Identification and Characterization of a Native Dichelobacter nodosus Plasmid, pDN1

Plasmid 43, 230 –234 (2000) doi:10.1006/plas.1999.1456, available online at http://www.idealibrary.com on

SHORT COMMUNICATION Identification and Characterization of a Native Dichelobacter nodosus Plasmid, pDN1 Gabrielle Whittle, 1 Margaret E. Katz, Edward H. Clayton, 2 and Brian F. Cheetham Molecular and Cellular Biology, School of Biological Sciences, The University of New England, Armidale, New South Wales 2351, Australia Received July 9, 1999; revised December 21, 1999 The gram-negative anaerobe Dichelobacter nodosus is the primary causative agent of ovine footrot, a mixed bacterial infection of the hoof. We report here the characterization of a novel native plasmid, pDN1, from D. nodosus. Sequence analysis has revealed that pDN1 has a high degree of similarity to broad-host-range plasmids belonging, or related, to Escherichia coli incompatibility group Q. However, in contrast to these plasmids, pDN1 encodes no antibiotic resistance determinants, lacks genes E and F, and hence is smaller than all previously reported IncQ plasmids. In addition, pDN1 belongs to a different incompatibility group than the IncQ plasmids to which it is related. However, pDN1 does contain the replication and mobilization genes that are responsible for the extremely broad host range characteristic of IncQ plasmids, and derivatives of pDN1 replicate in E. coli. In addition, the mobilization determinants of pDN1 are functional, since derivatives of pDN1 are mobilized by the IncP␣ plasmid RP4 in E. coli. © 2000 Academic Press Key Words: footrot; virulence; IncQ; broad-host-range; RSF1010.

The gram-negative anaerobic bacterium Dichelobacter nodosus is the principal causative agent of ovine footrot, a disease characterized by a mixed bacterial infection of the hoof (Beveridge, 1941). Two virulence-associated DNA regions have been isolated from the D. nodosus genome (Katz et al., 1991), designated the virulence-associated protein (vap) regions (Katz et al., 1992, 1994; Cheetham et al., 1995) and the virulence related locus (vrl, Haring et al., 1995). Only one native D. nodosus plasmid, pJIR896 (10 kb), has been reported (Billington et al., 1996). This plasmid consists of a circular form of vap region 1 from D. nodosus virulent reference strain A198 (Cheetham et al., 1995) together with a putative insertion sequence, IS1253. In this study we report the characterization of a second native D. nodosus plasmid, designated pDN1, which was identified after genomic DNA was isolated from D. nodosus strain 1311 1 Present address: Department of Microbiology, University of Illinois, Urbana, IL 61801-3709. 2 Present address: Animal Science, School of Rural Science and Natural Resources, The University of New England, Armidale, NSW 2351, Australia.

0147-619X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

(Whittle et al., 1999). Agarose gel electrophoresis revealed three DNA bands in addition to the genomic DNA, corresponding to the nickedcircle, linear, and covalently closed forms of a 5.1-kb plasmid, pDN1 (data not shown). Using laser densitometry the copy number of pDN1 was determined to be approximately 16 copies per copy of the D. nodosus chromosome. pDN1 was digested using restriction endonucleases with 6-bp recognition sequences to generate a restriction map (Fig. 1). However, there is a deficiency of 6-bp restriction sites in pDN1, a characteristic previously observed in broad-host-range plasmids (Dorrington and Rawlings, 1990). Two HindII fragments, of 2.1 and 3.0 kb (Fig. 1), from pDN1 were used to probe genomic DNA from 11 strains of D. nodosus. No hybridization to these fragments was observed for any strain other than 1311, in which three bands were detected corresponding to the three different conformations of the plasmid (data not shown). The complete nucleotide sequence of pDN1 was determined, using methods described pre-

