INTRODUCTION

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Motile bacteria are capable of sensing changes in the concentrations of certain chemicals that can be attractants or repellents to them (3, 5, 45). This is done by means of a chemosensory system which regulates motility by controlling the direction of flagellar rotation (34, 35, 45). Binding of an attractant or repellent ligand to a specific site in the periplasmic domain of a methyl-accepting chemotaxis protein (MCP) leads to a conformational change of its cytoplasmic domain, which is transmitted to the flagellar motor by means of a series of phosphorylation reactions (9, 18, 22). Adaptation of the microorganisms to the new environmental condition is achieved by increasing methylation or demethylation of specific glutamic acid residues in the cytoplasmic domain of the receptor (5, 16, 45).

Most of the industrially important bacteria that participate in bioleaching of minerals, such as the chemolithoautotrophic, acidophilic Thiobacillus ferrooxidans, Leptospirillum ferrooxidans, and Thiobacillus thiooxidans, are motile by means of flagella (14, 37). Therefore, they should possess chemotactic responses to sense and adapt to their environment. This is specially important since the microorganisms have to adhere to specific sites on the surface of the minerals which they will oxidize to obtain their energy. This attachment in turn could depend on the sensing by the microorganisms of a dissolved ion concentration gradient present in the immediate vicinity of the solid. As suggested by Sand et al. (42), this dissolution would probably be controlled by electrochemical processes (such as generation of an anode and a cathode due to charge imbalances, faults, and electron gaps, etc.).

We have previously demonstrated that, in fact, L. ferrooxidans possesses a chemotactic response to aspartate and Ni²⁺ which is opposite to that observed for Escherichia coli, since for the former aspartate acts as a repellent and Ni²⁺ acts as an attractant. In addition, Fe²⁺ is also an attractant for L. ferrooxidans (1, 2). On the other hand, a chemotactic response of T. ferrooxidans toward thiosulfate has been reported elsewhere (10).

L. ferrooxidans and T. ferrooxidans also possess methylatable proteins in the 60- to 80-kDa molecular mass range (1). The methylation of these proteins from L. ferrooxidans increases in the presence of Ni²⁺ and decreases in the presence of aspartate (2). Other experiments showed that the in vitro methylation of these putative L. ferrooxidans MCPs was stimulated in the presence of a membrane-free extract from E. coli. Interestingly, this response followed the pattern expected for L. ferrooxidans, i.e., increased methylation by Ni²⁺ and demethylation by aspartate (2). These results suggested to us the existence of sensory transducers in L. ferrooxidans having a common methylation domain with the E. coli methyl-accepting chemotaxis proteins.

In the present report, we have cloned and sequenced an L. ferrooxidans 2,262-bp chromosomal DNA fragment which showed a region with a high degree of identity with several MCPs genes from different microorganisms. The cloned DNA fragment was expressed both in vivo in E. coli and in vitro by using an E. coli DNA-dependent system in which transcription and translation were coupled.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and growth conditions. E. coli RP4372 (26), DH5 (46) HCB721 (48), and D-10 (47) were cultivated aerobically in Luria-Bertani medium at 37°C. The strains harboring plasmids were grown in the presence of ampicillin (100 µg/ml). Plasmid pNT201, in which the tar gene is under the control of P_tac (9), was kindly supplied by R. Bourret, California Institute of Technology, Pasadena, Calif. pNT201, pUC18, and pGEM-3Z and the recombinant plasmids were all maintained in E. coli DH5. L. ferrooxidans Z2, kindly supplied to us by A. Harrison, Jr., University of Missouri, Columbia, Mo., was grown at 30°C in modified Mackintosh medium (33).

