The plasmid-encoded hydrogenase gene cluster in Alcaligenes eutrophus

FEMSMicrobiologyReview-87 (1990)425-430 Publishedby Elsevier

425

FEMSRE 00205

The plasmid-encoded hydrogenase gene cluster

in Alcafigenes eutrophus B~rbel Friedrich lnstitutfur Pflanzenphysiologie und Mikrobiologie, Freie UniversitiitBerlin, Berlin, F.R.G.

Key words: Hydrogenase; NAD-reducing hydrogenase; Membrane-bound hydrogenase; Hydrogenase genes; Nucleotide sequence; Hydrogenase regulation; Sigma 54 of RNA polymerase

1. SUMMARY Alcaligenes eutrophus strain H16 harbors a 450 kilobase pairs (kb) conjugative plasmid which codes for the ability of the organism to grow lithoautotrophieally on hydrogen and carbon dioxide (reviewed in [1]). The genes for hydrogen oxidation, designated kox, are clustered on plasmid priG1 in a DNA region of approximately 100-kb in size ([2], Fig. 1). The hox genes and their organization have been analyzed by isolation of Hox-deficient mutants, by complementation analysis, by cloning of hox genes, identification of hox-encoded polypeptides and, most recently, by DNA sequencing. The hox cluster is flanked by the two structural gene regions, hoxS and hoxP; it contains a regulatory locus, hoxC, and additional genes like hoxN and hoxM whose products play a role in the formation of catalytically active hydrogenase proteins. Of four indigenous 1.3-kb insertion elements, two copies of IS,191 map in the box gene cluster. These elements may be involved in rearrangements and deletions which occur particularly frequently in this region of the

Correspondence to: B. Friedrich,Institutf~r Pflanzenphysiologie und Mikrobiologie, Freie Oniversit~itBerlin, K6niginLuise-Strasse12-16,1000 Berlin33, F.R.G.

megaplasrnid (Schwartz, Kortlfike and Friedrich, unpublished).

2. S T R U C T U R A L A N D HYDROGENASE GENES

ACCESSORY

The locus hoxS codes for a heterotetrameric NAD-reducing enzyme (HoxS) that contains FMN as a cofactor and resides in the cytoplasm [3]. In addition to HoxS, d. eutrophus harbors a second heterodimeric membrane-bound hydrogenase (HoxP) which is coupled to the electron transport chain via an unknown acceptor molecule, generating ATP [4]. Both enzymes belong to the group of iron-nickel-containing hydrogenase~s [5]. The DNA sequence of the previously identified hoxS and hoxP loci [6] has been determined and the deduced amino acid sequence revealed a number of interesting features, some of which are summarized in Table 1. (i) The four open reading frames (ORFs) of hoxS are tightly linked and appear to form an operon that is transcribed starting with the genes hoxF and hoxU coding for the NADH-oxidizing moiety of HoxS. The products of the distal genes h o x Y and hoxH are predicted to represent the nickel-containinghydrogen-activatingcenter of the HoxS holoenzyme [7].

0168-6445/90/$03.50© 1990Federationof EuropeanMicrobiologicalSocieties

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Table 1 Molecular structure of hox$ and hoxP genes from .4. eurrophus Charactedstic~ =

hoxS locus

Gene designation

hoxF

hot U

box Y

hoxH

hoxK b

hoxG

Subunlt designation

a

y30

small

63 67 1806 602 17

56 55 1464 487 6

31 35 951 317 + (43 aa) 13

larse 65 69 1854 617

12

8 26 23 624 209 9

~

M r ( k D a ) - S D S gel

+

0

0

Mr (kDa)-DNA sequence No. of base pair,~ No. of amino acids (aa) Leader peptide No. of Cys residues Potential iigation of Ni FMN No. of intergenic bp

hoxP locus

26 702 233

+

9 +

-

1"7

_ 41

The biochemical data are taken from [1]. The genetic data are referenced in the text. b Mature protein. -, not present; +, present.

