and rubredoxin-like proteins from many other bacteria. The latter proteins contain two ‘C-X-X-C’ motifs, which may serve as iron ligands for non-heme iron proteins involved as intermediate electron carriers or in the assembly process for Fe-S (or NiFe-S) clusters.

INTRODUCTION

The energy-demanding nitrogenase reaction intrinsically produces Hz as an obligate product. At least 25% of the energy (ATP) input into nitrogenase-catalyzed reaction is wasted due to the production of HZ. Fortunately, most N,-fixing bacteria possess hydrogenase (Hup) capable of recycling nitrogenase-evolved H, via an energyconserving respiratory electron transport chain. The structural genes for B. japonicum (Bj) Hup have been seCorrespondence to: Dr. R.J. Maier, Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA. Tel. (l-410) 5168276; Fax (l-410) 516-5213; e-mail: [email protected]

B., Bradyrhizobium; Bj. B. hydrogenase(s): hup, gene encoding Hup; kb. kilobase or 1000 bp; N, A or C or G or T; nt, nucleotide(s): ORF, open reading frame; RBS, ~bosome-binding site(s): [I. denotes plasmid-carrier state. Abbreviations:

aa,

amino

acid(s);

japanicum; bp, base pair(s): Hup, hydrogen-uptake

SSDI 0378-1~19(94)00193-V

quenced (Sayavedra-Soto et al., 1988). In addition to these structural genes, at least seventeen other hup-related genes located both downstream and upstream from the structural genes have been identified (Van Soom et al., 1993; Fu et al., 1993; Fu and Maier, 1994a,b). In this study, we report the sequence of five more genes (h~pG~~J~) located downstream from the Hup operon and located between h~pC~~ (Fu and Maier, 1994a) and hypAB (Fu and Maier, 1994b). Within this region (covering hupGHIJK) is a locus that was shown to be involved in Hup post-translational processing (Fu and Maier, 1993). Based on the nt sequence, hupGHZJK and hypAB (Fu and Maier, 1994b) are organized as a transcriptional unit. A sequence of S-TGGCACN,TTGCT very closely matching the consensus cr54-type promoter was present in front of the operon. A cosmid pHD4 containing the hup structural genes and other accessory genes compiements several types of Hup- mutants.

92 EXPERIMENTAL

(Fu

AND DISCUSSION

et al., 1994). In some

primers a region

stream from the hup structural 2.9-kb

of Hup

activity.

chromosomal

located

and

fragment

deletion

of DNA

The nt and deduced fragment

located

are shown

aa sequences

from the end of the hup structural

vealed the presence

of normal-sized

hupl, hupJ and hupK encoding

complete

loss of Hup activity

was concluded required

for Hup processing. of this genetic

subunits

(Fu and Maier,

genes. For DNA sequencing, the 2.9-kb

To further

region

lyzed the entire downstream (covering

Hup

and

started

EcoRI

investigate

we sequenced

the

and ana-

region of the hup structural a 3322-bp fragment

involved

products

hupG, hupH,

of 15.8, 30.7, 7.6,

by a RBS, terminated

with

TGA and were transcribed

in the same direction

hup structural

The stop codon of each ORF

following

gene operon. the

initiation

start

codon

(ATG)

gene by one nt, with the exception

as the of the

that the last

ORF (hupK) shared five nt with the further

in Hup

re-

(Fig. 2, Table I). All ORFs

with ATG preceded

overlapped

XcmI fragment

of sequence

of five ORFs, designated

18.1 and 38 kDa, respectively

1993). It

that this region encodes one or more genes

property

sequencing

of the 3322-bp XcmI

in Fig. 2. Analysis

2.2 kb downstream lack

for

(b) Sequence analysis

of a

genes

caused

used

down-

genes is needed for proper For example,

EcoRI

cases oligodeoxynucleotide

synthesized

junctions.

