Nucleotide Sequence, Structural Organization, and Functional Characterization of the Small Recombinant Plasmid pOM1 That Is Specific for Francisella tularensis

Plasmid 46, 86–94 (2001) doi:10.1006/plas.2001.1538, available online at http://www.academicpress.com on

Nucleotide Sequence, Structural Organization, and Functional Characterization of the Small Recombinant Plasmid pOM1 That Is Specific for Francisella tularensis Andrei P. Pomerantsev, Masatsugu Obuchi, and Yoshiro Ohara1 Department of Microbiology, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan Received January 22, 2001; revised July 5, 2001 pOM1 is a recombinant 4442-bp plasmid that includes the replicon of the Francisella novicida-like strain F6168 cryptic plasmid pFNL10 and the tetracycline resistance gene (tetC) of plasmid pBR328. pOM1 can stably replicate and is maintained in Francisella tularensis biovars tularensis, palaearctica, and palaearctica var. japonica. The replicon of pOM1 includes the ori region and the repA gene. The ori region, located upstream of the repA gene includes two sets of 31- and 13-bp direct repeats (DR), with AT-rich regions preceding each of the DRs. Two putative promoters of the repA gene were found connected with the DR regions. A 40-kDa protein was encoded by the repA gene and found essential for replication. Expression of the tetC gene is regulated by an Escherichia coli s70 -like promoter and is dependent on the F. tularensis strain and its environment. © 2001 Academic Press

The gram-negative bacterium Francisella tularensis, the causative agent of tularemia, is divided into two main biovars, the highly virulent F. biovar tularensis, found in North America, and the less virulent F. biovar palaearctica, found all over the northern hemisphere. Two other biovars have also been proposed, F. biovar mediaasiatica and F. biovar palaearctica var japonica. All of these biovars differ phenotypically in virulence and biochemical properties (Sandström et al., 1992). One of the outstanding features of F. tularensis is the absence of its own plasmids in any of these biovars. It is not clear whether this property is associated with the environment of F. tularensis or with the specificity of its genetic apparatus. It has been shown that heterologous plasmids, conjugative plasmid pSa replicating in gram-negative bacteria and plasmid pC194 specific for gram-positive bacteria, can replicate in F. tularensis. These plasmids, however, could be eliminated easily in media without antibiotic (Pomerantsev et al., 1991a,b). On the other hand, the recombinant plasmid pFNL200, constructed on the basis of two plasmids (4-kb cryptic plasmid pFNL10, originally 1

To whom correspondence should be addressed. Fax: 8176-286-3961. E-mail:[email protected]. 0147-619X/00 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

isolated from the Francisella novicida-like strain F6168 (Pavlov et al., 1994), and plasmid pBR328 of Escherichia coli), is reported to be stably inherited by F. tularensis (Pavlov et al., 1996). The DNA region responsible for replication of pFNL-like plasmids in F. tularensis has been identified (Norqvist et al., 1996). The deletion of the DNA fragment of pFNL200 plasmid just downstream of the tetC gene resulted in spontaneous appearance of a new small pOM1 plasmid in F. tularensis cells. The pOM1 inheriting the tetC gene, however, replicated in F. tularensis without segregation and without any genetic rearrangements. In addition, pOM21 was generated as a plasmid for the studying of F. tularensis transcriptional regulatory elements on the basis of pOM1 and the promoter region of F. tularensis chromosomal DNA (Pomerantsev and Pavlov, 1999). The final goal of our research is to develop cloning vectors for the genetic manipulation of F. tularensis, which will lead to the elucidation of the pathogenesis of F. tularensis. Therefore, in the present paper, we have sequenced and characterized the pOM1 plasmid as well as the promoter region of F. tularensis chromosomal DNA. We determined the transformation efficiency of pOM1-like plasmids in different strains of F. tu86

