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. 2010 Dec 1;12(1):57–71. doi: 10.1111/j.1364-3703.2010.00652.x

The Xanthomonas campestris pv. vesicatoria citH gene is expressed early in the infection process of tomato and is positively regulated by the TctDE two‐component regulatory system

DAFNA TAMIR‐ARIEL 1, TALLY ROSENBERG 1, SAUL BURDMAN ✉
PMCID: PMC6640381  PMID: 21118349

SUMMARY

Xanthomonas campestris pv. vesicatoria (Xcv) is the causal agent of bacterial spot disease of tomato and pepper. Previously, we have reported the adaptation of a recombinase‐ or resolvase‐based in vivo expression technology (RIVET) approach to identify Xcv genes that are specifically induced during its interaction with tomato. Analysis of some of these genes revealed that a citH (citrate transporter) homologous gene contributes to Xcv virulence on tomato. Here, we demonstrate that the citH product indeed facilitates citrate uptake by showing the following: citH is specifically needed for Xcv growth in citrate, but not in other carbon sources; the citH promoter is specifically induced by citrate; and the concentration of citrate from tomato leaf apoplast is considerably reduced following growth of the wild‐type and a citH‐complemented strain, but not the citH mutant. We also show that, in the Xcv–tomato interaction, the promoter activity of the citH gene is induced as early as 2.5 h after Xcv is syringe infiltrated into tomato leaves, and continues to be active for at least 96 h after inoculation. We identified an operon containing a two‐component regulatory system homologous to tctD/tctE influencing citH expression in Xcv, as well as its heterologous expression in Escherichia coli. The expression of hrp genes does not seem to be affected in the citH mutant, and this mutant cannot be complemented for growth in planta when co‐inoculated with the wild‐type strain, indicating that citrate uptake in the apoplast is important for the virulence of Xcv.

INTRODUCTION

The Gram‐negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is the causal agent of bacterial spot disease of tomato (Solanum lycopersicum L.) and pepper (Capsicum annuum) (Jones et al., 1998). Typical disease symptoms include necrotic lesions that are often surrounded by chlorosis on all above‐ground parts and, in some cases, defoliation. High temperature and relative humidity provide optimal conditions for the induction of this disease, which has a worldwide distribution. Although the reclassification of Xcv into four different species—X. vesicatoria, X. euvesicatoria, X. gardneri and X. perforans—has been proposed recently (2000, 2004), we refer here to the classical nomenclature, as it is still being broadly used in molecular studies on the interaction of this pathogen with its hosts.

As with many Gram‐negative phytopathogenic bacteria, Xcv relies on a functional hrp (hypersensitive response and pathogenicity)‐encoded type III secretion system (TTSS) to secrete effectors into the host cell, where they interact with host cellular processes to promote disease or to elicit defence responses in susceptible and resistant plants, respectively. In susceptible plants, type III effectors contribute collectively to the adaptation of the pathogen to the host tissue by interfering with host immune responses or mediating nutritional and virulence processes (Buttner and He, 2009; Mudgett, 2005; White et al., 2009). However, the establishment of bacterial pathogens in their host and their ability to cause disease expands beyond the ability to form this secretion system and to transfer effectors through it. The infectious process is very intricate, and requires the coordinated activity of a myriad of bacterial genes, whose identity and mode of action are still largely unknown.

In a previous report, we described an adaptation of recombinase‐ or resolvase‐based in vivo expression technology (RIVET), a promoter‐trap method, to identify Xcv genes that are specifically induced or overexpressed during its interaction with tomato (Tamir‐Ariel et al., 2007). In RIVET, promoter activity drives the expression of tnpR, which encodes a resolvase that specifically excises a genetic marker—typically an antibiotic resistance gene—flanked by resolvase recognition sequences (res sites). This reaction is named ‘resolution’ and leads to a phenotype switch that can be easily detected and screened. The rationale behind this approach is that selectively in vivo induced genes have the potential to contribute to the ability of the pathogen to survive and multiply in the host tissue. Among the identified genes was a citH homologue, Xcv3613, according to the Xcv strain 85‐10 genome annotation (GenBank accession AM039952; Thieme et al., 2005).

CitH possesses a CitMHS domain (citrate transporter, Pfam03600), and shows 61% and 54% identity with the characterized citrate transporters of Bacillus subtilis CitN (also annotated as CitH) and CitM, respectively (GenBank accessions P42308 and P55069) (Boorsma et al., 1996; Krom et al., 2000). Disruption of citH in Xcv leads to reduced virulence of the bacterium on tomato when compared with the wild‐type strain (Tamir‐Ariel et al., 2007).

The aims of this study were to further characterize citH and to gain an insight into its role in the Xcv–tomato interaction. We exploited RIVET to assess citH promoter activity (namely citH expression) in Xcv during the infection process. RIVET is a highly sensitive method, because a small pulse of resolvase expression is needed to mediate resolution; therefore, the method is exquisitely sensitive to low or transient gene expression (Angelichio and Camilli, 2002). This enabled us to detect citH expression also at the early stages of infection. We also questioned whether CitH specifically facilitates citrate uptake by assessing its contribution to growth in minimal medium with different carbon sources, as well as to citrate uptake in minimal medium and tomato apoplast extracts. We also assessed the activation of the citH promoter under various growth conditions, and explored how CitH could contribute to the virulence of Xcv by looking at several aspects of plant–microbe interactions. In this study, we also provide evidence demonstrating the involvement of a two‐component regulatory system (TCS) in the regulation of citH expression in culture and in planta.

RESULTS

Functional CitH is required for growth in minimal medium with citrate, but not with other carbon sources

A citH mutant has been generated previously in the background of Xcv strain 97‐2 by single recombination (Tamir‐Ariel et al., 2007). The vector used, pJPcit, contains an internal region of the citH gene (such that it does not span both the 5′ and 3′ ends of the transcriptional unit), and its chromosomal integration resulted in a cis‐merodiploid strain with each of the two copies of the citH gene lacking the 5′ or 3′ ends. As Xcv citH is part of a monocistronic locus, this mutation is not expected to affect downstream genes (Fig. 1).

Figure 1.

Figure 1

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The genomic region around citH (citrate transporter). (A) Genomic location of citH according to the Xanthomonas campestris pv. vesicatoria (Xcv) strain 85–10 annotation (GenBank accession AM039952; Thieme et al., 2005). Sequence analyses revealed a similar genomic structure for this region in strain 97‐2. tctD, tctE and Xcv3609 encode a response regulator, a histidine sensor kinase and a putative periplasmic iron‐binding protein, respectively, and oprP2 encodes phosphate‐selective porin P. (B) Vectors and cloned DNA fragments used in this study to generate the different strains: (1) pJPcit, containing an internal fragment of the citH open reading frame (ORF), used to generate the Xcv citH mutant (Tamir‐Ariel et al., 2007); (2) pHKmtctD, containing an internal fragment of the tctD ORF, used to generate the Xcv tct and XcvPcitHtct‐ mutants; (3) the amplified citH ORF and its promoter region used to generate the pMLcit complementation vector; (4) the amplified tctDE ORFs and their promoter region used to generate pUcittctDE and pUtctDE; (5) the entire tct‐Xcv3609 operon used to generate pUcitOP and pUOP.

We have shown that the citH mutant and the wild‐type do not differ in terms of their ability to grow in rich medium (nutrient broth, NB); hence, it appears that the mutant does not have a general metabolic defect (Tamir‐Ariel et al., 2007). In that study, we also reported that, unlike the wild‐type, the citH mutant is affected in its ability to grow on citrate as carbon source. Here, we show that pML122 carrying the citH gene with its native promoter (pMLcit; Table 1) restored the ability of the citH mutant to grow in M9 medium containing citrate (M9‐citrate) as carbon source, although, in most experiments, this ability was not fully restored (Fig. S1, see Supporting Information). Partial complementation by pMLcit in these and other experiments (see below) could be a result, at least in part, of a certain level of plasmid loss, although, in these growth curve experiments, gentamicin (Gm) was added to the medium. The citH mutant did not differ from the wild‐type in its ability to grow in M9 medium containing sucrose, fructose, malate or succinate as carbon source (Fig. S1). Together, these experiments strengthen the view that the citH product is specifically involved in citrate uptake.

Table 1.

Strains and plasmids used in this study.