(a) Cloning and sequencing Based on previous studies, expression

were

downstream

gene hJ]pA (Fig. 2; Fu and Maier, 1994b). A sequence of S-TGGCACNsTTGCT very clearly matching the consensus c?~ type promoter (Thony and Hennecke, 1989) was present in front of the hupG (Fig. 2). Apparently, hupGHIJK and hypAB are organized as one transcrip-

processing) located downstream from the hup operon (Fig. 1) was subcloned into the appropriate sites of pBluescript II KS(+) (Stratagene, La Jolla, CA, USA). Overlapping nested deletions were generated by using the ExoIII/Mung Bean deletion kit from Stratagene. Sequencing was conducted in both strands as described

tional

unit.

----_

(A) B

0.

E

00 .

E

.

BM . .

E.

Fig. 1. Genetic and physical maps of the Bj hup gene clusters. (A) Maps of plasmids pSH22, pHD4 and pJF18-4. The dashed line above pSH22 indicates the region that was involved in Hup processing (Fu and Maier, 1993). The arrows below pSH22 indicate the region of DNA (EcoRI fragment) that is not genetically linked to the rest of the DNA on pSH22 (Fu et al., 1994). Restriction sites in parentheses on pHD4 indicate from the vector. (B) Expanded region of hupGHIJK that was sequenced. ‘Al. AlwNI; B, BumHI; Bg, &/II; E, EcoRI; H. HindIII; Xc, XcmI.

sites

TABLE I of B. japonicum HupG,

Comparison B. japonicum Product

HupG HupH HupI HupJ HupK

Number of

Calculated Mol mass

aa

(kDa)

148 287 69 169 362

15.8 30.7 7.6 18.1 38.0

HupH,

HupI, HupJ

and HupK

with homologous

products

from several hydrogenase-containing

bacteriaa

A. eutrophus

A. uinelandii

E. coli

R. leguminosarum

R. capsulatus

Product

% Identity

Product

% Identity

Product

% Identity

Product

% Identity

Product

% Identity

HupG HupH HupI

48 51 70

HoxO HoxQ HoxR HoxT HoxV

36 41 51 34 41

HoxO HoxQ ORFl ORF2 0RF3

25 29 51 35 29

28 31

45 32

39 27 70 33 22

HyaE HyaF

HupJ HupK

HupG HupH HupJ (N-ter) HupJ (C-ter) HupK

“See section b or Fig. 3 legend for sources of references. Computer Group package (Madison, WI, USA).

The % identity

of the deduced

products

was based on the Gap program

of the Genetics

93 141 288 1:: 73 576 121

PLGAADEAGAIAVLLSAGDPWR~PXATDVAVVLPELIAAFGGRLRGAV AC

~m~ATGPXMCZ3ZAm

IARGDESALGQR?GVRVQ~I?VAXGBTLGLIAKIQDDSVYVDRITT lGGGApYcG

720 148

LIDRPRGQSAAVADTIVPQHRTQGVEL* Ru#%MKVG OP GE~SLIASKGSLTAAGALAXLATLDSAELARSCPNAVALLSRIADAYA qm~s~TTuxGA_ GQXADAPSQLFRLAWLWDLESKLIADVLGEGEVAGVVALPDGSLAQIQ AATCGZT~T~TACCTCGMA~~~~~TT LSVLAGIWRVRLBTDA~RliYLEIGAVPEIVKRAAADLTSADPEIGQVP ~~mBGTGUZmm%CATCAATTT EGA~lVLPVLAIIRERALAWRPGIRSQIIWFTLLP,,SPVD,,S~LEDTI Om~~%GACKCAmTp RUGPIQLVSRGYGTCRVLSTGIR~VWSVQFFNA,,DTIILDTLEVGGVP

PWD

A

PB

G

AE

V SVP Tc---

QP

P

I 8:: 10:: 117 1152 165 1296 213 1440 261 1584 287

TGGAmT mmTGAaTTTmm

P---mwAPP

BTG0ZZCAT

TVALAADEDPEDSAERLKBIIEAYVK* BupIl4

SAPB

NP

CP

GVR

QD

V

T

D

T RLE C TGGCGATCp#!CA-

V

CGAATTGC~

G

I 17:: 69

CWTVYDPADCDDVAQIAPGTPFAALPEXWRCPNCDAPXSEF"AIES*

aup.

