Characterization of the IncW Cryptic Plasmid pXV2 from Xanthomonas campestris pv. vesicatoria

Plasmid 44, 163–172 (2000) doi:10.1006/plas.2000.1468, available online at http://www.idealibrary.com on

Characterization of the IncW Cryptic Plasmid pXV2 from Xanthomonas campestris pv. vesicatoria Lii-Tzu Wu* and Yi-Hsiung Tseng† ,1 *Department of Microbiology, China Medical College, Taichung, Taiwan; and †Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan Received November 22, 1999; revised February 1, 2000 The gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria strain Xv2 harbors an indigenous, cryptic plasmid pXV2 of 14.6 kb. This plasmid can only be maintained in Xanthomonas and is incapable of self-transmission. However, incompatibility testing classified it in IncW, a group containing the smallest number of naturally occurring, broad-host-range, conjugative plasmids. A pXV2 derivative containing only a 5.5-kb PstI fragment is stably maintained. Deletion of a 3.0-kb region from the PstI fragment causes a loss of plasmid stability. Nucleotide sequencing of the 2.1-kb region essential for autonomous replication revealed a repA gene and a downstream noncoding region containing four iterons, two 17- and two 19-nt direct repeats, and an AT-rich region lying between the two sets of iterons. The sequence of the deduced RepA and the iterons shows homology to the RepA (39% identity) and the iterons, respectively, of the IncW plasmid pSa. Maxicell expression of the repA gene produced a protein of 35 kDa, a size similar to that deduced from the nucleotide sequence. Trans-complementation test confirmed that the repA gene and the iterons are indeed the essential elements for pXV2 replication. © 2000 Academic Press

Xanthomonas campestris, a gram-negative species of important plant pathogens, consists of more than 125 closely related pathovars (Vauterin et al., 1990). X. campestris pv. vesicatoria is the causative agent of foliage and fruit spot disease in pepper and tomato (Minsavage et al., 1990b). Most strains of this pathovar have been known to harbor plasmids in sizes ranging from 1.8 to over 200 kb (Dittapongpitch and Ritchie, 1993; Lazo and Gabriel, 1987; Weng et al., 1996). While some of these plasmids encode genes involved in host specificity and/or resistance to copper or streptomycin, the others are cryptic (Bender et al., 1990; Bonas et al., 1989; Cooksey et al., 1990; Dittapongpitch et al., 1989; Dittapongpitch and Ritchie, 1993; Kearney and Staskawicz, 1990; Lai et al., 1977; Minsavage et al., 1990a,b; Stall et al., 1986; Swanson et al., 1988; Weng et al., 1996). In addition, several of these plasmids are conjugative (Dittapongpitch et al., 1989; Lai et al., 1977; Stall et al., 1986). Previously, screening of 58 virulent strains of X. campestris pv. vesicatoria isolated in Taiwan 1

To whom correspondence should be addressed.

had shown that most of them harbor extrachromosomal DNA elements of different sizes (Lin and Tseng, unpublished results). Two of these elements had been characterized; one is filamentous phage ␾Xv specific to this pathovar (Lin et al., 1994, 1999), and the other is the cryptic miniplasmid pXV64 (1.8 kb) possessing a RepA 2 protein similar to the replication initiator protein (gene II protein) of Escherichia coli filamentous phage I2-2 (Weng et al., 1997). In this study, plasmid pXV2 (14.6 kb) from strain Xv2 was characterized and shown to be a new member of the IncW plasmids. In addition, the essential region required for autonomous replication, containing the repA gene and four iterons, was identified. MATERIALS AND METHODS Bacterial strains, plasmids, and cultural conditions. The 58 strains of X. campestris pv. 2 Abbreviations used: bp, base pair; Cm, chloramphenicol; Km, kanamycin; Km r, kanamycin resistance; nt, nucleotide; ORF, open reading frame; ori, origin of replication; RepA, replication protein; Tc, tetracycline; Tc r, tetracycline resistance.

