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Transposon mini-Tn7 vectors insert into the chromosome of several Gram-negative bacteria in a site-specific manner. Here, we demonstrated the application of mini-Tn7 as single copy site-specific integration vector system for Xanthomonas campestris pv. campestris. The transposition of the mini-Tn7 into the bacterial genome was detected at a Tn7 attachment (attTn7) site located downstream of glmS1. Furthermore, using a newly constructed vector pBBR1FLP2 containing the FLP recombinase for site-specific excision of the sequence between the FLP recognition target (FRT) sites, and a sacB counter selection marker, an unmarked mini-Tn7 insertion mutant was created. Mini-Tn7 insertion did not affect bacterial virulence on the tested plant. The mini-Tn7 and FLP-FRT systems also work well in X. oryzae pv. oryzae.
Transposons are mobile genetic elements that can transpose within the same, or other, genomes. Transposon Tn7 and its derivatives have been shown to exert a high frequency of transposition into specific target sites on the chromosome of several bacteria (Arciszewska et al., 1989). Integration of the transposable element requires the proteins encoded by Tn7 genes (tnsABCDE) and can occur using two different mechanisms (Waddell & Craig, 1989). TnsABC + TnsD promote transposition into a specific Tn7 attachment site, attTn7, with a high frequency, while TnsABC + TnsE promote insertion into non-attTn7 sites (Peters & Craig, 2001). Generally, attTn7 is located immediately downstream of glmS, a gene encoding glucosamine-fructose-6-phosphate aminotransferase, an enzyme that catalyzes the formation of glucosamine 6-phosphate. Most bacteria possess a single glmS gene and therefore a single attTn7 site. However, multiple glmS genes and attTn7 sites have been reported in some bacteria including Burkholderia spp. (Choi et al., 2006; Choi et al., 2008). Moreover, non-glmS-linked attTn7 sites have also been identified in Proteus mirabilis (Choi & Schweizer, 2006). Mini-Tn7 vectors have been developed and proven to be useful for integration of a cloned gene and genetic analysis of several Gram-negative bacteria (Choi & Schweizer, 2006; Choi & Schweizer, 2006; Choi et al., 2006; Choi et al., 2005; Peters & Craig, 2001; Waddell & Craig, 1989). These mini-Tn7-based vectors are a valuable genetic tool for examining gene complementation and expression analysis at either the transcriptional or translational level.
Xanthomonas spp. are Gram-negative aerobic bacteria that are soil and plant pathogens that cause destructive diseases in many economically important crops, including rice, citrus, cabbage, cauliflower and radish. Xanthomonas campestris is also the producer of industrially important Xanthan gum. The development of novel genetic tools to study these bacterial phytopathogens and industrially important bacteria could lead to a better understanding of the underlying pathophysiology, improve the treatment strategies and improve production of an industrial material Xanthan gum. However, one of the important problems for molecular characterization of genes in Xanthomonas is lacking of vectors for specific integration of genes into bacterial chromosome either for single copy gene complementation or in vivo promoter analysis. In this communication, we demonstrate the successful application of the mini-Tn7 vector system for integration of genes in X. campestris as well as X. oryzae. Although these bacterial species contain two glmS genes, insertion of the transposon was only detected at the glmS1-associated attTn7 site, and this insertion did not affect bacterial virulence. A new vector containing FLP recombinase that facilitates excision of FRT sites a sacB a counter section marker are useful for making unmarked mutation in Xanthomonas.
Xanthomonas strains were grown in Silva-Buddenhagen (SB) medium (Chauvatcharin et al., 2005) at 28°C with continuous shaking. Bacteria containing genetic elements expressing sacB were cultivated in modified SB in which glucose was used instead of sucrose; sacB-deficient, sucrose-resistant clones were then selected on SB medium supplemented with 5% sucrose. Antibiotic concentrations used were 5 µg−1 ml gentamicin (Gm) and 300 µg−1 ml carbenicillin (Cb).
pUC18-mini-Tn7T-Gm, pUC18-mini-Tn7T-Gm-lacZ and the pTNS2 helper plasmid have been described previously (Choi & Schweizer, 2006; Choi et al., 2006; Choi et al., 2005). The FLP-mediated excision plasmid, pBBR1-FLP2 was constructed by cloning the 5-kb Acc65I-SphI (blunt) fragment of pFLP2 (Hoang et al., 1998), which contained the cI857-FLP-sacB genes, into pBBR1MCS-4 (Kovach et al., 1995) digested with Acc65I and SmaI. The complete sequence of pBBR1-FLP2 was deposited to the GenBank databases under the accession number FJ797950.
pTn7T-PahpC::lacZ was constructed by PCR amplification of 270-bp ahpC (xcc0834) promoter fragment using two specific primers (BT2305: 5’TTGCCGTTGTGGTACGCG3’ and BT2306: 5’AGCCTCAGACATGCGGCA3’) designed from the Xcc genome sequence (da Silva et al., 2002). The PCR product was cloned into pDrive cloning vector (Qaigen, Germany) prior to the subcloning of the XhoI and Acc65I fragment into pUC18-mini-Tn7T-Gm-lacZ digested with the same restriction enzymes to generate pTn7T-PahpC::lacZ.