M

T

GGm

GARQIRTPTPWEGAISWPEAIGRSGXRA,4RDLPIYNDALGVEAVGFRA ~~TCA~~~~A~TATCGAGTKACCA~UGCGAG INGTIVGIHVTPWIUNVVLPASAVAQATSGATARIRFPAGDIEFTYSE Bv~TGmuxGTBpmGAGcmpw VGQIGRIASCSLFSPWIQPADWDAIRITABAARATAEAALAZL,,LPADSEEAVRR ~TCGATCGGCGT~TBG&%53ZA~~-TT~ REPATTPIDRRYPLRGTLTBRRG'

1a7: 20::

H"plr"SL cmpmBm_w QPRSRPPLTRLPAGXPAASLLPVLPRLFSLCSVAEQVAFLSAVEAAQG AGGKYiQXXX%CMEWTGGTCp QEAAPATVRBRLTVVVABRLTGiALLRGLPVGRRALDGTSAAA= Gppl+ZwTCAGGACpmp SALLGGPSBAVPQALRRDAVAQIRTAVGTLGISEDBALASGSALAASV -TGCGA~G~CGCGGCTAACGA-TAmm EGCDGRLVSRPLAEPSPLTAANDLDIVARLLADGAAYSDAPDLCGQIP AuccoocoTcpouEAcccwy~~mGoxAcx~~~GcGGA~ BTGVWARRABR~EISSTAAGPAARLRARIAEVAQLCTWLDHGDADLER GTA~A~~~TA~~TCGCAmTKC GIVASYRLGAGXGAAAVECARGRLYEAVVLDDEDRIVNI+SFLAPTEWN CTCGCCQXBCAGGA~YCC~ PHARGPLVQSLXGATLAAGRPGQDAVRALVGSFDPCVGPSLDFREAGR

SlRNE

ID

I

TVWL

SGAT

I

ADVA

I

CTATYCCGABBTKC%

T YmTcGc_GAGcccGacATG wFA

CA-TGG A l El E H-

Fig. 2. The nt sequences of the Bj hupGHIJK genes and deduced aa sequences. Potential RBS and consensus ~9” -type promoter Asterisks indicate stop codons. The sequence data reported in this paper have been submitted to the GenBank under the accession

The deduced

products

of the hupGHIJK

21:: 146 2304 169

TCGClXMXKGCGATCC

showed con-

siderable identity to some genes’ products from several hydrogenase-containing bacteria, such as Rhizobium leguminosarum (Rey et al., 1992), Rhodobacter capsulatus (Colbeau et al., 1993), Alcaligenes eutrophus (Kortltike et al., 1992), Azotobacter oinelandii (Menon et al., 1992; Chen and Mortenson, 1992a,b), and Escherichia coli (Menon et al., 1990b; see Table I). HupG showed 28% identity to HyaE of the hydrogenase-1 operon of E. coli (Menon et al., 1990b), which was shown to be involved in the processing of hydrogenase 1 (Menon et al., 1991). A processing function is also likely for Bj HupG (although not proved), since chromosomal deletion of the 2.9-kb EcoRI fragment covering hupGHIJK genes previously showed an effect on the Bj Hup processing (Fu and Maier, 1993). However, it cannot be ruled out that the hupG together with other genes are involved in Hup processing. HupI, in addition to the homology of the products listed in Table I, also showed 40-70% identity to rubredoxin or rubredoxin-like proteins from other bacteria (see Rey et al., 1992; Kortltike et al., 1992). All these proteins contain two ‘C-X-X-C’ clusters arranged as a ‘C-X-X-C-X,,_,,-C-P-X-C’. These conserved Cys could serve as iron-ligands for non-heme iron proteins (Beinert,

n

24:: 73 2592 121 2736 169 2880 217 3024 266 3168 313 3312 361 1 3322 362 4

are underlined. No. L25760.

