IBEX Pharmaceuticals Inc., 5485 Pare, Montreal, Quebec H4P 1P7, Canada1
Author for correspondence: Hongsheng Su. Tel: +1 514 336 7800. Fax: +1 514 336 7242. e-mail: hsu{at}theratech.com
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ABSTRACT |
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Keywords: Flavobacterium heparinum, integration vector, plasmid replication, heterologous DNA transfer
Abbreviations: Ap, ampicillin; Cm, chloramphenicol; Em, erythromycin; Gm, gentamicin; HT, trimethoprim-resistance gene (cassette); Km, kanamycin; Tc, tetracycline; Tp, trimethoprim
The GenBank accession number for the sequence reported in this paper is AF221716.
a Present address: Theratechnologies Inc., 2310 Alfred-Nobel Blvd, Saint Laurent, Quebec H4S 2A4, Canada.
b Present address: Merck Frosst Canada and Co., 16711 Trans Canada Hwy, Kirkland, Quebec, Canada H9H 3L1.
c Present address: The Millennium Three Group, 919 Conestoga Rd, Rosemont, PA 19010, USA.
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INTRODUCTION |
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The taxonomy of F. heparinum is not clear. It has been reclassified as Cytophaga heparinum by Christensen (1980) , Sphingobacterium heparinum by Takeuchi & Yokota (1992)
and lately, as Pedobacter heparinus by Steyn et al. (1998)
. Early 16S rRNA sequence analysis suggests that F. heparinum is more closely related to Bacteroides fragilis than to other species, such as Bacillus subtilis, Escherichia coli, Desulfovibrio desulfuricans and Agrobacterium tumefaciens (Weisburg et al., 1985
), even though these two bacteria differ in terms of their physiological characteristics and natural habitat. It is well known that the designation of Flavobacterium-Cytophaga-Bacteroides genera is permissive. In view of the unclear taxonomy, we have chosen to use the name Flavobacterium heparinum in this paper.
We wanted to develop a method for transfer of DNA into F. heparinum. Gram-negative bacterial broad-host-range plasmids belonging to several incompatibility groups have been widely used for the genetic manipulation of many micro-organisms. IncP group plasmids, such as RP4, R751 and their derivatives, have been successfully transferred and stably inherited among a large number of Gram-negative bacteria (Thomas & Helinski, 1989 ). IncQ group plasmids, such as RSF1010, are of small size, high copy number and have a wide host range. Such plasmids have been shown to replicate in most Gram-negative bacteria (Frey & Bagdasarian, 1989
) and in some Gram-positive actinomycetes such as Mycobacterium smegmatis and Streptomyces lividans (Gormley & Davies, 1991
). Furthermore, Bacteroides plasmid pB8-51 was shown to replicate in Prevotella ruminicola (Shoemaker et al., 1991
) and Porphyromonas gingivalis (Maley et al., 1992
), and its transposon, Tn4351, has been used for the genetic manipulation of bacteria which are members of Flavobacterium, Cytophaga, Flexibacter and Sporocytophaga species (McBride & Baker, 1996
; McBride & Kempf, 1996
). However, several commonly used broad-host-range Gram-negative bacterial plasmids from different incompatibility groups could not replicate in several Bacteroides strains (Shoemaker et al., 1986
), Pre. ruminicola (Shoemaker et al., 1991
), Por. gingivalis (Maley et al., 1992
) and Cytophaga johnsonae (McBride & Kempf, 1996
), and possibly many other bacteria of which we are unaware.
Plasmid pBBR1 (Antonie & Locht, 1992 ), from the Gram-negative bacterium Bordetella bronchiseptica, has been characterized as a small, high-copy-number plasmid with the ability to replicate in a broad range of Gram-negative bacteria including Acetobacter, Alcaligenes, Bartonella, Bordetella, Burkholderia, Caulobacter, Escherichia, Paracoccus, Pseudomonas, Rhizobium, Rhodobacter, Salmonella, Vibrio and Xanthomonas (Antonie & Locht, 1992
; Elizer et al., 1995
; Kovach et al., 1995
; DeShazer & Woods, 1996
). It has been shown not to belong to the IncP, IncQ or IncW groups of broad-host-range plasmids (Antonie & Locht, 1992
). DNA sequence analysis showed it to be a 2.6 kbp plasmid with two major ORFs, rep and mob, encoding proteins responsible for plasmid replication and mobilization, respectively.
