DNA sequence showed that the T. ferrooxidans ntrA gene coded for a protein of 475 amino acids (calculated Mr, .... I D E R G Y L A D S L E D L A A T M N V Q E D A L L A V. L. L. R. V ... mon to all four cosmid clones were observed (data not.
Vol. 172, No. 8
JOURNAL OF BACTERIOLOGY, Aug. 1990, p. 4399-4406 0021-9193/90/084399-08$02.00/0 Copyright © 1990, American Society for Microbiology
Complementation of Escherichia coli a54 (NtrA)-Dependent Formate Hydrogenlyase Activity by a Cloned Thiobacillus ferrooxidans ntrA Gene DAVID K. BERGER, DAVID R. WOODS, AND DOUGLAS E. RAWLINGS* Department of Microbiology, University of Cape Town, P B Rondebosch 7700, South Africa Received 29 December 1989/Accepted 14 May 1990 The ntrA gene of ThiobaciUusferrooxidans was cloned by complementation of an Escherichia coli ntrA mutant that was unable to produce gas via the r54 (NtrA)-dependent formate hydrogenlyase pathway. Analysis of the DNA sequence showed that the T. ferrooxidans ntrA gene coded for a protein of 475 amino acids (calculated Mr, 52,972). The T. ferrooxidans NtrA protein had 49, 44, 33, and 18% amino acid similarity with the NtrA proteins of KlebsieUa pneumoniae, Azotobacter vinelandii, Rhizobium meliloti, and Rhodobacter capsulatus, respectively. The ability of the T. ferrooxidans NtrA protein to direct transcription from a54-dependent promoters was demonstrated in E. coli by using fdhF-lacZ and nifl-lacZ fusions. An open reading frame coding for a protein of 241 amino acids (calculated Mr, 27,023) was situated 12 base pairs upstream of the T. ferrooxidans ntrA gene. Comparison of this protein with the product of the open reading frame ORF1, located upstream of the R. meliloti ntrA gene, showed that the two proteins had 55% amino acid similarity. The cloned T. ferrooxidans ntrA gene was expressed in E. coli from a promoter located within the ORF1 coding region.
wished to isolate the genes of regulators of nitrogen metabolism from T. ferrooxidans, including the ntrA gene. Since the assimilation of arginine by E. coli is aS4 dependent, previous workers have cloned the ntrA gene from bacteria such as Azotobacter vinelandii by complementation of E. coli ntrA mutants that are unable to grow on minimal medium and arginine (45). Attempts to use the same procedure to isolate the T. ferrooxidans ntrA gene were unsuccessful. We therefore devised a cloning strategy based on the observation that expression of the fdhF gene, which codes for a component of the E. coli formate hydrogenlyase pathway, is cr4 dependent (6). The formate hydrogenlyase pathway results in the evolution of H2 and CO2 gas when E. coli is grown under anaerobic conditions in the presence of formate (36). Since E. coli ntrA mutants are unable to produce gas, the ability to complement gas production in an E. coli ntrA strain was used in a novel screening technique for the presence of the cloned T. ferrooxidans ntrA gene. We report the cloning and nucleotide sequence of the T. ferrooxidans ntrA gene together with the associated upstream and downstream open reading frames (ORFs) and demonstrate biological activity of the T. ferrooxidans ntrA gene product.
Thiobacillus ferrooxidans is a gram-negative, acidophilic, diazotrophic, chemolithoautotrophic bacterium that obtains its energy from the oxidation of either ferrous iron to ferric iron or reduced sulfur compounds to sulfuric acid. The bacterium is ideally suited for growth in an inorganic mining environment, where it is used industrially to leach metals from a variety of ores (23). Eubacteria employ a number of sigma factors, which, when associated with RNA polymerase, alter the specificity of promoter recognition. Two sigma factors, or70 and r54, have been shown to direct the transcription of a physiologically diverse set of genes, whereas others, such as &32 and a28, direct transcription of genes whose products share a common physiological role (reviewed in reference 17). ua54 iS the product of the ntrA (rpoN, glnF) gene and is required for the transcription of promoters that have a characteristic -24 and -12 consensus recognition element. Promoters for which C54 is required (reviewed in reference 28) include the nitrogen-regulated glnA promoter of enteric bacteria, several of the nif and fix gene promoters of nitrogen-fixing bacteria, the arginine (argT) and histidine (dhuA) transport gene promoters of Salmonella typhimurium, and promoters for genes of the formate hydrogenlyase pathway of Escherichia coli. Genes involved in the nitrogen metabolism of T. ferrooxidans ATCC 33020 have recently been cloned and sequenced. These are the structural genes for glutamine synthetase (glnA) (4, 40) and nitrogenase (nifHDK) (37-39). A DNA sequence (-TTGGCACGGCCCTTGCA-) typical of u54 promoters was located in front of the T. ferrooxidans nifH gene (37). The binding of specific transcriptional activators to upstream activator sites appears to be a feature of u54-dependent promoters (28, 33). The transcriptional activator of most nif operons is the product of the nifA gene (16). The NifA binding sites have a TGT-N1O-ACA consensus sequence (10), and two tandem consensus activator sites are situated upstream of the T. ferrooxidans nifH gene (37). We *
MATERIALS AND METHODS Bacterial strains, plasmids, and media. The strains and plasmids used in this study are described in Table 1. E. coli was grown on LB or buffered TGYEP (pH 6.5) (5) complex medium under aerobic or anaerobic conditions, respectively. Aerobic minimal medium was glucose minimal medium supplemented with 0.2% arginine, and anaerobic minimal medium was NFDM (11) supplemented with 100 jig of glutamine per ml. When required, antibiotics were added at the following concentrations: ampicillin, 50 ,ug/ml; tetracycline, 15 jig/ml; chloramphenicol, 20 j,g/ml. DNA manipulations. The alkaline lysis method (8) was used for both small- and large-scale plasmid preparation. Standard methods (30) were used for restriction digests, gel electrophoresis, purification of DNA fragments from agarose
Corresponding author. 4399
4400
BERGER ET AL.