Strains and plasmids Characteristics Reference or source
Xanthomonas campestris pv. vesicatoria
 97‐2 Wild‐type, belongs to race T3 Astua‐Monge et al. (2000)
 110C Wild‐type, belongs to race T1 Sahin et al. (2003)
  Xcv citH mutant Kmr; strain 97‐2 with the citH ORF disrupted with insertion of plasmid pJPcit Tamir‐Ariel et al. (2007)
  Xcv citH comp Kmr, Gmr, Xcv citH‐complemented strain (carrying pMLcit) This work
 XcvRes Kmr, reporter strain containing the res cassette inserted within hpaG in 97‐2 Tamir‐Ariel et al. (2007)
 ResPcitH Kmr, Gmr, PcitH (citH promoter)::tnpR::uidA in XcvRes background Tamir‐Ariel et al. (2007)
 ResPcitHrev Kmr, Gmr, PcitHrev (citH promoter in reverse orientation)::tnpR::uidA in XcvRes Tamir‐Ariel et al. (2007)
 ResPhrpA Kmr, Cmr, PhrpA (hrpA promoter)::tnpR::uidA in XcvRes Tamir‐Ariel et al. (2007)
 ResPhrpG Kmr, Cmr; PhrpG (hrpG promoter)::tnpR::uidA in XcvRes Tamir‐Ariel et al. (2007)
 Xcv hrpA mutant hrpA knock‐out mutant in the background of strain 97‐2 Tamir‐Ariel et al. (2007)
 XcvPcitH Gmr; PcitH::tnpR::uidA in strain 97‐2 This work
 XcvPcitHrev Gmr; PcitHrev::tnpR::uidA in strain 97‐2 This work
 XcvPcitHtct‐ Kmr, Gmr, XcvPcitH with tctDE disrupted with plasmid pHKmtctD insertion This work
  Xcv tct mutant Kmr; Xcv 97‐2 with the tctDE disrupted with plasmid pHKmtctD insertion This work
Escherichia coli
 S17‐1 λ pir λ lysogenic S17‐1 derivative producing π protein for replication of plasmids carrying oriR6K; recA pro hsdR RP4‐2‐Tc::Mu‐Km::Tn7 λ‐ pir Simon et al. (1983)
 DH5α Cloning strain Hanahan (1983)
 DH5α/pUcit DH5α carrying plasmid pUcit This work
 DH5α/pUcittctDE DH5α carrying plasmid pUcittctDE This work
 DH5α/pUcitOP DH5α carrying plasmid pUcitOP This work
 DH5α/pUtctDE DH5α carrying plasmid pUtctDE This work
 DH5α/pUOP DH5α carrying plasmid pUOP This work
Acidovorax citrulli M6 Wild‐type, bacterial fruit blotch pathogen, isolated from melon Burdman et al. (2005)
Plasmids
 pTZ57R/T ApR; cloning vector Fermentas Inc.
 pHP45Ω Apr, Spr; suicide vector Prentki and Krisch (1984)
 pHPKm Apr, Kmr; pHP45Ω with the Kmr gene from pUC4K (38) replacing the Spr cassette This work
 pHKmtctD Apr, Kmr; pHKm containing an internal fragment of the tctD ORF This work
 pJPGm Gmr; pJP5603 (Penfold and Pemberton, 1992) with the Kmr gene replaced by a Gmr gene from pML122 Tamir‐Ariel et al. (2007)
 pJPcit Kmr; pJP5603 containing an internal fragment of the citH ORF This work
 pJPGmtnpR Gmr; pJPGm carrying a promoterless tnpR::uidA This work
 pPcitH Gmr; pJPGmtnpR with the citH promoter cloned upstream and in the same orientaton as tnpR::uidA to create a transcriptional fusion This work
 pPcitHrev Gmr; pBamGm1 with the citH promoter cloned upstream and in the opposite orientation as tnpR::uidA This work
 pML122 Gmr; broad host expression vector Labes et al. (1990)
 pMLcit Gmr; with the citH ORF including a 658‐bp fragment upstream of the translation start site, cloned into the SmaI site This work
 pUC18 Apr; cloning vector Yanisch‐Perron et al. (1985)
 pUcit Apr; pUC18 with the citH ORF and a 658‐bp fragment upstream of citH cloned into SmaI site This work
 pUcittctDE Apr; pUcit with tctDE cloned into the HindIII site This work
 pUcitOP Apr; pUcit with the entire tctDE‐Xcv3609 operon cloned into the HindIII site This work
 pUtctDE pUC18 with tctDE cloned into the HindIII site This work
 pUOP pUC18 with the entire tctDE‐Xcv3609 operon cloned into the HindIII site This work
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Apr, Cmr, Gmr, Kmr, Spr, ampicillin, chloramphenicol, gentamicin, kanamycin and streptomycin resistance, respectively; ORF, open reading frame.

In these experiments, it was also observed that growth of wild‐type and citH‐complemented strains in citrate minimal medium led to an increase in the pH of the medium from pH 7 to pH 8 (as measured after 48 h of growth). This may be indicative of citrate metabolism. In contrast, no pH change was observed in cultures of the citH mutant.

The wild‐type, but not the citH mutant, imports citrate from minimal and from tomato leaf apoplast‐enriched media

To further verify that CitH is involved in citrate uptake, the amounts of citrate left after the growth of the wild‐type, citH mutant and citH‐complemented strain were quantified in M9‐citrate and in extracts of tomato leaf apoplast. The latter medium was used as the apoplast is the site at which Xcv naturally populates the host. Citrate was quantified by spectrophotometric analysis of citrate lyase enzyme activity at the exponential and stationary phases of growth (after 24 and 48 h of growth, respectively). These analyses revealed that, in both media, citrate was imported by the wild‐type and the citH‐complemented strain, but not by the citH mutant (Fig. 2B,C,E,F). In M9‐citrate, however, complementation of the citH mutant by pMLcit was partial, as can be seen by the larger amounts of citrate left in the medium of the complemented strain relative to the wild‐type (Fig. 2B,E). In agreement with this and with the growth curves in M9‐citrate (Fig. S2, see Supporting Information), in these experiments, the complemented strain also showed intermediate growth between the citH mutant and the wild‐type in this medium (Fig. 2A,D).

Figure 2.

Figure 2

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Citrate uptake by the wild‐type, citH (citrate transporter) mutant and citH‐complemented strains. (A, D) Growth of the three strains in modified M9 minimal medium with 10 mm citrate and in apoplast extract. (B, E) Citrate concentration in modified M9 medium. (C, F) Citrate concentration in apoplast medium. (A–C) 24 h after inoculation (hai); (D–F) 48 hai. This experiment was repeated twice with similar results. One batch of apoplastic fluid was used in each experiment.

In contrast with M9‐citrate, no significant differences in growth between the three strains were observed in apoplast medium (Fig. 2A,D). Therefore, under the tested conditions, the impaired ability to import citrate in the citH mutant in the apoplast medium cannot be attributed to the reduced growth of this strain relative to the wild‐type.

The citH promoter is induced by citrate

So far, we have shown that, in Xcv, CitH functions as a transporter that specifically imports citrate into the cell. In several studies, the substrate itself has been shown to induce the expression of the corresponding transporter gene (Janausch et al., 2002; Warner and Lolkema, 2002). Therefore, we aimed to assess whether transcription of the citH gene is specifically induced in the presence of citrate, in comparison with other carbon substrates. We used RIVET to evaluate citH promoter activity in different growth conditions. As mentioned, RIVET relies on the induction of a promoterless resolvase gene (tnpR), which captures activated promoters (Camilli and Mekalanos, 1995). As TnpR‐mediated resolution is irreversible, the activation of the tested promoters at any time during growth can be detected with this assay.

A fragment containing the citH promoter was fused to the coding region of tnpR in both the correct and reverse orientations of transcription, and the transcriptional fusions were integrated into the chromosome of XcvRes, a modified 97‐2 strain carrying the res cassette (Tamir‐Ariel et al., 2007) to generate strains ResPcitH and ResPcitHrev, respectively. The res cassette confers kanamycin (Km) resistance; thus, TnpR‐mediated resolution, driven in this case by activation of the citH promoter, results in Km‐sensitive colonies. Therefore, citH promoter activity can be assessed with this system by evaluating the Km resistance versus sensitivity of the recovered cells. TnpR‐mediated resolution was tested by plating cultures grown under the different conditions on nutrient agar (NA) and NA with Km, and the percentage resolution was scored on the basis of the fraction of colony‐forming units (cfu) that were unable to grow in the presence of Km.

Under the majority of the conditions tested using modified M9 medium, resolution was shown to be affected only by the presence of citrate in the medium. Namely, tnpR induction via the citH promoter did not occur in the presence of other substrates (e.g. malate, succinate, isocitrate, fructose and glucose) as sole carbon sources. In contrast, when citrate was present in the medium as the sole carbon source or in combination with other carbon sources, full resolution of the res cassette was observed (Table 2). This was also the case when citrate and glucose were both added to the medium, indicating that the presence of glucose did not repress the induction of the citH promoter, as shown for citM of Bacillus subtilis (Warner and Lolkema, 2002; Yamamoto et al., 2000). Moreover, induction of the citH promoter by citrate appears to be highly specific, as the related isocitrate was not able to induce it (Table 2). No resolution was observed under the majority of the above conditions on strain ResPcitHrev (Table 2).