In this paper, we report the development of a genetic manipulation system for F. heparinum. A conjugative/integrative plasmid with a F. heparinum functional selective marker for the transfer of heterologous DNA into F. heparinum has been constructed. It was demonstrated that this plasmid integrates into the F. heparinum chromosome. In addition, a derivative of the broad-host-range plasmid pBBR1Cm with a F. heparinum functional selective marker has been shown to replicate in F. heparinum.
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METHODS |
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Conjugation.
E. coli strain S17-1 was used for plasmid conjugative transfer. Donor E. coli strains containing the plasmids were grown to mid-exponential phase in LB medium. Recipient F. heparinum was grown in FMH medium for 2 d, diluted fivefold in the same medium and grown for an additional 5 h. Aliquots of 0·1 ml donor and 1·0 ml recipient cells were mixed, concentrated and plated on a 2·5 cm diameter (0·45 µM pore size) HA filter (Millipore; catalogue no. HAWG 02500) which was placed on FMH/LB plates. After incubation overnight at 30 °C, the cells were harvested from the filters in 1 ml FM medium and plated on selective MHGKT (MH medium containing Gm, Km and Tp) plates. After 7 d incubation at 23 °C, antibiotic-resistant colonies appeared and were confirmed by streaking for single colonies on similar plates.
Molecular biology techniques.
Isolation of chromosomal DNA, cloning and DNA manipulation techniques were performed as described by Sambrook et al. (1989) . T4 DNA ligase, DNA polymerase I Klenow fragment and restriction endonucleases were purchased from New England Biolabs. DNA fragments destined for ligation were first separated by agarose gel electrophoresis and the DNA was extracted from the agarose with a GeneClean I kit (Bio101).
Electroporation.
DNA was introduced into E. coli by electroporation using a Gene Pulser electroporator (Bio-Rad) as described by the manufacturer. DNA from E. coli transformants was isolated for restriction analysis using the RPM kit (Bio101). Electrocompetent F. heparinum cells were prepared by growing them to exponential phase in FMH medium. The cells were harvested, washed once with ice-cold water, twice with ice-cold 10% (v/v) glycerol and concentrated 20-fold in 10% (v/v) glycerol solution. The cells were aliquoted and either used immediately or stored at -70 °C. Plasmid DNA (1 µg) was added to 50 µl cells in a 2 mm-electrode-gapped cuvette (Bio-Rad) and subjected to a 2·5 kV electrical pulse with a capacitance of 25 µF and an external resistance of 400 . Electroporated cells were immediately diluted into 1 ml LB medium with 1% heparin and incubated for 5 h at 23 °C. The cells were then harvested and plated on MHGKT plates. Antibiotic-resistant colonies normally appeared within 7 d and were confirmed by streaking for single colonies on MHGKT plates.
DNA hybridization.
DNA was prepared for Southern blotting (Southern, 1975 ) by digestion with appropriate restriction endonucleases, separated by electrophoresis on a 0·8% agarose gel and transferred to a nylon membrane (Hybond-N; Amersham). The probes were labelled with [
-32P]dCTP, using the Rediprime II kit (Amersham Pharmacia Biotech). Hybridization was performed with Rapid-hyb buffer (Amersham Pharmacia Biotech) as described by the manufacturer.
DNA amplification.
Oligonucleotide primer synthesis, preparation of F. heparinum genomic DNA or plasmid DNA for use as template and analysis of PCR-generated products were all conducted as previously described (Su et al., 1996 ). PCR amplification was performed according to Mullis et al. (1986)
with the modifications described previously (Su et al., 1996
). The annealing temperature was 55 °C.
DNA sequence analysis.