=

(ii) The two structural genes for the m e m b r a n e b o u n d hydrogenase hoxK and hoxG also a p p e a r to b e arranged in an operon, designated hoxP, in a similar order as reported for Bradyrhizobium japonicum [8] and Rhodobacter capsulatus [9]: the gene for the small subunit precedes the gene for the large one. T h e d e d u c e d a m i n o acid sequence for the small p o l y p e p t i d e predicts an N - t e r m i n a l leader p e p t i d e o f 43 a m i n o acids. A l i g n m e n t o f the a m i n o acid sequences o f A. eutrophus H o x K a n d H o x G a n d corresponding hydrogenase polyp e p t i d e s from B. japonicum [8] a n d R. capsulatus [9] revealed m o r e t h a n 80% homology. (iii) Despite substantial differences in catalytic properties, subunit composition, cofactor c o n t e n t a n d weak homology in the overall a m i n o acid sequence, the fl subunit o f H o x S contains two

highly conserved a m i n o acid stretches w h i c h are rich in cysteinyl residues a n d also occur in the c o r r e s p o n d i n g euhacterial a n d archaebacterial hydrogenase p o l y p e p t i d e s [7]. This conservation highlights the region as a significant structural, and potentially functional, m o t i f in h y d r o g e n a s e proteins. (iv) U n l i k e the small m e m b r a n e - b o u n d hydrogenase subunits from various sources, consisting o f an average of 300 a m i n o acids, the 6 polyp e p t i d e o f H o x S is considerably smaller (209 a m i n o acids). Nevertheless, three o f the n i n e cysteinyl residues are well conserved with respect to their N - t e r m i n a l location a n d spacings. (v) In the evolutionary c o c t e x t it is interesting to n o t e that the fl a n d ~ p r o t e i n sequences of H o x S show significant homology to the a a n d y hoxC region ' ,

,s.9, e "~oN

I~!11t!| hoxS locus

} .~ } e .~

IS491

N

[]

E','l E'.,~!B H Illl hoxP locus

i, lOkb

Fig. 1. Structure of the box gene cluster of megaplasmid priG1 ,,f A. eutrophus H16. Details are given in the text.

42"7

subunits of the methyl viologen-reducing hydrogenase (MVH) from Methanobacterium thermoautotrophicum [10]. Thu'~ HoxS appears to be closer related to MVH than t.~ HoxP. DNA sequence analysis has identified two new potential box genes, designated hoxZ and hoxM, downstream of the HoxP structural gene hoxG (Fig. 1). The hoxZ ORF predicts an extremely hydrophobic, probably membrane-bound polypeptide of 244 amino acids which may function as a membrane anchor for HoxP or as an electron transport component (Kortlfike, Horstmann and Friedrich, unpublished). HoxS-containing bacteria, including Nocardia opaca, exhibit a dominant 18.8-kDa polypeptide of unknown function designated HoxB, whose synthesis is regulated in coordination with the structural gene expression [11]. A hybrid cosmid which was used in complementation studies with HoxS-deficient mutants gave rise to HoxB production. Recent studies revealed that deletions downstream of the HoxS structural genes hoxF UYH abolished HoxB production as well as HoxS activity. This preliminary observation suggests that HoxB is implicated in the formation of catalytically active HoxS protein (BtJcker and Friedrich, unpublished). One class of mutants with deletions in the internal region (hoxN) of the gene cluster (Fig. 1) showed poor autotrophic growth on hydrogen unless the medium was supplemented with a high concentration of nickel chloride. Moreover, this nickel requirement was enhanced by increasing the concentration of magnesium ions. Since nickel-deficient mutants are altered in nickel uptake, it is concluded that hoxN codes for a highaffinity nickel uptake system [12]. The previous work of Lohmeyer and Friedrich [13] indicated that A. eutrophus contains two nickel transport systems, a high- and a low-affinity carrier. The latter also mediates the uptake of magnesium.

