The gene encoding ammonia monooxygenase subunit A exists in ...

FEMS Microbiology

Letters 139 (1996) 181-188

The gene encoding ammonia monooxygenase subunit A exists in three nearly identical copies in Nitrosospira sp. NpAV Jeanette M. Norton aT* , Jackie M. Low a, Martin G. Klotz b a Departmentof Plants,Soilsand Biometerology,UtahStateUniuersity, Logan, UT 84322-4820, b Deparhnentof Biology, University of Colorado, Denver, CO 80217-3364, Received

18 January

USA

USA

1996; revised 25 March 1996; accepted 25 March 1996

Abstract The gene encoding ammonia monooxygenase subunit A (AmoA) was found in three copies in the genome of the chemolithotrophic soil bacterium, Nitrosospira sp. NpAV. The open reading frame and flanking regions of the three copies were isolated from digested and size fractionated genomic DNA using oligodeoxyribonucleotide primers and polymerase chain reaction. The three gene copies of amoA were sequenced and the sequences compared to each other. The open reading frames and the upstream and downstream flanking regions were nearly identical in the three copies. All three copies were expressed in recombinant Escherichia coli strains from the indigenous promoter producing a product of approximately 30 kDa. All amoA copies encode 274 amino acid polypeptides which have similarity to the ammonia monooxygenase acetylene-binding protein from Nitrosonwnas europaea. Keywords:

Ammonia

monooxygenase;

Nitrosospira;

Nitrification;

Nitrosomonas;

1. Introduction Nitrosospira sp. NpAV is a chemolithotrophic soil bacterium which creates metabolic energy by the oxidation of ammonia to nitrite. The first enzyme in this nitrification pathway is ammonia monooxygenase (AMO) which is a membrane-bound multi-subunit enzyme responsible for the conversion of NH, to hydroxylamine [l]. An approximately 27 kDa sized membrane-associated peptide which contained the active binding site of AM0 [l] has been identified from Nitrosomonas europaea. The sequence of

* Corresponding author. Tel.: + 1 (801) 797 2166; Fax: (801) 797 3376; E-mail: [email protected] 0378-1097/96/$12.00 0 1996 Federation PII SO378-1097(96)00139-5

of European

+ 1

Microbiological

amoA; DNA sequence

a gene (amoA) coding for this peptide has been determined from two overlapping clones [2]. A second gene, amoB, extends directly downstream from the amoA gene in all strains examined [2,3]. The amoB gene from N. europaea has also been cloned in two independent overlapping fragments [4]. As of yet, there are no reports of recombinant constructs that contain a complete open reading frame of amoA or of the expression of ammonia oxidizer AmoA proteins in recombinant Escherichia coli host strains. Notably, N. europaea has multiple copies of the amoA gene [2] and the genes encoding the key enzymes of the subsequent steps in ammonia oxidation: hydroxylamine oxidoreductase (HA01 and cytochrome c554 (CYC) @I. We observed multiple copies of amoA and amoB in Nitrosospira sp. Societies. All rights reserved

NpAV, Nitrosolobus multifomzis (ATCC 25 196) and in five additional ammonia oxidizer strains representing the genera Nitrosomonas, Nitrosospira, Nitrosouibrio and Nitrosolobus [3]. Recently, we reported the sequence for one copy of amoA from Nitrosospira sp. NpAV and preliminary evidence that the gene exists in three copies in the genome [6]. Our objective for this study was to verify the existence of three functional copies of amoA and to determine and compare the sequences of these copies. In this paper, we present the sequences for the open reading frames (ORF) and the flanking regions for the three copies of the amoA genes in Nitrosospira sp. NpAV. Furthermore, we investigated the ability of the amoA genes to be expressed in the recombinant E. coli strains. Implications of the multiple copies of AM0 encoding genes are discussed in relation to the function and evolution of ammonia oxidizers.

1234 I

1

!@ 23.1 9.4 6.6 4.4

2.3 2.0

0.6 Fig. I. Southern anlysis of Nirrosospiru sp. NpAV genomic DNA with an umoA probe from N. ruropclea. DNA digested with EcoRI (lane I), SmaI and EcoRI (lane 2) and Sm01 (lane 3). Lane 3 contains a digoxigenin-labeled A Hind111 molecular weight marker (Boehringer Mannheim Corp., Indianapolis. IN).