DNA sequences were determined by the dideoxy chain termination method of Sanger et al. (1977) , using an ALF automated DNA sequencer (Amersham Pharmacia Biotech). Sequencing reactions were prepared with the Pharmacia Auto Read fluorescent labelling kit (Amersham Pharmacia Biotech). The DNA sequence of the hepA upstream region in plasmid pIB17, constructed by cloning a gel-purified 2·2 kbp BamHIHindIII DNA fragment containing the hepA gene from plasmid pIB10 into pUC21 was determined by the primer-walking method.
Insertion of the Tp-resistance gene cassette (HT) into various plasmids.
Plasmids pSUP106HT and pNJR12HT were constructed by inserting a gel-purified 1·1 kbp BamHIHindIII DNA fragment from pIB21 into the corresponding sites of pSUP106 and pNJR12, respectively. pCP11HT was constructed by cloning a gel-purified 1·1 kbp SpeI DNA fragment from pIB22 (formed by cloning a gel-purified 1·1 kbp EcoRI DNA fragment from pIB21 into pUC21) into the unique XbaI site of pCP11.
Plasmid pIBXF1 construction.
Primers OH1-1 (5'-GGGAATTCTTTCGCCGGTGAAAGGATTG-3') and OH1-2 (5'-GGCATATGTCTTTAGTTTTTATTGG-3') were used to amplify an 830 bp PCR fragment, corresponding to the upstream region of hepA, from plasmid pIB10. Primers RT-1 (5'-GGCATATGGACCAACACAACAATGGA-3') and RT-2 (5'-TTGAATTCTTAGGCCACGCGTTCAAGCG-3') generated a 250 bp PCR fragment corresponding to dhfrII from plasmid R751. The 830 and 250 bp fragments were cloned into plasmid pTZ/PC to create plasmids pIB18 and pIB19, respectively (as shown in Fig. 1). Both inserts were confirmed by DNA sequence analysis. A gel-purified 250 bp NdeIHindIII DNA fragment from pIB19 was inserted into the corresponding sites of pIB18, to yield plasmid pIB20. pIB21 was constructed by inserting a gel-purified 1·1 kbp BamHIHindIII DNA fragment from pIB20 into pUC13-oriT. Finally, a randomly chosen 10 kbp DNA fragment from HindIII-digested F. heparinum chromosomal DNA was cloned into the unique HindIII site of plasmid pIB20 to yield plasmid pIBXF1.
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RESULTS |
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Construction of a conjugation/integration plasmid for F. heparinum
To date, no publication has described any DNA transfer into F. heparinum. Our previous attempts to transfer broad-host-range plasmids and transposons into this micro-organism failed (data not shown). Therefore, a new system was developed based on the essential elements of DNA transfer, maintenance and selection. An E. coli conjugative plasmid and a randomly cloned DNA fragment from the F. heparinum chromosome would allow for both DNA transfer and stable DNA maintenance through homologous recombination. Obtaining a functional selective marker in F. heparinum was the main hurdle in the creation of this system.
Thus far, no selective marker has been demonstrated to be functional in F. heparinum. To ensure that a selective marker, e.g. an antibiotic-resistance gene, would be expressed in F. heparinum, a hybrid gene needed to be assembled which would consist of the desired antibiotic-resistance gene placed under the control of a F. heparinum regulatory region. Few genes from F. heparinum have been studied at the molecular level. DNA sequences are available only for the five glycosaminoglycan lyase genes hepA, B, C and cslA, B (Sasisekharan et al., 1993 ; Su et al., 1996
; Tkalec et al., 2000
). Among them, it appeared that hepA was expressed at the highest level when grown in heparin-only medium (Lohse & Linhardt, 1992
). Therefore, the hepA regulatory region was chosen to construct a hybrid antibiotic-resistance gene, which would allow for selection on heparin-only medium. A small region of 170 bp, 5' of the hepA start codon (upstream region), had been previously identified (Sasisekharan et al., 1993
). Within this sequence, no typical prokaryotic promoter elements were found. It was probable that this 170 bp hepA upstream region did not contain all the necessary elements that controlled hepA gene expression. Further sequence analysis of an 830 bp hepA upstream region in plasmid pIB17 was performed (GenBank accession no. AF221716). Although it did not reveal typical promoter elements, a partial putative ORF was identified, which translated in a direction opposite to, and 325 bp away from, the start codon of hepA. This suggested that the promoter region for hepA was probably located within this 325 bp region. To ensure that all the regulatory elements were included, the 830 bp hepA upstream region was used to create a hybrid antibiotic-resistance gene cassette.
