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Journal of Bacteriology, January 1999, p. 117-125, Vol. 181, No. 1
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Chromosome Map of Xanthomonas campestris pv. campestris 17 with Locations of Genes Involved in Xanthan Gum Synthesis and Yellow Pigmentation

Yi-Hsiung Tseng,* Ka-Tim Choy, Chih-Hsin Hung, Nien-Tsung Lin,dagger Jane-Yu Liu, Chih-Hong Lou, Bih-Ying Yang,Dagger Fu-Shyan Wen, Shu-Fen Weng, and Jung-Rung Wu§

Institute of Molecular Biology and Department of Botany, National Chung Hsing University, Taichung 402, Taiwan

Received 30 July 1998/Accepted 20 October 1998

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

No plasmid was detected in Xanthomonas campestris pv. campestris 17, a strain of the causative agent of black rot in cruciferous plants isolated in Taiwan. Its chromosome was cut by PacI, PmeI, and SwaI into five, two, and six fragments, respectively, and a size of 4.8 Mb was estimated by summing the fragment lengths in these digests. Based on the data obtained from partial digestion and Southern hybridization using probes common to pairs of the overlapping fragments or prepared from linking fragments, a circular physical map bearing the PacI, PmeI, and SwaI sites was constructed for the X. campestris pv. campestris 17 chromosome. Locations of eight eps loci involved in exopolysaccharide (xanthan gum) synthesis, two rrn operons each possessing an unique I-CeuI site, one pig cluster required for yellow pigmentation, and nine auxotrophic markers were determined, using mutants isolated by mutagenesis with Tn5(pfm)CmKm. This transposon contains a polylinker with sites for several rare-cutting restriction endonucleases located between the chloramphenicol resistance and kanamycin resistance (Kmr) genes, which upon insertion introduced additional sites into the chromosome. The recA and tdh genes, with known sequences, were mapped by tagging with the polylinker-Kmr segment from Tn5(pfm)CmKm. This is the first map for X. campestris and would be useful for genetic studies of this and related Xanthomonas species.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The gram-negative plant-pathogenic species Xanthomonas campestris has two common traits, yellow pigmentation and exopolysaccharide (xanthan gum) production which render the colonies yellowish and mucoid. This species consists of more than 123 pathovars, which can be differentiated on the basis of the host plants they infect (55). Most of the pathovars exhibit high degrees of host specificity in infection. X. campestris pv. campestris is the pathogen causing black rot in crucifers, resulting in heavy loss in agriculture worldwide (65). In addition, xanthan gum has a variety of applications in agriculture, petroleum production, and food industry as a stabilizing, viscosifying, emulsifying, thickening, and suspending agent (25, 43). Therefore, pathovar campestris has been the organism of choice for industrial production of xanthan gum.

The biochemistry and genetics of X. campestris are largely unknown. Methods for plasmid transformation by electroporation (57, 64, 71) and conjugal transfer of plasmids (14, 70) have been developed for gene cloning in this species, and a transducing phage has recently been characterized (60). However, these techniques have not been extended to other genetic studies of this species, and no genetic map has yet been reported.

Recently, physical maps for the chromosomes of many bacteria have been reported (13); such maps have been generated by application of techniques for preparation of intact chromosome from bacterial cells (46), rare-cutting restriction enzymes for obtaining long DNA fragments, and pulsed-field gel electrophoresis (PFGE) for separation of large DNA fragments (45). Therefore, physical maps of some bacterial chromosomes, for which no genetic maps are available, have been constructed as the first efforts to characterize the chromosomes (1-3, 7, 8, 27, 28, 31, 50, 63). Here, we report the construction of the physical map of X. campestris pv. campestris 17 chromosome based on restriction enzymes SwaI, PacI, PmeI, and I-CeuI. The size of the X. campestris pv. campestris 17 chromosome estimated from the sum total of the restriction fragments is about 4.8 Mb. Locations for 23 genetic loci were determined on the X. campestris pv. campestris 17 map.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Bacterial strains, plasmids, and phages. Bacterial strains and plasmids used in this study are listed in Table 1. M9 medium, LB broth, and LB agar have been described elsewhere (39). X. campestris was grown at 28°C, and Escherichia coli was grown at 37°C. Antibiotics ampicillin (50 µg/ml), chloramphenicol (25 µg/ml), rifampin (100 µg/ml), and kanamycin (50 µg/ml) were included when required.