Table 2.

Measurements of TnpR‐mediated resolution activated by the citH promoter under various conditions in culture.*

Growth conditions† Strains
XcvResPcitH XcvResPcitHrev XcvRes
NB No‡ No N.A.
XVM2 Complete§ 90% No
M9 + citrate Complete No No
M9 +l‐malate No No N.A.
M9 +l‐succinate No No N.A.
M9 + sucrose No No No
M9 + fructose No No No
M9 + glucose No No N.A.
M9 + glucose + citrate Complete No N.A.
M9 + isocitrate No No N.A.
M9 + sucrose + fructose 90% 65% No
M9 + sucrose + fructose + citrate Complete No No
M9 + sucrose pH 5.7, 6.2 or 6.7 No No No
M9 + sucrose pH 5 or 8 No No N.A.
M9 + citrate pH 5 or 8 Complete No N.A.
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*

The TnpR resolution assay is based on the fact that tnpR expression, activated by a tested promoter, leads to the excision of a KmR gene. Strains were grown in liquid medium under different conditions, and serial dilutions were plated on both nutrient agar (NA) (total cells) and NA/kanamycin (Km) (unresolved cells). The percentage resolution is determined from [(average total cfu − average unresolved cfu)/average total cfu] × 100. Tested strains were as follows: XcvResPcitH, tnpR under the control of the citH promoter; XcvResPcitHrev, the citH promoter in the opposite orientation relative to tnpR; and XcvRes, carrying the res cassette but no tnpR.

†

Where not indicated, the pH of the medium was 7.

‡

‘No’ indicates no resolution and means that the differences in colony‐forming unit (cfu) counts between Km‐lacking versus Km‐containing plates were smaller than 5%.

§

‘Complete’ indicates complete resolution (differences between counts in Km‐lacking versus Km‐containing plates were greater than 95%). Resolution was assessed for each strain in at least two independent experiments with similar results for each medium, with the exception of N.A. (not assessed).

Surprisingly, in XVM2 medium, which contains sucrose and fructose, but does not contain citrate, resolution was observed. However, the resolution observed under these conditions does not appear to be citrate responsive, as a high level of resolution (90%) was also observed in strain ResPcitHrev. The same resolution pattern was observed when sucrose and fructose were both added to M9 medium, although no resolution was observed when sucrose and fructose were used separately in this medium (Table 2). Interestingly, when citrate was added to M9 modified medium containing sucrose and fructose, no resolution occurred in the ResPcitHrev strain, but the ResPcitH strain resolved completely. Thus, the presence of citrate seems to make this resolution event specific.

citH is expressed from the early stages of tomato infection by Xcv

We have shown that the citH promoter is strongly induced in planta, being induced by more than two orders of magnitude over the hrpA (hrcC) promoter, 48 and 72 h after inoculation (hai) at 106 cfu/mL by syringe infiltration into tomato leaves (Tamir‐Ariel et al., 2007). As the β‐glucuronidase (GUS) reporter gene (uidA) was used for these experiments, we could not technically detect expression prior to 48 hai, because of a lack of sensitivity of this assay at early times of infection. Here, we exploited the high sensitivity of RIVET to detect citH induction during the first hours after inoculation. It should be noted, however, that, unlike the GUS assay, this analysis detects the percentage of cells from the total population, which irreversibly resolved from the time of inoculation to the given point of detection. For this analysis, we used strains ResPcitH, ResPcitHrev, ResPhrpA and ResPhrpG, containing the promoter of the citH gene in the correct and reverse orientations, and the promoters of hrpA and hrpG, respectively, fused to promoterless tnpR‐uidA in the background of the XcvRes strain.

The strains were grown previously in rich medium (NA) with Km to ensure that the cells were in the unresolved state before inoculation. They were then syringe infiltrated into tomato leaves at 106 cfu/mL, and resolution of the res cassette was assessed by evaluating the Km resistance versus sensitivity of the recovered strains. The activity of the citH promoter was detected as early as 2.5 hai (Fig. 3). Assays performed at an earlier time (1.5 hai) showed no or low resolution levels for all treatments, with relatively high variability between replicates (not shown). The pattern of citH promoter activity was similar to that of hrpG and hrpA: although the level of resolution mediated by the citH promoter was lower during the first few hours after inoculation (2.5 and 4.5 hai), most ResPcitH cells had already been resolved at 4.5 hai. At 24 hai, no or very low resolution levels were still observed for the negative control strain ResPcitHrev (Fig. 3).

Figure 3.

Figure 3

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In planta resolution of the res (resolvase recognition) cassette induced by the citH (citrate transporter) promoter fused to the tnpR‐uidA transcriptional fusion (strain ResPcitH) relative to the hrpG, hrpA and reverse citH promoters (ResPhrpG, ResPhrpA and ResPcitHrev, respectively). The activity of the citH promoter in the reverse orientation was only measured at 24 h after inoculation (hai) as this time point represents the cumulative amount of resolved bacteria, and preliminary experiments showed no or very low resolution levels for this strain. Tomato (cv. H7998) leaves were syringe infiltrated with bacterial suspensions at 106 cfu/mL, which were grown in nutrient broth (NB) supplemented with kanamycin (Km) to ensure that inocula contained only unresolved cells. The percentage resolution was measured several times after inoculation by extracting bacteria from leaves and plating them onto nutrient agar (NA) and NA/Km. At each time point, four plants per treatment were sampled. Data (averages and standard errors) from one experiment of two with similar results are shown.

The Xcv TctDE TCS is sufficient to direct heterologous expression of citH in Escherichia coli

In Xcv, citH is closely linked to an operon containing two genes encoding components of a typical prokaryotic TCS, annotated as tctD‐tctE, and a third gene annotated as a putative periplasmic iron‐binding protein (genes Xcv3611, Xcv3610 and Xcv3609, respectively, according to strain 85‐10 annotation; GenBank accession AM039952; Thieme et al., 2005) (Fig. 1). Based on open reading frame (ORF) predictions and sequence analysis, it is likely that tctD, tctE and Xcv3609 are part of a single transcriptional unit, and their products are highly similar to putative gene products of other related xanthomonads. TctD is a predicted response regulator (RR) of the OmpR family, and TctE is a predicted histidine sensor kinase (HK). Table S2 (see Supporting Information) summarizes the similarities of the TctDE components to the putative gene products of other xanthomonads, as well as to experimentally characterized proteins.

In prokaryotes, TCSs regulate a wide variety of biological processes, including the expression of toxins and other proteins related to virulence and pathogenicity (Stock et al., 2000; West and Stock, 2001). To assess whether this operon and, in particular, the TctDE system, has a role in the regulation of citrate uptake, we used Escherichia coli DH5α carrying the Xcv citH gene with its indigenous promoter in several pUC18‐modified plasmids. Escherichia coli was chosen as it is unable to use citrate as carbon source. Thus, this bacterium was a suitable background to assess whether citH may confer the ability to grow on citrate, and to examine whether this ability is regulated by the TctDE system.

First experiments revealed that DH5α/pUcit (carrying the Xcv citH gene with its indigenous promoter) showed impaired growth in M9 medium with citrate as sole carbon source [Fig. 4A; note that the optical density (OD) scale is logarithmic and OD for this strain did not exceed 0.01]. This strain was also unable to alter the pH of Simmons citrate medium, where alkalinization caused by citrate uptake by the cells leads to medium bluing (Fig. 4B,C). However, when either the entire tctDE‐Xcv3609 operon, or only tctDE, was introduced, together with citH and its promoter region (in plasmids pUcitOP and pUcittctDE, respectively), DH5α cells were able to grow well in both media and to change the pH in Simmons medium (Fig. 4). As controls, strains DH5α/pUOP and DH5α/pUtctDE, containing the entire operon or only tctDE, respectively, but without the citH gene, were created. These strains were not able to utilize citrate (Fig. 4). These results demonstrate that the tctDE part of tctDE‐Xcv3609 is sufficient to direct citrate utilization in E. coli via the citH gene.

Figure 4.

Figure 4

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Heterologous expression of Xanthomonas campestris pv. vesicatoria (Xcv) citH (citrate transporter) and tctDE in Escherichia coli DH5α. Escherichia coli strains were grown in M9‐citrate liquid medium (A) and in Simmons citrate broth (B) and solid medium (C). In Simmons citrate medium, citrate utilization is accompanied by an increase in pH and, consequently, bluing of the medium. The strains were as follows: DH5α/pUcittctDE containing citH and tctDE (1), DH5α/pUcitOP containing citH and the entire tctDE‐Xcv3609 operon (2), DH5α/pUcit containing only citH (3), DH5α/pUtctDE containing only tctDE (4) and DH5α/pUOP containing only the tctDE‐Xcv3609 operon (5). Xcv genes were moved into E. coli with their indigenous promoters via pUC18‐modified plasmids. All experiments were carried out twice with similar results.