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larensis by using cryotransformation and electroporation methods and examined whether the tetC gene expression of these plasmids depends on the F. tularensis strain and its environment. MATERIALS AND METHODS Bacterial Strains and Plasmids E. coli strain JM-109 (Yanisch-Perron et al., 1985) was used as a host for creating recombinant plasmids. The vaccine strain LVS (Eigelbach and Downs, 1961), the virulent Schu, Ebina 1 and 2, the low-virulent Ebina 3 and 4, and the avirulent Hashimoto 1 strains of F. tularensis (Sato et al., 1992; Fujita et al., 1995) were used as recipients for transformation and gene-expression studies. All F. tularensis strains were supplied from the Laboratory of Ohara General Hospital (Fukushima, Japan). Plasmids used in this study are listed in the Table 1. Media E. coli JM-109 was grown in L broth or on LB agar (Ausubel et al., 1997). For selection of recombinant plasmids, ampicillin (Amp),2 chloramphenicol (Cm), or tetracycline (Tc) were added to final concentrations of 50, 30, and 15 mg/ml, respectively. F. tularensis was grown in Eugon broth or on Eugon agar (Difco Laboratories, Detroit, MI) supplemented if needed with 8% whole human blood (Fujita et al., 1995). Cm or Tc was added to a final concentration of 5 mg/ml for selection of transformants. Cells of bacteria (104–105) were spotted on agar with different concentrations of Cm (2–128 mg/ml for E. coli and 2–32 mg/ml for F. tularensis) in order to determine minimal inhibitory concentration (MIC). The MIC value was estimated as the minimal concentration of antibiotic that inhibits growth of bacteria at 37°C. DNA Manipulations Plasmid DNAs were isolated by using QIAGEN Plasmid Mini Kit (QIAGEN Inc., Valen2 Abbreviations used: Amp, ampicillin; Cm, chloramphenicol; Tc, tetracycline; MIC, minimal inhibitory concentration; ORF, open reading frame; ORFtet, open reading frame for the tetC gene; DR, direct repeats; NHS, normal human serum.

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cia, CA). QIAquick Gel Extraction Kit (QIAGEN Inc.) was also used for the recovery of DNA fragments from an agarose gel. For chromosomal DNA isolation, cells were treated with 10% sodium dodecyl sulfate and proteinase K (100 mg/ml), followed by selective precipitation with cetyltrimethylammonium bromide. A highmolecular-weight DNA was recovered by isopropanol precipitation (Ausubel et al., 1997). DNA Sequencing and Data Analysis Nucleotide sequencing was performed by the dideoxy chain-termination technique with a Taq dye primer cycle sequencing kit and an ABI 373 DNA sequencer (Perkin–Elmer Japan, Urayasu, Japan). In the case of pOM1, primer H of the tetC gene of plasmid pBR322 (Takara Shuzo Co., Ltd., Otsu, Japan) was used initially; primers complementary to the determined sequence were subsequently used. For sequencing of the Sau3A fragment of chromosomal DNA of F. tularensis in pOM22, primer M13 of pUC18 (Takara Shuzo Co., Ltd.) and synthetic primer 5⬘-CAACGGTGGTATATCCAGTG-3⬘ complementary to the beginning of the cat gene of transposon Tn9 (Alton and Vapnek, 1979) were used. Sequencing data were analyzed by the Mac DNASIS program (Hitachi Software Engineering Co., Ltd., Yokohama, Japan). The FASTA and BLAST programs of the GCG package were used for homology searches in the GenBank and SWISS Protein databases. Transformation Cryotransformation (Pavlov et al., 1996) and electroporation methods were used for transformation of F. tularensis. The Gene Pulser II Electroporation System (Bio-Rad, Hercules, CA) was used for electroporation. The recipient cells were washed twice with 0.5 M sucrose, 1 mM EDTA, pH 7.5 (Pomerantsev and Pavlov, 1999), and suspended in the same buffer at a concentration of 1010–1011 cells/ml. After addition of DNA, 200 ml of suspension was incubated for 10 min at room temperature, transferred into a Bio-Rad Gene Pulser Cuvett (interelectrode distance, 0.2 cm), and subjected to electroporation (standard set voltage, 2.5 kV; set capacitance 25 mF; resistance, 100 ohm). The electroporated samples were diluted with 1 ml of Eugon broth,

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POMERANTSEV, OBUCHI, AND OHARA TABLE 1 Plasmids Used in This Study