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vesicatoria were obtained from S.-T Hsu, National Chung Hsing University (Weng et al., 1996). X. campestris pv. campestris 17 has been described previously (Yang and Tseng, 1988). E. coli DH5␣ was used for gene cloning (Sambrook et al., 1989). The other bacterial strains, including X. oryzae pv. oryzae, Pseudomonas aeruginosa, Erwinia carotovora, Agrobacterium tumefaciens ATB2, and Rhizobium japonicum RJ110, were the collections of our laboratory. LB broth and L agar (Miller, 1972) were used as the general-purpose media for growing E. coli (37°C), X. campestris strains (28°C), and the other bacteria (30°C). As a selective pressure for plasmid, antibiotics were added: ampicillin (50 ␮g/ml) for pUC4K, chloramphenicol (Cm, 20 ␮g/ml) for pKT248, kanamycin (Km, 50 ␮g/ml) for pOK12, and tetracycline (Tc, 15 ␮g/ml) for pLAFR1. Resistance of the pXV2harboring strain, Xv2, was tested in L agar plate containing CuSO 4 (150 ␮g/ml) or with an antibiotic disc containing streptomycin (10 ␮g), chloramphenicol (30 ␮g), or kanamycin (30 ␮g). DNA techniques. Restriction endonucleases and the other enzymes were purchased from New England Biolabs (Beverly, MA) and Promega (Madison, WI) and were used by following the instructions provided by the suppliers. Plasmid DNA was isolated by the alkaline lysis method (Birnboim and Doly, 1979). Standard protocols (Sambrook et al., 1989) were followed for agarose gel electrophoresis, preparation of labeled DNA probes, Southern hybridization, and transformation of E. coli. All other bacteria were transformed by electroporation (Wang and Tseng, 1992). DNA sequence analysis. The nucleotide sequence of the 2.1-kb PstI fragment was determined for both strands by the dideoxy chain termination method (Sanger et al., 1977), using single-stranded M13 clones or double-stranded pUC clones as the templates. Sequence analysis was carried out using release 6.01 of PC/GENE (IntelliGenetics). Tfasta was used to search in the Genetics Computer Group for homologous sequences. The sequence reported here has been registered in GenBank under the Accession No. AF201825.

Stability test. The pXV2 derivatives, pOKE34, p55PK, p25PK, and p21PK, were separately electroporated into Xv65. To test for stability, a single colony of a test strain was picked from a selective plate and grown overnight. The culture was diluted 1000-fold into fresh LB broth without antibiotics and grown to saturation (OD 550 of approximately 5.0). The dilution and growth in fresh medium was performed 10 times in which each cycle constituted approximately 10 generations. After each growth saturation, aliquots of the cells were diluted and spread onto LB agar without Km. Two hundred colonies were patched on LB agar plates with or without Km. The percentages of cells able to grow in presence of Km were calculated. The rapid screening method of Weng et al. (1996) was adopted to detect the presence of plasmids. Incompatibility test. For incompatibility tests, double-plasmid containing strains were constructed by electroporating Xc17 harboring p55PK with one of the three test plasmids, pLAFR1 (Friedman et al., 1982), pKT248 (Wohlfarth and Winkler, 1988), and pSa3 (Tait et al., 1982). Transformants were selected with the antibiotic selecting for the incoming plasmid. One single colony of a test strain was grown to saturation, and the culture was subjected to the same dilution and growth cycles as in stability tests. The presence of plasmids was scored by antibiotic resistance and plasmid extraction (Weng et al., 1996) followed by agarose gel electrophoresis. Expression of RepA protein in E. coli maxicell. The 2.1-kb Sau3A1 fragment inserted in p21PK was cloned into the P15A-derived pOK12 (Vieira and Messing, 1991) to generate pOK21PK. pOK21PK was transformed into E. coli LCD44 (Li and Cronan, 1992) and the resultant strain LCD44(pOK21PK) was used for maxicell expression with LCD44(pOK12) as the control. Preparation of maxicell, induction with IPTG (0.1 mM), and labeling the gene product with [ 35S]methionine were performed by the method of Sancar et al. (1979). The cellular proteins were subjected to SDS–polyacrylamide gel electrophoresis (Laemmli, 1970) followed by detection with autoradiography.