J. BACTERIOL. TABLE 1. Bacterial strains and plasmids used
Strain or plasmid
Relevant characteristics
E. coli YMC1O TH1 FM911
Plasmids pEcoR251 pEcoR252 pUC19 Bluescript KS' Bluescript SK+ pACYC184 p184t pT3 pT20 pT30 pT30-D pT40 pT41
pT50 pFB71 pK50 pCK3 pMB1 pMC1403 pIMP11 pHlac
Source or reference
A1acU169 AlacU169 ntrA AfdhF recA56
3 F. Ausubel 49
Apr EcoRI Derivative of pEcoR251 with PstI site within amp gene mutated Apr Apr Apr Clmr Tetr Clmr Tet' pACYC184 tet gene deleted at the HindlIl and Hindll sites pHC79 cosmid clone containing T. ferrooxidans ntrA pEcoR252 with 5.5-kbp BamHI insert from pT3 pUC19 with T. ferrooxidans ntrA on a 2.9-kbp SaIl-BgIIl insert Exonuclease III shortening of pT30 from the BgIlI end Bluescript KS' with T. ferrooxidans ntrA on a 1.69-kbp ClI-EcoRI insert from pT30-D Bluescript SK+ with T. ferrooxidans ntrA on a 1.69-kbp CIaI-EcoRI insert from pT30-D Clmr Tets pACYC184 tet gene replaced at the ClaI and HindII sites by T. ferrooxidans ntrA on ClaI-EcoRI fragment from pT30-D Clmr Tetr K. pneumoniae ntrA Clmr Tets p184t with K. pneumoniae ntrA on a 1.69-kbp ClaI insert Tetr K. pneumoniae nifA constitutively expressed, Inc P Apr K. pneumoniae nifH-lacZ Apr lac'ZYA Apr T. ferrooxidans nifH Apr T. ferrooxidans nifH-lacZ on the vector pMC1403
47 P. Janssen 34 Stratagene, San Diego Stratagene 13 This work This work This work This work This work This work
This work This work 14 This work 24 9 12 38 This work 7
Apr fdhF-lacZ
pBN208
gels, ligations, the filling in of 5' sticky ends, and Southern ifiter hybridization. DNA sequencing. DNA sequencing was carried out by the dideoxy-chain termination method (43) with overlapping templates generated by exonuclease III shortening (18). Sequencing reactions were carried out by using the Sequenase kit (version 2.0) from U.S. Biochemical Corp., Cleveland, Ohio. The entire sequence of the T. ferrooxidans DNA insert in pT30 was determined from both strands. Sequence analysis. The DNA sequences were analyzed with IBM XT computer DNA tools and Genepro (version 4.1) programs. Predicted amino acid sequences were analyzed and compared with the Genepro (version 4.1) protein alignment subroutine. Isolation of the T. ferrooxidans ntrA gene. A T. ferrooxidans
T
II
I
ORFI
ntrA
I
s
ATCC 33020(pHC79) cosmid library was transduced into the E. coli ntrA deletion mutant THL. Transductants were plated onto TGYEP medium plus 30 mM formate with antibiotic selection and overlaid with TGYEP containing 0.8% agar. Plates were incubated anaerobically ovemight at 35°C. Complementation of ntrA product activity was identified by a pocket of gas surrounding a colony in the agar. Plasmid constructions. Construction of the gene bank of T. ferrooxidans ATCC 33020 DNA in the cosmid vector pHC79 has been described previously (R. Ramesar, Ph.D. thesis, University of Cape Town, South Africa, 1988). A 5.5kilobase-pair (kbp) BamHI fragment from pT3 was cloned into the BglII site of the vector pEcoR252 to produce pT20. A 2.9-kbp Sall-BglII fragment from pT20 was cloned into the SalI site of pUC19 to generate pT30 (Fig. 1). pT30 was used
00-
B
B4
I
c
.Zlol m
C R FIG. 1. Proposed ORFs on the 2.9-kbp SaII-BglI T.ferrooxidans DNA insert in the construct pT30. The single line represents the region of sequence that is presented in Fig. 2. Numerals refer to nucleotides from the Sail site and match those in Fig. 2. The 1.69-kbp ClaI-EcoRI fragment from pT30-D used to construct pT40, pT41, and pT50 is shown by a hatched box (T. ferrooxidans DNA) and a filled box (pUC19 DNA). Proposed ORFs are indicated by open boxes. B, BglII; C, ClaI; R, EcoRI; S, Sall.