Plasmid pCW5

pOM1

pOM2

pOM3

pOM4

pOM5

pOM6

Description

Reference

Promoterless cat gene of transposon Tn9 introduced into the SmaI site of the multiple cloning site of the pUC18 plasmid. AmpR, CmS (E. coli), does not replicate in F. tularensis. Deleted variant of the pFNL200 plasmid. TcR (F. tularensis), does not replicate in E. coli. Recombinant plasmid of pOM1 BglII and pCW5 BamHI plasmids. The cat gene is introduced into the ⫹strand of the pOM1 plasmid. CmS (E. coli), replicates in E. coli and does not replicate in F. tularensis. Recombinant plasmid of pOM1 BglII and pCW5 BamHI plasmids. The cat gene is introduced into the ⫺strand of the pOM1 plasmid. CmR (E. coli), replicates in E. coli and does not replicate in F. tularensis. Recombinant plasmid of pOM1 XbaI and pCW5 XbaI plasmids. The cat gene is introduced into the ⫹strand of the pOM1 plasmid. CmS (E. coli, F. tularensis), replicates in E. coli and in F. tularensis. Recombinant plasmid of pOM1 XbaI and pCW5 XbaI plasmids. The cat gene is introduced into the ⫺strand of the pOM1 plasmid. CmR (E. coli, F. tularensis), replicates in E. coli and in F. tularensis Recombinant plasmid of pOM1 ClaI and pCW5 AccI plasmids. The cat gene is introduced into the ⫹strand of the pOM1 plasmid. CmR (E. coli, F. tularensis), replicates in E. coli and in F. tularensis.

Pomerantsev and Pavlov (1999) Pomerantsev and Pavlov (1999) Present study

Present study

Pomerantsev and Pavlov (1999) Pomerantsev and Pavlov (1999) Present study

pOM7

Recombinant plasmid of pOM1 ClaI and pCW5 AccI plasmids. The cat gene is introduced into the ⫺strand of the pOM1 plasmid. CmS (E. coli, F. tularensis), replicate in E. coli and in F. tularensis.

Present study

pOM21

Sau3A fragment of chromosomal DNA of F. tularensis (Schu) introduced into the BamHI site of the pOM4 plasmid. CmR (E. coli, F. tularensis), replicates in E. coli and in F. tularensis. pOM21 plasmid without pOM1 plasmid, deleted by XbaI digestion. AmpR, CmR (E. coli), does not replicate in F. tularensis. PstI fragment of the pOM21 plasmid. TcR (F. tularensis), does not replicate in E. coli Recombinant plasmid of pOM8 PstI and pCW5 PstI plasmids. The cat gene is introduced into the ⫹strand of the pOM8 plasmid. CmR (E. coli, F. tularensis), replicates in E. coli and in F. tularensis. Recombinant plasmid of pOM8 PstI and pCW5 PstI plasmids. The cat gene is introduced into the ⫺strand of the pOM8 plasmid. CmR (E. coli, F. tularensis), replicates in E. coli and in F. tularensis.

Pomerantsev and Pavlov (1999) Present study

pOM22 pOM8 pOM81

pOM82

incubated for 2 h at 37°C with shaking, and plated on Eugon agar containing an appropriate antibiotic. After 2–4 days, colonies were counted. Nucleotide Sequence Accession Numbers The nucleotide sequences encoding the promoter region of F. tularensis chromosomal DNA and plasmid pOM1 were submitted to the GenBank database. The registered Accession Nos. were AF055344 and AF055345, respectively.

Present study Present study

Present study

RESULTS Sequence Analysis and Genetic Organization of pOM1 The genetic map of pOM1 is shown in Fig. 1. The size of the genome is 4442 bp long. The overall A⫹T content (without the tetC gene region, which belongs to pBR328) is high (68%), in agreement with previously published sequence information on F. tularensis (Ericsson et al., 1997; Golovliov et al., 1997).

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TABLE 2 Chloramphenicol Resistance of E. coli and F. tularensis MIC (mg/ml) of Cm Plasmid

E. coli

pOM2 pOM3 pOM4 pOM5 pOM6 pOM7

⬍32 ⬎128 ⬍32 ⬎128 ⬎128 ⬍32

F. tularensis — — ⬍2 ⬎32 ⬎32 ⬍2

Note. MIC, minimal inhibitory concentration; Cm, chloramphenicol. FIG. 1. The genetic map of the pOM1 plasmid. The promotorless cat gene from pCW5 was inserted into the various ORFs and allowed to determine the direction of transcription of the ORFs. This confirmed the transcription directions inferred from sequence analysis. The directions of transcription are indicated by the arrows. The transcription termination site (Ter) is indicated by the closed triangle, and the origin of plasmid replication (Ori) is marked by the closed box.