3. REGULATION OF ~.~OX GENE EXPRESSION

A. eutrophus is one of the model organisms for studies on the regulation of box gene expression.

Over the last two years our detailed understanding

of the underlying molecular mechanisms has developed considerably. At least two genes, hno (or rpoN or ntrA) and hoxA, control the expression of the hoxS and hoxP operons in response to the redox status of the cell and the environmental temperature (reviewed in [14]). The rpoN-fike gene maps on the chromosome of A. eutrophus H16, and mutations in this gene give rise to extremely pleiotropic mutants affected in at least eight diverse metabofic functions including H 2 oxidation, CO2 fixation and denitrification [15]. The initial observation that rpoN from A. eutrophus complements RpoN- mutants of enteric bacteria [16] led to the assumption that the rpoN (hno) gene product is an RNA polymerase (RNAP) sigma factor (o 54) which directs core RNAP to recognize -24/-12 promoter sequences. Recent data substantiated our previous analysis (Warrelmann, RSmermann and Friedrich, unpublished): (i) rpoN has been sequenced from A. eutrophus and the predicted amino acid sequence of RpoN is distinct from that of the major family of bacterial sigma factors but highly homologous to RpoN from Klebsieila pneumon~,ae, Rhizobium meliloti and Azotobacter ~inelandii (cited in [17]). (ii) Furtl~er analysis of the rpoN (hno) flanking sequence identified two contiguous ORFs, one of which is homologous to ORF1 reported to be conserved in location and predicted amino acid sequence in Salmonella typhimurium, K. pneumoniae and R. meiilot;. The second ORF ties downstream of rpoN (hnc) and is comparable to an ORF downstream of rpoN (ntrA) in K. pneumoniae, R. meliloti and A. vinelandii (cited in [17]). The precise roles of these highly conserved ORF products and their mode of action are still unknown. (iii) The upstream region of the HoxS and HoxP structural genes are devoid of - 3 5 and - 1 0 sequences usually found in prokaryotic promoters but contain DNA sequence motifs which resemble the ntr-activatable promoters characterized by the consensus sequence 5'-TI'GCA-3' at - 15 to - 10 and the sequence 5'-CTGG-3' at - 2 6 to - 2 3 (reviewed in [lg,]). These preliminary observations are in good agreement with the proposal that box

428

gene expression is positively regulated by an ntrlike system. In all cases transcriptional activation of RpoNcontrolled genes requires the product of an additional system-specific regulatory gene. Mutant analysis and complementation studies revealed that expression of the HoxS and HoxP structural genes strictly depends on the hoxA gene which is a constituent of the plasmid-encoded box gene cluster and located in the hoxC region (Fig. 1). hoxC extends over 10 kb, and sequence analysis of the left part of this D N A fragment (Fig. 1) has identified the hoxA O R F encoding a putative product of 53.5 kDa (Eberz and Friedrich, unpubfished). The deduced amino acid sequence of the HoxA protein identified several domains with a high level of homology to previously characterized regulatory proteins, including a potential nucleotidebinding pocket in the central domain and a helixturn-helix motif at the carboxy terminus. It is not yet known whether HoxA is subject to covalent modification as described for NtrC which interacts as part of a two-component system with a sensory protein, the product of ntrB (reviewed in [19]). Experimental evidence suggest that HoxA responds to the physiological signals redox status and temperature which control the expression of the two hydrogenases of A. eutrophus (reviewed in [141). The two loci hoxD and hoxE, located adjacent to hoxA (Fig. 1), were initially supposed to be solely involved in the regulation of hydrogenase synthesis, since mutants.defective in this stretch of D N A had lost HoxS and HoxP activities [20!. Detailed immunological and genetic studies pre-

dieted a more complex function of the hoxD and hoxE gene products. Mutants carrying Tn5 insertions at various sites of the hoxC region were tested for their ability to activate a hoxS-lacZ fusion that had been introduced in trans on a broad-host-range vector (Table 2). As expected, mutations in hoxA completely abolished hydrogenase and fl-galactosidase activities. Mutations in hoxD and hoxE, however, rendered the cells catalytically H o x S - and H o x P - while hydrogenase-specific antigen was retained, although at a reduced level. Moreover, H o x D - a n d HoxEmutants also exhibited diminished activity of figalactosidase activity (50 and 15~, respectively). These results suggest that the produc~s of hoxD and hoxE are primarily essential for the synthesis of catalytically active HoxS and HoxP proteins and secondarily also involved in the regulation of structural gene expression. Since HoxS and HoxP are affected in a similar fashion, H o x D and HoxE must be involved in a process common to both enzymes, e.g. iron or nickel incorporation.