2. Materials and methods

2. I. Bacterial

strains, plasmids

and culture condi-

tions The bacterial strains and plasmids used in this study are summarized in Table I. Ammonia oxidizers were cultivated in 500 ml static batch cultures of ATCC medium 929 [7] at 30°C with periodic pH adjustment based on the phenol red indicator. Cells were harvested by centrifugation (5000 X g) after approximately 3 weeks of growth or when growth slowed as assessed from a decrease in acid production. Cultures were examined for heterotrophic contamination by plating on nutrient broth agar plates which were incubated for 1 month following harvest, and by microscopic examination [8]. 2.2. DNA isolation, hybridization cation and sequencing

analysis,

amplifi-

Genomic DNA (gDNA) was isolated from the ammonia oxidizer cells following the procedure of McTavish [2]. The gDNA was restriction digested following the manufacturer’s directions (Boehringer Mannheim) and electrophoresed on a 0.8% agarose gel in 1 X TAE and blotted to a nylon membrane [9].

A N. europaea amoA DNA probe of 792 bp (see Table 1) was prepared by random labelling with digoxigenin, hybridized to the membrane and the hybridizing fragments detected following the manufacturer’s recommendations (Genius” System, Boehringer Mannheim). Molecular masses of hybridizing fragments were determined using the RFLPscan program (Scanalytics/CSPI, Billerica, MA). We examined the restriction digests of Nitrosospira sp. NpAV gDNA by Southern analysis and found that gDNA digested with EcoRI yielded three high molecular mass, easily separable fragments which hybridized to the amoA probe (Fig. 1). In order to isolate DNA fragments that contained only one of the three copies, gDNA digested with EcoRI was electrophoresed and small blocks of agar containing the fragments of approximately 4.6, 5.7 or 16.8 kb (Fig. 1) were cut out. DNA was extracted from the agar blocks using the GENECLEAN@ 1 kit (Bio 101, La Jolla, CA). These size fractionated DNAs were then used as templates for the polymerase chain reaction (PCR) employing DNA polymerase and oligonucleotide primers designed using published N. europaea [2] and Nitrosospira sp. NpAV sequences [6]. The primer sequences

pNA-5

pNA-3

pNA-

I

amoA-p

Plasmids pCR’“I1

1

Niirosomonas europaea ATCC 19 178 Nitrosobbus multiformis ATCC 25 196 E. coli InvaF’

or genotype

from N. pCR”I1 from the pCRT”II from the pCR”I1 from the

colEI,

europaea amoA position 282- 1074 with insert of amoA1 amplicon 16.8 kB EcoRI fragment with insert of amoA2 amplicon 5.7 kB EC&I fragment with insert of amoA3 amplicon 4.6 kB EcoRI fragment

ampR, kanR, lac promoter,lacZa, T7 and Sp6 promoters pCR’“I1 with insert of amplicon

source of anoA probe sequence type strain originally isolated from soil endAI,recAl, hsdR17(r-km+k, supE44, thi- I, gyrA, relA1, ~lacZAM15A(lacZYA-argF), deoR, F’, One Shot’” competent cells

ammonia oxidizer from soil

wild-type

sp. NpAV

Relevant nhenotvne

Strains Nitrosospira

used in this study

Strain or nlasmid

Table 1 Bacterial strains and plasmids

[2]

San Diego. CA

San Diego, CA

D. Arp and N. Hommes (Oregon State University) Genbank LO8050 this study Genbank U38250 this study, [6] Genbank U20644 this study Genbank U38250

Invitrogena,

Invitrogen~,

[71

E. Schmidt (University of Minnesota) via M.A. Bruns (Michigan State University), [23] D. Arp and N. Hommes (Oregon State University),

Source or reference

[2]