_ _ _ _ _ _ _ _ _ _ _ %v _
VOL. 172, 1990
ISOLATION OF THE T. FERROOXIDANS ntrA GENE
GTCGACGCGATTGCCGGCAAGGTGCATCAGGCTACGGCGGTGGGGAG
1
48
4401
CCCGGTCACCTTCACCATGTCCGCAGCGCAGCGCCACGGCTATGGCGACACCCTGGTCTACMGCCTGGCAAGGGCGAGATCGTGCTCACGGGTAACGCCCATCTCTGGCAGAAAAAGAA
168 CGAGATCAGTGGGCAGCAGGTCACTTATTTCCTGCAGACTCAGCAAACCGCCGTCACCGCGGCACCCGGTAAGCGTGTGCAATCGATCTTTTATCCCGCTGCGGCGGGCCGCTCCGCTGG 168 288 CGGTGGGAGGCCATGAGCGAACTGCTCCAGGCGCAATCGCTGTTCAAGTCGTACCGGCGGCGCGTCGTGGTGCGCGATGTCTCGGTGCAGGTGGCCACTGGCGAAGTGGTAGGGCTGCTC -------------M S E L L Q A QS L F K S Y R R RV V V R D V S V Q V A T G E V V G L L 408 GGCCCCAACGGGGCAGGCAAGACCACGACCTTTTACATGATGGTCGGGCTGGTGCGTCCCGATCGCGGTCATATTTTTTTGCAGCAGCGCGATATTACCGCGCTGCCCATGCATGAACGC G P N G A G K T T T F Y M M V G L V R P D R G H I F L Q Q R D I T A L P M H E R 528 GCCCGTATGGGTCTGGGTTATCTCCCTCAGGYSCCTTCCGTTTTCCGCCAGATGAGCGCCGCCGATCGTCCTTGCGGTGCTGGAAACTTTGCCCCTGAGCCCCGTCGAGCGGCAGGAG A R M G L G Y L P Q E P S V F R Q M S A A D N V L A V L E T L P L S P V E R Q E 648 R Q E Q L L S E L H L H A L R D T K G H S L S G G E R R R V E I A R A L A M S P 768 CGTTTTATCCTGCTCGATGAGCCATTCGCTGGTATCGATCCTATTTCCGTGCTGGAAATTCAACGCCTGATCCGGGACCTGCGCGCGCGCGGCATCGGGGTTCTGATAACCGATCATAAC R F I L L D E P F A G I D P I S V L E I Q R L I R D L R A R G I G V L I T D H N 888
GiTG;CGCGAGACGCTTAGGCATTTGCGAGCGCGCCTATATACTGCATGATGGTAAAGTGCTGACGGCGGGCzAGCCCGCAGAAATCGTTGATGiATCCCATGGCTGCGGCAGGTTACCtTGGGA V R E T L G I C E R A Y I L H D G K V L T A G S P E I V
1008
GATCAGTTTCAAATCTGATTTTGGGATATTATGAAACAAGGCTTAGAACTCAAGCTCGGCCAGCATCTGGCCATGACGCCGCAACTGCAACAGGCGATTCGTCTGCTGCAGTTGTC-TACG D F Q I * M K
1368
CCGGATTTTGAATCGCGCAACAGCCGCACCCAGAGCCTGCAGGACTACCTCCGCTGGCAGGCCGACATGACGCACTTCACTGCTGACGAACGCAACATGGCGGAGTTGATCATCGACGCC
Q
D
Q V Y L G
P M V R
D
Q G L E L K L G Q H L A M T P Q L Q Q A I R L L Q L S T Q 1128 GTCGATCTGCAGCAAGAGGTTCAAGGTATGCTGGAGAGCAACCCGCTGCTTGATGAAGAAACCGGAGACGAAGGCGGTGGCGGGCCGATCCCGGAGACCGTGGAACTTCCTTCCGAAGAG V D L Q Q E V Q G M L E S N P L L D E E T G D E G G G G P I P E T V E L P S E E 1248 CGCCAGTTGGATCTGGCGGCTGAGAATATCCTGCCCGACGAGTTGCCGGTAGACAGCCAGTGGGACGACATCTTCGACATGGGAACCTCGGGCTCCGGTAATGGTTCCGACGAGGATCTG R Q L D L A A E N I L P D E L P V D S Q W D D I F D M G T S G S G N G S D E D L P
1488
D
E
S
R
N
S
R
T
Q
S
L
Q
D
Y
L
R
W
Q
A
D
M
H F
T
T
A
E
D
R
M
N
A
L
E
I
D
I
A
E
R
G
Y
L A
D
S
L
E
D
L
A A
T
M
N
Q
V
E
D
A
L
L
A
V
L
L
R
Q
V
D
F
D
P
G
P
V
GGCGCGCGCAATCTCAGCGAGTGCCTCCTGCTGCAGCTGAAGCAGATGGTGGAAAAAGATGACGCGCACGTGTTGCTGGCCCAGCGGATTGTGAAGGACCATCTGCAAGCCTTGGGCCGT G
1728
F
ATTGATGAGCGTGGTTATCTCGCAGACAGCCTGGAAGACCTCGCCGCCACAATGAATGTGCAGGAGGATGCGCTGCTGGCCGTGCTGCTCCGTGTGCAGGACTTCGACCCGCCGGGTGTT I
1608
D
A
R
N
L
S
E
C
L
L
L
Q
L
K
Q
M
V
E
K
D
D
H
A
V
L
L
Q
A
R
I
V
D
K
H
Q A L G R
L
CATGATTACCCGCGTCTGTGCACGGTGCTGGGCGTGGATGAAGCGGCATTGCGTGCGGCGATGGCGCTGATTTCCGCACTGAATCCCAAGCCCGGTGAAGATGTGGGCACCGAGAGCACC H
D
Y
P
R
L
C
T
V
L
G V
D
E
A A
L
R
A A M A L
S
I
A
L
N
P
K
G
P
D
E
G
V
T
S T
E
1848 GAGTATGTGATCCCCGATGTGATCGTGCGCTGGGCCGGCAGCCGCCTGCGCACCGATCTGAMATCCCGAGGCCATGCCCAAATTACGCATCAATCGTCACTATGCCGACATGGCGGGCGGG E Y V I P D V I V R W A G S R L R T D L N P E A M P K L R I N R H Y A D M A G G
1968 AAAGACGCGGCGCATAAATACATCCAGGATCAGCTTAATGAAGCGCGCTGGTTTATCAAAAGCCTGCAAAGCCGCCAAGACACCATATTGAAGGTGGCGCGCGCCATTGTCGAACGGCAG K D A A H K Y I Q D Q L N E A R W F I K S L Q S R Q D T I L K V A R A I V E R Q 2088
AAGGATTTTTTTGCCAACGGGCCCGAATCCATGCGGCCCATGGTTCTGCGTCACATCGCCGATGCAGTGGAAATGCATGAGTCTACGGTGTCGCGGGTCACCAACCAGAAATACATGATC