Swarm assays. Cells were grown to the mid-exponential phase in tryptone broth (1% tryptone, 0.5% NaCl) supplemented with ampicillin when necessary. Tryptone swarm plates were 0.3% agar in the same broth but without antibiotic (49). A 2-µl aliquot of the culture (approximately 10⁶ cells) was placed on the surface of a swarm plate near its center, and the plate was incubated at 31°C in a humid chamber. The radial displacements of the microorganisms were determined after 40 h in duplicate assays.

pLf13 plasmid construction. We employed Southern blotting to analyze the chromosomal DNA from L. ferrooxidans using a 719-bp probe coding for part of the tar gene, including the methylated amino acid residues present in two regions of the Tar cytoplasmic domain (from amino acids 255 to 494). When the chromosomal DNA from L. ferrooxidans Z2 was digested with HindIII, a 3.5-kb DNA fragment hybridizing with the probe was obtained. This fragment was cloned into the vector pUC18 yielding the pLf3.5 recombinant plasmid. The pLf3.5 plasmid was digested with EcoRI enzyme, resulting in a 2.3-kb fragment still hybridizing with the probe. This fragment was subcloned into the expression vector pGEM-3Z to yield plasmid pLf13. The orientation of the 2.3-kb fragment in pLf13 was specified by digestion with HindIII.

In vitro DNA-dependent system of gene expression. The in vitro protein synthesis was done with a complete system for transcription and translation, with plasmid DNA as the template (25, 39). The DNA plasmids employed were purified by a CsCl gradient (41). The assay was done in the presence of 20 µCi of a mixture of both [³⁵S]methionine (70%) and [³⁵S]cysteine (25%) (specific activity, 1,190 Ci/mmol), 200 µg of E. coli D-10 S-30 proteins, and 3 µg of DNA template in a 30-µl final volume. The reaction mixture was incubated for 50 min at 37°C, supplemented with 10 µl of L-methionine (8 mg/ml), and then it was incubated for another 5 min.

SDS-PAGE of proteins. E. coli cytoplasmic fraction was prepared essentially as described by Booth and Curtis (8). The cytoplasmic membrane fractions (between 10 to 20 µg of total protein) from each strain were boiled in Laemmli's buffer for 5 min and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To analyze the polypeptides synthesized in the in vitro DNA-dependent system, 4 µl from the reaction mixture was added to 4 µl of Laemmli's buffer, and the new mixture was boiled for 5 min. Proteins were separated on an SDS-10% PAGE gel as described by Laemmli (30), followed by Coomassie blue staining or by staining followed by fluorography and autoradiography (25).

Immunoprecipitation assay. The Tar protein was obtained by using E. coli HCB721 (48) carrying plasmid pNT201. The overproduced Tar protein was separated by SDS-PAGE of the E. coli membrane fractions (17), followed by its excision from the gels. To obtain a polyclonal antiserum specific against Tar, 100 µg of the Tar protein was mixed with complete Freund's adjuvant (2 ml) and the mixture was injected into a New Zealand White rabbit subcutaneously. Two months later, Tar protein (100 µg) was mixed with incomplete Freund's adjuvant (2 ml) and the mixture was again injected subcutaneously into the rabbit. The final serum was obtained 17 days later.

DNA sequencing. The L. ferrooxidans DNA fragment contained in the pLf13 recombinant plasmid was sequenced by the dideoxy chain termination method (43) with the Sequenase version 2.0 kit (U.S. Biochemicals Co.). Nucleotide sequences for both strands were determined. For DNA sequencing, we employed the T7 and SP6 vector primers, as well as synthetic oligonucleotide primers constructed on the basis of the sequence being obtained. Computer analysis of the nucleotide sequence was performed with the PC-Gene program. Homology searches were conducted against the GenBank, EMBL, DDBJ, and PDB data- bases by using the BLAST (4) and FASTA (38) programs.

Nucleotide sequence accession number. The nucleotide sequence of the 2,262-bp DNA region containing the lcrI gene is available in the EMBL database under accession no. AJ002392.