J.M. Norton et al./FEMS

Microbiology

(‘AmolO’: 5’-CTGACCGAYGTGGTYTGGACC and ‘Amos’: 5’-TINACRTTNAGCATNGCRTGCAT) used for DNA amplification with PCR are more than 20 bp upstream and 50 bp downstream, respectively, of the sequence in Fig. 2. The following PCR conditions were used: Perkin Elmer DNA cycler and PCR reagents; 25 cycles (1 min at 94°C 1 min at 55°C and 2 min at 72°C) with a 7 min extension at 72°C; optimum concentrations were 2.5 mM MgCl, and 0.2 JLM for primer. The PCR product was ligated into pCRT”II plasmid and transformed into One Shot’” competent E. coli cells (Table 1) according to the manufacturer’s directions (TA Cloning@ system, Invitrogen, San Diego, CA). The DNA sequences were obtained from the double-stranded insert templates, using T7, SP6 and synthetic primers based on the existing sequence and DNA polymerase for dideoxy dye-primer cycle sequencing (ABI 373A, USU Biotechnology Center) following the instructions of the manufacturer. Sequences were aligned and percentage identities were calculated using the NCBI Blast program [lo]. The sequences for amoA1 and amoA3 genes were deposited in GenBank under accession numbers U38250 and U38251 and the sequence for amoA2 was updated in entry U20644. 2.3. Expression nant E. coli

of the amoA gene copies in recombi-

To examine expression of the AmoA proteins in cells, the three recombinant pCR”I1 based (kanamycinR , ampicillinR) plasmid subclones were transformed into E. coli strain DHSa (F- @80 AZlacZ Ml5 recAZ SmR ; BRL, Bethesda, MD) by the Ca2+/heat shock method [9]. Transformants were selected on LB agar plates supplemented with the appropriate antibiotics and screened for resident

Letters 139 (1996) 181-188

185

plasmid using the CTAB-plasmid miniprep method [ll]. To characterize the recombinant expression products, mid-exponential growth phase cultures of E. coli DHSa carrying a recombinant plasmid (pNA-1, pNA-2 or pNA-3), or the vector alone (pCRW), were harvested by centrifugation, washed with 20% (v/v) glycerol and suspended in l/50 of the original volume of 50 mM potassium phosphate buffer, pH 7.0. The bacterial suspensions were divided into two equal volumes: the first was used to obtain total cellular lysates and the second to prepare fluids of the cytoplasmic and periplasmic fractions as described in [ 121. In brief, the first volume was pulse sonicated with a Virtis Sonifier for 5 min in order to fragment the cytoplasmic lamellae and membrane, into which the functional AM0 protein is believed to incorporate. The sonicates were clarified by centrifugation (6000 X g, 20 min, 4°C) to remove cell debris and the supematants were filter sterilized and used as total lysates [121. The second volume was used to obtain periplasmic and cytoplasmic fluids. Following chloroform extraction and clarification by centrifugation [12], the periplasmic fluids were obtained in the supematant fraction. After removal of the supernatants (representing the periplasmic fluids), the pellets were resuspended in potassium phosphate buffer (pH 7.0), briefly pulse sonicated and again clarified by centrifugation (16 000 X g for 20 min, 4°C). These supematants were filter sterilized and used as cytoplasmic fluids [ 121. The protein concentrations in the lysates were determined using the BCA protein kit (Sigma, St. Louis, MO) and bovine serum albumin as a standard. For the determination of the molecular mass of the putative recombinant AmoA proteins, lysates were prepared for denaturing polyacrylamide gel electrophoresis (SDS-PAGE) following the protocol of Laemmli [ 131. Proteins (100 Fg per lane) were

Fig. 2. Nucleotide sequence for the three copies of amoA and flanking regions from Nitrosospira sp. NpAV (anwA1, Genbank No. U38250; u1noA2, Genbank No. U20644; and amoA3, Genbank No. U38251) compared to the nucleotide sequence of amoA and amoB from N. europaea (Genbank No. LOSOSO). Putative transcriptional (’ - 35’ and ‘ - 10’ promoter regions and start point ‘ + 1’) and translational (S/D and start codon) control sequences are underlined; in-frame translational stop codons are marked by an asterisk. The start of the AmoA and AmoB ORF is indicated over the ATG start codon. Dots are used to indicate identical nucleotides in comparison to the sequence of amoA2 (partially given in Klotz and Norton, 1995). Vertical lines (1) represent base deletions with respect to the alignment.

186

J.M. Norton

et trl./

FEMS Microbiology

separated in a denaturing (SDS) gel (12% T; 2.7% C) by electrophoresis, followed by staining for protein with Coomassie R-250. [ 121.