                              
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  		TABLE 1.   Bacterial strains and plasmids used in this study

Isolation of mutants by Tn5 mutagenesis. pUT-Tn5(pfm)CmKm is a mobilizable derivative of R6K carrying mini-Tn5 (16), which contains within its inverted repeats the chloramphenicol resistance and kanamycin resistance (Cmr and Kmr) genes and a polylinker with unique rare-cutting sites for M · XbaI-DpnI, SwaI, PacI, NotI, SfiI, BlnI, SpeI, and XbaI (66). For mutagenesis, pUT-Tn5(pfm)CmKm was mobilized by conjugation from E. coli S17-1::lambda pir to X. campestris pv. campestris 17R as described previously (70). Auxotrophic mutants were first isolated as the Cmr and Kmr transconjugants that could not grow in the M9 medium containing glucose (80 mM), and their amino acid requirements were determined in the crossed pool plates (15). Nonmucoid and low-mucoid mutants were recognized by their dry colony morphology on M9 medium supplemented with sucrose (4%). The pigmentation mutants were identified by the grayish color of the colonies on the LB agar. Mutant XKB was verified by its resistance to kanamycin on LB agar containing kanamycin and the presence of Tn5 sequence in chromosome by Southern hybridization.

Gene tagging. The locations of tdh and recA genes were determined by a gene-tagging strategy. The 2-kb XbaI-EcoRI fragment containing the promoter and the N terminus of the tdh gene from plasmid pX727 (44) was cloned into the multiple cloning sites of pNEB193 (New England Biolabs, Beverly, Mass.); then the 3-kb PvuII fragment from pUT-Tn5(pfm)CmKm, containing the rare-cutting sites and the Kmr gene, was ligated into the EcoRV site adjacent to the tdh fragment. The resulting plasmid was integrated into the X. campestris pv. campestris 17 chromosome by a single crossover through the homologous tdh sequences. The recA gene was tagged in a similar way except that the 267-bp PstI fragment internal to the recA coding region (30) was used. Southern hybridization was carried out to verify integration of the whole plasmid into the target gene.

DNA techniques. The methods described by Sambrook et al. (42) were used for preparation of plasmid and chromosomal DNAs, restriction digestion, DNA ligation, preparation of alpha -32P-labeled probes by random priming, Southern hybridization, conventional agarose gel (0.7%) electrophoresis (0.5× Tris-acetate-EDTA buffer), and transformation of E. coli. X. campestris was transformed by electroporation (57). To detect plasmid in X. campestris, we used three methods: the rapid screening method described by Weng et al. (61), the alkaline lysis method of Birnboim and Doly (4), and the method of Kado and Liu (24) for extraction of large plasmids.

Preparation of chromosomal DNA. Cells of X. campestris, grown in M9 medium containing 20 mM glucose until mid-log phase (optical density at 600 nm of 0.5), were pelleted and resuspended directly in molten (37°C) agarose (SeaPlaque agarose; FMC Corp.), which was allowed to solidify in a syringe 3 mm in diameter (1 ml). The gel cylinder was blown out of the syringe through the larger opening and treated with lysozyme, RNase, and proteinase K by procedures described by Smith et al. (46). The DNA-containing gel was stored in 0.5 M EDTA at 4°C and then sliced into disks of 1.0 mm prior to restriction enzyme digestion.

Restriction digestion with rare-cutting endonucleases. SwaI (purchased from Boehringer Mannheim GmbH, Mannheim, Germany) and the other restriction enzymes (from New England Biolabs or Promega [Madison, Wis.]) were used as instructed by the suppliers. Each agarose disk was preincubated in the enzyme mixture for 3 h at 4°C to ensure sufficient diffusion of enzyme and buffer into the agarose disk. Each disk was (i) incubated with 5 to 10 U of restriction enzyme and incubated for 4 to 12 h at 37°C to obtain complete digestion or (ii) incubated with 0.5 to 2 U of enzyme for 5 to 30 min to obtain partial digestion. Sequential double digestion was performed sequentially by washing and equilibrating the agarose disks or the gel strips from first-dimensional PFGE in appropriate buffers prior to digestion.

PFGE. DNA samples were separated in 0.9 to 1.3% agarose gel (SeaKem LE agarose; FMC) in 0.5× Tris-borate-EDTA buffer refrigerated throughout the experiment. When two-dimensional separations were performed, 1% low-melting-point agarose (SeaPlaque agarose; FMC) was used for the first dimension. Unless otherwise indicated, PFGE was performed with a Bio-Rad CHEF-DRII system. Since different conditions were used for PFGE, the details are given below.