2208
ACACCCCGCGGCCTCTACGAGTTCAAATATTTTTTTTCCAGTCATGTCGGTACCGACAGCGGGGGCTC TGCGTCGGCCACTGCCATTCGCGCGTTGCTCATCAAGATGACGCAGGCGGAA
2328
GACGCACAACACCCCCTCAGCGACGCGGAAATTGCCCGGGTGCTGGCCGATCAGGGCATTCAGATCGCGCGGCGMACGGTGGCCAAGTACCGCGAAGCGGCCAATGTCCCACCGGCGAGC
2448
CAGCGCCGTCGTTTGTGAGGGCGTGGGGCCAGGGTTTTTTGCTGGCGCCGTTGGCGTTACACTGAAGC CGGGGCGCACCGTGGGACGCGGGTGCGCATCATCACCTGGATAGGAGAACGC
K
D
T
D
P
A
F
R
Q
F A N G P
G
H
L
P
Y
L
E
F
S
E S M R P M V L R H
K
D
A
Y
F
F
S
S
H
V
G
T
I A D A V
D
I A R V L A D Q G
E
S
G
G
E
S
M
A
E
H
S
A
S
T
T V
A
S
R
I
R
A
V
L
L
Q I A R R T V A K Y R E
I
A
T N I
Q K Y M I
K
A
M
N
V
T
Q A
P
P
A
E
S
Q R R R L * 2568 MOA Dl*ALTA YLADACACCTAT DTNVSACGG M Q I T I DSALK T G Q H L D LIGGCRGLTGCRTYAAACAG T D S I K N Y AFCG D E K I G ACGTTACTTCGATCACGTAGCACGCTCAGGTGGT R L G R Y F D H V S N A Q V V 2688 GCTAAAGCACCTCCCCCACGAAAAACTGAGCAACGTCGTGGACATTACGGTCAATGCTCCGGGGCACG TCTTCCATGCGGAGGTGCATGATGCCGATATGTACACGGGCATAGATCT* ITITG0
L
K
H
L
P
H
E
K
L
L
S
K
N
V
V
D
I
T
V
N
A
P
G
H
K
V
F
H
A
E
V
H
D
A
D
M
Y
T
G
I
D
FIG. 2. Nucleotide sequence of the 2.9-kbp SalI-BglII fragment containing T. ferrooxidans ORF1, ntrA, and ORF3, with predicted translation products. Nucleotides are numbered from the first base of the Sall site. Putative ribosome-binding sites are indicated with asterisks. A putative -10 promoter sequence for ntrA is shown by a dashed overline. The inverted repeat sequence at nucleotide 491 is indicated by arrows. These data have been submitted to Genbank and have been assigned the accession number M33831. to generate exonuclease III-shortened clones for sequencing. Plasmid pT30-D was isolated as a result of shortening from the BglII end to nucleotide 2491 (Fig. 1). The T. ferrooxidans ntrA gene together with its putative promoter region on a ClaI (position 802 in Fig. 2)-EcoRI 1.69-kbp fragment from pT30-D was subcloned into the Clal-EcoRI sites of Bluescript KS' and Bluescript SK+ to generate pT40 and pT41, respectively (Fig. 1). The tetracycline resistance gene of the vector pACYC184 was replaced by this 1.69-kbp ClaI-EcoRI fragment, to produce pT50. p184t was a derivative of pACYC184 deleted for the tetracycline resistance gene and a region recently implicated in destabilization of the vector pACYC184 (26). Deletion of the tetracycline resistance gene was necessary, since this was the marker carried on the plasmid pCK3 carrying the Klebsiella pneu-
moniae nifA gene. The K. pneumoniae ntrA gene was cloned into p184t to produce pK50. A 1.2-kbp EcoRI-XhoI fragment from pIMPll (38) was cloned into the EcoRI site of pMC1403 (12) to produce a translational fusion between the T. ferrooxidans nifH and the E. coli lacZ genes. RESULTS Isolation of the T. ferrooxidans ntrA gene. The T. ferrooxidans cosmid bank was transduced into the gas-negative E. coli TH1 mutant; 4 gas-producing colonies were isolated out of 2,000 transductants screened. Transformation of cosmid DNA from each colony into E. coli TH1 confirmed the gas-positive phenotype. Restriction enzyme fragments common to all four cosmid clones were observed (data not
4402
BERGER ET AL.
J. BACTERIOL.
TABLE 2. Percent similarity of predicted ntrA products Bacterium
% Similaritya of predicted ntrA products R. meliK. pneuA. vineT. ferromoniae landii loti oxidans
T. ferrooxidans K. pneumoniae A. vinelandii R. meliloti R. capsulatus
49 44 33 18
50 32 27
36 27
29
a Based on identical amino acids.