IncW PLASMID pXV2 FROM Xanthomonas campestris

RESULTS AND DISCUSSION pXV2 is cryptic. Because several plasmids of pv. vesicatoria have been shown to encode genes involved in pathogenicity and resistance to streptomycin or copper (Bender et al., 1990; Bonas et al., 1989; Cooksey et al., 1990; Dittapongpitch et al., 1989; Kearney and Staskawicz, 1990; Minsavage et al., 1990a,b; Stall et al., 1986; Swanson et al., 1988), we tested pXV2 on these properties. Originally isolated from infected plants, the 58 X. campestris pv. vesicatoria strains used for plasmid screening were all virulent. Among these strains, only two harbored pXV2 and one harbored the homologous plasmid, pXV17, as detected by Southern hybridization (data not shown). Since the other strains without pXV2 were still virulent, this observation indicates that pXV2 is not required for pathogenicity. In addition, strain Xv2 harboring pXV2 was sensitive to copper and antibiotics including streptomycin, chloramphenicol, and kanamycin. These results indicated that pXV2 is cryptic. Restriction map of pXV2. The first steps we took to characterize pXV2 were the estimation of the plasmid size and construction of a physical map bearing sites for various restriction enzymes. In these experiments, pXV2 DNA was cut into one, two, and eight fragments by SmaI, KpnI, and PstI, respectively; into three fragments by BamHI, BglII, EcoRV, or HindIII; and into four fragments by EcoRI, MluI, SalI, or SphI. The sum totals, calculated from each of the digests, had an average of 14.6 kb representing the size of pXV2. The restriction sites were mapped primarily by single, double, or triple enzyme digestion. Some larger fragments from pXV2 were cloned for determination of the sites contained. When necessary, Southern hybridization was performed to confirm the relative positions of the fragments. These analyses resulted in the construction of the pXV2 physical map shown in Fig. 1. Identification of essential region for autonomous replication of pXV2. To clone the minimal region required for autonomous replication, a procedure similar to that used by Sobecky et al.

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(1998) was employed. pXV2 was digested with PstI and the fragments generated were ligated with the 1.3-kb Km r cartridge (encoding neomycin phosphotransferase II) from pUC4K (Vieira and Messing, 1982). After electroporation of the ligation mixture into X. campestris pv. vesicatoria 65 (Xv65), several transformants capable of growing in LB agar containing Km were obtained. Restriction analysis showed that all of these transformants contained the identical PstI fragment of 5.5 kb (Fig. 1). One of them, designated p55PK, was selected and used for further studies. Since Xv65 harbored other plasmids and p55PK could be maintained in the plasmid-free X. campestris pv. campestris 17 (Xc17) (Tseng et al., 1999) as well, Xc17 was used as the host for the experiments hereafter. To narrow down the region essential for replication, deletion clones derived from p55PK were tested for maintenance in Xc17. In the first series, made by ligating Sau3A1 partial fragments with the Km cartridge, p21PK and p25PK containing a 2.1- and a 2.5-kb overlapping insert (Fig. 1), respectively, were maintained in Xc17. The 2.1-kb insert from p21PK (Fig. 2A) was further deleted by treatment with either Bal31 nuclease or exonuclease III, resulting in series pD4 through pD7 and series pB1 through pB3, respectively (Fig. 2B). These data indicated that the minimal region required for autonomous replication was contained in a 1.5-kb fragment within the p21PK insert (Fig. 2C). The sequencing data obtained after these experiments indicated that the pB3 insert had a 5⬘ end corresponding to position ⫺65 relative to the repA start codon, whereas the pD5 insert had a 3⬘ end corresponding to bp 493 after the repA stop codon (see below). Nucleotide sequence of the pXV2 region essential for autonomous replication. The nucleotide sequence was determined for both strands of the 2.1-kb p21PK insert, from which a total of 2014 bp was obtained (Fig. 3A). In this fragment, we found a possible open reading frame (orf319) of 957 nt which was followed by a noncoding region. orf319, starting with ATG at nt 249 and ending at nt 1026 with two consecutive TGA, was predicted to encode a polypeptide of 319 amino acid residues with a