(i) Three different PFGE conditions were used to separate the SpeI digests of the X. campestris pv. campestris 17 chromosome. Fragments larger than 140 kb were separated in 1.2% agarose gel, at 160 V with pulses 40 to 35 s for 7 h followed by 20 s for 24 h. The fragments between 140 and 6 kb were separated in 1.3% agarose gel, at 160 V with a pulse of 10 s for 10 h followed by pulses 8 to 3.5 s for 19 h. The fragments between 8 and 1 kb were separated in 1.3% agarose gel, at 130 V with a pulse of 10 s for 3 h followed by pulses of 8 to 3 s for 11 h. Two-dimensional PFGE was performed to separate the subfragments generated by sequential digestion of the SpeI fragments in gel with SspI or XbaI. The first dimension was run with the migration parameters described above. The second dimension was run in a GeneLine II (Beckman) apparatus in 1% agarose at 375 mA with a pulse of 24 s for 25 h followed by 12 s for 20 h and then 6 s for 15 h (to separate the XbaI digests) or with a pulse of 8 s for 40 h followed by 4 s for 20 h (to separate the SspI digests).

(ii) The SwaI, PmeI, and PacI fragments were separated in 0.9% agarose, at 170 V with pulses 180 to 150 s for 11.5 h followed by 85 to 5 s for 12 h. To separate the fragments generated by sequential double digestion in gel of the SwaI or PacI fragments with SpeI, second-dimensional PFGE was performed with a CHEF-DRIII or CHEF-DRII apparatus for fragments with sizes of 432 to 26 kb or 154 to 5 kb, respectively. The parameters for the former were 1.2% agarose, at 4.9 V/cm with pulses 40 to 35 s for 5 h followed by 20 to 15 s for 15 h and then 10 to 1.5 s for 13 h; the latter were at 160 V with pulses 15 to 10 s for 12 h followed by 8 to 2 s for 20 h.

(iii) For the other PFGE experiments, the migration parameters are detailed in the figure legends.

Lambda DNA concatemers, HindIII-digested lambda  DNA, and Saccharomyces cerevisiae YPH80 chromosomes, purchased from New England Biolabs, were used as molecular size markers.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mutants isolated by Tn5(pfm)CmKm mutagenesis of X. campestris pv. campestris 17R. Thirty-four auxotrophic mutants (13 adenine, 2 guanine, 2 uracil, 1 cysteine, 1 glutamine, 1 glycine, 6 histidine, 2 threonine, and 6 tryptophan auxotrophic strains) were isolated. One strain each of mutant is described in Table 1.

Twenty-three nonmucoid or low-mucoid mutants were isolated and divided into eight groups according to complementation test and chromosomal locations (Table 1); these eight loci were designated eps1 to 8 (eps denotes exocellular polysaccharide synthesis). The eps1 mutants (BL81E and 12 others) were complemented by cosmid clone pP2402B carrying a ca. 24-kb insert which contained the rfbCDAB gene cluster required for lipopolysaccharide synthesis and the pmi gene involved in xanthan synthesis (26, 51-53). The eps3 mutants (BG44E and AG60E) were complemented by pSD701 carrying the UDP-glucose dehydrogenase gene (33). The eps6 mutant (G76E) was complemented by clone pEK6 carrying the gene encoding UDP-glucose pyrophosphorylase (58, 59). The eps7 mutants (AH53E and AL11E) were complemented by cosmid clone pP3DA01B with a ca. 25-kb insert (69) containing the gum gene cluster required for assembly of the glycosyl carrier lipid-bound pentasaccharide repeating units and translocation of xanthan across the bacterial membranes (GenBank accession no. U22511 [10, 11, 35, 38, 54, 56, 72]). The remaining four mutants, eps2 (BB15E), eps4 (BM23E), eps5 (AY61E), and eps8 (AU56E and BJ100E), carried the Tn5(pfm)CmKm insertion at different locations (see below).

Ten pigmentation mutants (D57W and nine others) forming grayish colonies were isolated and used for the mapping work in this study. The defect in normal pigmentation of these mutants was presumably due to the failure in synthesis of the yellow pigment xanthomonadins (49).

Detection of plasmid and selection of restriction enzymes. Several strains of X. campestris have been found to carry plasmids of various sizes (9, 12, 29, 32, 47, 48, 67). However, in this study, no plasmid in X. campestris pv. campestris 17 was detected by using three different methods of extracting circular plasmids (4, 24, 61).