1990). In Pseudomonas oleovorans, rubredoxin was demonstrated to serve as an electron-carrier component of the alkane hydroxylate systems (Kok et al., 1989). Specific functions of bacterial rubredoxins in other bacteria are not known. It is possible that this protein in HIutilizing bacteria functions as an intermediate electron carrier, or in the assembly process for Fe/S or Ni/Fe/S clusters. Based on the method of Klein et al. (1985), both HupJ and HupK were classified as integral proteins, each with one transmembrane segment. Specific functions of HupJ, HupK, HupH and their homologues in those H,oxidizing bacteria are not known; nevertheless, HyaF of the E. coli hydrogenase operon was suggested to enhance nickel incorporation into hydrogenase (Menon et al., 1991). (c) Isolation of a pHD4 cosmid Due to the DNA rearrangement on pSH22 (Fu et al. 1994) we isolated a new cosmid, pHD4, from the Bj gene library USDA 110 (kindly provided by Drs. G. Stacey and J.Y. Chun), which was constructed by partial digestion of genomic DNA with Sau3A and cloned into the BamHI site of pLAFR3 (G. Stacey and J.Y. Chun, personal communication). Cosmid pHD4 (Fig. 1) complemented the previously-described mutants SR139

94

EC

Fig. 3. Schematic figure indicates

comparison

of the Bj hup gene cluster

the transcriptional

direction

in pSH22

with other homologous

(all genes listed in the figure are transcribed

genes. Boxes representing genes are not drawn proportionally been sequenced. References: B. japonicurn (Bj): Sayavedra-Soto

genes in several bacteria. in the same direction).

The arrow

Vertical

at the top of the

lines indicate

homologous

to the genes’ actual sizes. The hatched region in Bj indicates the area which has not et al. (1988) (hupSL), Fu and Maier (1994a) (hupCDF), this study (hupCHIJK), Fu

and Maier (1994b) (hypAB), Van Soom et al. (1993) (hypD’E, hoxXA);

R. leguminosarum

(RI): Hidaigo

et al. (1990) (hupSL), Hidalgo

et al. (1992)

(hupCDF). Rey et al. (1992) (hupGHIJK); Rey et al. (1993) (hypABCDE). R. capsularus (Rc): Leclerc et al. (1988) (hupSL), Richaud et al. (1991) (hupRl), Colbeau et al. (1993): Vignais and Toussaint (1994) (hupCDFGHIJK. hypABDE); A. eutrophus (Ae): Kortltike et al. (1992) (hoxKGZMLOQRTI/),

Eberz and Friedrich

(1991) (hoxA), Dernedde

et al. (1993) (hypABCDE);

A. uinelandii (Av): A.L. Menon et al. (1990a) (hoxKGZ),

A.L. Menon et al. ( 1992) (hoxMLOQ), Chen and Mortenson (1992a)(ORFl-2), Chen and Mortenson (1992b) (ORF3-8); E. co/i (EC): N.K. Menon et al. (1990b) (hyaABCDEF), Lutz et al. (1991) and Jacobi et al. (1992) (hypABCDE andfhlA). Some homologous genes from A. chroococcum (Ford et al.. 1990; Tibelius et al., 1992) are not shown.

TABLE

11

Hydrogenase

activity

after derepression

of various

strains

in the pres-

ence of 5 uM NiClza H, uptake

Bj strain

activity

(nmol HZ/h per 10’ cells) JH (wild type)

180 0

JH47 JH47[pHD4]

181

JHKm4

0 160

JHKm4[pHD4] SR (wild type)

83

SR139

2 146

SR139[PHD4] &Strains were derepressed as previously

described

for 20 h and then assayed (Fu and Maier,

for Hup activity

1993). Data are the average

of

duplicates.