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FIG. 1. Restriction map of pXV2. The unique SmaI site is set as kb unit 0/14.6. The arrow indicates the position of repA. Hatched region represents the insert of p55PK. Inner and outer lines are the inserts of p21PK and p25PK, respectively.

calculated molecular weight of 34,765. A consensus ⫺10 sequence 5⬘-AATATT-3⬘ (nt 222) was present in the orf319 upstream region, but no consensus ⫺35 sequence or Shine–Dalgarno sequence was found (Fig. 3A). Without the consensus sequences for transcription/translation initiation, the repA gene probably exhibits a low level of expression. Comparison of the deuced protein product of orf319 revealed 39% amino acid sequence identity to RepA of the IncW plasmid pSa (Okumura and Kado, 1992). As shown in Fig. 4, the highest degree of identity is shared in the C-terminal 240 residues of Orf319 with the N-terminal three-fourths of the pSa RepA protein. With sequence similarity to that of the pSa RepA and as the only gene present in the minimal region required for autonomous replication, orf319 was thus proposed to be the repA gene of pXV2.

In iteron-containing plasmids, three common motifs comprise the origins of replication (ori): a region with directly repeated sequences, termed iterons, which are the binding sites for the plasmid-encoded Rep proteins and which have the control properties; an AT-rich region which serves as the site of helix destabilization; and one or more sites where the host DnaA protein binds (del Solar et al., 1998). In the DNA sequences downstream from orf319, there were a putative DnaA box and four direct repeats similar to iterons (Fig. 3A). The putative DnaA box, with a sequence (9 bp) of 5⬘TTATCCACG-3⬘ similar to the E. coli DnaA binding site, overlapped with the first direct repeat by five nucleotides. The first two direct repeats were 18 bp with a sequence of 5⬘CCACGCATAAACGGTCAG-3⬘ located at nt positions 1448 –1465 and 1478 –1495, and the

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FIG. 2. Deletion mapping of the essential region for pXV2 replication. (A) The p21PK insert, from which deletions were made as described under Materials and Methods. (B) The deletion clones maintained in Xc17 are indicated by “⫹” and those not maintained by “⫺.” Numbers above the 5⬘ and 3⬘ ends of each deletion fragment indicate the nucleotide positions relative to the sequenced 2014-bp fragment. Only seven of the clones are shown. (C) The determined essential region for pXV2 autonomous replication encompasses bp 184 to 1698. (D) The minimal ori-containing fragment capable of replication with the repA gene being provided in trans.

last two were 19 bp with a sequence of 5⬘TACGCATAAACGGACAGAT-3⬘ located at positions 1575–1593 and 1598 –1616 (Fig. 3A). The two sets of direct repeats were separated by an intervening sequence of 77 bp, a multiple of 11 bp which is close to the helical periodicity of the DNA double helix (del Solar et al., 1998). In addition, this intervening sequence is rich in adenine and thymine (A ⫹ T, 70%). Regions rich in adenine and thymine have low thermal stability and therefore are known to be common to a variety of replicating origins (Stalker et al., 1979; Grosschedl and Hobom, 1979). These structural features suggest these four direct repeats to be the binding sites for the RepA protein of pXV2 and serve as the origin (ori) of plasmid replication. It is also worth noting that the direct repeat sequences of pXV2 are homol-

ogous to the seven iterons of pSa (Fig. 3B; Okumura and Kado, 1992). Trans-complementation of the pXV2. To test whether the pXV2 fragment containing the direct repeat sequences possessed ori function, a trans-complementation was performed, with the repeated sequences and the repA gene being cloned in separate plasmids. The plasmid carrying the putative ori sequence, pDR1, was constructed using the P15A-derived narrowhost-range pOK12 (Vieira and Messing, 1991) by cloning the 431-bp fragment, cut down as a HindIII–SphI fragment, from the downstream of the pD5 insert as shown in Figs. 2D and 3A. This insert, spanning nt 1268 –1698 of the sequenced 2014-bp Sau3A1 fragment, contained all four repeats. The repA-containing plasmid, pRK1, was constructed in the RK2-derived