The X. campestris genome has a G+C content of approximately 64% (6). Thus, for cutting the X. campestris pv. campestris 17 chromosome, several restriction enzymes which recognize AT-rich sequences were tested. The X. campestris pv. campestris 17 chromosome was cut by PacI (TTAATTAA), SwaI (ATTTAAAT), PmeI (GTTTAAAC), and SpeI (ACTAGT) into 5, 6, 2, and 37 bands, respectively. DraI, XbaI, and SspI each generated several large fragments and many small fragments too small for convenient analysis.

Estimation of chromosome size. PFGE of the SpeI digests of the X. campestris pv. campestris 17 chromosome produced bands ranging from 1.4 to 432 kb in size. Different conditions were used for maximum resolution of the fragments in different size ranges. In total, 37 ethidium bromide-stained bands were observed and designated, in descending alphabetical order, SA to SZ, followed by SAA to SAK (Fig. 1). The fluorescence intensities in some bands suggested that they might contain multiple molecular species. To resolve the multiplets, the DNA in the band was isolated and digested with a second restriction enzyme, SspI or XbaI, and separated by a second-dimensional PFGE. A singlet would generate subfragments with a total size similar to that of the original fragment, whereas a multiplet would give multiple sizes. By these methods, a total of 50 SpeI fragments were observed. DNA bands SI, SS, and SU contained triplets, SJ, SK, SN, SV, SW, SY, and SZ contained doublets, and the remaining bands had single species. The sum of the size of these SpeI fragments was 4,832 kb.


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  		FIG. 1.   PFGE of SpeI digests of the X. campestris pv. campestris 17 chromosome. Migration parameters are described in Materials and Methods. SA to SAK are designations of the SpeI fragments. Numbers in parentheses denote fragment sizes in kilobases; numbers on the left indicate sizes of molecular markers. One asterisk and two asterisks indicate doublet and triplet, respectively. M1, M2, and Y are HindIII-digested lambda  DNA, lambda  ladder, and S. cerevisiae chromosomes, respectively.

The six DNA bands formed by SwaI digests of the X. campestris pv. campestris 17 chromosome were designated WA to WF, and the five bands formed by PacI digests were designated PA to PE (Table 2). We could determine the sizes of all of these fragments except WA and PA, which migrated in the compressed zone containing DNA of 1,640 kb or larger, under the PFGE conditions used in these experiments. Therefore, for better size estimation, we used PacI plus PmeI or SwaI plus PmeI to cut the chromosomes of X. campestris pv. campestris 17 and mutant A48A(trp-1). In X. campestris pv. campestris 17, fragments PA and WA were each cut into three subfragments, the two larger ones forming a double band in both digests. The doublets from PA were designated (P/M)A and (P/M)B (1,375 kb), and those from WA were designated (W/M)A and (W/M)B (1,180 kb) (Table 2). The (P/M)A and (W/M)A from A48A(trp-1) were each further cut into two fragments due to the presence of Tn5(pfm)CmKm and thus were differentiated from (P/M)B and (W/M)B, respectively (Table 2). All fragments generated by PacI/PmeI digestion of the A48A(trp-1) chromosome were shown to contain single species by further restriction digestion with SpeI (data not shown). The sizes were calculated to be 3,270 and 3,253 kb for PA and 2,880 and 2,888 kb for WA, respectively, from the digests of X. campestris pv. campestris 17 and A48A(trp-1). In addition to the PacI/PmeI and SwaI/PmeI double digests, we were able to obtain PacI/SwaI double digests of the X. campestris pv. campestris 17 chromosome. In the latter digests, fragments PB, PC, PE, WA, WD, WE, and WF were not affected, but we observed four new fragments of 175, 108, 48, and 36 kb, which were designated (P/W)A, (P/W)B, (P/W)C, and (P/W)D, respectively.

                              
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  		TABLE 2.   Sizes of restriction fragments from X. campestris pv. campestris 17 and mutant A48A(trp-1) chromosomes

The sums from the PacI/PmeI and SwaI/PmeI digests of the X. campestris pv. campestris 17 and A48A(trp-1) chromosomes were used to represent independent estimations, and values of 4,799, 4,782, 4,789, and 4,797 kb were obtained (Table 2). Taking together these values and those estimated from the SpeI fragments, we calculated the size of the X. campestris pv. campestris 17 chromosome to be 4,800 ± 17 kb.