(Moshiri et al., 1983), JH47 (Kim and Maier, 1990) and JHKm4 (Fu and Maier, 1993; also see Table II), indicating that pHD4 contains hup structural genes and some accessory genes required for H2 oxidation. Based on Southern blotting (data not shown), nt sequence and physiological experiments, it was concluded that pHD4 encodes a continuous fragment of DNA from the chromosome. (d) Organization of the hup gene cluster In addition to the Bj hup structural genes (hupSL), at least 17 other genes located upstream and downstream have been identified. These genes (including hupSL) are

organized as at least three (or four) different operons and all are transcribed in the same direction (Fig. 1). The operon consisting of hupNOP is involved in the incorporation of nickel into the Hup protein (Fu et al., 1994). The structural gene operon consists of five genes, hupS, hupL, hupC, hupD and hupF. The hupS and hupL encode small (33-35 kDa) and large subunits (65 kDa) of Hup (Sayavedra-Soto et al., 1988). The hupCDF (or one of those) may be involved in the stabilization or postranslational regulation of Http (Fu and Maier, 1994a). The third operon consists of at least seven genes hupGHZJK and hypAB (Fu and Maier, 1994b). Based on the previous studies (Fu and Maier, 1993), at least one of the genes (possibly hupG) in the third operon may be involved in Hup processing. The hypB encodes a protein with an extremely His-rich region (24 His residues within a 39-aa stretch) and GTP-binding domains (Fu and Maier, 1994b), probably involved in nickel-binding accumulation and mobilization into the Hup in a GTP-requiring reaction. Sequencing further downstream from hypB (approx. 2.3 kb from the hypB) by Van Soom et al. (1993) identified four additional ORFs, hypD’, hypE, hoxX and 1~0.~4 (these may be within a different operon). Comparison of the Bj hup gene cluster in pSH22 with other homologous genes from several bacteria are shown in Fig. 3. Homologs of all these accessory L?j genes have been found in most other HZ-utilizing bacteria, thus these genes must be important for bacterial H, metabolism. However, each bacterium has its unique genes and/or genetic organization with regard to H, metabolism (see

9.5

Fig. 3). For example (see Fig. 3), in R. ~eg~mi~o~ar~rn hail is present between he’d and huff whereas no hupE has been found in Bj. Also the hup operon of R. capsulatus contains three genes (hupSLC), whereas the Bj hup operon consists of five genes (hupSLCDF). To further understand roles of these genes in the molecular biology of Hz oxidation, in-frame deletions for each gene are needed.

ACKNOWLEDGEMENT

This work was supported by Department Grant number DE-FG02-89ER14011.

of Energy

NOTE ADDED IN PROOF

Additional

B. jagonicum

hydrogenase-related genes (hupCDFG) have been recently sequenced (Van Soom et al., J. Mol. Biol. 234 (1993) 508-512).