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FIG. 3. (A) Nucleotide sequence of the 2014-bp insert of p21PK and the predicted amino acid sequence of RepA protein. The consensus ⫺10 sequence is in bold. The boxed region represents the predicted DnaA binding site. The four direct repeats (iterons) are indicated by half arrows under the sequences. The two bent arrows confine the 431-bp fragment containing ori, between bp 1268 and 1698. (B) The four direct repeats in the pXV2 ori region (left) and the six iterons of pSa. The consensus sequences are shown at the bottom.

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FIG. 4. Alignment of the amino acid sequence deduced from the pXV2 repA (upper lines) with that of the pSa RepA protein (lower line). Dots indicate conservative substitutions. Dashes are introduced for optimal alignments.

broad-host-range pRK415 (Keen et al., 1988) by cloning the upstream region of the pD5 insert as a PstI–BglII fragment. This region corresponded to nt 1–1256 of the sequenced 2014-bp Sau3A1 fragment (Fig. 3A). Using pRK1 for electroporation, tetracycline-resistant transformants of Xc17, Xc17(pRK1), were readily obtainable; however, no transformant was obtained by electroporation of Xc17 with pDR1 indicating that, as expected, this P15A derivative could not replicate in Xanthomonas. Upon electroporation of Xc17(pRK1) with pDR1, Tc rKm r transformants were obtained. Analysis of the plasmids by restriction digestion followed by agarose gel electrophoresis indicated that these transformants indeed contained both pRK1 and pDR1. These results indicate that this 431-bp fragment contained the pXV2 ori. It has been shown that the defective replication of pSa3 can be partially complemented by R6K (Tait et al., 1983). In this study, we electroporated pDR1 into Xc17 carrying pSa3;

however, no Km r transformant was obtained. This result indicates that, although the DNA replication elements are homologous, the pSa3 RepA cannot complement the pXV2 ori for replication. In vitro expression. To express the product encoded by the pXV2 repA, plasmid pOK21PK was used for maxicell expression in E. coli LCD44. A distinct protein band with a molecular mass of 35 kDa was visualized in the autoradiogram after SDS–polyacrylamide gel electrophoresis (Fig. 5). The molecular size of this protein is similar to the value predicted for pXV2 RepA from the nucleotide sequence. Stability of pXV2 derivatives. The stability test was carried out with Xv65 carrying the pXV2-derived plasmid, pOKE34, p55PK, p25PK, or p21PK. These plasmids have been described above except that pOKE34 was a pXV2 derivative carrying the EcoRI-linearized pOK12 cloned into the EcoRI site near kb unit 1.8 of pXV2 (Fig. 1). As shown in Fig. 5,

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cells still carried both plasmids after 50 generations (Fig. 6B). In these experiments, plasmid analysis showed two plasmids of the expected sizes, giving no evidence of recombination between two plasmids (data not shown). Based on these results, it can be concluded that pXV2 belongs to the same incompatibility group as pSa3, IncW, the smallest group of naturally occurring, broad-host-range, conjugative plasmids. It has been demonstrated that some miniplasmids of pv. vesicatoria are capable of conjugal

FIG. 5. Expression of the pXV2 repA gene in E. coli LCD44. Cellular proteins were subjected to SDS–polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue (left) and the labeled proteins were detected by autoradiography (right). Lanes: A, maxicell LCD44; B, vector-only control LCD44(pOK12); C, LCD44(pOK21PK); D, molecular weight markers. The 35-kDa product is indicated by an arrow.