TctDE affects the ability of Xcv to grow on citrate

Next, we generated an Xcv tct mutant strain, impaired in the tctDE‐Xcv3609 operon (Fig. 1B), to assess whether this mutant is compromised during growth in M9‐citrate. Growth curve experiments revealed that the growth of the tct mutant on citrate was compromised relative to the wild‐type. However, this mutant was not as impaired as the citH mutant in its growth ability (Fig. 5A). In agreement with these results, the tct mutant showed an intermediate uptake of citrate between the citH mutant and the wild‐type in Simmons citrate medium: although bluing of the medium started to become visible after 48 h of growth in the case of the wild‐type, it started to become visible only after 5 days (120 h) of growth, and became clearer after 7 days (168 h), in the case of the tct mutant. As expected, no bluing was observed in the medium of the citH mutant throughout these experiments (Fig. 5B). These findings confirm that the TctDE system positively regulates citH expression and citrate uptake in Xcv.

Figure 5.

Figure 5

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Growth and citrate uptake of Xanthomonas campestris pv. vesicatoria (Xcv) citH (citrate transporter) and tct mutants, and wild‐type strain, in citrate medium. (A) Growth curves of strains in M9‐citrate. As growth media were not supplemented with kanamycin (Km), dilutions from cultures of the citH and tct mutants were plated at the end of the growth period on plates with and without Km to confirm that the integrated plasmids were stable during the experiments. Averages and standard errors from one experiment of three independent experiments with similar results are shown. (B) Growth of the same strains in Simmons citrate medium. Photographs of representative tubes (three for each strain per experiment of two with similar results) were taken after 48, 72, 120 and 168 h of growth.

The TctDE system regulates the expression of Xcv citH in culture and in planta

To verify whether the TctDE system affects citH expression in Xcv, citH promoter activity was tested in the background of the tct mutant containing the citH promoter fused to tnpR‐uidA (strain XcvPcitHtct‐). GUS activity in this strain was assessed following growth in M9‐citrate, in comparison with strain XcvPcitH, which contains the same transcriptional fusion but possesses intact tctDE. In XcvPcitHtct‐, citH promoter activity was reduced significantly compared with that in strain XcvPcitH (Fig. 6A).

Figure 6.

Figure 6

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β‐Glucuronidase (GUS) expression in XcvPcitH and XcvPcitHtct‐ in M9‐citrate medium (A) and in planta (B). For assays with bacteria grown in culture, cells were collected at an optical density at 640 nm (OD640) of approximately 0.2. Inoculations of tomato (cv. H7998) leaves were performed by syringe infiltration at 106 cfu/mL. At each time point, three samples from three plants were taken for each treatment. GUS assays were performed using the substrate 4‐methylumbelliferyl‐β‐d‐glucuronide (MUG). Averages and standard errors from one experiment of two with similar results are shown.

The effect of the TctDE system on citH expression was also assessed in planta following syringe infiltration of tomato leaves with strains XcvPcitHtct‐, XcvPcitH and XcvPcitHrev. The latter served as a negative control, as it carries the transcriptional fusion of the citH promoter in the opposite orientation relative to the tnpR‐uidA fusion. At all tested time points (48, 72 and 96 hai), the citH promoter activity in XcvPcitHtct‐ was approximately two orders of magnitude lower than that in XcvPcitH (Fig. 6B). GUS activity in the negative control was too low to be detected reliably (not shown). Together, these results confirm that TctDE positively regulates the expression of citH, although its disruption does not completely abolish citH transcription.

To test whether the disruption of tctDE affects the virulence of Xcv on tomato, symptom development and in planta growth of the tct mutant were compared with those of the wild‐type and the citH mutant. Under the tested conditions (dip inoculation with bacterial suspensions at 105 cfu/mL and syringe infiltration at various concentrations), we were unable to detect significant differences in these parameters between the tct mutant and the wild‐type (not shown).

Expression of hrp genes is not affected by the citH mutation

Xcv pathogenicity depends on a functional Hrp TTSS. In Pseudomonas syringae pv. phaseolicola, the addition of citrate to the medium resulted in a reduction in expression of several hrp genes (Rahme et al., 1992). Therefore, we set out to assess whether the reduced virulence of the citH mutant could be associated with a negative effect on hrp gene transcription.

The expression of hrpA, hrpX and hrpG genes in the citH mutant was compared with that in the wild‐type in XVM2, an hrp‐inducing medium (Wengelnik et al., 1996a), by semiquantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR). hrpA encodes an outer membrane structural component of the Hrp TTSS that belongs to the PulD superfamily of proteins (Wengelnik et al., 1996a). hrpX encodes an AraC‐type regulator of several hrp genes (Wengelnik and Bonas, 1996), and hrpG encodes a global regulator, which belongs to the OmpR family of RRs of typical TCSs (Wengelnik et al., 1996b). Under the tested conditions, the levels of transcription of these hrp genes were probably unaffected by the citH mutation (Fig. S2A).

As Hrp TTSS is essential for hypersensitive response (HR) induction in incompatible interactions, we assessed the ability of the citH mutant to induce HR in resistant pepper and H7981 tomato plants. These experiments were performed as a means to assess whether the citH mutation affects hrp expression in planta. The tested strains were infiltrated at concentrations of 105 and 108 cfu/mL. In pepper, wild‐type 97‐2 and Acidovorax citrulli M6 served as positive controls for HR induction, whereas the nonpathogenic Xcv 97‐2 hrpA mutant, defective in its ability to form the Hrp TTSS, served as a negative control. At the lowest inoculation dose, visible HR symptoms could not be detected (not shown). At the highest inoculation dose, faint HR symptoms started to appear at 24 hai for the positive controls, as well as for the citH mutant, with HR becoming clearer at 48 hai, and with no differences between the citH mutant and the wild‐type (Fig. S2B).

In H7981 tomato plants, A. citrulli M6 and Xcv 97‐2 served as positive controls for HR induction, whereas the 97‐2 hrpA mutant served as a negative control. The Xcv T1 race strain 110C is virulent on this tomato cultivar, and thus enabled the comparison between compatible and incompatible interactions. Again, the lowest inoculation dose resulted in no visible HR, but, at the highest inoculum concentration, HR induced by A. citrulli M6, Xcv 97‐2 and the Xcv citH mutant was clearly visible at 48 hai, whereas no symptoms were detected following inoculation with the hrpA mutant and strain 110C at this early time point (Fig. S2B). Together, these results indicate that disruption of citH probably does not affect hrp gene expression in Xcv.

The citH mutant is not complemented for growth in planta by the wild‐type

The citH mutant is compromised in its ability to cause disease symptoms and to grow in planta relative to the wild‐type (Tamir‐Ariel et al., 2007). Here, several experiments were carried out in which the wild‐type and the citH mutant were co‐inoculated using different inoculum concentrations to assess whether there was an interaction when both strains coexisted in the same niche. The occurrence of such an interaction could point to a possible mechanism by which citH influences Xcv virulence.

In co‐inoculation experiments using relatively low bacterial concentrations (103 cfu/mL of each strain), the mutant grew to significantly lower levels than the wild‐type, and the growth patterns did not differ from those observed in individual inoculations (not shown), suggesting that the presence of the wild‐type does not complement the mutant under these conditions. Macho et al. (2007) have reported that the inoculation dose influences the complementation of growth in planta of a mixed population of phytopathogenic bacteria. Their findings indicate that the complementation of a mutant affected in growth in planta may occur when highly concentrated inocula are used. Therefore, we also co‐inoculated the citH mutant and the wild‐type using a mixture of 107 cfu/mL from each strain. These experiments also indicated that, when co‐inoculated, the growth of the citH mutant could not be complemented by the wild‐type (Fig. 7).

Figure 7.

Figure 7

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Individual and mixed inoculation of the citH (citrate transporter) mutant and wild‐type 97‐2 by syringe infiltration of tomato (cv. H7998) leaves at 107 cfu/mL. Three plants were inoculated with an equal mixture of wild‐type and mutant strains (or with each strain alone). Five leaf discs were removed at each time point from each plant and macerated for serial dilutions and plating. Averages and standard errors from one experiment of three with similar results are shown. (A) In planta growth of the citH mutant and wild‐type strains, individually inoculated (left panel) or co‐inoculated (right panel). t 0, t 2 and t 3 represent 0, 2 and 3 days after inoculation (dai), respectively. (B) Competitive index (CI) and relative increase ratio (RIR) values, calculated as described by Macho et al. (2007). CI is defined for co‐inoculations as the mutant‐to‐wild‐type output ratio divided by the mutant‐to‐wild‐type input ratio. RIR is calculated with the same formula but for individual inoculations. No significant differences (P = 0.05) were observed between individual and mixed inoculations at t 2 and t 3 as determined by Student's t‐test.