	RESULTS

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Sequence of the lcrI gene and properties of the LcrI protein. The L. ferrooxidans DNA fragment contained in the recombinant plasmid pLf13 was sequenced in both strands. One complete open reading frame (ORF; LcrI) was found in the 2,262-bp EcoRI/HindIII insert of pLf13 by codon analysis, starting with an AUG codon in nucleotide 412 and stopping with a UGA codon in nucleotide 2,143. Identity searching in databases with the BLAST and FASTA programs indicated a strong similarity of this ORF to several chemotactic receptor genes. Therefore, it was called lcrI (Leptospirillum chemotactic receptor I). It was preceded by a plausible ribosome binding site with an AAAGAAAG core located upstream from the initiating AUG codon (nucleotides 397 through 404). Upstream of this ribosome binding site, a ²⁸-like promoter sequence (TAAA N₁₅ CTCGAACT) similar to the consensus sequence for ²⁸ (TAAA N₁₅ GCCGATAA) (20) was present. This promoter type is specific for the expression of flagellar genes, motility genes, and chemotactic genes in several microorganisms. A plausible E. coli ⁷⁰-like promoter sequence overlapping with the ²⁸-like promoter sequence could also be considered (nucleotides 196 through 201 and 218 through 223 for the 35 and 10 regions, respectively). Downstream of the translational stop codon of LcrI, we could not find an inverted repeat that could function as a rho-independent transcription terminator. In addition, there was another ORF (ORF2) which could be cotranscribed with lcrI. This started with a GTG codon at nucleotide 2,169 and was interrupted on the 3' end by the restriction site used to clone this DNA. This ORF2 was also preceded by a plausible ribosome binding site.

View larger version (56K): [in this window] [in a new window]   FIG. 1.   Analysis of the amino acid sequence of LcrI from L. ferrooxidans. (A) Comparison of the amino acid sequences of Tar from E. coli (27) and LcrI from L. ferrooxidans. In general, the sequences are shown in italic letters. The following features are indicated by normal and underlined letters: two potential membrane-spanning regions in LcrI (residues 8 through 26 and 164 through 180) and two membrane-spanning regions in Tar (residues 7 through 37 and 189 through 212), the signaling domain or HCD from LcrI and Tar (residues 444 through 488 and 361 through 405, respectively), putative methylation regions in LcrI (residues 213 through 221 and 552 through 560), and the methylated residues in Tar (Q296, E302, Q309, and E491). Asterisks, amino acids identical in both proteins. (B) Comparison of the amino acid sequence of the putative HCD region present in LcrI from L. ferrooxidans with the amino acid sequences of HCD regions of chemoreceptors from different microorganisms. The HCD amino acid sequences (45 amino acids) of the following proteins were aligned (sources indicated in parentheses): Tar, Tap, and Tsr (27) and Trg (7) from E. coli; Tas and Tse (11) from E. aerogenes; Tcp (50) from Salmonella typhimurium; PctA (28) from Pseudomonas aeruginosa; DcrA (15), DcrH (13), and DcrI (12) from D. vulgaris Hildenborough; MCPA (19) from B. subtilis; FrzCD (36) from Myxococcus xanthus; HtrI (51), Htp3, Htp4, Htp5, and Htp6 (40) from H. salinarium; vHtrII from Halobacterium vallismortis; and pHtrII (44) from N. pharaonis. Underlining, amino acids identical to LcrI; asterisks, residues conserved in all of the MCPs; dots, residues conserved in most MCPs, including LcrI. In the right columns, the percentages of identity (I) and similarity (S) with LcrI are shown.

View larger version (8K): [in this window] [in a new window]   FIG. 2.   Comparison of the consensus sequences containing the methylatable residues of chemoreceptors from different microorganisms with putative methylation sites from LcrI. The possible methylation sites in LcrI (residues 213 through 221 and 552 through 560) are compared with the consensus sites of methylation present in the MCPs from E. coli (27) and B. subtilis (19) and with those proposed for DcrA and DcrH from D. vulgaris Hildenborough (13, 15). The methylatable residues and the postulated ones in LcrI (residues 217 and 556) are indicated in boldface.

Expression of pLf13 in vitro. To study the expression of the putative mcp gene from L. ferrooxidans, we used a DNA-dependent system for in vitro synthesis of the encoded proteins. Under our conditions, the E. coli Tar protein (Fig. 3A, arrowhead in lane f) and -lactamase (arrow in Fig. 3A) were synthesized from plasmid pNT201. The expression of pLf13 DNA resulted in the synthesis of the following major proteins: a 58-kDa band (asterisk) and bands of 51, 39, and 30 kDa (Fig. 3A, lane c). The 30-kDa protein band corresponded to the -lactamase encoded by the expression vectors pGEM-3Z (lane b) and pUC18 (lane e). When plasmid pLf3.5 was employed in this assay (lane d), the same protein bands of 58, 51, 39, and 30 kDa were obtained. In addition, a 62-kDa protein band was also synthesized. This bigger polypeptide could correspond to a product resulting from transcription-translation of a different encoded gene (see below).