shown). One clone, pT3, was chosen for further analysis. E. coli TH1 containing pT20, a subclone of pT3, was able to produce gas when grown on formate. A 3.7-kbp BglHl fragment internal to the pT20 insert hybridized to a T. ferrooxidans chromosomal fragment of the same size, confirming the origin of the cloned fragment (data not shown). Nucleotide sequence of the T. ferrooxidans ntrA gene. Analysis of the sequence data revealed three ORFs (Fig. 1). The central ORF (1,428 bp) coded for an acidic protein with a calculated Mr of 52,927. The predicted amino acid sequence of this ORF was aligned with those of the K. pneumoniae (31), A. vinelandii (32), Rhizobium meliloti (41), and Rhodobacter capsulatus (2, 22) ntrA gene products. Regions of amino acid sequence homology, reported previously for ntrA gene products (32), were observed and identified this ORF as the T. ferrooxidans ntrA gene. The percentage of similarity between the various ntrA gene products is shown in Table 2. A weak ribosome-binding site, GGGA, was present at position 1030 (Fig. 2). There were no clearly identifiable -35 promoter sequences, although a putative -10 promoter sequence (TATATA) at position 921 was detected (Fig. 2). Upstream of the T. ferrooxidans ntrA gene a second ORF, ORFi (nucleotides 300 through 1025, Fig. 1), was identified. The predicted amino acid sequence of the T. ferrooxidans ORF1 product showed 55% amino acid similarity to the predicted product of R. meliloti ORFi (Fig. 3), which is located upstream of the R. meliloti ntrA gene (1). This was considerably greater than the number of identical residues shared between the ntrA gene products of the two bacteria (33%) (Table 2). On the basis of the amino acid sequence similarity to the R. meliloti ORFi product and the occurrence of a strong ribosome-binding sequence (GGGAGG) at Tf Rm
Tf Rm
position 292, the ATG codon at position 300 is the most likely start of the T. ferrooxidans ORFi (Fig. 2). The predicted product of T. ferrooxidans ORFi is 29 amino acids shorter at its N-terminal end than is the R. meliloti ORFi product. The translation products of all three ORFs upstream of nucleotide 300 (Fig. 2) shared no homology with the N terminus of R. meliloti ORF1. T. ferrooxidans ORF1 terminated 12 bp from the proposed start codon of the T. ferrooxidans ntrA gene. A sequence with potential to form a stem-loop RNA secondary structure was present within the coding region of T. ferrooxidans ORFI (position 491, Fig. 2). This stem-loop structure (AG, -18.6 kcal [ca. -77.8 kJ]/mol [42]) was preceded by 7 U's at position 480 (Fig. 2). No ORF equivalent to R. meliloti ORFO (1) was detected upstream of T. ferrooxidans ORF1. A third ORF, called ORF3, was located downstream of the T. ferrooxidans ntrA gene (Fig. 1). The proposed ATG start codon of ORF3 was at position 2569 (Fig. 2) and was preceded by a consensus ribosome-binding site (AGGAG) at position 2558 (Fig. 2), and ORF3 extended beyond the BgIII cloning site at position 2804 (Fig. 2). Alignment of the predicted 75 N-terminal amino acids with the partial sequences of ORF3 products from A. vinelandii (32), K. pneumoniae (31), and R. meliloti (41) showed 38, 29, and 20% amino acid similarity, respectively. Expression of the T. ferrooxidans ntrA gene in E. coli. E. coli TH1 cells containing the T. ferrooxidans ntrA gene cloned in both orientations with respect to the Bluescript vector lacZ gene (pT40, pT41) produced gas and reduced benzylviologen when grown with formate (Table 3). We concluded that the T. ferrooxidans ntrA gene was expressed from its own regulatory region in E. coli. Biological activity of the T. ferrooxidans ntrA gene product in E. coli. Experiments were carried out to determine whether the pattern of formate and nitrate regulation of the formate hydrogenlyase pathway were the same in the mutant E. coli TH1 strain containing the cloned T. ferrooxidans ntrA gene as in the parent strain, E. coli YMC10. E. coli cells grown anaerobically on glucose produce formate, which is metabolized either via the fermentative formate hydrogenlyase pathway or via the respiratory nitrate-linked route (reviewed in reference 44). The gas-producing formate hydrogenlyase pathway is induced by formate and repressed by nitrate (7, 48). Reduction of benzylviologen is characteristic of the formate hydrogenlyase pathway (15, 29). E. coli TH1
MSELLQAQSLFKSYRRRVVVRDVSVQVATGE * * * **** * ** **I * ** MQIPFLHKRKRGKKPSAAAAAARAVDKARYDGTLIARGLTKSYRSRRVVNGVSLVVRRGE
31
VVGLLGPNGAGKTTTFYMMVGLVRPDRGHIFLQQRDITALPMHERARMGLGYLPQEPSVF ************* *** *** * * * 11 *1* ** *** *t****** *1* AVGLLGPNGAGKTTCFYMITGLVPVDEGSIEINGNDVTTMPMYRRARLGVGYLPQEASIF
91
60
120 151
Rm
RQMSAADNVLAVLETLPLSPVERQERQEQLLSELHLHALRDTKGHSLSGGERRRVEIARA * ** * * ** * ****I** ** *** RGLTVEDNIRAVLEVHDENVDRRESKLNDLLGEFSITHLRKSPAIALSGGERRRLEIARA
Tf
LAMSPRFILLDEPFAGIDPISVLEIQRLIRDLRARGIGVLITDHNVRETLGICERAYILH
211
Rm
I** *1* * *****************I I****I* LATDPTFMLLDEPFAGVDPISVADIQALVRHLTSRGIGVLITDHNVRETLGLIDRAYIIH
Tf
DGKVLTAGSPQEIVDDPMVRQVYLGDQFQI
241
Tf
** * * ********I*****
II***I** Rmn*R***T*
I****I* AGEVLTHGRANDIVTNPDVRRLYLGDNFSL
180
240 270
FIG. 3. Alignment of T. ferrooxidans ORF1 with R. meliloti ORF1. Alignment was made with the IBM XT computer Genepro (version 4.1) protein alignment subroutine. Identical amino acids are marked with asterisks, and conservative substitutions are marked with vertical bars. These are based on the following groups: I, L, V, and M; D and E; K, R, and H; Q and N; S and T; G and A; and F and Y. Regions with similarity to the nucleotide-binding pocket are underlined.