pOKE34 and p55PK were stable in Xv65 under nonselective conditions; after 100 generations over 98% of the cells retained the plasmids. In contrast, the other plasmids were not stably maintained; after 100 generations, only 35% of the cells retained p25PK and 15% of the cells retained p21PK (Fig. 6A). A similar degree of stability was also observed for these pXV2 derivatives when they were each electroporated into Xc17 (data not shown). Since p55PK was maintained with higher stability than that of p21PK and p25PK, it appears that the factor(s) required for plasmid stability is contained within the region next to the minireplicon in the 5.5-kb fragment. Incompatibility and transmissibility of pXV2. We examined the incompatibility between p55PK and plasmids from IncP (pLAFR1; Friedman et al., 1982), IncQ (pKT248; Wohlfarth and Winkler, 1988), and IncW (pSa3; Tait et al., 1982). Results showed that p55PK coexisted with pLAFR1 and pKT248 in Xc17 during the 100-generation testing period. However, p55PK was incompatible with pSa3; while all cells maintained pSa3 for at least 100 generations, only 40% of the cells maintained p55PK after 10 generations and less than 5% of the

FIG. 6. (A) Stability of pXV2 derivatives under nonselective growth conditions. Symbols: closed circle, p55PK; closed square, p25PK; open diamond, p21PK; open triangle, pOKE34. (B) Incompatibility of plasmid p55PK (closed circle) with pSa3 (open triangle). The percentages of plasmid retention in both tests were measured as described under Materials and Methods.

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transfer (Dittapongpitch et al., 1989; Dittapongpitch and Ritchie, 1993). To test for self-transmissibility, we used Km r-resistant derivatives of pXV2, pOKE34, and pOKV12, that were constructed by integrating p21PK into the homologous region of pXV2. Like pOKE34, pOKV12 was also stably maintained in pathovars campestris and vesicatoria. These plasmids were separately electroporated into Xc17 and the resultant strains were used as the donor cells for conjugation with Tc7, a tetracycline-resistant uvrB mutant of Xc17. No transconjugants were obtained in these experiments, indicating that pXV2 is not self-transmissible. Thus, pXV2 is different from the miniplasmids in pv. vesicatoria and the other IncW plasmids which are conjugative. Host range and homology with other plasmids. To test for host range, p55PK was electroporated into different bacteria, using Xc17 and Xv65 as the controls. The plasmid was able to transform the control cells and X. oryzae pv. oryzae with high efficiency. However, no transformants were obtained after electroporating P. aeruginosa, E. carotovora, A. tumefaciens ATB2, R. japonicum RJ110, or E. coli DH5␣. These results indicate that pXV2 is narrow host range and can only replicate in closely related hosts. This is different from other IncW plasmids which can be maintained in a wide range of gram-negative bacteria (Tait et al., 1982). Factors involved in determination of host range for plasmids are largely unknown. However, at least one report has described that in the IncP-1 plasmid R18, host range can be affected by reduced repA promoter activity (Krishnapillai et al., 1987). Since no typical promoter and ribosome binding site were found in the upstream region of pXV2 repA, it is possible that the weak transcription/translation initiation structures cause the failure of pXV2 to replicate in hosts other than Xanthomonas. Among the plasmids we screened in different strains of X. campestris pv. vesicatoria, at least six in addition to pXV2 could be differentiated by their sizes (Lin and Tseng, unpublished results). To detect homology, we carried out Southern hybridization with the probe prepared from pXV2. The hybridization signal was found to be associated with pXV17 (about 15 kb)

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only, and not with pXV7, pXV12, pXV15, pXV64, or pXV65 (data not shown). ACKNOWLEDGMENTS This research was supported by NSC85-2311-B-005-028 from the National Science Council, Republic of China. We are thankful to Dr. A. Tokudarian for critically reading the manuscript.