Order of SwaI fragments and circularity of the X. campestris pv. campestris 17 chromosome. The order of three of the SwaI fragments, WC-WD-WF, was established by partial digestion of the X. campestris pv. campestris 17 chromosome with SwaI, followed by Southern hybridization with probes each specific for one of the fragments. The three probes used were pPPE3, pPWDR3, and pW2Y2 (Table 1). pPPE3 and pPWDR3 contained inserts cloned from fragments PE (contained within WC) and WD (contained within PD), respectively. pW2Y2 was a cosmid clone containing a ca. 38-kb insert with unique sites for SwaI and PacI and could hybridize to fragments WB, WF, PC, and PD (see below). By using about 2 U of SwaI and incubation for 5 to 30 min to cut the X. campestris pv. campestris 17 chromosome, three partial bands, i.e., Wpa, Wpb, and Wpc, were visualized in the gel (Fig. 2A). The size of Wpa (442 kb) suggested that it consists of WC (300 kb), WD (125 kb), and WF (17 kb); this was confirmed by Southern hybridization, which showed that all three probes hybridized to Wpa (Fig. 2B to D). Wpb exhibited a size equal to the sum of those of WC plus WD and hybridized to pPPE3 and pPWDR3 (Fig. 2B and C), indicating that these two fragments are linked. Wpc, having a size equal to the sum of those of WD plus WF, hybridized to pPWDR3 and pW2Y2 (Fig. 2C and D), indicating that WD and WF are connected. Therefore, the fragment order WC-WD-WF was established.


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  		FIG. 2.   Southern hybridization of SwaI fragments of X. campestris pv. campestris 17 with probes specific to fragment WC, WD, or WF or probes prepared from large pieces of DNA containing fragment junctions. (A) PFGE of partial digests of the X. campestris pv. campestris 17 chromosome. Lanes 1 to 4 represent fragments obtained by cutting with SwaI (2 U) for 5, 10, 15, and 30 min, respectively; lane 5 is the complete digest for comparison. PFGE was performed with a CHEF-DRIII apparatus in 1% agarose gel at 4 V/cm with pulses of 150 to 130 s for 8 h followed by 70 to 5 s for 14 h. The gel was Southern transferred onto a nylon membrane. The same membrane was repeatedly used, after appropriate washing before each use, for hybridization with probes prepared from pPPE3 (B), pPWDR3 (C), and pW2Y2 (D). Wpa, Wpb, and Wpc are partial fragments WC-WD-WF, WC-WD, and WD-WF, respectively. Gels on the left in panels E to G represent PFGE of the SwaI complete digests of the X. campestris pv. campestris 17 chromosome, which were Southern hybridized with the probes (right-hand gels) prepared from pW2Y2 (E), the PAsura-1 fragment of A48A(ura-1) (F), and the PAsthr-1 fragment of BG49A(thr-1) (G).

The fragment orders WF-WB, WC-WA, and WA-WE-WB were established by hybridization using large DNA fragments which showed linkage relationships between these fragments. In Southern hybridization, the pW2Y2 probe reacted with fragments WB and WF (Fig. 2E), indicating that these two fragments are linked. Fragment PA of AS20A(ura-1) was cut by PacI into two subfragments, PAbura-1 (larger than 1,640 kb) and PAsura-1 (210 kb), due to the presence of Tn5(pfm)CmKm. The probe prepared from PAsura-1 (recovered from agarose gel) hybridized to WA and WC of the X. campestris pv. campestris 17 chromosome (Fig. 2F), indicating that WA and WC are linked. Fragment PA from mutant BG49A(thr-1) was cut by PacI into two subfragments, PAbthr-1 (larger than 1,640 kb) and PAsthr-1 (425 kb). The PAsthr-1 probe hybridized to fragments WA, WE, and WB of the X. campestris pv. campestris 17 chromosome (Fig. 2G), indicating that WE is connected with WA and WB in the order WA-WE-WB. Based on the linkages described above, we proposed the fragment order WB-WE-WA-WC-WD-WF-WB, which implies that X. campestris pv. campestris 17 has a circular chromosome (Fig. 3).


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  		FIG. 3.   Physical map of the X. campestris pv. campestris 17 chromosome. PA to PE, WA to WF, and MA and MB represent the fragments generated by digestion of the chromosome with PacI, SwaI, and PmeI, respectively. Lines outside the circles pW2Y2, PAsura-1, and PAsthr-1 were used as the probes for hybridization to link WB/WF, WA/WC, and WA/WE/WB, respectively. The approximate positions of the clones used as probes for hybridization are indicated by arrows outside the circles.

Alignment of PacI fragments by overlapping with the SwaI fragments. In this study, a partial fragment (583 kb) with a size equal to the sum of the sizes of PC and PD was observed many times in the PacI digests of X. campestris pv. campestris 17, indicating its partial resistance to the digestion. pW2Y2 probe hybridized to this partial fragment as well as to PC and PD (data not shown), suggesting that PC and PD are linked. In addition, pPWDR3 (from WD) hybridized to PD, and pPPE3 (from PE) hybridized to WC (data not shown), suggesting a PD-PE linkage. Combining these results, we established the order PE-PD-PC.