230

SHORT COMMUNICATION

231

FIG. 1. Map of plasmid pDN1 compared with related plasmids pIE1107 (Tietze, 1998) and RSF1010 (Scholz et al., 1989). The numbers indicate the distances (kb) in all three sequences. Restriction enzyme sites shown for pDN1 include AccI (Ac), AvaI (Av), BglII (B), ClaI (C), EcoRI (E), EcoRV (EV), FspI (F), HindII (H), KpnI (K), PvuII (P), and SmaI (S). The major open reading frames present in pDN1, pIE1107, and RSF1010 are indicated by black arrows. Antibiotic resistance genes are represented by unshaded arrows. Those genes present in pDN1 and pIE1107 (spotted arrows) but replaced by genes E and F (crossed arrows) in RSF1010 are also distinguished. Key to map: oriV, vegetative origin of replication; oriT, origin of conjugative transfer; mobA, mobB, and mobC correspond to mobilization proteins genes A, B, and C respectively; repA, repB, and repC indicate replication protein genes A, B, and C; aphA, aminoglycoside-3⬘-phosphotransferase, kanamycin, and neomycin resistance determinants; sat3, streptothricin-acetyltransferase-3, streptothricin resistance determinant; sul, sulfonamide resistance protein gene; strA/strB, streptomycin resistance protein genes A and B, respectively; F, repressor protein F. In pIE1107 sul is truncated.

viously (Whittle et al., 1999). Sequence comparison revealed that pDN1 (5112 nt) has a high degree of similarity to plasmids belonging, or related, to the Escherichia coli incompatibility group Q (Fig. 1). pDN1 has 96.5% identity over 5112 nt to the IncQ-related plasmid pIE1107 from Pseudomonas putida (Tietze, 1998). Nucleotides 1–3064 and 3339 –5112 from pDN1 have 65.7 and 89.0% identity, respectively, to the E. coli IncQ plasmid RSF1010 (Scholz et al., 1989). However, pDN1 lacks the antibiotic resistance genes found on pIE1107 and RSF1010. The G ⫹ C content of pDN1 (62%) is very similar to those of RSF1010 (61%) and pIE1107 (62%), but is considerably higher than the 45% G ⫹ C content of the D. nodosus chromosome (La Fontaine and Rood, 1990), suggesting that the plasmid was originally derived from a different host. Three 20-bp repeats were identified in the pDN1 sequence, and comparison with the sequence of pIE1107 suggests that these are part

of a vegetative origin of replication, oriV. The first base of the putative oriV was chosen as nucleotide number 1 (Fig. 1). All orfs identified in pDN1 except one (orf78) were found to have a very high degree of amino acid identity to RSF1010 gene products known to be involved in plasmid replication (repA–C) and mobilization (mobA–C; Haring and Scherzinger, 1989; Scholz et al., 1989). The order and orientation of the orfs in pDN1 are the same as those in pIE1107 and RSF1010 (Fig. 1). In pDN1 a 300-nt sequence containing orf78 separates the mobA and repA genes. The corresponding region in RSF1010 consists of a different sequence of approximately 500 nt, which contains genes E and F. A putative protein that is identical to that encoded by orf78 is encoded by orf29 of pIE1107. The function of gene E in RSF1010 is not known; however, gene F has been demonstrated to have a role in the negative feedback control of repA and repC (Maeser et al., 1990; Scholz et al., 1989). The mobA–C and

232

SHORT COMMUNICATION TABLE 1 Sequence Analysis of D. nodosus Native Plasmid pDN1

Gene

Coordinates 5⬘–3⬘ (nt)

Size aa a

mobC

476–772 (comp)

98

% Identity to putative protein b 100% (98 aa) 75.9% (87 aa) 39.6% (91 aa)

mobA

953–3064

703

100% (703 aa) 71.8% (710 aa) 38.0% (295 aa) 26.9% (171 aa) 28.8% (184 aa)

Accession No.

P(n) c

Pseudomonas putida plasmid pIE1107 Escherichia coli IncQ plasmid RSF1010 MobC protein Thiobacillus ferooxidans plasmid pTF1 mobilization region

Z74787 M28829

1.4 ⫻ 10 ⫺39 1.5 ⫻ 10 ⫺25

X52699

7.3 ⫻ 10 ⫺6

P. putida plasmid pIE1107 E. coli IncQ plasmid RSF1010 MobA protein Salmonella typhimurium plasmid pSC101 T. ferooxidans plasmid pTF-FC2 MobS protein Agrobacterium tumefaciens It plasmid TraA protein

Z74787 M28829

0 0

X01654

2.3 ⫻ 10 ⫺17

M64981

2.8 ⫻ 10 ⫺8

U43674

4.5 ⫻ 10 ⫺1

Homologue description

mobB

1701–2111

136

100% (136 aa) 59.8% (132 aa)