View larger version (40K): [in this window] [in a new window]   FIG. 3.   In vitro expression of pLf13 and pLf3.5 in a DNA-dependent system and immunoprecipitation of the products synthesized with serum against Tar from E. coli. (A) Proteins synthesized in the absence of DNA (lane a) or in the presence of 3 µg of pGEM-3Z (lane b), pLf13 (lane c), pLf3.5 (lane d), pUC18 (lane e), or pNT201 (lane f). After incubation of the reaction mixtures in the presence of [35S]methionine-cysteine, the products were separated by SDS-PAGE, followed by autoradiography as described in Materials and Methods. (B) Proteins synthesized in vitro from pLf13 (lanes a and b) or from pNT201 (lane c) after incubation of the reaction mixtures of the DNA-dependent system in the presence of [35S]methionine-cysteine and immunoprecipitation of the synthesized products with the preimmune serum (lane a) or with the serum against Tar (lane b and c). When pLf13 was used, the products from five pooled reaction mixtures were immunoprecipitated. The radioactive proteins were then separated by SDS-PAGE, followed by autoradiography. Arrowhead, migrating position of the E. coli Tar protein (the arrow indicates the position of the -lactamase); asterisk, position of a 58-kDa protein from L. ferrooxidans mentioned in the text. Numbers to the left of the gels are molecular mass markers (in kilodaltons).

Expression of pLf13 in vivo. To establish whether the cloned gene from L. ferrooxidans was expressed in E. coli, cells harboring the plasmids of interest were grown and their cytoplasmic membrane fractions were obtained and analyzed by SDS-PAGE followed by Coomassie blue staining as shown in Fig. 4. As a control, we employed the E. coli DH5 strain possessing the normal chemotactic system (lane a) and the RP4372 strain, which is entirely lacking in chemotactic receptors (lane b). Both of these strains showed very faint bands in the 60-kDa region, and it was not possible to distinguish between them under the conditions employed. When the RP4372 strain containing the plasmid pNT201 with the tar gene was used, a very faint Tar protein band was seen (arrowhead in lane c). However, when the expression of this plasmid was induced by isopropyl--D-thiogalactopyranoside (IPTG), a great amount of Tar was present in the cytoplasmic membrane fraction (arrowhead in lane d). When the E. coli RP4372 strain transformed with plasmid pLf13 was employed, a 58-kDa band was observed in the cytoplasmic membrane (asterisk in lane e), in agreement with the 58-kDa protein synthesized in vitro. The expression of this protein was not stimulated in the presence of IPTG (lane f), suggesting that the 2.3-kb fragment has its own promoter and that it is functional in E. coli. The E. coli strain containing only the pGEM-3Z vector did not show the 58-kDa protein in cytoplasmic membranes either in the absence or in the presence of IPTG (lanes g and h, respectively). These results strongly suggest the in vivo expression of a 58-kDa protein from L. ferrooxidans in E. coli and its association with the cytoplasmic membrane from E. coli.

View larger version (83K): [in this window] [in a new window]   FIG. 4.   Detection of proteins coded by pLf13 in the cytoplasmic membrane fraction from E. coli. E. coli strains were grown for 4 h in the presence or in the absence of 1 mM IPTG added at the half-logarithmic phase of growth. Proteins present in the cytoplasmic membrane fractions of each strain were separated by SDS-PAGE and stained with Coomassie blue. The bacterial strains employed were DH5 (lane a), RP4372 (lane b), RP4372/pNT201 (lane c), RP4372/pNT201 in the presence of IPTG (lane d), RP4372/pLf13 (lane e), RP4372/pLf13 in the presence of IPTG (lane f), RP4372/pGEM-3Z (lane g), and RP4372/pGEM-3Z in the presence of IPTG (lane h). The bands corresponding to Tar (arrowheads) and the 58-kDa L. ferrooxidans protein (asterisks) are indicated. Numbers to the left are molecular mass markers (in kilodaltons).