It was shown above that the PAsura-1 from mutant AS20A(ura-1) overlaps WC and WA (Fig. 2F), indicating that PA is next to PE. Adding this linkage relationship to the order established above, we propose the fragment order PA-PE-PD-PC. Since the X. campestris pv. campestris 17 chromosome is circular, fragment PB can be placed between PA and PC, resulting in the order PA-PE-PD-PC-PB-PA (Fig. 3). The overlapping relationships between WB and PC, WB and PB, and WA and PA were confirmed by cutting the chromosomes of several Tn5(pfm)CmKm-induced mutants (see below).

Location of the PmeI sites. The X. campestris pv. campestris 17 chromosome was cut by PmeI into two fragments, MA (larger than 1,640 kb) and MB (520 kb), suggesting that it may have two PmeI sites (Table 2). The PA fragment from X. campestris pv. campestris 17 was cut by PacI plus PmeI into three subfragments, (P/M)A, (P/M)B, and MB, with (P/M)A and (P/M)B (1,375 kb) forming a double band. Since no other PacI fragment was affected, these results indicate that there are two PmeI sites both residing within the PA fragment. (P/M)A and (P/M)B were differentiated by double digestion of the A48A(trp-1) chromosome with PacI plus PmeI, where the (P/M)A was cleaved into two subfragments, Mmtrp-1 (748 kb) and PAstrp-1 (610 kb) (Table 2). To determine the locations of the PmeI sites, hybridization was carried out with probes pSD701 and pREF, which carried a 5.3-kb insert from WE near the PA/PB junction and a 0.3-kb insert from the overlapping region of WC and PA near the PA/PE junction, respectively (Table 1). Probes pSD701 and pREF were found to hybridize to (P/M)B and the 610-kb PAstrp-1 fragment, respectively (data not shown). These results suggest that one PmeI site is 1,380 kb from the PA/PE junction and the other is 1,375 kb from the PA/PB junction with a spacer of 520 kb, equal to the size of MB (Fig. 3).

Location of genetic markers. Fragments WB and PC of the mutant XKB chromosome were cut into WBbXKB (1,230 kb) plus WBsXKB (147 kb) by SwaI and into PCbXKB (242 kb) plus PCsXKB (111 kb) by PacI, respectively, indicating that the Tn5(pfm)CmKm insertion in XKB occurred in the WB/PC overlapping region (data not shown). Southern hybridization using the pW2Y2 probe showed signals with WBsXKB and PCsXKB, indicating that XKB is ca. 147 kb from the WF/WB interface and ca. 111 kb from the PC/PD interface (Fig. 3). Using a similar strategy with the pW2Y2 probe, eps1, ade-1, and eps2 were localized within WB at ca. 438, 513, and 734 kb from WF/WB interface and within PB at ca. 45, 120, and 345 kb from the PC/PB interface, respectively (Fig. 3).

The PA fragment of mutant BG44E(eps3) was cut by PacI into two fragments (one 150 kb and the other larger than 1,640 kb), whereas the WE fragment was cut into two fragments with similar sizes (ca. 42 kb) (data not shown). These results indicate that WE and PA are overlapping and suggest eps3 to be near the middle of WE and ca. 150 kb from the PA/PB interface (Fig. 3).

The recA gene, eight auxotrophic markers, and five eps loci were localized in the PA/WA overlapping region. Due to the presence of Tn5(pfm)CmKm, the PA and WA fragments of these mutants were each cut by PacI and SwaI, respectively, into two subfragments. In mutants XKLR1(recA), BG49A(thr-1), T2A(his-1), BC3A(gly-1), BM23E(eps4), AY61E(eps5), AM39A(gua-1), G76E(eps6), and AH53E(eps7), the subfragments derived from PA having sizes of 353, 420, 425, 440, 500, 630, 866, 872, and 1,020 kb, respectively, were found to hybridize to the pSD701 probe (data not shown). These sizes represent the distances between the respective mutants and the PA/PB junction (Fig. 3). In mutants AS20A(ura-1), AN32A(gln-1), A48A(trp-1), AU56E(eps8), and G56A(cys-1), the subfragments derived from PA having sizes of 210, 295, 610, 620, and 1,120 kb, respectively, were found to hybridize to the pREF probe. These sizes represent the distances between the respective mutants and the PA/PE junction. Furthermore, these fragments are ca. 175 kb larger than the corresponding smaller subfragments derived from WA, indicating that PA overhangs WA at this end by about 175 kb (Fig. 3).