P. putida plasmid pIE1107 E. coli IncQ plasmid RSF1010 MobB protein

Z74787 M28829

0 6.6 ⫻ 10 ⫺26

repB⬘

2087–3064

325

100% (325 aa) 70.6% (330 aa)

P. putida plasmid pIE1107 E. coli IncQ plasmid RSF1010 RepB⬘ protein T. ferooxidans plasmid pTF-FC2 RepB primase

Z74787 M28829

0 0

M64981

8.9 ⫻ 10 ⫺15

P. putida plasmid pIE1107 E. coli IncQ plasmid RSF1010 RepA protein T. ferooxidans plasmid pTF-FC2 RepA primase

Z74787 M28829

0 0

M64981

0

P. putida plasmid pIE1107 E. coli IncQ plasmid RSF1010 RepC protein T. ferooxidans plasmid pTF-FC2 RepC protein

Z74787 M28829

0 0

M64981

0

P. putida plasmid pIE1107

Z74787

4.8 ⫻ 10 ⫺36

26.7% (281 aa) repA

3434–4273

279

99.3% (279 aa) 92.5% (279 aa) 44.4% (286 aa)

repC

4260–5112

283

100% (283 aa) 92.2% (283 aa) 66.1% (283 aa)

orf78

3133–3369

78

100% (78 aa)

a

aa, amino acids. The length over which the putative proteins (aa) from pDN1 have identity to homologous proteins. c The number of sequences with this level of similarity that would be expected in a database of this size by chance alone. b

repA–C genes have a strong preference for a G or C residue in the third position (77.5%), while in orf78 only 56.4% of codons have a G or C in the third position (data not shown), suggesting that orf78 has been incorporated into pDN1 more recently. Similarly, in RSF1010 (Scholz et al., 1989) the genes encoding plasmid replication, maintenance, and mobilization functions have a strong codon bias for a G or C residue in the third position of a codon (78.3%), while genes encoding antibiotic resistance and gene E

have a much lower preference for G or C in that position (61.0%). Four putative promoter sequences were identified in pDN1 (Fig. 1). Although the consensus sequences of these promoter regions differ from those identified as P1, P2, P3, and P4 (Scholz et al., 1989) in RSF1010, their locations are the same, suggesting that they have an equivalent function. No putative transcriptional terminator sequences were identified in pDN1. It is therefore possible that the oriV may act as a tran-

SHORT COMMUNICATION

scriptional terminator, due to the dyad symmetry characteristic of this region. The oriT region of RSF1010 (positions 3083 to 3170) was localized to an 88-bp segment (Derbyshire et al., 1987) and has 76% identity to pDN1 (positions 804 to 891). The corresponding region of pIE1107 (positions 893 to 980) has 100% identity to the oriT region of pDN1. The putative origin of replication (oriV) from pDN1 (positions 1 to 453) was compared to the oriV of RSF1010 (positions 2335 to 2778) and the two oriV regions identified in pIE1107, oriQa (positions 145 to 553) and oriQb (positions 5202 to 5672). The D. nodosus oriV and pIE1107 oriQb regions have three direct repeats of a 20-nt sequence (iterons), while pIE1107 oriQa has four copies and RSF1010 has three complete copies and one partial copy. All four oriV sequences have a set of inverted repeats, each containing a plasmid-specific single-strand DNA replication initiation (ssi) signal (Sakai and Komano, 1996). Sequence analysis indicated that oriV of pDN1 is most similar to oriQb of pIE1107. These are both located immediately after the stop codon of repC and are not separated by antibiotic resistance genes. The high nucleotide similarity between oriV of pDN1 and oriQb of pIE1107 is significant because oriQb is essential for the replication of pIE1107 and oriQb and oriV of RSF1010 belong to two different incompatibility groups, whereas oriQa is dispensable for the replication of pIE1107 and incompatible with the RSF1010 oriV (Tietze, 1998). These observations indicate that pIE1107 belongs to a different incompatibility group from that of the related IncQ plasmid RSF1010, and hence pDN1 may also belong to a different incompatibility group. To confirm this experimentally, we constructed the pDN1 derivatives pDN3, which has the tetracycline resistance determinant from pBR322 (Sutcliffe, 1978) located between repC and oriV, and pDN4, which contains the ampicillin and kanamycin resistance determinants from plasmid pKT240 (Bagdasarian et al., 1983). E. coli host cells carrying the tet R pDN1 derivative pDN3 were transformed either with pKT240, an IncQ RSF1010-derived plasmid, or with the amp R, kan R pDN1 derivative, pDN4.