The Tp-resistance gene, dhfrII, from plasmid R751 (Meyer & Shapiro, 1980 ), was chosen for the hybrid antibiotic-resistance cassette construction. This gene has been shown to confer very high levels of Tp resistance in E. coli. In addition, the gene was very small, only 250 bp, increasing the likelihood that it would be expressed and confer appreciable levels of antibiotic resistance in F. heparinum. The Tp-resistance (HT) cassette was constructed by fusing two PCR-generated DNA fragments consisting of the 830 bp hepA upstream region and a 250 bp dhfrII gene. Plasmid pIB20, containing the HT cassette, was introduced into E. coli but failed to confer Tp resistance, even when heparin was added to the growth medium. These data suggested that the hepA promoter was not functional in E. coli.
The final conjugative/integrative plasmid was constructed as shown in Fig. 1. The HT cassette and a randomly cloned 10 kbp F. heparinum chromosomal HindIII DNA fragment were added to pUC13-oriT. The features of the resulting plasmid, pIBXF1, include mobility conferred by pUC13-oriT, chromosomal integration through homologous recombination facilitated by the 10 kbp F. heparinum chromosomal DNA fragment, and selection conveyed by the HT cassette.
Introduction of pIBXF1 into F. heparinum
pIBXF1 was transferred into F. heparinum by conjugation as described in Methods. After more than 7 d incubation, colonies were isolated which displayed Kmr, Gmr and Tpr, as well as the ability to grow on FMH medium. One such strain was named FIBX1. The frequency of stable Tpr transconjugants per recipient cell was approximately 10-10.
To confirm that strain FIBX1 was a derivative of F. heparinum, it was purified to a single colony three times and further characterized by biochemical analysis. It was shown that FIBX1 could grow in FMH medium, was a Gram-negative bacterium, produced yellow pigment when grown on solid agar medium and had heparin and heparan sulfate degrading activity profiles similar to wild-type F. heparinum (data not shown). Together, these results confirmed that FIBX1 was a derivative of the wild-type F. heparinum strain.
It was possible that the Tpr phenotype of FIBX1 was the result of a mutation, and/or selection of F. heparinum cells exhibiting a low level of Tpr. To exclude this possibility and to confirm chromosomal plasmid integration, Southern analysis was performed. As shown in Fig. 3, HindIII/EcoRI digests of genomic DNA isolated from strain FIBX1 and plasmid pIBXF1 both hybridized to plasmid pIB21. Hybridization signals of 3·0 kbp and 1·1 kbp, corresponding to the vector pUC13-oriT and the HT cassette, respectively, were observed (Fig. 3b
, lanes 2 and 3). No such signals were seen for genomic DNA isolated from wild-type F. heparinum (Fig. 3b
, lane 1). These data clearly demonstrated the integration of plasmid pIBXF1 into the chromosome of strain FIBX1. More importantly, the successful recovery of Tpr transconjugants indicated that the HT gene cassette could be used as a selective marker in F. heparinum.