3. Results and discussion The amoA gene exists in three nearly identical copies in the genome of Nitrosospiru sp. NpAV. Each copy is preceded by a functional cT7’-like promoter that is recognized by the transcriptional machinery of E. coli. The existence of three copies was first indicated by Southern hybridization of an N. europaea amoA DNA probe to three high molecular mass fragments (approximately 16.8, 5.7 and 4.6 kB) of genomic DNA restriction digested with EcoRI (Nitrosospiru sp. NpAV) (Fig. I). While three high molecular mass bands were observed when genomic DNA was restricted with EcoRI only one band appeared in digests using SmaI + EcoFU or SmaI alone (Fig. I). This indicates that the observed three bands were not the result of EcoRI restriction sites inside the amoA gene but rather three individual DNA fragments, each of which contained a copy of an amoA gene. Further, at least two SmaI restriction sites are conserved within the EcoRI fragments containing the amoA gene but outside the three amoA gene copies. Nucleotide sequence data (Fig. 2) support this interpretation as there are no SmaI or EcoRI restriction sites within the sequence (Fig. 2). Similarly, with Nitrosolobus multiformis ATCC 25 196 we observed three high molecular mass bands with Sac1 or with BamHI, but only one band with EcoRI and one band with either Sac1 + EcoRI or BamHI + EcoRI. Because there are no restriction sites for these enzymes in the ORF of N. multiformis (Klotz and Norton (1994) Nucleotide sequence of a gene encoding ammonia monooxygenase (AmoAI in Nitrosolobus multiformis. GenBank accession number Ul5733), we conclude that amoA also exist in three copies in this organism. The three copies in the Nitrosospiru sp. NpAV genome are practically identical for the ORF of amoA and, unexpectedly, highly similar for the 165 bases upstream of the ORF and the downstream partial amoB sequence. A comparison of the three sequences from Nitrosospira sp. NpAV with the amoA and partial amoB sequences from N. euro-

Letten

139 (19961 181-188

paea is shown in Fig. 2. The sequences are 75% identical within the amoA ORF, 67% identical within the partial ORF of amoB and 47% identical upstream of the amoA ORF with the published N. europaea sequences. It appears that the genes coding for the key enzymes in ammonia oxidation are present in multiple (two or three) copies in all strains of ammoniaoxidizing bacteria that have been examined to date ([2,4,6,14,15], this article). The amoA gene exist in two copies in the genome of N. europaea but three copies in Nitrosospiru sp. NpAV and N. multi,formis. While our method of DNA isolation (copy template separation followed by individual PCR amplification, cloning and sequencing) allowed us to obtain complete independent sequences for all three amoA gene copies present in Nitrosospira sp. NpAV, the amoA sequence reported for N. europaea [2] was cloned in two overlapping fragments. Because N. europaea has two copies of the amoA gene [2], it is not yet clear whether the presented sequence represents the nucleotide sequence of one of the copies or is a chimerical sequence with fragments from both copies. The AM0 enzyme is related to the particulate methane monooxygenase (pMM0) which is found in the methane oxidizers such as Methylococcus capsulatus [Bath] [16]. AM0 and pMM0 are the first representatives of a new class of copper containing monooxygenases [ 161. The pMM0 subunits are encoded by the genes designated pmoA and pmoB in analogy to the AM0 encoding genes [ 171. The pmoA and pmoB genes are found in two copies in M. capsulatus [Bath] and in Methylobacter albus BG8 [17]. As with the amoA and amoB genes from N. europaea, the pmoA and pmoB sequences from M. cupsulatus [Bath] were cloned in several overlapping fragments. While the entire individual copies of pmoA and pmoB have not yet been sequenced, Southern analysis and partial sequencing suggest that they are highly similar [ 171. A common observation in all the studies [2,6,17] which involve genes encoding these monooxygenases, is that library cloning of complete sequences, especially amoB and pmoB, is problematic, possibly due to toxicity of the gene products to the E. coli expression hosts. Our method of isolation of the copies before amplification, cloning and sequencing allowed us to obtain com-

J.M. Norton et al. / FEMS Microbiology Letters 139 (1996) 181-188

kDa 97.4 ) 66.2 )

31.0 *

putative AmoA

21.5 ) 14.5 * Fig. 3. SDS PAGE gel of proteins (100 /~g per lane) from recombinant E. coli DH5a (pNA-x). Lane 1, low molecular mass marker (Bio-Rad); lane 2, total lysate from DHSa containing pCR’“II; lane 3, total lysate from DHSLY containing pNA-1; lane 4, total lysate from DHSO containing pNA-3; and lane 5, total lysate from DH5a containing pNA-5. Right arrow indicates putative AmoA peptide produced in strains containing the amoA gene from Nitrosospira sp. NpAV.