233

After approximately 36 generations in the absence of antibiotic selection (Crosa et al., 1994), 100% of pDN3/pKT240 cotransformants maintained both plasmids. In contrast, pDN3/pDN4 cotransformants contained either pDN3 or pDN4, but not both plasmids, as would be expected for two plasmids belonging to the same incompatibility group. Since pDN3 and the IncQ derivative pKT240 are not incompatible in the same host cell, they belong to different E. coli incompatibility groups. Comparison of the iteron regions of pDN1 and related plasmids indicates that the observed difference in incompatibility may be attributed to a 4-bp change within this region, which is responsible for plasmid incompatibility (Nordstrom, 1990). IncQ plasmids are mobilized by broad-hostrange self-transmissible plasmids from E. coli incompatibility Group P. To test whether pDN1 derivatives were also mobilized by this group of plasmids, the rifampicin-resistant E. coli strain S17-1␭pir (Simon et al., 1983), which contains the integrated IncP␣ plasmid RP4, was transformed with pDN4, pDN2 (which contains the ampicillin resistance determinant from pUC18) (Norrander et al., 1983), or pKT240 (Bagdasarian et al., 1983) and mated with rif R DH5-␣. pDN4, pDN2, and pKT240 were found to be mobilized at frequencies of 4.1 ⫻ 10 ⫺2, 9.0 ⫻ 10 ⫺2, and 6.1 ⫻ 10 ⫺2 per donor cell, respectively. The frequency of transfer observed for pKT240 compares very well with the frequency of transfer reported for the transconjugation of pKT230 (7.5 ⫻ 10 ⫺1 per donor cell; Franklin, 1985), of which pKT240 is a derivative. We have characterized the plasmid pDN1 and shown that it does not belong to incompatibility group Q and that pDN1 derivatives are mobilized at high frequency by derivatives of the conjugative plasmid RP-4 from E. coli incompatibility group P␣. Transformation of D. nodosus has been achieved recently (Kennan et al., 1998), but the plasmid which was introduced failed to replicate independently. The pDN1 derivatives described here would greatly facilitate the molecular analysis of pathogenesis of D. nodosus if they were found to replicate independently after transformation, or if they could be introduced into D. nodosus by mobilization from an E. coli host strain carrying an

234

SHORT COMMUNICATION

integrated IncP␣ plasmid. In particular, the functions of genes from the vap and vrl regions could be investigated. ACKNOWLEDGMENTS We thank J. Druitt for excellent technical assistance and gratefully acknowledge the support of the Australian Research Council. G.W. is the recipient of an Australian Postgraduate Research Award.

REFERENCES Bagdasarian, M. M., Amann, E., Lurz, R., Ruckert, B., and Bagdasarian, M. (1983). Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida: Construction of broad-host-range, controlled expression vectors. Gene 26, 273–282. Beveridge, W. I. B. (1941). Footrot in sheep: A transmissible disease due to infection with Fusiformis nodosus. Counc. Sci. Ind. Res. Bull. 140, 1– 40. Billington, S. J., Sinitaj, M., Cheetham, B. F., Ayres, A., Moses, E. K., Katz, M. E., and Rood, J. I. (1996). Identification of a native Dichelobacter nodosus plasmid and implications for the evolution of the vap regions. Gene 172, 111–116. Cheetham, B. F., Tattersall, D. B., Bloomfield, G. A., Rood, J. I., and Katz, M. E. (1995). Identification of a bacteriophage-related integrase gene in a vap region of the Dichelobacter nodosus genome. Gene 162, 53–58. Crosa, J. H., Tolmasky, M. E., Actis, L. A., and Falkow, S. (1994). Plasmids. In “Methods for General and Molecular Bacteriology” (P. Gerhardt, R. G. E. Maurray, W. A. Wood, and N. R. Krieg, Eds.), pp. 365–386. American Society for Microbiology, Washington, DC. Derbyshire, K. M., Hatfull, G., and Willets, N. (1987). Mobilization of non-conjugative plasmid RSF1010: A genetic and DNA sequence analysis of the mobilization region. Mol. Gen. Genet. 206, 161–168. Dorrington, R. A., and Rawlings, D. E. (1990). Characterization of the minimum replicon of the broad-host-range plasmid pTF-FC2 and similarity between pTF-FC2 and the IncQ plasmids. J. Bacteriol. 172, 5697–5705. Franklin, F. C. H. (1985). Broad-host-range cloning vectors for gram-negative bacteria. In “DNA Cloning: A Practical Approach” (D. M. Glover, Ed.), Vol. 1. IRL Press, New York. Haring, V., Billington, S. J., Wright, C. L., Huggins, A. S., Katz, M. E., and Rood, J. I. (1995). Delineation of the virulence-related locus (vrl) of Dichelobacter nodosus. Microbiology 141, 2081–2089. Haring, V., and Scherzinger, E. (1989). Replication proteins of IncQ plasmids. In “Promiscuous Plasmids of Gram Negative Bacteria” (C. M. Thomas, Ed.), pp. 95–124. Academic Press, New York. Katz, M. E., Howarth, P. M., Yong, W. K., Riffkin, G. G., Depiazzi, L. J., and Rood, J. I. (1991). Identification of the three gene regions associated with virulence in Dich-