The insertion site of plasmid pIBXF1 in the 10 kbp HindIII DNA fragment on the genome was confirmed by PCR and Southern analysis. If plasmid pIBXF1 inserted within the 10 kbp HindIII region, then the vector pIB21 should be flanked by the 10 kbp HindIII DNA fragments at both ends and in the same orientation. A pair of primers complementary to the ends of the randomly cloned 10 kbp HindIII insert from plasmid pIBXF1 (Fig. 1) should amplify the 4·1 kbp pIB21 vector from FIBX1 chromosomal DNA. As expected, DNA fragments corresponding to the size of pIB21 were generated from FIBX1 chromosomal DNA and plasmid pIBXF1 (Fig. 3a
, lanes 8 and 9, respectively), but not from wild-type F. heparinum chromosomal DNA (Fig. 3a
, lane 7). When these PCR products were digested with EcoRI, a 1·1 kbp band corresponding to the HT cassette and a 3·0 kbp band corresponding to plasmid pUC13-oriT were obtained (Fig. 3a
, lanes 5 and 6), both of which hybridized to plasmid pIB21 (Fig. 3b
, lanes 5 and 6). To exclude the possibility that plasmid pIBXF1 integrated in another region of the F. heparinum chromosome sharing homology with the 10 kbp HindIII DNA fragment, FIBX1 genomic DNA digested with HindIII was hybridized with that fragment but no additional signals were observed (data not shown). These results clearly demonstrate that FIBX1 carries an insertion of plasmid pIBXF1 within the 10 kbp HindIII DNA fragment of the F. heparinum chromosome.
Plasmid replication in F. heparinum
Two broad-host-range plasmids, pSUP106 (Frey & Bagdasarian, 1989 ), belonging to IncQ and pNJR12, an RSF1010 derivative containing a Bacteroides plasmid pB8-51 replicon (Maley et al., 1992
), and an E. coliC. johnsonae shuttle plasmid, pCP11 (McBride & Kempf, 1996
), were introduced into F. heparinum by conjugation but failed to yield selectable transconjugants (data not shown). We have demonstrated that the HT cassette is a functional selective marker and that plasmid DNA can be introduced by conjugation in F. heparinum. Therefore, it now became possible to define the replicative function of these plasmids in F. heparinum. Derivatives of pSUP106, pNJR12 and pCP11 were constructed by addition of an HT cassette and introduced into F. heparinum by conjugation. No stable Tpr colonies were obtained. These plasmids lacked the ability to replicate in F. heparinum. An alternative broad-host-range plasmid, pBBR1Cm, was used to transform F. heparinum by electroporation but no Cmr colonies were obtained (data not shown). To confer conjugative and selective functions to pBBR1Cm, the mob, Apr and HT elements were incorporated to form plasmid pIBXF2 as shown in Fig. 2
. The plasmid was transferred into F. heparinum by conjugation. Several Tpr colonies were obtained, which displayed characteristics similar to F. heparinum. The conjugation rate was similar to that obtained with the integrative plasmid pIBXF1. One of the new isolates was designated strain FIBX2.
Demonstration of the existence of plasmid DNA in FIBX2 was necessary to verify that the Tpr phenotype was a result of episomal replication of pIBXF2 in F. heparinum. Plasmid DNA was isolated using the RPM method (see Methods), from a culture of FIBX2 grown in FMH medium, and analysed by electrophoresis. No plasmid DNA was visualized after ethidium bromide staining. Similar results were obtained when a highly concentrated plasmid preparation was subjected to restriction analysis (data not shown). However, when this DNA solution was electroporated into E. coli, Apr colonies were obtained. The digestion pattern of the plasmid DNA purified from these Apr colonies was identical to that of pIBXF2 (data not shown). These results confirmed the existence of plasmid pIBXF2 in strain FIBX2. The inability to visualize plasmid DNA from FIBX2 may be due to its existence as a low-copy-number plasmid in F. heparinum. Plasmid DNA from a FIBX2 culture was purified using Qiagens low-copy-number plasmid purification method and analysed by electrophoresis (Fig. 4a, lane 3). No plasmid DNA band corresponding to pIBXF2 (Fig. 4a
, lane 2) was seen. However, when the same DNA preparation was digested with EcoRI or XhoI (Fig. 4a
, lanes 6 and 9, respectively), the restriction patterns were identical to that of pIBXF2 treated similarly (Fig. 4a
, lanes 5 and 8). Hybridization of the restriction fragment with pIBXF2 identified them as the elements of the plasmid (Fig. 4b
, lanes 5, 6, 8 and 9). The analysis also revealed the presence of a hybridization signal in the undigested pIBXF2 DNA preparation from FIBX2 (Fig. 4b
, lane 3), that appeared to co-migrate with chromosomal DNA. The hybridization signals corresponding to the 2 kbp molecular mass marker (Fig. 4b
, lanes 2, 5 and 8), which was not seen in the ethidium bromide stained gel, were probably due to partial plasmid degradation occurring during purification. E. coli transformed with pIBXF2 DNA yeilded Apr colonies and identical plasmid digestion patterns, as described previously (data not shown). The data from this study clearly show that pBBR1-based plasmids can replicate in F. heparinum.