VOL. 172, 1990
ISOLATION OF THE T. FERROOXIDANS ntrA GENE
4403
TABLE 3. Phenotypic tests for formate hydrogenlyase activity 30 mM formate Benzylviologen reduction
40 mM nitrate Benzylviologen reduction
Strainb
Relevant genotype
Gas formation
TH1(pT40) TH1(pT41) TH1(Bluescript SK+) TH1(p184t) TH1(pT50) TH1(pK50) YMC10(pl84t) FM911(p184t) FM911(pT50) FM911(pK50)
T. ferrooxidans ntrA T. ferrooxidans ntrA AntrA AntrA T. ferrooxidans ntrA K. pneumoniae ntrA E. coli ntrA E. coli ntrA AfdhF T. ferrooxidans ntrA AfdhF K. pneumoniae ntrA LfdhF
+ + -
+ + -
ND ND ND
ND ND ND
+ + +
+ + +
-
-
Gas formation
a Gas production was scored positive (+) on formation of a gas pocket around a colony growing beneath a TGYEP agar overlay. Benzylviologen reduction was tested by the benzylviologen overlay technique (29). Reduction of benzylviologen was scored positive (+) on the conversion of the benzylviologen dye from colorless to purple within 1 min of overlaying colonies grown anaerobically overnight on TGYEP agar. ND, Not determined. b Strains were grown anaerobically on TGYEP agar, pH 6.5 (5), containing either 30 mM formate or 40 mM nitrate.
containing the plasmid vector p184t was unable to produce reduce benzylviologen when grown with formate or nitrate (Table 3). In contrast, E. coli YMC10 and TH1 containing either the T. ferrooxidans or the K. pneumoniae ntrA gene were gas positive and were able to reduce benzylviologen when grown with formate (Table 3). In the presence of nitrate, both gas production and benzylviologen reduction were repressed (Table 3). The inability of the E. coli fdhF mutant FM911 containing either the T. ferrooxidans or the K. pneumoniae ntrA gene to produce gas or reduce benzylviologen indicated that a functional fdhF gene was required for ntrA complementation of formate hydrogenlyase phenotypes (Table 3). Several clones of the T. ferrooxidans ntrA gene (pT3, pT20, pT30, pT40, pT41, and pT50) were transformed into E. coli TH1 and plated onto minimal medium and arginine. The amount of growth obtained was equivalent to that shown by E. coli TH1 transformed with vector DNA and much less than the parent E. coli YMC10 strain (data not shown). fdhF-lacZ and nil-lacZ fusions were expressed in the presence of the T. ferrooxidans ntrA gene. The biological activity of the T. ferrooxidans ntrA gene product in E. coli was tested by investigation of the expression of translational fusions between ,-galactosidase and the E. coli fdhF gene and two nifH genes. Positive controls were the cloned K. pneumoniae ntrA gene and the parent E. coli YMC10 strain, which has a chromosomal ntrA gene. When E. coli TH1(pBN208) cells containing the T. ferrooxidans ntrA gene on a compatible plasmid were grown anaerobically with formate, the expression of the fdhF-lacZ fusion was increased sevenfold above the basal level of expression obtained in the absence of an ntrA gene (Table 4). A similar increase in P-galactosidase activity was obtained in the presence of either the K. pneumoniae or the E. coli ntrA gas or
(Table 4). P-Galactosidase activity was repressed when the cultures were grown anaerobically with nitrate (Table 4). Low basal levels of 3-galactosidase activity were obtained from E. coli TH1 containing the vector p184t and compatible plasmid vectors carrying a constitutively expressed K. pneumoniae nifA gene (pCK3) and either the K. pneumoniae nifl-lacZ fusion (pMB1) or the T. ferrooxidans nifH-lacZ fusion (pHlac) when the bacteria were grown anaerobically in nitrogen-limited medium (Table 5). Replacemnent of the vector p184t by the plasmid pT50 (carrying the T. ferrooxidans ntrA gene) increased expression of both nifH-iacZ fusions more than 50-fold above basal levels (Table 5). The cloned T. ferrooxidans ntrA gene product was less efficient at directing expression of the K. pneumoniae nifH-lacZ fusion than was the cloned K. pneumoniae ntrA gene product or the chromosomally encoded E. coli ntrA gene product (Table 5). All three ntrA gene products directed expression of the T. ferrooxidans nifH-1acZ fusion to similar levels of gene
3-galactosidase activity (Table 5). DISCUSSION We isolated the T. ferrooxidans ntrA gene by complementation of an E. coli ntrA mutant for u54-dependent expression of the formate hydrogenlyase pathway. An agar overlay technique was used as a simple and effective procedure for screening a large number of individual colonies for the ability to produce gas. The cloned T. ferrooxidans ntrA gene was also able to complement an E. coli ntrA mutant for the ability to reduce benzylviologen; both activities were repressed by nitrate, a characteristic of the formate hydrogenlyase pathway.
The predicted protein product of the T. ferrooxidans ntrA acidic polypeptide of 475 amino acids with a
gene was an
TABLE 4. Effect of T. ferrooxidans ntrA on expression of ,-galactosidase activity from a fdhF-lacZ fusion plasmid in E. coli
f3-Galactosidase activity (Miller units) Straina
TH1(pBN208, pT50) TH1(pBN208, pACYC184) TH1(pBN208, pFB71) YMC10(pBN208, pACYC184) a
Relevant genotype
T. ferrooxidans ntrA cF(fdhF-1acZ) 4¢(fdhF-lacZ)
K. pneumoniae ntrA 4(fdhF-iacZ) E. coli ntrA '1(fdhF-iacZ)
Strains were grown anaerobically in TGYEP medium (pH 6.5) with 0.2% (wt/vol) glutamine.
in cells grown on glucose plus: 30 mM formate
40 mM nitrate
2,267
322
180 102
2,514
260 359
3,761
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BERGER ET AL.