Molecular genetic techniques including genomic and plasmid DNA preparations, polymerase chain reaction (PCR), restriction endonuclease digestion, DNA ligation, transformation in Escherichia coli, bacterial crude lysate preparation, gel electrophoresis and Southern blotting analysis were performed using standard protocols (Sambrook et al., 1989). Transformation of X. campestris pv. phaseoli (Xcc) was performed by electroporation, as previously described (Mongkolsuk et al., 1996). DNA sequences were determined on an ABI 310 automated DNA sequencer (Applied Biosystems, USA).
The virulence of Xcc was determined on Chinese radish (Raphanus sativus), a compatible host plant, using leaf-clipping methods (Dow et al., 2003) with some modifications. Overnight cultures of Xanthomonas strains in AB minimal medium (Chilton et al., 1974) supplemented with 0.1 % (w/v) casamino acid were diluted to an optical density at 600 nm (OD600) of 1.0 in fresh AB medium. Three leaves per plant were inoculated by leaf clipping, and five leaves were inoculated for each bacterial strain. The lesion lengths were measured at 14 days post-inoculation. Experiments were done in triplicate. The difference between strains was analyzed by t-test, and P-values less than 0.05 were considered to be significantly different.
Crude bacterial lysates were prepared and protein assays were performed as previously described (Panmanee et al., 2002). The total protein concentration in the cleared lysate was determined using dye-binding method (Bio-Rad, USA). β-galactosidase assays were performed as previously described and expressed as international units defined as the amount of enzyme capable of releasing 1 µmol p-nitrophenol generated at 25°C per min (Panmanee et al., 2002).
In order to examine the utility of Tn7-based genetic tools vectors for the study of Xanthomonas campestris pv. campestris (Xcc), we tested whether a mini-Tn7 vector could be used in Xcc. pUC18-mini-Tn7T-Gm and pTNS2 (the helper plasmid encoding the TnsABCD site-specific transposition pathway) were transformed into wild-type Xcc. The transformants were selected on SB plates containing 5 µg−1 ml gentamicin. The analysis of the annotated Xcc genomic sequence using the BLASTP algorithm (Altschul et al., 1997) and Pseudomonas aeruginosa glmS as the query sequence revealed that Xcc contains two putative glucosamine-fructose-6-phosphate aminotransferase genes, glmS1 (xcc0569) and glmS2 (xcc3411). Therefore, the insertion of the mini-Tn7 was verified using PCR with glmS1- or glmS2-specific forward primers and a reverse primer that located in the mini-Tn7 (Fig. 1a). The results demonstrate that the mini-Tn7-Gm was inserted downstream of glmS1 in all 37 transformants that were analyzed in this study. Fig. 1b shows the PCR products of 398 bp and 235 bp amplified from a representative transformant using glmS1Up (5’ACGACCGCCTGCTGGAAAA3’) and Tn7R (5’-CACAGCATAACTGGACTGATT3’) primers, and glmS1Down (5’-ACGGGATGGCTGCGGCTT3’) and Tn7L (5’-ATTTGCTTACGACGCTACACC3’) primers, respectively. Amplification with glmS1Up and Tn7L or glmS1Down and Tn7R primer pairs resulted in no PCR products (Fig. 1b). The transposition of mini-Tn7 was orientation-specific with Tn7R located immediately downstream of and facing glmS1 (Fig. 1a). Site- and orientation-specific transposition is a common feature of Tn7 insertions into other bacterial chromosomes (Choi & Schweizer, 2006; Choi & Schweizer, 2006; Choi et al., 2006; Choi et al., 2005; Choi et al., 2008; Waddell & Craig, 1989). Possible insertions of mini-Tn7 at the glmS2 site were assessed using PCR amplification with glmS2Up (5’GATGGCGACCTGCCGCTG3’) and glmS2Down (5’GCATCGTCGGCCGCGACA3’) (Fig. 1a). Amplification from all strains resulted in a 283 bp PCR product that indicates that no insertion of mini-Tn7 occurred at the glmS2 site (Fig. 1b). Based on the DNA sequence analysis of the PCR products from five reactions, the insertion site of mini-Tn7 was located 25 nucleotides downstream of the glmS1 stop codon (Fig. 1c). The analysis of the 3’ glmS1 nucleotide sequence identified a putative attTn7 site that was very similar to the attTn7 sites identified in E. coli (Waddell & Craig, 1989) and P. aeruginosa (Choi & Schweizer, 2006) (Fig. 1c). No putative attTn7 site could be identified at glmS2 (Fig. 1c). These results support the experimental findings that mini-Tn7 insertions seemed to be confined to the glmS1-linked attTn7 site with no insertions observed at glmS2.