DISCUSSION

We have previously identified citH as a novel virulence gene of Xcv (Tamir‐Ariel et al., 2007). Here, several experiments were performed that demonstrate that citH encodes a specific citrate transporter. The citH mutant was considerably impaired in growth compared with the wild‐type and the citH‐complemented strains when citrate was the carbon source. In contrast, the citH mutant was able to grow as well as the wild‐type on other tested carbon sources, including other tricarboxylic acid (TCA) cycle intermediates, confirming that this mutant is not impaired in general metabolism, and that the citH mutation specifically affects its ability to grow on citrate.

As many of the characterized di‐ and tricarboxylate transporter genes are induced by the presence of their substrates (Janausch et al., 2002; Warner and Lolkema, 2002), we aimed to determine the conditions under which the citH promoter is activated. In particular, we asked whether citH transcription is specifically induced by citrate. Indeed, using RIVET, we showed that the induction of the citH promoter occurred almost exclusively in the presence of citrate. Nevertheless, a surprising finding from these experiments was that, although citH induction was not observed in the presence of sucrose or fructose as sole carbon source, it did occur with a combination of both compounds. Interestingly, in the presence of combined sucrose and fructose, resolution also occurred in the ResPcitHrev strain, carrying the citH promoter in the opposite direction relative to citH transcription. This finding suggests the possible occurrence of a cryptic promoter towards the opposite direction of the citH neighbour gene, oprP2 (Fig. 1); however, this hypothesis should be verified further. The resolution pattern observed in the presence of sucrose and fructose remains unclear at this point.

We also report novel regulatory elements of the citrate transporter belonging to a TCS. TCSs have been shown to regulate the transporters of organic acids in bacteria (Janausch et al., 2002; Yamamoto et al., 2000). Thus, we looked at an operon containing a predicted TCS, annotated as tctD‐tctE, which is closely linked to citH in the Xcv chromosome. tctD and tctE encode putative RR and HK, respectively, showing homology to TctD and TctE of Salmonella enterica serovar typhimurium. In this bacterium, the TctDE system regulates the tctI operon, encoding the structural components of a tricarboxylate transporter (Valdivia et al., 2000; Widenhorn et al., 1989). Here, characterization of a tct mutant of Xcv, as well as heterologous expression of the Xcv citH and tctDE genes in E. coli, demonstrated that the TctDE system positively regulates the expression of citH. Importantly, disruption of tctDE did not completely abolish citH expression in Xcv; thus, it appears that, in the absence of functional TctDE, there is a basal level of citH expression, which could be mediated by other, yet unidentified, regulatory elements.

Under the tested conditions, the tct mutant did not show reduced virulence compared with the wild‐type strain. It is well known that many virulence determinants in plant pathogenic bacteria possess a subtle phenotype. This is often a result of a lack of sensitivity of the available virulence assays, combined with functional redundancy and/or a relatively slight contribution to virulence of each determinant (Alfano and Collmer, 1996).

To explore how citH contributes to the virulence of Xcv, we looked at several aspects of the plant–pathogen interaction. In P. syringae pv. phaseolicola, the expression of hrp genes is affected by carbohydrates, amino acids and organic acids. In this bacterium, when citrate was added to minimal medium as the sole carbon source, the expression of hrpC was reduced; when it was added in tandem with fructose or sucrose, the expression of hrpAB, hrpC and hrpD was reduced (Rahme et al., 1992). In Xcv, under the tested conditions in culture and in planta, hrp gene expression was not affected by the citH mutation, indicating that the effects of citrate transport on Xcv virulence are probably independent of the secretion of virulence effectors through Hrp TTSS.

Our data indicate that the citH mutation does not affect the HR‐inducing ability of Xcv. These findings are similar to those recently reported by Mellgren et al. (2009) for P. syringae pv. tomato. The authors found that mqo, encoding the TCA enzyme malate:quinone oxidoreductase, and dctA1, probably encoding a dicarboxylate transporter, were required for normal growth in planta of this bacterium on Arabidopsis thaliana. HR assays revealed that the mqo mutation did not affect hrp expression in P. syringae pv. tomato, and the defective phenotypes of both mqo and dctA1 mutants were attributed to a nutritional deficiency (Mellgren et al., 2009). In our study, the lack of differences in growth ability between the citH mutant and the wild‐type in apoplast extracts could suggest that the citH mutation does not affect the virulence of Xcv through a nutritional defect. However, the possibility that CitH confers a nutritional advantage to Xcv cannot be discarded. First, these were in vitro assays, which may not reflect the complexity of a dynamic plant–pathogen interaction. Second, both mutant and wild‐type grew relatively poorly in apoplast extracts under the tested conditions. Third, we also showed that the citH mutant could not be complemented for growth by the wild‐type in co‐inoculation experiments, a fact that may point to a nutritional deficiency of the former.

As the pH of the citrate medium increases during the growth of Xcv, it can also be hypothesized that citrate uptake by Xcv in planta could contribute to create more favourable conditions for infection. This stems from the fact that alkalinization of the host apoplast is required for the successful establishment of many phytopathogenic bacteria (Alfano and Collmer, 1996, 2004). However, if this was the case, one would expect that the citH mutant would benefit from the presence of the wild‐type in the plant apoplast, which did not occur in co‐inoculation experiments. Therefore, it is not likely that the role of CitH in virulence is associated with apoplast alkanization.

As the timing of gene induction during the infection process may provide clues with regard to the role played by a specific gene product in pathogenicity, we attempted to determine when the citH promoter is activated in planta. Induction of the citH promoter was compared with that of the promoters of two previously characterized hrp genes, hrpG and hrpA (1996a, 1996b). hrpG and hrpA must be activated early on in the disease process to allow the pathogen to go undetected by the host defence mechanisms and to gain access to host intracellular reservoirs. Our analyses showed that, under the conditions tested, the citH promoter was also induced early during the first hours of the infection process. Previously, we have shown that citH expression continues for at least 96 hai (Tamir‐Ariel et al., 2007). Taken together, it appears that CitH might be needed from early on in the infection process until later stages of the interaction with the host tissue.

This report expands our knowledge on a novel virulence factor of Xcv, and sheds some light on its regulation. Although, from co‐inoculation experiments, it appears that CitH‐mediated citrate uptake may confer a nutritional advantage to Xcv during infection, the literature provides some clues about other possible roles for citrate that can be studied in the future. Citrate is known to be a good chelating agent. It forms complexes with metal ions, thereby allowing a more efficient transport of these micronutrients (Lopez‐Bucio et al., 2000). In addition, citrate can potentially participate in the maintenance of osmotic and ionic (in addition of pH) balances (Lopez‐Bucio et al., 2000). Another possibility is that citrate is needed for the functionality of as yet unidentified molecules in Xcv that contribute to its virulence. Finally, recent studies have implicated the TCA cycle as playing a role in the maintenance of the balance of reactive oxygen species (ROS) (Mailloux et al., 2007). Thus, if Xcv competes for the citrate reservoirs, the plant may be less efficient in producing ROS, and the pathogen may be better equipped to deal with the oxidative stress imposed by the plant.

Support for the importance of citrate in plant–microbial interactions comes from several studies. Genes encoding TCA cycle enzymes have been identified in several other IVET‐based studies of bacteria in diverse plant niches, such as Ralstonia solanacearum in the xylem, Erwinia amylovora in fruit tissue, P. syringae pv. syringae in the phyllosphere and P. fluorescens in the rhizosphere (Brown and Allen, 2004; Marco et al., 2003; Rainey, 1999; Zhao et al., 2005). In contrast, these genes were not detected in IVET studies of bacterial interactions with animal hosts, possibly pointing to their specificity for plant–microbe interactions (Brown and Allen, 2004). Importantly, two recent studies point to the importance of citrate in plant–microbe interactions. In one study, Rico and Preston (2008) demonstrated that the metabolic pathway that enables citrate utilization by P. syringae pv. tomato is specifically induced in tomato leaf apoplast. A second study showed that the interruption of a citrate transporter in Pectobacterium atrosepticum results in reduced virulence of this pathogen on potato tubers (Urbany and Neuhaus, 2008).

Finally, it is important to mention that the requirements of functional citH to achieve both wild‐type levels of virulence in planta and to grow on citrate as sole carbon source were observed in our experiments despite the fact that, according to the Xcv genome annotation, this bacterium contains another gene encoding a putative citrate transporter (Xcv3602, encoding a protein that is 72% homologous to a citrate:H+ symporter from Klebsiella pneumoniae). If this gene indeed encodes an additional citrate transporter, whether it possesses a role in the Xcv–host interaction, and under what conditions it is expressed, are questions we intend to study in the future.