TABLE 5. Effect of T. ferrooxidans ntrA on expression of 3-galactosidase activity from nifH-lacZ fusion plasmids in the presence of the K. pneumoniae nifA gene in E. coli Strain"
Relevant genotype
T. ferrooxidans ntrA O(K. pneumoniae nafH-lacZ) K. pneumoniae nifA TH1(pCK3, pMB1, pT50) 4O(K. pneumoniae nifl-lacZ) K. pneumoniae nifA TH1(pCK3, pMB1, p184t) K. pneumoniae ntrA I(K. pneumoniae nifHf-lacZ) K. pneumoniae nifA TH1(pCK3, pMB1, pK50) E. coli ntrA 4D(K. pneumoniae niff1-lacZ) K. pneumoniae nifA YMC10(pCK3, pMB1, p184t) T. ferrooxidans ntrA W(T. ferrooxidans nfWH-lacZ) K. pneumoniae nifA TH1(pCK3, pHlac, pT5O) 4(T. ferrooxidans nif-lacZ) K. pneumoniae niUA TH1(pCK3, pHlac, p184t) K. pneumoniae ntrA 4(T. ferrooxidans njfH-lacZ) K. pneumoniae nifA TH1(pCK3, pHlac, pK50) E. coli ntrA O(T. ferrooxidans nifH-lacZ) K. pneumoniae nifA YMC1O(pCK3, pHlac, p184t) a Strains were grown anaerobically in nitrogen-limiting conditions as described by Ow and Ausubel (35).
calculated Mr of 52,927. The term &'4 reflects the Mr of the product of the ntrA gene of enteric bacteria; however, the Mr of ntrA gene products vary considerably. From previously published nucleotide sequences the Mrs of other ntrA gene products have been calculated as 53,926, 56,916, 57,814, and 46,328 for K. pneumoniae (31), A. vinelandii (32), R. meliloti (41), and R. capsulatus (2, 22), respectively. The regions of conserved amino acids previously reported for the NtrA proteins of R. meliloti, K. pneumoniae, and A. vinelandii (32) were also present in the sequence of the T. ferrooxidans ntrA gene product. A striking feature is the conservation of the sequence -ARRTVAKYR- near the C terminus of the NtrA proteins from all five organisms. On the basis of amino acid sequence conservation, it is possible to divide the NtrA proteins for which sequence information is available into three groups. The NtrA proteins of A. vinelandii, K. pneumoniae, and T. ferrooxidans are clearly more closely related to each other than to the NtrA proteins of R. meliloti and R. capsulatus (Table 2). These two proteins share relatively little sequence similarity with each other and appear to belong to two separate groups. Biological activity of the cloned T. ferrooxidans NtrA protein was demonstrated in an E. coli ntrA mutant by an increase in expression offdhF-lacZ and nipfl-lacZ fusions. T. ferrooxidans NtrA was able to promote expression from both the E. colifdhF promoter and the T. ferrooxidans nifH promoter to levels equivalent to that achieved by either the E. coli or the K. pneumoniae NtrA protein. The weak expression from the K. pneumoniae nifH promoter directed by the T. ferrooxidans NtrA could be due to either poor recognition of the naifH promoter by the heterologous T. ferrooxidans NtrA complexed to the E. coli core RNA polymerase or inefficient interaction between the T. ferrooxidans NtrA protein and the K. pneumoniae NifA protein. However, strict comparisons of NtrA efficiency should not be made, because the nttA genes are expressed at different levels and expression of each fusion is dependent on NifA, NtrA, and core RNA polymerase from different bacteria. The reason why the cloned T. ferrooxidans ntrA gene was unable to complement the E. coli ntrA mutant for growth on minimal medium plus arginine is unknown. This complementation has been demonstrated for the cloned K. pneumoniae (14), A. vinelandii (45), and Pseudomonas putida ntrA genes (25). A possible explanation is that arginine utilization is tested on minimal medium and is essential for growth, whereas gas production is tested on a rich medium and is not required for growth. T. ferrooxidans NtrA may promote transcription from the E. coli &-4-dependent arginine utilization promoter(s) too weakly to permit growth on arginine. However, the sensitivity of detecting gas bubble formation enabled identification of transductants in which transcription
activity (M-Galactosidase (Miller units) 1,484 28 13,333 10,113
1,642 32 2,042 2,044
of the E. colifdhF promoter was directed by T. ferrooxidans NtrA. Immediately upstream of the T. ferrooxidans ntrA gene an ORF equivalent to the ORFi located upstream of the R. meliloti ntrA gene was detected. The linkage of ORFi to ntrA has been reported also to occur in K. pneumoniae (1, 31) and S. typhimurium (1) and therefore appears to be a feature of bacteria of very different physiological types. The biological function of ORF1 and the reason for its linkage to the ntrA gene are uncertain. Transcription of ORFi and ntrA was reported to be uncoupled in R. meliloti (1). The observation that subclones of the T. ferrooxidans ntrA gene from which most of ORF1 had been deleted (pT40 and pT41) were able to complement the E. coli ntrA mutant is evidence that transcription of the T. ferrooxidans ntrA gene is independent of transcription through ORF1. In vitro studies on CfS4_ dependent promoters have indicated that NRl-activated transcription from the S. typhimurium ginA (20), E. coli glnA (21), K. pneumoniae n#fLA, and K. pneumoniae npfHDK (46) promoters requires only the cr54-RNA polymerase holoenzyme. Involvement of the ORF1 product in the function of Cr54 iS therefore unlikely. Albright et al. (1) tried unsuccessfully to insertionally inactivate ORF1 by using transposon mutagenesis, and they suggested that ORFi may code for an essential housekeeping protein. The predicted amino acid sequence of T. ferrooxidans ORF was aligned with the R. meliloti protein, the only other ORFi for which a complete sequence has been published (Fig. 3). The two sequences have a high level of similarity over their entire length, although the N-terminal end of the predicted T. ferrooxidans protein was 29 amino acids shorter. The amino acid sequence of R. melioti ORFi was compared with that of a family of ATP-binding proteins (19), and two regions that had homology to an ATP-binding pocket were identified (1). These