plete sequences for all three of the amoA copies present in Nitrosospira sp. NpAV. Multiple copies of genes encoding functional proteins are relatively unusual in prokaryotes. Examples include those which encode enzymes with slightly different amino acid sequences and hence, an altered function (e.g. the three aro genes in E. coli [lS]) and those in which the copies encode identical amino acid sequences (possibly regulated on the transcriptional level) such as the multiple copies of the rbcL and rbcS genes in Thiobacillus ferroonidans [19]. Our results indicate that the amoA genes are in the second group. The upstream flanking regions of each of the copies contain a Shine Delgarno sequence, an in-frame translational stop codon preceding the translational start codon (ATG), putative E. coli cT7’-type promoter sequences [20] and a transcriptional start point (Fig. 2). A comparison of the SDS-PAGE profiles of total lysates from E. coli strain DHSo (pCRI1) (Fig. 3, lane 2) with those from recombinant DHSa (pNA- 1, pNA-2 and pNA-3) (Fig. 3, lanes 3, 4 and 51, shows unique, approximately 30 kDa protein bands (putative AmoA) in me lysates from the

187

recombinant E. coli strains. In contrast, periplasmic and cytoplasmic fluids of the recombinant strains lacked this protein (data not shown) suggesting that the nascent putative AmoA proteins are incorporated into the membrane immediately after translation. Because no N-terminal signal sequence 1211 was detected in any of the amoA copies, incorporation seems to occur without the chaperoning aid of the SecA protein. The fact that the recombinant host cells survived with nitrifier putative AmoA proteins being incorporated in their membrane also indicates that E. coli does not complement for the other protein subunits that would form a functional, potentially toxic (hydroxylamine-producing) AMO. The three recombinant E. coli strains containing the individual amoA gene copies yielded a protein product despite the opposite orientation of placZ and the amoA ORFs in the vector and the absence of IPTG. All three cloned amoA genes appear to have functional promoters (identified in Fig. 2 sequence) which are recognized by E. coli transcription factor(s). Because the regions sequenced in the three sets of the genes were nearly identical, we could not determine from the current information whether all copies were being transcribed in Nitrosospira sp. NpAV under a specified culture condition. Since ammonia oxidizers such as Nitrosospira sp. NpAV are obligamly autotrophic and absolutely dependent on AMO, they may not need to regulate the, transcription of multiple copies independently [22]. Alternatively, a constitutive expression of all three amoA gene copies may be too expensive for the catabolic efficiency of ammonia oxidizers and ,differential expression may occur. The almost complete identity of the multiple gene copies in a given strain but significantly lower similarity between am0 genes of different strains suggests that the multiple copies are the products of gene duplication rather than horizontal .gene transfer. This duplication of the gene sequences may have occurred relatively recently in the evolution ,of the ammonia oxidizers. Alternatively, a rectification mechanism may be responstble for maintaining the high level of sequence identity of the multiple copies. This hypothesis is supported by the observation that the sequences are nearly identical and lack third base degeneracy which should have developed after the gene duplication event due to mutations. Further

188

J.M. Norton et ul./ FEMS Microbiolog\

investigations are needed to determine the function of the identical multiple copies of the genes encoding AA40 in ammonia-oxidizing bacteria.

Acknowledgements This work was supported by the Utah Agricultural Experiment Station, Utah State University and approved as journal paper no. 4858. Support from the University of Colorado at Denver, CLAS, is acknowledged. We thank Daniel Arp and Norman Hommes for supplying the clone containing amoA from N. europaea, Mary Anne Bruns and Edwin Schmidt for supplying ammonia oxidizer strains and Bradley Kropp for use of his Perkin-Elmer thermocycler.