We have reported that X. campestris pv. campestris 17 has two rrn operons ca. 715 kb apart on the chromosome (34). The X. campestris pv. campestris 17 chromosome was cut into five, two, and seven fragments by PacI, I-CeuI, and PacI plus I-CeuI, respectively. In the double digests, one fragment of 200 kb (PAsrrn) was released from PA, and PC was cleaved into two subfragments, PCbrrn (220 kb) and PCsrrn (135 kb). The pRRNSM3 probe, spanning the I-CeuI site (36) in the X. campestris pv. campestris 17 23S rRNA, hybridized to PA and PC from the PacI digests and to the four subfragments derived from PA and PC in the PacI/I-CeuI double digests (data not shown), indicating that one rrn operon (rrnA) is in PC and the other (rrnB) is in PA. When the same DNA fragments were hybridized with pW2Y2, the signal was associated with PC, PD, and PCsrrn (data not shown), indicating that rrnA is ca. 135 kb from the PC/PD interface (Fig. 3). On the other hand, with the pREF probe, the signal was found to associate with PAsrrn (data not shown), indicating that rrnB is ca. 200 kb from the PA/PE interface (Fig. 3).

The chromosomes of the 10 pigmentation mutants were each cut within WF by SwaI and within PD by PacI. The sizes of the smaller subfragments derived from PD ranged from 20 to 35 kb, whereas those of the larger subfragments ranged from 190 to 205 kb (data not shown), indicating the pigmentation genes to be clustered (ca. 15 kb) in the WF/PD overlapping region.

With the pX727 probe (Table 1) containing the X. campestris pv. campestris 17 tdh gene for Southern hybridization with the PacI and SwaI digests of the X. campestris pv. campestris 17 chromosome, the signal was found to associate with PA and WB, indicating that the tdh gene is in the PA/WB overlapping region. In the digests of the gene-tagged tdh mutant MTDH chromosome, two subfragments of ca. 92 kb (PAstdh) and 15 kb (WAstdh), released from PA and WB, respectively, were observed (data not shown). These results suggest that the tdh gene is ca. 92 kb from the PA/PB junction and ca. 15 kb from the WB/WE junction (Fig. 3).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

As a first step to study the X. campestris chromosome, we have accomplished the following in this study. Using three different methods for extracting circular plasmids, we detected no plasmid in X. campestris pv. campestris 17. The size of the X. campestris pv. campestris 17 chromosome was estimated to be 4.8 Mb. All restriction fragments of PacI and SwaI from the X. campestris pv. campestris 17 digests were assembled into a single circle, and then the PmeI and I-CeuI sites were localized. In addition, 23 loci dispersing around the chromosome have been identified. These loci include the genes conferring the two most important traits common to Xanthomonas, synthesis of xanthan gum and normal pigmentation. This is the first map for X. campestris reported and would be useful for genetic studies of this and related Xanthomonas species.

The chromosomes of bacteria have sizes ranging from 580 kb for Mycoplasma genitalium (17) to 9,450 kb for Myxococcus xanthus (8). Thus, the size estimated here places the X. campestris pv. campestris 17 chromosome among the genomes in group 4 (genome sizes larger than 4.5 Mb) of the bacterial chromosomes (13). This size is similar to the 5.0 Mb estimated for the chromosome of X. campestris DSM1049 (18) and the 4.6-Mb E. coli chromosome (5) but is 1.1 Mb smaller than the chromosome of Pseudomonas aeruginosa (41), a robust saprophyte of the same family.

The chromosome of X. campestris, having a G+C content of 64% (6), is suitable for restriction analysis with enzymes that recognize the sequences rich in A+T. Accordingly, PacI, PmeI, and SwaI were used in this study. For easier analysis, we also carried out PacI/PmeI, SwaI/PmeI, and PacI/SwaI double digestions. However, fragments PA and WA of the X. campestris pv. campestris 17 chromosome generated by these digestions were still too large to be resolved. Therefore, we used mutant A48A(trp-1), carrying in the PA/WA overlapping region a Tn5(pfm)CmKm insertion, which caused the PA and WA fragments to be cut into sizable subfragments. In addition, introduction of unique PacI and SwaI sites by Tn5(pfm)CmKm insertion in the chromosomes of various mutants helped identify the mutual overlapping relationships between the PacI and SwaI fragments. Furthermore, these insertions enabled us not only to calculate the approximate distances between the mutations but also to determine the gene order on the chromosome.