REFERENCES Bender, C. L., Malyick, D. K., Conway, K. E., George, S., and Pratt, P. (1990). Characterization of pXV10A, a copper resistant plasmid in Xanthomonas campestris pv. vesicatoria. Appl. Environ. Microbiol. 56, 170 –175. Birnboim, H. C., and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513–1523. Bonas, U., Stall, R. E., and Staskawicz, B. (1989). Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria. Mol. Gen. Genet. 218, 127–136. Cooksey, D. A., Azad, H. R., Cha, J. S., and Lim, C. K. (1990). Copper resistance gene homologs in pathogenic and saporphytic bacterial species from tomato. Appl. Environ. Microbiol. 56, 431– 435. del Solar, G., Giraldo, R., Ruiz-Echevarria, M. J., Espinosa, M., and Diaz-Orejas, R. (1998). Replication and control of circular bacterial plasmids. Microbiol. Mol. Biol. Rev. 62, 434 – 464. Dittapongpitch, V., Ritchie, D. F., and Upchurch, D. R. (1989). Small, conjugatable plasmid in copper-resistant strains of Xanthomonas campestris pv. vesicatoria. Phytopathology 79, 1192. Dittapongpitch, V., and Ritchie, D. F. (1993). Indigenous miniplasmids in strains of Xanthomonas campestris pv. vesicatoria. Phytopathology 83, 95–964. Friedman, A. M., Long, S. R., Brown, S. E., Buikema, W. J., and Ausubel, F. M. (1982). Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene 18, 289 –296. Grosschedl, R., and Hobom, G. (1979). DNA sequences and structural homologies of the replication origins of lambdoid bacteriophages. Nature 277, 621– 627. Kearney, B., and Staskawicz, B. J. (1990). Characterization of IS476 and its role in bacterial spot disease of tomato and pepper. J. Bacteriol. 172, 143–148. Keen, N. T., Tamaki, S., Kobayashi, D., and Trollinger, D. (1988). Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene 70, 191–197. Krishnapillai, V., Wexler, M., Nash, J., and Figurski, D. H. (1987). Genetic basis of a Tn7 insertion mutation in the trfA region of the promiscuous IncP-1 plasmid R18 which affects its host range. Plasmid 17, 164 –166. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685.

172

WU AND TSENG

Lai, M., Panopoulos, N. J., and Shaffers, S. (1977). Transmission of R plasmids among Xanthomonas spp. and other plant pathogenic bacteria. Phytopathology 67, 1104 –1050. Lazo, G. R., and Gabriel, D. W. (1987). Conservation of plasmid DNA sequences and pathovar identification of strains of Xanthomonas campestris. Phytopathology 77, 448 – 453. Li, S. J., and Cronan, J. E., Jr. (1992). The gene encoding the biotin carboxylase subunit of Escherichia coli acetylCoA carboxylase. J. Biol. Chem. 267, 855– 863. Lin, N. T., You, B. Y., Huang, C. Y., Kuo, C. W., Wen, F. S., Yang, J. S., and Tseng, Y. H. (1994). Characterization of two novel filamentous phages of Xanthomonas. J. Gen. Virol. 75, 2543–2547. Lin, N. T., Liu, T. J., Lee, T. C., You, B. Y., Yang, M. H., Wen, F. S., and Tseng, Y. H. (1999). The adsorption protein genes of Xanthomonas campestris filamentous phages determining host specificity. J. Bacteriol. 181, 2465–2471. Miller, J. H. (1972) “Experiments in Molecular Genetics.” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Minsavage, G. V., Canteros, B. I., and Stall, R. E. (1990a). Plasmid-mediated resistance to streptomycin in Xanthomonas campestris pv. vesicatoria. Phytopathology 80, 719 –723. Minsavage, G. V., Dahlbeck, D., Whalen, M., Kearney, B., Bonas, U., Staskawicz, B. J., and Stall, R. E. (1990b). Gene-for-gene relationships specifying disease resistance in Xanthomonas campestris pv. vesicatoria–pepper interactions. Mol. Plant–Microbe Interact. 3, 41– 47. Okumura, M. S., and Kado, C. I. (1992). The region essential for efficient autonomous replication of pSa in Escherichia coli. Mol. Gen. Genet. 235, 55– 63. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) “Molecular Cloning: A Laboratory Manual,” 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sancar, A., Hack, A. M., and Rupp, W. D. (1979). Simple method for identification of plasmid-coded proteins. J. Bacteriol. 137, 692– 693. Sanger, F., Nicklen, S., and Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467. Sobecky, P. A., Mincer, T. J., Chang, M. C., Toukdarian, A., and Helinski, D. R. (1998). Isolation of broad-hostrange replicons from marine sediment bacteria. Appl. Environ. Microbiol. 64, 2822–2830. Stalker, D. M., Kolter, R., and Helinski, D. R. (1979). Nucleotide sequence of the region of an origin of replication of the antibiotic resistance plasmid R6K. Proc. Natl. Acad. Sci. USA 76, 1150 –1154.