4. C O N C L U S I O N S The studies summarized here show that the structural genes for the two hydrogenases HoxS and HoxP are located in a gene cluster extending over 100 kb on megaplasn-Ad priG1. The regulation of hox gene expression in A. eutrophus is complex and shows features of regulatory circuitries described for nif gene expression. Our current model is presented in Fig. 2. The mode of

Table 2 Functional activationof a hoxS.lacZ fusion in mutants defectivein the h,~xC locus Phenotype

Growth on H2/Oz/CO2 a

HoxA+, HoxD+, HoxE+ HoxA-, HoxD+, HoxE+ HoxA+, HoxD-, HoxE+ HoxA+, HoxD+, HoxE-

+ -

Enzymeactivity(U/mg of protein) b HoxS /~-Galactosidase 4.7 (+ + ) 11.1 0 (-) 0 0 (+) 5.1 0 (+) 2.4

" Figuresin parenthesis refer to the content of anti HoxScross-reactingmaterial. + +, high level; +, reduced level; +, low level; +, growth; - , no growth. b Enzyme activitieswere determined from cells grown heterotrophicallywith fructos~ and glycerol as carbon sources (reviewedin 1141).

429

rpoN (hno) Chromosomal DNA

hoxS

l

"

hoxA hoxD hoxE

hoxP

1

"

I

t

,ll

r

Fig. 2. Current model of the regulation of the two hydrogenase operons hoxS and hoxP in Alcaligeneseuteophus HI6. hoxA codes for a putative 50-kDa transcriptional activator; the product of rpoN is proposed to be the minor sigma factor 54 of RNA polymerese. More details are given in the text. a c t i o n o f t h e r e g u l a t o r H o x A a n d its c o o r d i n a t i o n w i t h u n k n o w n p r o d u c t s o f hoxD and hoxE rem a i n to b e elucidated. Likewise, t h e p r o d u c t s o f t h e o t h e r a c c e s s o r y g e n e s hoxB, hoxN, h o x M and h o x Z a n d their precise f u n c t i o n h a v e to be identified.

ACKNOWLEDGEMENTS M y co-workers C h r i s t i n e Hogrefe, A n k e N i e s a n d U t e W a r n e c k e w h o h a v e c o n s i d e r a b l y cont r i b u t e d to t h e a d v a n c e m e n t s o f this project in t h e p a s t are gratefully a c k n o w l e d g e d . I t h a n k m y cow o r k e r s C h r i s t i a n BiScker, G i i n t h e r Eherz, T h o m a s Eitinger, M a r i t a Feldotte, K a r i n H o r s t m a n n , C h r i s t i a n e Kortliike, D e t l e f RiSmermann, F_xlward Schwartz, A n d r e a T r a n - B e t c k e a n d Jiirgen W a r r e l m a n n for p r o v i d i n g results p r i o r to publication. T h e project w a s s u p p o r t e d b y g r a n t s f r o m t h e Deutsche Forschungsgemeinschaft.

REFERENCES [1] Bowien, B. and Schlegel, H.G. (1981) Physiology and biochemistry of aerobic hydrogen-oxidizing bacteria. Annu. Rev. Microbiol. 35, 405-452.