EXPERIMENTAL PROCEDURES

Bacterial strains, plasmids and media

The bacterial strains and plasmids used in this study are listed in Table 1. Wild‐type Xcv 97‐2 (race T3; X. perforans, according to the new nomenclature; Jones et al., 2004) and mutant strains were grown on NA/NB (Becton, Dickinson and Co., Sparks, MD, USA), XVM2 minimal medium (Wengelnik et al., 1996a) or modified M9 minimal medium at 28 °C. M9 medium contained 2.5 mg/mL NaCl, 5 mg/mL NH4Cl, 0.3% casamino acid, 5 mm MgSO4, 0.1 mm CaCl and different carbon sources that were added to a final concentration of 10 mm. M9 medium was buffered with appropriate buffers according to the desired pH as follows. For in vitro resolution experiments, phosphate buffers were used for media in the pH range 5.7–8, and potassium hydrogen phthalate buffer (0.01 m potassium hydrogen phthalate and 0.0045 m NaOH) was used when the desired pH of the medium was pH 5. For growth curves of Xcv strains in M9 medium with different carbon sources (including citrate), and for the quantification of citrate remaining in M9‐citrate following growth, media were buffered to pH 7 with phosphate buffer (0.02 m monobasic sodium phosphate, 0.03 m dibasic sodium phosphate; pH 7). Escherichia coli strains were routinely grown on Luria–Bertani (LB) medium at 37 °C. Liquid and solid Simmons citrate media (Simmons, 1926) were used to qualitatively assess the pH change in the medium following citrate uptake by E. coli and Xcv strains. Antibiotics were added at the indicated concentrations: Km, 25 µg/mL; cephalexin (Cp), 20 µg/mL; Gm, 30 µg/mL in solid medium and 10–15 µg/mL in broth; chloramphenicol (Cm), 25 µg/mL; ampicillin (Ap), 100 µg/mL.

Genetic manipulations

Routine molecular manipulations and cloning procedures were carried out as described previously (Sambrook et al., 1989). Unless stated otherwise, enzymes were purchased from Fermentas (Burlington, ON, Canada). Kits for plasmid and PCR product extraction and purification were purchased from Real Biotech Corporation (Taipei, Taiwan). Genomic DNA from Xcv was prepared with the GenElute™ Bacterial Genomic DNA Kit (Sigma‐Aldrich, St. Louis, MO, USA). Southern blot hybridization was performed using the ECL Direct Nucleic Acid Labelling and Detection System (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). Oligonucleotide primers used in this study were purchased from Hy Laboratories (Rehovot, Israel) and are listed in Table S1 (see Supporting Information).

Generation of a citH‐complemented strain

This strain was created by amplification of the citH region from Xcv 97‐2 by PCR using FBamPcitH and RCitcompNewSma as primers, followed by TA cloning into pTZ57R/T (Fermentas). The cloned fragment was then verified, excised with SmaI and cloned into pML122, previously digested with the same enzyme and dephosphorylated. This vector, named pMLcit, was verified by restriction digests and electroporated into the citH mutant to create a citH‐complemented strain (Xcv citH comp; Fig. 1).

Extraction of tomato leaf apoplast and bacterial growth in apoplast medium

Tomato leaf apoplast was extracted by submerging detached leaflets in water under light and alternately applying and releasing vacuum until the leaflets were completely infiltrated. The leaflets were then blotted dry, rolled into 5‐mL syringes and placed in 15‐mL Falcon tubes. The tubes were centrifuged at 4 °C for 6 min at 1000 g. A fraction of the liquid obtained was immediately used for analysis of the activity of the cytoplasmic enzyme glucose‐6‐phosphate dehydrogenase (G6PD), as an indicator for cytoplasmic contamination of the samples. The rest of the extract was stored at −80 °C until use as growth medium, at which point it was filtered through a 0.4‐µm‐pore‐size nitrocellulose membrane, and the appropriate antibiotic was added. G6PD was assayed spectrophotometrically (340 nm) at 25 °C. To 0.05 mL of extract, 0.05% β‐mercaptoethanol, 10 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0) and a protease inhibitor tablet (Roche, Indianapolis, IN, USA) were added. This mix was then added to 0.89 mL of 50 mm glycylglycine buffer (pH 7.5) containing 0.05 mL of nicotinamide adenine dinucleotide phosphate (NADP) (6 mm). The reaction was started by the addition of 0.05 mL of glucose‐6‐phosphate (40 mm). Activity was measured as the rate of increase in absorbance at 340 nm (ΔA 340). The activity of the apoplast extract measured in multiple trials was determined to be approximately 1% of that of whole leaf extract. Cultures were grown in 0.6 mL of apoplast medium in 15‐mL tubes. Samples of 0.11 mL were removed during the exponential and stationary phases of growth, and placed into 96‐well plates for spectrophotometric measurement of growth at 640 nm. The same samples were used for the quantification of citrate as described below.

Quantification of citrate in medium after bacterial growth

The quantification of citrate in growth medium was determined enzymatically using the ENZYTEC™ Citric Acid Kit (Scil Diagnostics GmbH, Viernheim, Germany). The kit was adapted for use in 96‐well microplates, such that, to 0.1 mL of kit solution 1, 0.1 mL of apoplast extract, or 0.02 mL of minimal medium (diluted 10‐fold), was added. Double‐distilled water was then added to a final volume of 0.3 mL. The mixture was mixed and, after 5 min at 25 °C, A1340 (the absorbance at 340 nm) was read. Then, 0.002 mL of kit solution 2 was added and mixed and, after 10 min at 25 °C, A2340 was read. Each microplate had a blank (medium without bacteria) and standard solutions. ΔA citrate was determined as follows: ΔA citrate = (A1340 − A2340)sample/standard − (A1340 − A2340)blank. The concentration of citrate in µg/µL was determined based on the standard curve.

Generation of Xcv mutants for the assessment of tnpR‐mediated resolution driven by the citH promoter

A 658‐bp DNA fragment from strain 97‐2, upstream of the citH ORF (thus containing the citH promoter), was amplified using primers FBamPcitH and RBamXhoPcitH, and cloned upstream of a promoterless tnpR‐uidA, to create a transcriptional fusion, as follows: the amplified PCR product was digested with BamHI and cloned into pJPGmtnpR, which was cut with the same enzyme and dephosphorylated. The orientation of the cloned fragment was determined by restriction analysis, as well as by PCR and sequencing of the cloned fragment. In pPcitH, the citH promoter was cloned in the same orientation as the transcription of tnpR‐uidA, whereas, in pPcitHrev, the promoter was cloned in the opposite orientation to tnpR‐uidA. The two vectors were conjugated into Xcv 97‐2 to obtain XcvPcitH and XcvPcitHrev, respectively, and into XcvRes, to obtain ResPcitH, and ResPcitHrev, respectively.

Resolution of the res cassette for assessment of promoter activity in culture

Cells were grown in 5 mL of NB supplemented with Km for 48 h at 28 °C with shaking. Then, a 1:500 dilution was made in 5 mL of fresh medium to be tested, and cells were grown at similar conditions to the stationary growth phase. Three replicates were made for each growth medium. Serial dilutions were plated from each replicate onto NA (to obtain the total number of cfu) and onto NA supplemented with Km (to obtain the number of unresolved cfu). To obtain the number of resolved cfu, the average number of unresolved cfu was subtracted from the average of the total number of cfu. The percentage resolution was determined based on the ratio of resolved cfu to the total number of cfu: % resolution = [(average total cfu − average unresolved cfu)/average total cfu] × 100.

Plant material and inoculation procedures

Tomato (S. lycopersicum L.) cultivar Hawaii 7998 (H7998; Yu et al., 1995) was grown from seeds in a glasshouse (25–28 °C). Inoculation of plants for the assessment of growth in planta was carried out by dipping 4–5‐week‐old plants into bacterial suspensions of 105 cfu/mL containing 0.02% of the surfactant Silwet L‐77 and 10 mm MgCl2, as described previously (Tamir‐Ariel et al., 2007). Serial dilutions of plant extracts were plated on plates supplemented with appropriate antibiotics. Some of the dilutions were also plated on medium with no antibiotics, which confirmed that there was no loss of the integrated plasmids in the mutants (similar tests were also performed for in vitro experiments). In co‐inoculation experiments, growth in planta was determined following syringe infiltration of leaves of 4–5‐week‐old plants with bacterial suspensions of 103 or 107 cfu/mL in 1 mm MgCl2. Leaf discs were extracted and treated as described previously (Tamir‐Ariel et al., 2007). The assessed strains were discriminated on the basis of antibiotic resistance (the citH mutant is resistant to Km, whereas the wild‐type is not). Competitive index (CI) and relative increase ratio (RIR) values were obtained as described by Macho et al. (2007). Briefly, CI and RIR were calculated from mixed and individual inoculations, respectively. CI was calculated as the mutant‐to‐wild‐type output ratio divided by the mutant‐to‐wild‐type input ratio. RIR was calculated with the same formula using the data from individual inoculations. CI and RIR values were analysed by Student's t‐test. To determine promoter induction in planta by resolution or GUS assays, plants were syringe infiltrated with bacterial suspensions of 106 cfu/mL in 1 mm MgCl2. HR assays were performed on 4–5‐week‐old plant leaves of pepper (C. annuum var. annuum California type) and tomato cv. Hawaii 7981 (H7981; Yu et al., 1995) by syringe infiltration with bacterial suspensions of 105 or 108 cfu/mL in 1 mm MgCl2.