REFERENCES Beinert. H.: Recent developments in the field of iron-sulfur proteins. FASEB J. 4 (1990) 2483-2491. Chen, J.C. and Mortenson, L.E.: Two open reading frames (ORFs) identified near the hydrogenase structural genes in Azotobucter uinelandii. the first ORF may encode for a polypeptide similar to rubredoxins. Biochim. Biophys. Acta 1131 (1992a) 122-124. Chen, J.C. and Mortenson, L.E.: Identification of six open reading frames from a region of the Azotobacter oine~and~~genome likely involved in dihydrogen metabolism. Biochim. Biophys. Acta 1131 (1992b) 199-202. Colbeau, A., Richaud, P., Toussaint, B.. Caballero, F.J., Elster, C., Delphin, C., Smith, R.L., Chabert, J. and Vignais, P.M.: Organization of the genes necessary for hydrogenase expression in Rhodobacter capstrlatus. Sequence analysis and identification of two hyp regulatory mutants, Mol. Microbial. 8 (1993) 15-29. Dernedde. J., Eitinger, M. and Friedrich, B.: Analysis of a pleiotropic gene region involved information of catalytically active hydrogenases in Alcaligenes eutrophus H16. Arch. Microbial. 159 (1994) 545-553. Eberz, G. and Friedrich. B.: Three frans-acting regulatory functions control hydrogenase synthesisin Alcaligenes eutrophus. J. Bacterial. 173 (1991) 1845-1854. Ford, CM., Garg, N., Garg, R.P., Tibelius, K.H., Yates, M.G., Arp, D.J. and Seefeldt, L.C.: The identification, characterization, sequencing and mutagenesis of the genes (hupSL) encoding the small and large subunits of the Hz-uptake hydrogenase of Azotobacter chroococcum. Mol. Microbial. 4 (1990) 999-1008. Fu, C. and Maier, R.J.: A genetic region downstream from the hydrogenase structural genes of Bradyrhizobiumjaponicum that is required for hydrogenase processing. J. Bacterial. 175 (1993) 295-298. Fu, C. and Maier, R.J.: Sequence and characterization of three additional genes within the hydrogenase gene cluster of Bradyrhizobium japonicum. Gene 141 ( 1994a) 47-52. Fu, C. and Maier, R.J.: Nucleotide sequences of two hydrogenaserelated genes (hypA and hypB) from Bradyrhizobiurn ja~onicu~n, one

of which (h~~~~)encodes an extremety histidine-rich region and guanine nuc~eotide-binding domains. Biochim. Biophys. Acta 1184 (1994b) 135-138. Fu, C., Javedan, S., Moshiri, F. and Maier, R.J.: Bacterial genes involved in incorporation of nickel into a hydrogenase enzyme. Proc. Natl. Acad. Sci. USA 91 C1994) 509995103. Hidalgo, E., Leyva, A. and Ruiz-Argtieso, T.: Nucleotide sequence of the hydrogenase structural genes from Rhizobium leguminosarum. Plant Mol. Biol. 15 (1990) 367-370. Hidalgo. E.. Palacios. J.M., Murillo, J. and Ruiz-Argtteso, T.: Nucleotide sequence and characterization of four additional genes of the hydrogenase structural operon from Rh~zob~~rn~egz~m~nosarum bv. viciae. J. Bacterial. 174 (1992) 4130-4139. Jacobi, A., Rossmann, R. and Bock, A.: The hyp operon gene products are required for the maturation of catalytically active hydrogenase isoenzymes in Escherichia coli. Arch. Microbial. 158 ( 1992) 444451. Kim, H. and Maier. R.J.: Transcriptional regulation of hydrogenase synthesis by nickel in Bradyrhizobium japonicum. J. Biol. Chem. 265 (1990) 18729-18732. Klein, P., Kanehisa. M. and DeLisi, C.: The detection and classification of membrane-spanning proteins. Biochim. Biophys. Acta 815 (1985) 468-476. Kok, M., Oldenhuis, R., Van den Linden, M.P.G., Meulenberg, C.H.C., Kingma, J. and Witholt, B.: The Pseudomonas oleocorans alkBAC operon encodes two structurally related rubredoxins and an aldehyde dehydrogenase. J. Biol. Chem. 264 (1989) 5442-5451. Korthike, C., Horstmann, K., Schwartz, E., Rohde, M., Binsack, R. and Friedrich, B.: A gene complex coding for the membrane-bound hydrogenase of Akaligenes eutrophus H16. J. Bacterial. 174 (1992) 627776289. Leclerc, M., Colbeau, A., Cauvin, B. and Vignais, P.M.: Cloning and sequencing of the genes encoding the large subunits of the H, uptake hydrogenase (hup) of Rhodobacter capsulatus. Mol. Gen. Genet. 214 (1988) 977107, Erratum 215 (1989) 368. Lutz, S., Jacobi, A., Schlensog, V., Bohm, R., Sawers, G., and BKc k, A.: Molecular characterization of an operon (hyp) necessary for the activity of the three hydrogenase isoenzymes in Escherichia coli. Mol. Microbial. 5 (1991) 123-125. Menon, A.L., St&s, L.W., Robson. R.L. and Mortenson~ L.E.: Cloning, sequencing and characterization of the [NiFe] hydrogenaseencoding structural genes (hoxli: and hoxG) from Azotobacter uinelandii. Gene 96 (1990a) 67-74. Menon. N.K., Robbins, J., Peck Jr., H.D., Chatelus, C.Y., Choi, ES. and Przybyla, A.E.: Cloning and sequencing of putative Escherichia coli [NiFe] hydrogenase-1 operon containing six open reading frames. J. Bacterial. 172 (1990b) 1969-1977. Menon, N.K., Robbins, J., Wendt. J.C., Shanmugam, K.T. and Przybyla, A.E.: Mutational analysis and characterization of the Escherichia coli hya operon, which encodes [NiFe] hydrogenase 1. J. Bacterial. 173 (1991) 4851-4861. Menon, A.L., Mortenson, L.E. and Robson, R.L.: Nucleotide sequences and genetic analysis of hydrogen oxidation @OX) genes in Azotobacter uj?ze~andii.J. Bacterial. 174 (1992) 4549-4557. Moshiri, F., Stults, L., Novak, P. and Maier, R.J.: Nif- Hup- mutants of Rhizobium japonicum. J. Bacterial. 155 (1983) 9266929. Rey. L.. Hidalgo, E., Palacios, J. and Ruiz-Argtteso, T.: Nucleotide sequence and organization of an HZ-uptake gene cluster from Rhizobium leguminosanum bv. viciae containing a rubredoxin-like gene and four additional open reading frames. J. Mol. Biol. 228 (1992) 9981002. Rey, L., Murillo, J.. Hernando, Y., Hidalgo, E., Cabrera, E., Imperial, J. and Ruiz-Argtieso, T.: Molecular analysis of a microaerobically induced operon required for hydrogenase synthesis in Rhizobium ~egu~n~nosarumbiovar viciae. Mol. Microbial. 8 (1993) 471.-481.