[2] Kortliike, C., Hogrefe, C., Eberz, G., Plihler, A. and Friedrich, B. (1987) G e e s of fithoautotrophic metabolism are clustered, on the megaplasmid priG1 in Alcaligenes eutrophus. Mol. Gen. Genet. 210,122-128. [3] Schneider, K., Cammack, R., Schlegel, H.G. and Hall D.O. (1979) The iron-sulphur centers of soluble hydrogcnas¢ from Alcaligeneseutrophus.Biochim. Biophys. Acta 578, 445-461. [4] Schink, B. and Schlegel, H.G. (1979) The membranebound hydrogenase of Alcaligeneseutrophus. 1. Solubilization, purification, and biochemical properties. Biochim. Biophys. Acta 567, 315-324. [5] Friedrich, C.G., Schneider, K. and Friedrich, B. (1982) Nickel in the cata)ytically active hydrogenase of AIcaligenes eutrophus. J. Bacteriol. 152, 42-48. [6] Eberz, G., Hogrefe, C., Kortliike, C., Kamienski, A. and Friedrich, B. (1986) Molecular cloning of structural and regulatory bydrogenase (box) genes of Alcaligenes eutrophus H16. J. Bacteriol. 168, 636-641. [7] Tran-Betcke, A., Wamecke, U., BiScker, C., Zaborosch, C. and Friedrich, B. (1990) Cloning and nucleofide sequence of the genes for NAD-redueing hydrogenase of AIcaligenes eutrophus strain H16, J. Bacteriol. 172, 29202929. [8] Sayavedra.Soto, L.A., Powen, G.K., Evans, HJ. and Morris, R.O. (1988) Nucleotide sequence of the genetic loci encoding subunits of Bradyrhizobiumjaponicum uptake hydrogenase. Proc. Natl. Acad. SCi. USA 85, 8395-8399. [9] Leclcrc, M., Colbeau, A., Cauvin, B. and Vignais, P.M. (1988) Cloning and sequencing of the gems encoding the large and the small subunits of the H 2 uptake hydrogenase hup of Rhodobacter capstdatus. Mol. Gen. Genet. 214, 97-107. Corrected, 1989. Mol. Gen. Genet. 215, 368.

430 [I0] Reeve, J.N., Beclder, G.S., Cram, D.S., Hamilton, P.T., Brown, J.W., Kxzyckl J.A., Kolodziej, A.F., Alex, L., Orme-Johnson, W.H. and Walsh, C.T. (1989) A hydrogenase linked gene in Methano&Jcterium thermoautotrophicum AH encodes a poly-ferredoxin. Proc. Natl. Acad. Sci. USA 86, 3031-3035. [11] KIh'st, U., Suetin, S. and Friedrich, C.G. (1987) Purification and properties of a protein finked to the soluble hydrogenase of hydrogen-oxidizing bacteria. J. Bacteriol. 169, 2079-2085. [12] Eberz, G., Eitinger, T. and Friedrich, B. (1989) Genetic determinants of a nickel-specific transport system are part of the plasmid-encoded hydrogenase gene cluster in Ab cal/genes eutrophus. J. Bacteriol. 171,1340-1345. [13J Lohmeyer, M. and Friedrich, C.G. (1987) Nickel transport in AIcaligenes eutrophus. Arch. Microbiol. 149,130-135. [14] Friedrich, B. and Friedrich, C.G. (1990) Hydrogenases in fithoautotrophic bacteria. In: G.A. Codd et al. (ed), Advances in autotrophic microbiology and one-c.arbon metabolism, pp. 59-99. Kluwer Academic Publishers, Dordrecht. [15] RSmermann, D., Lohmeyer, M., Friedrich, C.G. and

Friedrich, B. (1988) Pleiotropic mutants from Alcaligenes eutrophus defective in the metabolism of hydrogen, nitrate, urea and fumarate. Arch. Microbiol. 149, 471-475. [16] R~mermann, D., Warrelmann, J., Bender, R.A. and Friedrich, B. (1989) An rpoN-like gene of AIcaligenes eutrophus and Pseudomonas facilis controls expression of diverse metabofic pathways, including hydrogen oxidation. J. Bacteriol. 171,1093-1099. [17] Merrick, M., Gibbins, J. and Toukdarian, A. (1987) The nucleotide sequence of the sigma factor ntrA (rpoN) of Azotobacter vinelondii: Analysis of conserved sequences in NtrA proteins. Mol. Gen. Genet. 210, 323-330. [18] Ausubel, F.M. (1984) Regulation of nitrogen fixation genes. Cell 37, 5-6. [19] Kustu, S., Santero, E., Keener, J., Popham, D. and Weiss, D. (1989) Expression of o ~ (ntrA)-dependent genes is probably united by a common mechanism. Microbiol. Rev. 53, 367-376. [20] Hogrefe, C., R~mermann, D. and Frledrich, B. (1984) AIcaligenes eutrophus hydrogenase genes (Hox). J. BacterioL 158, 43-48.