pUC18-mini-Tn7T-Gm, a mini-Tn7 vector, contains two FLP recombinase target (FRT) sites flanking aacC1 (Gmr) (Fig. 1a). These FRT sites allow for flipase (FLP) mediated excision of aacC1. To assess whether an unmarked mini-Tn7 insertion could be generated in Xcc, pFLP2 (Hoang et al., 1998) was introduced into three representative Xcc transformants (Xcc::mini-Tn7T-Gm). The pFLP2 plasmid encodes the Saccharomyces cerevisiae FLP recombinase enzyme whose expression is driven by a λ promoter under the control of temperature sensitive λ repressor (cI857). After several attempts to transform pFLP2 into Xcc::mini-Tn7T-Gm, no transformants could be obtained, suggesting that the plasmid may not be able to efficiently replicate and be maintained in Xcc. Hence, a cI857-FLP-sacB containing fragment of pFLP2 was sub-cloned on a Acc65I-SphI (blunt) fragment into the broad-host range pBBR1MCS-4 plasmid which contains a carbenicillin resistance (Cbr) gene. The resulting expression vector pBBR1-FLP2 (Fig. 2) is capable of expressing cloned genes and replicating in Xanthomonas, yielding pBBR1-FLP2. This plasmid was subsequently electroporated into Xcc::mini-Tn7T-Gm and Cbr transformants were selected. Excision of the Gm resistance cassette resulted in Cbr and Gms transformants, and the excision event was confirmed by PCR analysis. PCR analyses using Xcc::mini-Tn7T-Gm genomic DNA and primers glmS1Up and glmS1Down generated a PCR product of 2,270 bp (Fig. 3a and b). Excision of aacC1 by FLP-mediated recombination should result in a PCR product of 1,312 bp when amplification is performed using glmS1Up and glmS1Down primers and DNA from Cbr and Gms colonies. As expected, 1,312 bp PCR products were detected (Fig. 3b). Hence, pBBR1-FLP2 could be efficiently used to mediate FLP-FRT recombination in order to generate an unmarked Tn7 insertion in Xanthomonas. Moreover, the advantage of using pBBR1MCS and its derivative plasmids is that they can stably replicate at a moderate copy number in a number of Gram-negative bacteria, including α- and γ-proteobacteria (Khan et al., 2008; Kovach et al., 1995; Vattanaviboon et al., 2007). Also, these plasmids can be mobilized through the IncP1 conjugative transfer system into many Gram-negative bacteria (Kovach et al., 1995). The removal pBBR1-FLP2 plasmid was cured from Xcc::mini-Tn7T was by growing the strain on SB medium containing 5% sucrose. pBBR1-FLP2 contains a counter-selection gene, sacB, which encodes Bacillus subtilis levansucrase (Gay et al., 1985). The presence of sucrose in the medium causes cell death in Gram-negative bacteria due to production of a toxic high-molecular-weight fructose polymer. Cells which survived and grew on the sucrose-supplemented medium were selected for further characterization and were shown to be Cbs indicating plasmid loss. The pBBR1-FLP2-cured clones could be obtained after a single passage of cells through SB broth supplemented with sucrose. Therefore, the presence of the sacB counter-selection marker in pBBR1-FLP2 allows for the simple and rapid elimination of the plasmid from the Xcc host.
Xcc is a causative agent of black rot disease in cruciferous crops. The usefulness of the mini-Tn7 system as a genetic tool in Xanthomonas and in the investigation of plant--microbe interactions depends on the premise that mini-Tn7 insertion should not affect the virulence of the bacteria on the compatible host plant. Wild-type Xcc and Xcc::mini-Tn7T strains were inoculated into Chinese radish (Raphanus sativus) leaves by the leaf clipping method (Dow et al., 2003). The results demonstrate that the lesion length in both Xcc::mini-Tn7T and the isogenic wild-type strains was not significantly different (P-value > 0.05). This suggests that the transposition of the mini-Tn7 into the glmS1 site has no effect on the virulence of Xcc on the tested host plant (Fig. 4). Thus, the mini-Tn7 offers a potential shuttle vector system for the site-specific chromosomal integration for single copy complementation experiments, as well as gene expression analysis in X. campestris pv. campestris using reporter gene constructs.