The main feature of the nucleotide sequence is the presence of four open reading frames (ORFs), which account for about 86% of the plasmid (Fig. 1). One of these is the tetC gene (ORFtet); the details of the other three ORFs are still unknown. ORF1 and ORF2 are located on the ⫺strand of pOM1, and ORF3 is located on the ⫹strand, immediately downstream of ORFtet. The promoterless cat gene of pCW5 was introduced in each of the unidentified ORFs in order to determine the direction of transcription. The recombinant plasmids pOM2 and pOM3, with an insertion in the BglII site, which is located within ORF1, failed to replicate in F. tularensis (Table 2), in agreement with previous data (Norqvist et al., 1996). The strict dependence of replication on the integrity of ORF1 suggested that ORF1 codes for the RepA protein essential for plasmid replication. A SWISSprot database search found homology of ORF1 with two proteins essential for plasmid replication, the RepE protein of the E. coli F and R27 plasmids (Disque-Kochem et al., 1986; Saul et al., 1988) and the RepA protein of the Neisseria gonorrhoeae pAF3 plasmid (Gilbride and Brunton, 1990). No homologies were found

between the predicted amino acid sequences of ORF2 and ORF3 and any other protein associated with DNA replication. The close proximity of ORF1 to ORF2 as well as ORFtet to ORF3 suggested that these genetic structures may be arranged as bicistronic operons. There were no promoter regions identified between ORF1 and ORF2 and between ORFtet and ORF3. The detailed functions of ORF2 and ORF3 still remain unknown. Organization of the Replication Domain of pOM1 A region of note was observed in the intergenic region between ORF1 and ORFtet (⬃600 bp), which probably includes the promoter regions of both the operons and the origin of replication of the pOM1 plasmid. It contained approximately two-and-a-half 13-bp direct repeats (DR) without spacers and a 100% A⫹T-rich 13bp region located downstream of the repeats. In addition, a 31-bp DR with a 100% A⫹T-rich 19bp sequence (Fig. 2, double underline) is located between ORF1 and a 13-bp DR. A region resembling the consensus sequence of the s70 (E. coli) promoter was found inside the 31-bp DR. In addition, the motif of the sc (Bacillus subtilis) promoter (Haldenwang, 1995) was found in the region including the 13-bp DR (Fig. 2). Sequence analysis using the GenBank database showed that the plP404 plasmid of Clostridium perfringens (Garnier and Cole, 1988a) showed homology with pOM1, precisely in the replication regions of both plasmids. Both plasmids did not share sequence homology with

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FIG. 2. The nucleotide sequence of the 600-bp fragment encompasing the putative origin of pOM1 plasmid replication and the putative promoter regions for RepA and TetC protein expression. The upper sequence of double-stranded DNA is the ⫹strand, and the lower sequence is the ⫺strand. The 31- and 13-bp direct repeats (DR) are indicated by the arrows. A⫹Trich segments are noted with double underlines. The -35 and -10 regions of the s70 (E. coli)-like promoter are located inside the 31-bp DR, and the -35 and -10 regions of the sc (B. subtilis)-like promoter are connected with a 13-bp DR. The initiation site of tet gene transcription is indicated by the open circle for E. coli.

the consensus sequences of rolling circle replicating plasmids (Gruss and Ehrlich, 1989), suggesting that pOM1 and plP404 may not exploit a single-strand intermediate. A region similar to the consensus sequence of the s70 (E. coli) promoter was found upstream of the tetC gene. The initiation start of tetC gene transcription, identified previously as a G (Brosius et al., 1982), was also observed in this region (Fig. 2).