References and ” CO,Ill Hyman, M.R. and Arp, D.J. (1992) “CIHz labeling studies of the de nova synthesis of polypeptides by Nitrosomonas europea during recovery from acetylene and light inactivation of ammonia monooxygenase. J. Biol Chem. 267, 1534-1545. 121McTavish, H., Fuchs, J.A. and Hooper, A.B. (1993) Sequence of the gene coding for ammonia monooxygenase in Nitrosomonas europaea. J. Bacterial. 175. 24362444. among 131 Norton, J.M. and Klotz, M.G. (1995) Homology ammonia monooxygenase genes from ammonia-oxidizing bacteria. Abstracts of the 95th General Meeting of the American Society for Microbiology. American Society of Microbiology, Washington, DC. 141 Bergmann, D.J. and Hooper, A.B. (1994) Sequence of the gene, amoB, for the 43-kDa polypeptide of ammonia monooxygenase of Nitrosomonns europaea. B&hem. Biophys. Res. Commun. 204, 759-762. [51 McTavish, H., LaQuier, F., Aciero, D., Logan, M.. Mundfrom, G., Fuchs, J.A. and Hooper, A.B. (1993) Multiple copies of genes coding for electron transport proteins in the bacterium Nitrosomonas europaea. J. Bacterial. 175, 24452447. [61 Klotz, M.G. and Norton, J.M. (1995) Sequence of an ammonia monooxygenase subunit A-encoding gene from Nitrosospira sp. NpAV. Gene 163, 159- 160. [71 Gema, R., Cote, R. and Pienta, P. (1992) American Type Culture Collection Catalogue of Bacteria and Phages. American Type Culture Collection, Rockville, MD. b31Schmidt, E.L. and Belser, L.W. (1994) Nitrifying bacteria. In: Methods of Soil Analysis Microbiological and Biochemical Properties, 3rd edn. (Weaver, R.W., Angle, S., Bottom-

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ley, P., Bezdicek, D., Smith. S.. Tabatabai, A. and Wollum, A., Eds.), pp. 159-177. Soil Science Society of America, Madison, WI. [9j Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [lOI Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403-10. [l II Del Sal, G., Manfioletti, G. and Schneider, C. (1989) The CTAB-DNA precipitation method: a common mini-scale preparation of template DNA from phagemids, phages or plasmids suitable for sequencing. Biotechniques 7, 5 14-5 19. [I21 Klotz, M.G., Kim, Y.C., Katsuwon, J. and Anderson, A.J. (I 995) Cloning, characterization and phenotypic expression in Escherichia coli of catF, which encodes the catalytic subunit of catalase isozyme CatF of Pseudomonas .s.vringae. Appi. Microbial. Biotechnol. 43, 656-666. [131 Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680-685. [141 Bergmann, D.J.. Arciero, D.A. and Hooper, A.B. (1994) Organization of the hao gene cluster of Nitrosomonas europaea: genes for two tetraheme c cytochromes. J. Bacterial. 176, 3148-3153. L. A., Hommes, N.G. and Arp, D.J. (1994) [151 Sayavedra-Soto, Characterization of the gene encoding hydroxylamine oxidoreductase in Nitrosomonus europea. J. Bacterial. 176, 504-5 10. [I61 Holmes, A.J., Costello, A.. Lidstrom, M.E. and Murrell, J.C. (1995) Evidence that particulate methane monooxygenase and ammonia monooxygenase may be evolutionarily related. FEMS Microbial. Lett. 132, 203-208. A., Lebron, J., Costello, A.. [171 Semrau, J.D.. Chistoserdov, Davagnino, J., Kenna, E., Holmes, A.J., Finch, R., Murrell, J.C. and Lidstrom, M.E. (1995) Particulate methane monooxygenase genes in methanotrophs. J. Bacterial. 177, 3071-3079. [I81 Riley, M. (1993) Functions of the gene products of Escherichia coli. Microbial. Rev. 57, 862-952. 1191 Kusano, T., Takeshima, T., moue, C. and Sigawara, K. (1991) Evidence for two sets of structural genes coding for ribulose biphosphate carboxylase in Thiobacillus ferroo.ridans. J. Bact. 22, 7313-7323. [201 Hawley, D.K. and McClure, W.R. (1983) Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 11, 2237-2255. [211 Pugsley, A.P. (1993) The complete secretory pathway in Gram-negative bacteria. Microbial. Rev. 57, 50- 108. [221 Bowien, B., Bednarski, R., Kusian, B., Windhovel, U., Freter, A., Schaferjohann, J. and Yoo, J.G. (1992) Genetic regulation of CO, assimilation in chemoautotrophs. In: Microbial Growth on-Cl Compounds (Murrell, J.C. and Kelly, D.P., Eds.), Intercept, Andover. [231 McCaig, A.E., Embley, T.M. and Presser, J.I. (1994) Molecular analysis of enrichment cultures of marine ammonia oxidisers. FEMS Microbial. Lett. 120, 363-368.