We have characterized ahpC, a gene encoding alkyl hydroperoxide reductase, in X. campestris pv. phaseoli (Loprasert et al., 2000; Mongkolsuk et al., 1997). The expression of ahpC is inducible by H2O2, organic hydroperoxides and superoxide generators (menadione and paraquat) in an OxyR dependent manner. In an attempt to evaluate the use of mini-Tn7 vector in gene expression analysis in Xcc, ahpC promoter was transcriptionally fused to lacZ in pUC18-mini-Tn7T-Gm-lacZ as described in the Materials and Methods. The recombinant plasmid pTn7T-PahpC-lacZ was introduced into Xcc wild-type and the transformants were selected for Gmr phenotype. Insertion of Tn7 elements containing ahpC promoter-lacZ fusion and curing of pBBR1-FLP2 from the transformants giving Xcc::mini-Tn7T-PahpC-lacZ, was verified as described in an earlier section. The ahpC promoter activity was monitored by measuring β-galactosidase activity. Xcc::mini-Tn7T-PahpC-lacZ was cultivated in SB medium until the cells reached exponential phase of growth before bacterial cultures were being challenged 100 µM menadione (MD), 100 µM H2O2, 100 µM cumene hydroperoxide (CHP) 100 µM tert-butyl hydroperoxide (BHP). As illustrated in Fig. 5, treatment with MD, H2O2, CHP and BHP increased β-galactosidase activity by 3.0, 2.4, 4.5 and 3.3 folds, respectively, relative to wild-type level. The expression pattern of ahpC in Xcc was similar to previously characterized and closely related ahpC from X. campestris pv. phaseoli (Mongkolsuk et al., 1997). Our data presented the usefulness of mini-Tn7 vectors as tools to assess gene expression profile in plant pathogenic Xanthomonas.
We further tested the functionality of the mini-Tn7 system in other Xanthomonas spp., specifically Xanthomonas oryzae pv. orzyae (Xoo). The pUC18-mini-Tn7T-Gm delivery plasmid and pTNS2 helper plasmid were introduced into Xoo by electroporation using previously described conditions (Mongkolsuk et al., 1996). The mini-Tn7 insertion sites were determined in 35 transformants by PCR using genomic DNA templates isolated and two pairs of primers (glmS1XoUp, 5’-CGACCGCCTGCTGGAAAA3’, and Tn7R or glmS1Xo Down, 5’-ATCTTCGACGCTCAACAG3’, and Tn7L). Amplification of a 280 bp fragment demonstrated that insertion of the mini-Tn7T-Gm element in all transformants occurred downstream of glmS1 (xoo0746). Furthermore, sequence analysis of the PCR products indicated that the mini-Tn7 insertion site (attTn7) was 25 bp downstream of glmS1. No insertions were observed at glmS2 (xoo3917)(data not shown). We also analyzed the published genomic sequences of all Xanthomonas spp. and determined that X. campestris pv. vesicatoria (Thieme et al., 2005), X. oryzae pv. oryzae (Xoo) (Lee et al., 2005), and X. axonopodis pv. citri (da Silva et al., 2002) all contain two putative glmS genes. However, upon searching for putative attTn7 sites surrounding the two glmS genes, attTn7 sites could only be identified downstream of the respective glmS1 genes (Fig. 1c). Based on the results obtained with the mini-Tn7 system in Xcc and Xoo, it is likely that the mini-Tn7 system will work in other Xanthomonas spp. and that the mini-Tn7 will probably insert downstream of glmS1. Additionally, the excision of aacC1 from Xoo::mini-Tn7T-Gm was tested by transforming the strain with pBBR1-FLP2. The results from the PCR analysis using glmS1XoUp and glmS1XoDown primers and DNA from Xoo::mini-Tn7T/pBBR1-FLP2 demonstrated the excision of the Gmr marker through FLP-FRT recombination (data not shown). As described in the earlier section 3.2, pBBR1-FLP2 could be cured from the resulting strain with sucrose counter-selection. Taken together, our data suggest the possible applications of mini-Tn7 system and a new vector for FLP-FRT system for making unmarked mutation in a variety species of xanthomonads. This will be useful for genetic analysis of industrially important and phytopathogenic bacteria, Xanthomonas spp.
The research was supported by grants (BT-B-01-PG-14-5112) from the National Center for Genetic Engineering and Biotechnology (BIOTEC), Mahidol University and from the ESTM under the higher Education Development Project of the Ministry of Education. T.J. was supported by a Royal Golden Jubilee Scholarship PHD/0222/2547 from the Thailand Research Fund. HPS was supported by NIH grants AI058141 and AI065357.