Terminator of Transcription The presence of approximately a 200-nucleotide sequence between two operon structures transcribed in opposite directions in pOM1 indicated that the transcription terminator for both operons is present in this region. In addition, pOM4, in which the cat gene was inserted into the ⫹strand at the XbaI site of ORF2, was not expressed in F. tularensis (Table 2). pOM7, in which the cat gene was inserted into the

pOM1 SPECIFIC FOR Francisella tularensis

⫺strand at the ClaI site of ORF3, was not expressed in F. tularensis, either. The DNASIS program showed that the region forms dyad symmetries, indicating that the region is a factor-independent transcription terminator. Some hairpins are presented within a large hairpinloop structure located in this region (data not shown). The DNASIS program also showed that the energy of formation of the whole structure is ⫺34.6 kcal/mol. In a preliminary experiment, an 880-nucleotide fragment of F. tularensis was cloned into the shuttle vector (pOM21, Table 1). The preliminary analysis suggested that it contained the 5⬘ part of an unknown ORF. Additionally, SWISS Protein database analysis showed that the predicted amino acid sequence of the ORF shares homology with some putative proteases of E. coli, Haemophilus influenzae, Helicobacter pylori, Metanococcus jannashii, and the collagenase precursor of Porphyromonas gingivalis. Detailed characterization of this chromosomal DNA of F. tularensis is further required. Plasmid Transformation of F. tularensis We next examined and compared the transformation efficiency of pOM21 into various strains of F. tularensis. For virulent Schu, Ebina 1 and 2, and low-virulent Ebina 3 and Ebina 4, there were only a few single colonies in all strains that were examined when selected on Eugon agar without blood. Therefore, the presence of blood in Eugon agar is an extremely important factor for transformation efficiency. Low-virulent Ebina 3 and Ebina 4 and avirulent Hashimoto strains demonstrated only a few colonies or no colonies when selected on Eugon agar with blood by cryotransformation (in both Cm and Tc selections). In the case of electroporation, Tc selection dramatically decreased the efficiency of transformation of the low-virulent Ebina 3 and 4 and the avirulent Hashimoto 1 strains, although the transformation efficiency of these strains was similar to that of the virulent (Schu, Ebina 1, and Ebina 2) and LVS strains in the case of Cm selection. The nucleotide sequences of pOM21 plasmids isolated from Hashimoto 1 transformants obtained on blood agar (Cm resistant) or on pure Eugon agar (Cm

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and Tc resistant) were identical (data not shown). DISCUSSION This study presented the first complete nucleotide sequence of a plasmid of Francisella origin. The information obtained will be valuable for a better understanding of plasmid replication mechanisms and for the construction of F. tularensis cloning vectors. The genetic organization of the pOM1 plasmid defines two operon structures (four ORFs). The first is involved in the expression of the phenotype of Tc resistance and the second is required for the replication of the plasmid. Unlike ColE1, the synthesis of a plasmid-encoded protein (RepA) is required for replication. Functions have been attributed to two ORFs (ORFtet and ORF1) identified on pOM1, and it seems likely that the products of the remaining two ORFs are involved in plasmid mobilization, transfer, or incompatibility. Many procaryotic replication regions contain an A⫹T-rich region closely associated with DR sequences and a sequence encoding a replication initiation protein (Filutowicz et al., 1994). Comparison of the replication region of the pOM1 plasmid with those of pSa (Okumura and Kado, 1992) and pC194 (Gruss and Ehrlich, 1989), which can replicate in F. tularensis, and with plP404 (Garnier and Cole, 1988b), which shares homology with pOM1, demonstrated one of these general features. Although all these plasmids code for a protein necessary for their replication, targets for interaction with the protein are different. The Ori region of pSa includes six 17-bp DR sequences, which are located downstream of the RepA coding region and are the binding sites of RepA. In plP404, a number of DRs are also located downstream of the protein coding region. All pC194-type rolling circle plasmids contain a double-(plus) strand origin 5⬘ to the rep gene without a DR. Within this origin, a highly conserved nicking site is recognized by the Rep protein (Pillidge et al., 1996). pOM1 Ori contains a set of DRs as well, but the arrangement and the size of the monomers are different from those of pSa and plP404. No homology was found with consensus sequences of ⫹Ori of pC194-type plasmids. It is of interest