96 Richaud,

P.. Colbeau,

and sequence

A., Toussaint,

analysis

B. and Vignais, P.M.: Identification

of hupRZ gene which

regulator of the NtrC family required in Rhodobacter capsulatus. J. Bacterial. Sayavedra-Soto, Nucleotide

L.A.. Powell, sequence

G.K..

of the

Bradyrhizobium japonicum uptake

FEMS

Microbial.

loci

H.J. and

Morris,

encoding

hydrogenase.

Sci. USA 85 (1988) 8395-8399. Thony. B. and Hennecke. H.: The - 12/-

a response

Tibelius, K.H.. Du. L., Tito. D. and Stejskal, F.: Azotobacter chroococcum hydrogenase gene cluster: nucleotide sequences and genetic analysis

for hydrogenase expression 173 (1991) 5928-5932.

Evans,

genetic

encodes

R.O.:

subunits

Proc.

of

Natl. Acad.

of four accessory

genes (hupA. hupB. hupY and hupC). Gene

127 (1993) 53-61. Van

Soom.

C.. Verreth.

C., Sampaio,

M.J.

and

Vanderleyden,

J.:

Identification of a potential transcriptional regulator of hydrogenase activity in free-living Bradyrhizobium japonicum strains. Mol. Gen. Genet. 239 (1993) 2355240.

24 promoter

Rev. 63 (1989) 341-357.

comes of age.

Vignais, P.M. and Toussaint. B.: Molecular biology of membranebound HZ uptake hydrogenases. Arch. Microbial. 161 (1994) l-10.