Generation of E. coli strains for heterologous expression of citH

The Xcv citH gene with its indigenous promoter was excised from plasmid pMLcit using SmaI, and cloned into pUC18, previously digested with SmaI and dephosphorylated. The resulting vector, named pUcit, was electroporated into DH5α to create DH5α/pUcit. For the generation of strain DH5α/pUcittctDE, tctDE with its indigenous promoter was PCR amplified with the proofreading enzyme Phusion™ Hot Start (Finnzymes, Espoo, Finland) using primers FHindtctDE and RHindtctDE. The resulting fragment was treated with HindIII and cloned into pUcit previously treated with the same enzyme. Strain DH5α/pUcitOP was created in a similar way but, in this case, the entire tctDE‐Xcv3609 operon was PCR amplified using primers FHindtctDEoperon and RHindtctDEoperon, and cloned into pUcit, following treatment with HindIII. As controls, tctDE and tctDE‐Xcv3609 were similarly cloned into pUC18 (without citH) to generate plasmids pUtctDE and pUOP, and strains DH5α/pUtctDE and DH5α/pUOP, respectively (Fig. 1).

Generation of tct and XcvPcitHtct‐ mutants

tctDE mutant strains were generated in the background of wild‐type 97‐2 and XcvPcitH containing the PcitH::tnpR::uidA transcriptional fusion. Primers FtctDSac and RtctDSac were designed to amplify an internal region within the tctD ORF. The PCR product was sequenced and cloned into pHPKm, a pHP45Ω derivative containing a Km resistance gene instead of the original streptomycin resistance cassette (Tamir‐Ariel et al., 2007). The resulting suicide vector, designated pHKmtctD, was transformed into strains 97‐2 and XcvPcitH to generate Xcv tct and XcvPcitHtct‐ mutants, respectively (Fig. 1), which were verified by Southern blot.

Growth curves in M9 medium

Growth curve experiments with Xcv strains were performed in modified M9 medium with different carbon sources (at 10 mm, as described above). Xcv strains were previously grown for 24 h in NB, and then washed twice by centrifugation (3000 g, 4 °C, 10 min) and resuspended in the corresponding M9 medium. Cells were then transferred to 30 mL of fresh medium in 100‐mL Erlenmayer flasks (three per treatment) to an initial OD640 of about 0.005, with the exception of growth curves in M9‐citrate with Gm (see below), where the initial OD640 was ∼0.05. Cultures were grown at 28 °C with shaking (200 r.p.m.), and growth was assessed using a Helios Gamma (Thermo Spectronic, Cambridge, UK) spectrophotometer. For growth curves on M9‐citrate that included the citH‐complemented strain (citH mutant carrying pMLcit), wild‐type and citH mutant strains, both carrying pML122, were used, and Gm was added at 10 µg/mL to both NB and M9‐citrate. Growth curves of E. coli strains carrying pUC18‐derived plasmids in M9‐citrate were performed similarly, with the following modifications: E. coli strains were previously grown in LB, Ap (100 µg/mL) was added to the medium and the strains were grown at 37 °C.

Assessment of citH promoter activity by GUS assays

The effect of the tct mutation on citH promoter activity was assessed in planta and in culture by GUS (uidA) assays with the substrate 4‐methylumbelliferyl‐β‐d‐glucuronide (MUG). For assays with bacteria grown in culture, cells were grown in M9‐citrate and collected at OD640 ∼ 0.2. Assays were performed as described previously (Tamir‐Ariel et al., 2007). For in planta assays, bacteria at 106 cfu/mL in 1 mm MgCl2 were inoculated into leaves of 5‐week‐old plants as described above. Each mutant was inoculated into three plants, and samples from each plant were collected at 48, 72 and 96 hai, with a 0.8‐cm‐diameter corkscrew, and macerated. Macerated tissues were centrifuged at low speed (1000 g) for 30 s to pellet the plant material. The supernatants were transferred to a clean tube and centrifuged again at 12 000 g for 2 min to pellet bacterial cells. The pellets were then resuspended in double‐distilled water, and samples from each treatment were diluted and plated for cfu counts. The remaining cells were stored at −80 °C until use for GUS activity measurements, as described previously (Jefferson et al., 1987).

Semiquantitative RT‐PCR to assess the expression of hrp genes

The citH mutant and the wild‐type were grown in NB, and then diluted 1:1000 into XVM2, from which cultures were collected during exponential growth. XVM2 was chosen as it is known to induce the expression of hrp genes (Wengelnik et al., 1996a), and has also been shown to induce the expression of citH (Tamir‐Ariel et al., 2007; this study). RNA extraction was carried out using the MasterPure™ RNA Purification Kit (Epicentre Biotechnologies, Madison, WI, USA). A further DNA degradation treatment was conducted using TURBO DNase (MO BIO Laboratories Inc., Carlsbad, CA, USA). cDNA was prepared with random hexamers using an ImProm‐II(tm) Reverse Transcription System (Promega, Madison, WI, USA). PCRs were carried out in an Eppendorf (Hamburg, Germany) thermal cycler using REDTaq PCR ReadyMix (Sigma‐Aldrich). Specific primer sets, F16sRNA‐RT/R16sRNA‐RT, FhrpA‐RT/RhrpA‐RT, FhrpXv‐RT/RhrpXv‐RT and FhrpG‐RT/RhrpG‐RT, were used for amplification of 16sRNA, hrpA, hrpX and hrpG cDNA, respectively. The PCR proceeded for 30 cycles of denaturation at 95 °C for 4 min, annealing at 57 °C for 45 s and elongation at 72 °C for 50 s. Samples were collected after 10, 15, 20, 25 and 30 cycles, and separated by electrophoresis in 1% agarose gels. The gels were stained in an ethidium bromide solution (0.5 µg/mL) for 20 min and photographed with transmitted UV light at 295 nm.

Supporting information

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ACKNOWLEDGEMENTS

We thank Tamar Zimmermann for technical support and Edouard Jurkevitch for valuable comments during the preparation of the manuscript. This work was supported by grant #975/07 from the Israel Science Foundation (ISF).