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that targets for the RepA protein of pFA3 and the RepE protein of the F and R27 plasmids homologous to the RepA of the pOM1 plasmid are also present in DRs (Disque-Kochem et al., 1986; Saul et al., 1988; Gilbride and Brunton, 1990). The pOM1 repA gene (ORF1) is preceded by two putative promoter regions, which are closed to consensus sequences of s70 (E. coli) and sc(B. subtilis) promoters (Haldenwang, 1995). It is not clear why two different promoters are present in a single gene. The origin of pOM1 is the cryptic pFNL10 plasmid of the F. novicida-like strain F6168, which has been isolated from man (Hollis et al., 1989). Apparently, the human body is an optimal environment for multiplication of the bacteria. At the same time, F. novicida as well as F. tularensis may survive for prolonged periods in water. In this situation, the microbe has to survive in a competitive, nutrient-limited environment. The survival of bacteria in both environments may be ensured by modulation of protein synthesis via variation of s-subunits of DNA-dependent RNA polymerase, which uses different promoters, such as B. subtilis or Pseudomonas aeruginosa, for example (Deretic et al., 1994; Haldenwang, 1995). In this case, the presence of two promoters of the repA gene of pFNL10-like plasmids may ensure the stable autonomous maintenance of these plasmids in F. novicida (Hollis et al., 1989) and in F. tularensis (Pavlov et al., 1996), in comparison with heterologous pSa and pC194 plasmids (Pomerantsev et al., 1991a,b). The repA gene putative promoter regions of the pOM1 plasmid overlap with 31-bp s70 (E. coli) and 13-bp sc (B. subtilis) DRs. This overlapping may be involved in autoregulation of repA gene expression, such that the transcription of the repA is inhibited by the binding of the RepA protein to DR sequences, thereby blocking transcription. This mechanism of autoregulation is suggested in the replication control of pSa and pFA3 (Gilbride and Brunton, 1990; Okumura and Kado, 1992). The experiments on transformation showed that transformation efficiency depends on the method, the strain of F. tularensis, and the medium used for clone selection. The F. tularen-

sis cells were transformed more effectively with Cm than with Tc selection. Apparently, the mechanism of resistance to Cm is different from that to Tc. Resistance to Cm is ensured by the synthesis of chloramphenicol acetyltransferase, which hydrolyzes the antibiotic (Gaffney et al., 1978). In contrast, resistance to Tc is related to the production of the TetC membrane protein, which confers a resistance phenotype by pumping the antibiotic out of the cell (Speer et al., 1992). The membrane location of the TetC protein may be a crucial factor in the dramatic decrease in transformation efficiency of the lowvirulent and avirulent strains in the case of selection on blood agar with Tc. It was shown that low-virulent and avirulent mutants of F. tularensis are capsule-deficient (Cap⫺) and exhibit enhanced sensitivity to killing by normal human serum (NHS) (Sandström et al., 1988; Sorokin et al., 1996). The capsule covering the surface of virulent cells blocks the adsorption of NHS antibodies to the cell surface (Pavlovich et al., 1996), and the TetC protein is allowed to insert into the membrane and to pump Tc from the cell into the medium. The outer membrane of the Cap⫺ cells is covered by antibodies, and the TetC protein either does not insert into the membrane or, being inserted, does not pump the antibiotic from the cells. Apparently, the capsule protects F. tularensis cells against damage caused by the freezing–thawing procedure. There is another possible explanation for the absence of the TcR phenotype when the Hashimoto 1 strain is grown on blood agar. The adaptation of F. tularensis to various hostile environments involves the modulation of its protein synthesis (Ericsson et al., 1994; Golovliov et al., 1997). The contact with blood may be a powerful signal for the expression of unusual proteins in the Cap⫺ cells, directly interacting with NHS antibodies. In this case, the promoter of the tet C gene, which is very similar to the s70 (E. coli) consensus sequence, may not interact with new transcriptional factors, which can use different promoter regions. Another promoter of pOM21 plasmid sc (B. subtilis) for the repA gene and the F. tularensis chromosome promoter for the cat gene may be adapted to stress.

pOM1 SPECIFIC FOR Francisella tularensis

The silence of the tet gene of the pOM1 plasmid in Cap⫺ F. tularensis cells on blood agar with Tc can be interrupted by cloning of the cap region of F. tularensis chromosomal DNA into Cap⫺ F. tularensis on pOM1-like plasmids containing this gene. This approach opens up the possibility for study of the genetics of capsule forming of F. tuleransis and, at long last, for clarifying the virulence of this enigmatic bacterium. ACKNOWLEDGMENT This work was supported by a Grant for Promoted Research from Kanazawa Medical University (S00-1).

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