Stall, R. E., Loschke, D. C., and Jones, J. B. (1986). Linkage of copper resistance and avirulence loci on a self-transmissible plasmid in Xanthomonas campestris pv. vesicatoria. Phytopathology 76, 240 –243. Swanson, J., Kearney, B., Dahlbeck, D., and Staskawicz, B. (1988). Cloned avirulence gene of Xanthomonas campestris pv. vesicatoria complements spontaneous racechange mutants. Mol. Plant–Microbe Interact. 1, 5–9. Tait, R. C., Close, T. J., Rodriguez, R. L., and Kado, C. I. (1982). Isolation of the origin of replication of the IncWgroup plasmid pSa. Gene 20, 39 – 49. Tseng, Y. H., Choy, K. T., Hung, C. H., Lin, N. T., Liu, J. Y., Lou, C. H., Yang, B. Y., Wen, F. S., Weng, S. F., and Wu, J. R. (1999). Chromosome map of Xanthomonas campestris pv. campestris 17 with locations of genes involved in xanthan gum synthesis and yellow pigmentation. J. Bacteriol. 181, 117–125. Vauterin, L., Swings, J., Kersters, K., Gillis, M., Mew, T. W., Schroth, M. N., Palleroni, N. J., Hildebrand, D. C., Stead, D. E., Civerolo, E. L., Hayward, A. C., Maraite, H., Stall, R. E., Vidaver, A. K., and F., B. J. (1990). Towards an improved taxonomy of Xanthomonas. Int. J. Syst. Bacteriol. 40, 312–316. Vieira, J., and Messing, J. (1982). The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259 –268. Vieira, J., and Messing, J. (1991). New pUC-derived cloning vectors with different selectable markers and DNA replication origins. Gene 100, 189 –194. Wang, T. W., and Tseng, Y. H. (1992). Electrotransformation of Xanthomonas campestris by RF DNA of filamentous phage ␾Lf. Lett. Appl. Microbiol. 14, 65– 68. Weng, S. F., Lin, N. T., Fan, Y. F., Lin, J. W., and Tseng, Y. H. (1996). Characterization of the 1.8-kb plasmid pXV64 from Xanthomonas campestris pv. vesicatoria. Bot. Bull. Acad. Sin. 37, 93–98. Weng, S. F., Fan, Y. F., Tseng, Y. H., and Lin, J. W. (1997). Sequence analysis of the small cryptic Xanthomonas campestris pv. vesicatoria plasmid pXV64 encoding a Rep protein similar to gene II protein of phage 12-2. Biochem. Biophys. Res. Commun. 231, 121–125. Wohlfarth, S., and Winkler, U. K. (1988). Chromosomal mapping and cloning of the lipase gene of Pseudomonas aeruginosa. J. Gen. Microbiol. 134, 433– 440. Yang, B. Y., and Tseng, Y. H. (1988). Production of exopolysaccharide and levels of proteases and pectinase activity in pathogenic and nonpathogenic strains of Xanthomonas campestris pv. campestris. Bot. Bull. Acad. Sin. 29, 93–99. Communicated by Donald Helsinki