elobacter nodosus, the causative agent of ovine footrot. J. Gen. Microbiol. 137, 2117–2124. Katz, M. E., Strugnell, R. A., and Rood, J. I. (1992). Molecular characterization of a genomic region associated with virulence in Dichelobacter nodosus. Infect. Immun. 60, 2– 4. Katz, M. E., Wright, C. L., Gartside, T. S., Cheetham, B. F., Doige, C. V., Moses, E., and Rood, J. I. (1994). Genetic organisation of the duplicated vap region of the Dichelobacter nodosus genome. J. Bacteriol. 176, 2663–2669. Kennan, R. M., Billington, S. J., and Rood, J. I. (1998). Electroporation-mediated transformation of the ovine footrot pathogen Dichelobacter nodosus. FEMS Microbiol. Lett. 169, 383–389. La Fontaine, S., and Rood, J. I. (1990). Evidence that Bacteroides nodosus belongs in subgroup gamma of the class Proteobacteria, not in the genus Bacteroides: Partial sequence analysis of a B. nodosus 16S rRNA gene. Int. J. Syst. Bacteriol. 40, 152–159. Maeser, S., Scholz, P., Otto, S., and Scherzinger, E. (1990). Gene F of plasmid RSF1010 codes for a low-molecularweight repressor protein that autoregulates expression of the repAC operon. Nucleic Acids Res. 18, 6215– 6222. Nordstrom, K. (1990). Control of plasmid replication— How do DNA iterons set the replication frequency? Cell 63, 1121–1124. Norrander, J. M., Kempe, T., and Messing, J. (1983). Construction of improved M13 vectors using oligonucleotidedirected mutagenesis. Gene 26, 101–106. Sakai, H., and Komano, T. (1996). DNA replication of IncQ broad-host-range plasmids in gram-negative bacteria. Biosci. Biotech. Biochem. 60, 377–382. Scholz, P., Haring, V., Wittmann-Liebold, B., Ashman, K., Bagdasarian, M., and Scherzinger, E. (1989). Complete nucleotide sequence and gene organization of the broad host range plasmid RSF1010. Gene 75, 271–288. Simon, R., Priefer, U., and Puhler, A. (1983). A broad-hostrange mobilization system for in vivo genetic engineering: Transposon mutagenesis in gram-negative bacteria. Biotechnology 30, 33– 44. Sutcliffe, J. (1978). Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proc. Natl. Acad. Sci. USA 75, 3737–3741. Tietze, E. (1998). Nucleotide sequence and genetic characterization of the novel IncQ-like plasmid pIE1107. Plasmid 39, 165–181. Whittle, G., Bloomfield, G. A., Katz, M. E., and Cheetham, B. F. (1999). The site-specific integration of genetic elements may modulate thermostable protease production, a virulence factor in Dichelobacter nodosus, the causative agent of ovine footrot. Microbiology, 145, 2845–2855. Willetts, N. S., and Crowther, C. (1981). Mobilization of the non-conjugative IncQ plasmid RSF1010. Genet. Res. 37, 311–316. Willetts, N. S., and Wilkins, B. M. (1984). Processing of plasmid DNA during bacterial conjugation. Microbiol. Rev. 48, 24 – 41. Communicated by C. Smith