Plasmid pIBXF2 is stably maintained in strain FIBX2. This was demonstrated by growing strain FIBX2 in the presence or absence of Tp and examining the Tpr phenotype. Three hundred colonies from each group were tested and all of them showed resistance to Tp. Twelve colonies from the group growing in FH without Tp were cultured for isolation of plasmid DNA and E. coli transformation. All produced E. coli Apr colonies. In addition, 12 colonies from four of these 12 DNA preparations (three colonies each from four transformations) were grown and the plasmid DNA was isolated. Restriction analysis indicated that the plasmid DNA from these 12 isolates was identical to that of pIBXF2 (data not shown). These results, along with those described previously indicated that pIBXF2 could replicate in F. heparinum without selective pressure and be stably maintained.
Electroporation of pIBXF2
A method to introduce plasmid DNA into F. heparinum by electroporation was also developed. Typically, 2050 F. heparinum Tpr colonies could be obtained on selective plates within 7 d. As was the case with the transconjugants, undigested plasmid DNA isolated from these transformants could not be visualized by ethidium bromide staining of an agarose gel, but could be used to obtain Apr E. coli transformants.
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DISCUSSION |
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In our system, the rate of transconjugants produced with pIBXF1 was 10-10 per recipient. This low rate may be the result of a combination of factors including conjugation, homologous recombination and factors affecting conjugation efficiency. Surprisingly, a similar rate of transconjugant recovery was seen for pIBXF2, a derivative of the broad-host-range plasmid pBBR1, even though homologous recombination was no longer a required event. The possibility that a mutation in pFIBX2, either in the plasmid or in the host chromosome, that allowed maintenance of the plasmid in F. heparinum, was selected for during the conjugative process was considered. However, when pIBXF2 isolated from E. coli was reintroduced into F. heparinum or when pIBXF2 was introduced into the FIBX2 strain that had been cured of its plasmid, no increase in conjugation frequency was seen in either case (data not shown). The low efficiency of pIBXF2 conjugation in F. heparinum remains unexplained.
pIBXF2 replicated in F. heparinum irregularly. The plasmid copy number was significantly lower compared to pBBR1Cm as a high-copy-number plasmid in E. coli (Antonie & Locht, 1992 ), and 10 copies per cell described for Brucella species (Elizer et al., 1995
). In addition, the plasmid was somehow altered in strain FIBX2, as shown by it co-migrating with chromosomal F. heparinum DNA, while it retained a similar restriction pattern. Furthermore, its irregular plasmid form was also confirmed when examining plasmids recovered from E. coli after successive F. heparinum passages. Several large-size plasmids were observed (data not shown). Nevertheless, this study clearly demonstrates that pIBXF2 can replicate in F. heparinum. In a subsequent study, this plasmid system was used for high-level expression of both cslA and cslB in F. heparinum (F. Blain & others, unpublished results).
Plasmid pSUP106, a derivative of the broad-host-range IncQ plasmid RSF1010, was unable to replicate in F. heparinum. The pSUP106 derivative containing a F. heparinum functional selective marker, the HT cassette, also failed to generate F. heparinum transconjugants. This, along with other reported cases [Bacteriodes strains (Shoemaker et al., 1986 ), C. johnsonae (McBride & Kempf, 1996
), Por. gingivalis (Maley et al., 1992
) and Pre. ruminicola (Shoemaker et al., 1991
)] demonstrated the inability of plasmid RSF1010 and its derivatives to replicate in some Gram-negative bacteria. One possible explanation is that one or all of the repABC genes (Scherzinger et al., 1984
), involved in plasmid replication, are not functionally expressed in these organisms.
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ACKNOWLEDGEMENTS |
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Received 30 October 2000;
accepted 16 November 2000.