A gene-tagging technique, instead of Tn5(pfm)CmKm transposition, was used to determine the locations of recA and tdh genes which have known nucleotide sequences (30, 37, 62). These genes were tagged by cloning the segment including the PacI-SwaI sites and the drug markers from Tn5(pfm)CmKm into a plasmid carrying a fragment of the target genes, which was then integrated into the chromosome by homologous recombination via single crossover. After these steps of manipulation, the gene of interest was tagged by the drug markers and the rare-cutting enzyme sites. So far, more than 40 Xanthomonas genes, other than the ones whose locations have been determined in this study, have been sequenced. Our method would be useful to tag these genes for determination of their chromosomal locations.

Biosynthesis of xanthan gum involves multiple steps and a multitude of enzymes. It requires (i) synthesis of sugar nucleotide precursors UDP-glucose, UDP-glucuronic acid, and GDP-mannose, (ii) sequential transfer of these sugars to a glycosyl carrier lipid to form pentasaccharide repeating-unit diphosphate lipid, (iii) acetylation and pyruvylation of the mannose residues, (iv) polymerization of the pentasaccharide repeating units, and (v) secretion of the polymeric molecule (21-23, 56). Defects in any of these steps can cause the failure of xanthan synthesis, resulting in a nonmucoid or low-mucoid phenotype. Harding et al. (19) isolated seven nonoverlapping xps (xanthan polysaccharide synthesis) regions essential for xanthan gum synthesis in X. campestris pv. campestris NRRL B-1459. Although the functions have been identified for four of them, none of their chromosomal locations have been determined. It is known that (i) xpsI, containing the gum gene cluster, encodes functions required for assembly of the lipid-bound repeating unit, (ii) xpsIII contains the gene conferring phosphoglucomutase/phosphomannomutase and mannose isomerase/phosphomannoisomerase activities, (iii) xpsIV contains the UDP-glucose pyrophosphorylase gene, and (iv) xpsVI contains the UDP-glucose dehydrogenase gene (19). In this study, we have localized eight chromosomal loci involved in xanthan synthesis. These loci were designated eps instead of xps to avoid confusion with the Xanthomonas protein secretion genes (20). The four loci eps1, -3, -6, and -7, whose functions are known, are analogous to the xpsIII, -VI, -IV, and -I of strain B-1459, respectively. Together, these findings suggest that there may be more than eight chromosomal regions required for the synthesis of xanthan gum. The other four loci determined in this study, which remain to be characterized, should include the genes specifying the functions required for gum polymerization, secretion, and regulation of the genes involved in gum synthesis.

Colonies of the genus Xanthomonas are yellow in color due to production of the membrane-bound, brominated aryl-polyene pigments called xanthomonadins (49). Since such pigmentation is unique to this genus, it is commonly used as a marker for chemotaxonomy (49). The genes responsible for the synthesis of xanthomonadins have been identified within an 18.6-kb fragment containing seven transcriptional units (pigA through pigG) in X. campestris pv. campestris B-24 (40). All of the 10 pigmentation mutants that we isolated in this study were found to be located within the same region extending for at least 15 kb on the X. campestris pv. campestris 17 chromosome, indicating clustering of the related genes. In addition, since the mutation in each of the 10 mutants was caused by a single event of transposon insertion, this DNA region appears to contain the major pig gene clusters. Therefore, it seems safe to conclude that this locus is analogous to the pig gene clusters of B-24.

    ACKNOWLEDGMENTS

This study was supported by grants NSC 83-0203-B-005-071 and NSC 83-0211-B-005-041 from National Science Council, Republic of China.

We thank Carton W. Chen for very helpful discussion and editing the manuscript, and we thank K. K. Wong and M. McClelland for donating pUT-Tn5(pfm)CmKm.

    FOOTNOTES

* Corresponding author. Mailing address: Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan. Phone: 886-4-285-1885. Fax: 886-4-287-4879. E-mail: yhtseng{at}dragon.nchu.edu.tw.

dagger Present address: Department of Microbiology, Tzu-Chi College of Medicine and Humanities, Hualien 970, Taiwan.

Dagger Present address: Biotechnology Center, University of Connecticut, Storrs, CT 06269-3149.

§ Present address: Center for Human Genome Research, Los Alamos National Laboratory, Los Alamos, NM 87544.

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Abstract
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Discussion
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Journal of Bacteriology, January 1999, p. 117-125, Vol. 181, No. 1
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