are also conserved in the predicted T.ferrooxidans ORF1 protein (Fig. 3). The spacing between T. ferrooxidans ORF1 and the ntrA gene was different from that of R. meliloti. R. meliloti ORF1 terminated 176 bp upstream of the ntrA gene, whereas T. ferrooxidans ORFi terminated only 12 bp from the start codon of the ntrA gene. Since expression of the T. ferrooxidans ntrA gene was independent of orientation and most of ORF1, the ntrA promoter must be located within the carboxy-terminal coding region of ORF1. The existence of a putative region of RNA stem-loop secondary structure (A&G, -18.6 kcal [ca. -77.8 kJ]/mol [42]) preceded by seven U's within the coding region of T. ferrooxidans ORF1 (Fig. 2) is interesting, since a similar region of potential secondary structure (AG, -17.1 kcal [ca. -71.5 kJ]/mol [42]) preceded by an A+U-rich region is also present in the R. meliloti ORF1 nucleotide sequence (position 635 in Fig. 1 of reference 1). These
VOL. 172, 1990
regions of potential secondary structure are situated within an area of low amino acid conservation on the C-terminal side of the first component of the ATP-binding pocket of the two predicted ORF1 products. A similar region of secondary structure has been shown to reduce translation efficiency in another bacterial system (27). The identification of an ntrA gene in the chemolithoautotrophic bacterium T. ferrooxidans and the ability of the product of this gene to direct expression from the T. ferrooxidans nifH promoter region in E. coli is interesting, since no Uf4 consensus promoter has been identified upstream of the T. ferrooxidans ginA gene (40), which codes for a key nitrogen metabolism enzyme, glutamine synthetase. The predicted amino acid sequences of five ntrA genes have now been determined, and these data provide a basis for modelling of the general mechanism whereby oS4 interacts with both the core RNA polymerase and the consensus GG-10 bp-GC promoter. ACKNOWLEDGMENTS We thank R. Ramasar for providing the T. ferrooxidans ATCC 3020 cosmid gene bank, C. Kennedy for plasmids pCK3 and pMB1, F. Ausubel for strain TH1 and plasmid pFB71, and A. Bock for strain FM911 and plasmid pBN208. LITERATURE CITED 1. Albright, L. M., C. W. Ronson, B. T. Nixon, and F. M. Ausubel. 1989. Identification of a gene linked to Rhizobium meliloti ntrA whose product is homologous to a family of ATP-binding proteins. J. Bacteriol. 171:1932-1941. 2. Alias, A., F. J. Cejudo, J. Chabert, J. C. Willison, and P. M. Vignais. 1989. Nucleotide sequence of the wild-type and mutant nifr4 (ntrA) genes of Rhodobacter capsulatus-identification of an essential glycine residue. Nucleic Acids Res. 13:5377. 3. Backman, K., Y. M. Chen, and B. Mgasanik. 1981. Physical and genetic characterization of the ginA-glnG region of the Escherichia coli chromosome. Proc. Natl. Acad. Sci. USA 78:3743-3747. 4. Barros, M. E., D. E. Rawlings, and D. R. Woods. 1986. Purification and regulation of a cloned Thiobacillus ferrooxidans glutamine synthetase. J. Gen. Microbiol. 132:1989-1995. 5. Begg, I. A., J. N. Whyte, and B. A. Haddock. 1977. The identification of mutants of Escherichia coli deficient in formate dehydrogenase and nitrate reductase activities using dye indicator plates. FEMS Microbiol. Lett. 2:47-50. 6. Birkmann, A., R. G. Sawers, and A. Bock. 1987. Involvement of the ntrA gene product in the anaerobic metabolism of Escherichia coli. Mol. Gen. Genet. 210:535-542. 7. Birkmann, A., F. Zinoni, G. Sawers, and A. Bock. 1987. Factors affecting transcriptional regulation of the formate-hydrogenlyase pathway of Escherichia coli. Arch. Microbiol. 148:44-51. 8. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 9. Buck, M., H. Khan, and R. Dixon. 1985. Site directed mutagenesis of the Klebsiella pneumoniae nifL and nifH promoters and in vivo analysis of promoter activity. Nucleic Acids Res. 13: 7621-7638. 10. Buck, M., S. Miller, M. Drummond, and R. Dixon. 1986. Upstream activator sequences are present in the promoters of nitrogen fixation genes. Nature (London) 320:374-378. 11. Cannon, F. C., R. A. Dixon, J. R. Postgate, and S. B. Primrose. 1974. Chromosomal integration of Klebsiella nitrogen fixation genes in Escherichia coli. J. Gen. Microbiol. 80:227-239. 12. Casadaban, M. J., A. Martinez-Arias, S. K. Shapira, and J. Chen. 1980. 1-Galactosidase gene fusions for analyzing gene expression in Echerichia coli and yeast. Methods Enzymol. 100:293-308. 13. Chang, A. C. Y., and S. N. Cohen. 1978. Construction and characterization of amplifiable multicopy DNA cloning vehicles
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4326-4333. 26. Kolot, M. N., M. V. Kashlev, A. I. Gragerov, and I. A. Khmel. 1989. Stability of the pBR322 plasmid is affected by the promoter region of the tetracycline-resistance gene. Gene 75: 335-339. 27. Kubo, M., and T. Imanaka. 1989. mRNA secondary structure in an open reading frame reduces translation efficiency in Bacillus subtilis. J. Bacteriol. 171:4080-4082. 28. Kustu, S., E. Santero, J. Keener, D. Popham, and D. Weiss. 1989. Expression of o-J5 (ntrA)-dependent genes is probably united by a common mechanism. Microbiol. Rev. 53:367-376. 29. Mandrand-Berthelot, M. A., M. Y. K. Wee, and B. A. Haddock. 1978. An improved method for the identification and characterization of mutants of Escherichia coli deficient in formate dehydrogenase activity. FEMS Microbiol. Lett. 4:37-40. 30. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 31. Merrick, M. J., and J. R. Gibbons. 1985. The nucleotide sequence of the nitrogen regulation gene ntrA of Klebsiella pneumoniae and comparison with conserved features in bacterial sigma factors. Nucleic Acids Res. 13:7607-7620. 32. Merrick, M. J., J. R. Gibbons, and A. Toukdarian. 1987. The nucleotide sequence of the sigma factor gene ntrA (rpoN) of Azotobacter vinelandii-analysis of conserved sequences in
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