In vivo complementation by LcrI of chemotaxis in E. coli RP4372. To test whether LcrI could restore chemotactic ability to the mutant E. coli RP4372 cells that lack all of the chemotactic receptors (26), we transformed this strain with plasmid pLf13 (expressing LcrI) or pNT201 (expressing Tar). The resulting transformants were inoculated at the center of tryptone swarm plates, and the displacements of the outermost edges of their swarms were compared. Only wild-type E. coli cells and those containing plasmid pNT201 showed chemotaxis under these conditions (results not shown).

	DISCUSSION

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Analysis and comparison of the lcrI sequence with those of mcp genes from different microorganisms indicate that the codified LcrI protein corresponds to an MCP. The MCPs from several bacterial species have been shown to contain functionally significant conserved regions. All of these features are present in LcrI in the following expected regions: (i) two hydrophobic transmembrane segments, (ii) an HCD, and (iii) two probable methylation sites. In addition, the protein not only possessed the expected molecular mass for a chemoreceptor but showed antigenic cross-reaction with Tar from E. coli and was localized in the cytoplasmic membrane of E. coli when expressed in this bacterium. Since HCD is the region of the chemotactic receptor that is supposed to interact with CheA and CheW in E. coli (31), the results obtained for LcrI strongly suggest the existence of similar proteins in the signaling pathway of L. ferrooxidans.

As transmembrane regions for LcrI, we proposed TM1 from residues 8 through 26 and TM2 from residues 164 through 180. If these regions insert into the membrane in a way similar to that of MCPs of enterobacteria, there would exist a cytoplasmic N terminus of seven amino acid residues with a positive charge such as the one that occurs in the equivalent fragment from E. coli MCPs (11).

The presence of a ²⁸-like promoter, which is characteristic of flagellar operons from E. coli and other microorganisms (6, 20, 29), strongly suggests that the protein from L. ferrooxidans encoded in the sequenced gene participates in chemotaxis. The lcrI gene sequence also showed a putative ⁷⁰ promoter overlapping with the ²⁸-type promoter. Whether one or both of these putative promoters function in the cell under different growth conditions remains to be seen. In the case of another chemolithotrophic bacterium, D. vulgaris Hildenborough, which possesses two completely described genes coding for MCPs, dcrA and dcrH, a putative ⁷⁰ promoter upstream of the first AUG codon rather than a ²⁸-type promoter has been reported (12, 13, 15). On the other hand, the mcp and che genes from E. coli possess only a ²⁸ promoter (6, 21, 32).

The postulated cytoplasmic domain of LcrI has an isoelectric point similar to those from the cytoplasmic domains of MCPs from several microorganisms. This was expected, since all of these bacteria, including L. ferrooxidans, would have similar intracellular pH values. On the other hand, the proposed periplasmic domain of LcrI, which would contain 14 fewer amino acids than the one corresponding to Tar, would be exposed to an acidic pH of 2 to 3 in the periplasm of an acidophilic microorganism such as L. ferrooxidans (24). This putative periplasmic domain of LcrI has an isoelectric point of 10.43, which is very high compared with the isoelectric points of most periplasmic domains in MCPs from several microorganisms. At neutral pH, if one assigns to the cationic amino acids arginine and lysine each a charge of +1, to the cationic amino acid histidine a charge of +0.5, and to the anionic amino acids glutamic acid and aspartic acid each a charge of 1, one can calculate the net charges of the periplasmic domains as the sum of the charges. This charge for the periplasmic domain of LcrI (residues 27 through 163; Fig. 1A) at pH 2.5 would be highly positive (+21). On the other hand, for a nonacidophilic bacterium such as E. coli, with a periplasmic pH of approximately 7, a net charge of 0.5 can be calculated for the periplasmic domain of a receptor such as Tar (residues 38 through 188; Fig. 1A). This difference in charge may represent a special adaptation of acidophilic microorganisms such as T. ferrooxidans and L. ferrooxidans to sense effectors at the very low pH present in their periplasm.

It was not possible to show a chemotactic receptor function for LcrI expressed in E. coli. This was probably due to the fact that the periplasmic pH of E. coli does not allow the right conformation for the LcrI periplasmic domain, as already discussed. In addition, the lack of recognition by LcrI of the common E. coli chemotactic effectors is also possible. The lack of a genetic system in acidophilic chemolithoautotrophic bacteria and the appropriate L. ferrooxidans mutants currently makes it difficult to extend studies of the mechanisms of L. ferrooxidans sensing and adaptation.