REFERENCES

  1. Alfano, J.R. and Collmer, A. (1996) Bacterial pathogens in plants: life up against the wall. Plant Cell, 8, 1683–1698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alfano, J.R. and Collmer, A. (2004) Type III secretion system effector proteins: double agents in bacterial disease and plant defense. Annu. Rev. Phytopathol. 42, 385–414. [DOI] [PubMed] [Google Scholar]
  3. Angelichio, M.J. and Camilli, A. (2002) In vivo expression technology. Infect. Immun. 70, 6518–6523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Astua‐Monge, G. , Minsavage, G.V. , Stall, R.E. , Davis, M.J. , Bonas, U. and Jones, J.B. (2000) Resistance of tomato and pepper to T3 strains of Xanthomonas campestris pv. vesicatoria is specified by a plant‐inducible avirulence gene. Mol. Plant–Microbe Interact. 13, 911–921. [DOI] [PubMed] [Google Scholar]
  5. Boorsma, A. , Van Der Rest, M.E. , Lolkema, J.S. and Konings, W.N. (1996) Secondary transporters for citrate and the Mg(2+)–citrate complex in Bacillus subtilis are homologous proteins. J. Bacteriol. 178, 6216–6222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown, D.G. and Allen, C. (2004) Ralstonia solanacearum genes induced during growth in tomato: an inside view of bacterial wilt. Mol. Microbiol. 53, 1641–1660. [DOI] [PubMed] [Google Scholar]
  7. Burdman, S. , Kots, N. , Kritzman, G. and Kopelowitz, J. (2005) Molecular, physiological and host‐range characterization of Acidovorax avenae subsp. citrulli isolates from watermelon and melon in Israel. Plant Dis. 89, 1339–1347. [DOI] [PubMed] [Google Scholar]
  8. Buttner, D. and He, S.Y. (2009) Type III protein secretion in plant pathogenic bacteria. Plant Physiol. 150, 1656–1664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Camilli, A. and Mekalanos, J.J. (1995) Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection. Mol. Microbiol. 18, 671–683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557–580. [DOI] [PubMed] [Google Scholar]
  11. Janausch, I.G. , Zientz, E. , Tran, Q.H. , Kroger, A. and Unden, G. (2002) C4‐dicarboxylate carriers and sensors in bacteria. Biochim. Biophys. Acta Bioenerg. 1553, 39–56. [DOI] [PubMed] [Google Scholar]
  12. Jefferson, R.A. , Kavanagh, T.A. and Bevan, M.W. (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5, 387–405. [Google Scholar]
  13. Jones, J.B. , Stall, R.E. and Bouzar, H. (1998) Diversity among xanthomonads pathogenic on pepper and tomato. Annu. Rev. Phytopathol. 36, 41–58. [DOI] [PubMed] [Google Scholar]
  14. Jones, J.B. , Bouzar, H. , Stall, R.E. , Almira, E.C. , Roberts, P.D. , Bowen, B.W. , Sudberry, J. , Strickler, P.M. and Chun, J. (2000) Systematic analysis of xanthomonads (Xanthomonas spp.) associated with pepper and tomato lesions. Int. J. Syst. Evol. Microbiol. 50, 1211–1219. [DOI] [PubMed] [Google Scholar]
  15. Jones, J.B. , Lacy, G.H. , Bouzar, H. , Stall, R.E. and Schaad, N.W. (2004) Reclassification of the xanthomonads associated with bacterial spot disease of tomato and pepper. Syst. Appl. Microbiol. 27, 755–762. [DOI] [PubMed] [Google Scholar]
  16. Krom, B.P. , Warner, J.B. , Konings, W.N. and Lolkema, J.S. (2000) Complementary metal ion specificity of the metal‐citrate transporters CitM and CitH of Bacillus subtilis . J. Bacteriol. 182, 6374–6381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Labes, M. , Puhler, A. and Simon, R. (1990) A new family of RSF1010‐derived expression and lac‐fusion broad‐host‐range vectors for gram‐negative bacteria. Gene, 89, 37–46. [DOI] [PubMed] [Google Scholar]
  18. Lopez‐Bucio, J. , Nieto‐Jacobo, M.F. , Ramirez‐Rodriguez, V.V. and Herrera‐Estrella, L. (2000) Organic acid metabolism in plants: from adaptive physiology to transgenic varieties for cultivation in extreme soils. Plant Sci. 160, 1–13. [DOI] [PubMed] [Google Scholar]
  19. Macho, A.P. , Zumaquero, A. , Ortiz‐Martín, I. and Beuzón, C.R. (2007) Competitive index in mixed infections: a sensitive and accurate assay for the genetic analysis of Pseudomonas syringae–plant interactions. Mol. Plant Pathol. 8, 437–450. [DOI] [PubMed] [Google Scholar]
  20. Mailloux, R.J. , Beriault, R. , Lemire, J. , Singh, R. , Chenier, D.R. and Hamel, R.D. (2007) The tricarboxylic acid cycle, an ancient metabolic network with a novel twist. PLoS ONE, 2, e690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Marco, M.L. , Legac, J. and Lindow, S.E. (2003) Conditional survival as a selection strategy to identify plant‐inducible genes of Pseudomonas syringae . Appl. Environ. Microbiol. 69, 5793–5801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mellgren, E.M. , Kloek, A.P. and Kunkel, B.N. (2009) Mqo, a tricarboxylic acid cycle enzyme, is required for virulence of Pseudomonas syringae pv. tomato strain DC3000 on Arabidopsis thaliana . J. Bacteriol. 191, 3132–3141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mudgett, M.B. (2005) New insights to the function of phytopathogenic bacterial type III effectors in plants. Annu. Rev. Plant Biol. 56, 509–531. [DOI] [PubMed] [Google Scholar]
  24. Penfold, R.J. and Pemberton, J.M. (1992) An improved suicide vector for construction of chromosomal insertion mutations in bacteria. Gene, 118, 145–146. [DOI] [PubMed] [Google Scholar]
  25. Prentki, P. and Krisch, H.M. (1984) In vitro insertional mutagenesis with a selectable DNA fragment. Gene, 29, 303–313. [DOI] [PubMed] [Google Scholar]
  26. Rahme, L.G. , Mindrinos, M.N. and Panopoulos, N.J. (1992) Plant and environmental sensory signals control the expression of hrp genes in Pseudomonas syringae pv. phaseolicola . J. Bacteriol. 174, 3499–3507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rainey, P.B. (1999) Adaptation of Pseudomonas fluorescens to the plant rhizosphere. Environ. Microbiol. 1, 243–257. [DOI] [PubMed] [Google Scholar]
  28. Rico, A. and Preston, G.M. (2008) Pseudomonas syringae pv. tomato DC3000 uses constitutive and apoplast‐induced nutrient assimilation pathways to catabolize nutrients that are abundant in the tomato apoplast. Mol. Plant–Microbe Interact. 21, 269–282. [DOI] [PubMed] [Google Scholar]
  29. Sahin, F. , Abbasi, P.A. , Ivey, M.L. , Zhang, J. and Miller, S.A. (2003) Diversity among strains of Xanthomonas campestris pv. vitians from lettuce. Phytopathology, 93, 64–70. [DOI] [PubMed] [Google Scholar]
  30. Sambrook, J. , Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press. [Google Scholar]
  31. Simmons, J.S. (1926) A culture medium for differentiating organisms of typhoid‐colon aerogenes groups and for isolation of certain fungi. J. Infect. Dis. 39, 209. [Google Scholar]
  32. Simon, R. , Priefer, U. and Puhler, A. (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram‐negative bacteria. Biotechnology, 1, 784–791. [Google Scholar]
  33. Sperandio, V. , Torres, A.G. and Kaper, J.B. (2002) Quorum sensing Escherichia coli regulators B and C (QseBC): a novel two‐component regulatory system involved in the regulation of flagella and motility by quorum sensing in E. coli . Mol. Microbiol. 43, 809–821. [DOI] [PubMed] [Google Scholar]
  34. Stock, A.M. , Robinson, V.L. and Goudreau, P.N. (2000) Two‐component signal transduction. Annu. Rev. Biochem. 69, 183–215. [DOI] [PubMed] [Google Scholar]
  35. Tamir‐Ariel, D. , Navon, N. and Burdman, S. (2007) Identification of genes in Xanthomonas campestris pv. vesicatoria induced during its interaction with tomato. J. Bacteriol. 189, 6359–6371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Thieme, F. , Koebnik, R. , Bekel, T. , Berger, C. , Boch, J. , Buttner, D. , Caldana, C. , Gaigalat, L. , Goesmann, A. , Kay, S. , Kirchner, O. , Lanz, C. , Linke, B. , McHardy, A.C. , Meyer, F. , Mittenhuber, G. , Nies, D.H. , Niesbach‐Klosgen, U. , Patschkowski, T. , Ruckert, C. , Rupp, O. , Schneiker, S. , Schuster, S.C. , Vorholter, F.J. , Weber, E. , Puhler, A. , Bonas, U. , Bartels, D. and Kaiser, O. (2005) Insights into genome plasticity and pathogenicity of the plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria revealed by the complete genome sequence. J. Bacteriol. 187, 7254–7266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Urbany, C. and Neuhaus, H.E. (2008) Citrate uptake into Pectobacterium atrosepticum is critical for bacterial virulence. Mol. Plant–Microbe Interact. 21, 547–554. [DOI] [PubMed] [Google Scholar]
  38. Valdivia, R.H. , Cirillo, D.M. , Lee, A.K. , Bouley, D.M. and Falkow, S. (2000) mig‐14 is a horizontally acquired, host‐induced gene required for Salmonella enterica lethal infection in the murine model of typhoid fever. Infect. Immun. 68, 7126–7131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Warner, J.B. and Lolkema, J.S. (2002) Growth of Bacillus subtilis on citrate and isocitrate is supported by the Mg2+‐citrate transporter CitM. Microbiology, 148, 3405–3412. [DOI] [PubMed] [Google Scholar]
  40. Wengelnik, K. and Bonas, U. (1996) HrpXv, an AraC‐type regulator, activates expression of five of the six loci in the hrp cluster of Xanthomonas campestris pv. vesicatoria . J. Bacteriol. 178, 3462–3469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wengelnik, K. , Marie, C. , Russel, M. and Bonas, U. (1996a) Expression and localization of HrpA1, a protein of Xanthomonas campestris pv. vesicatoria essential for pathogenicity and induction of the hypersensitive reaction. J. Bacteriol. 178, 1061–1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wengelnik, K. , Van den Ackerveken, G. and Bonas, U. (1996b) HrpG, a key hrp regulatory protein of Xanthomonas campestris pv. vesicatoria is homologous to two‐component response regulators. Mol. Plant–Microbe Interact. 9, 704–712. [DOI] [PubMed] [Google Scholar]
  43. West, A.H. and Stock, A.M. (2001) Histidine kinases and response regulator proteins in two‐component signaling systems. Trends Biochem. Sci. 26, 369–376. [DOI] [PubMed] [Google Scholar]
  44. White, F.F. , Potnis, N. , Jones, J.B. and Koebnik, R. (2009) The type III effectors of Xanthomonas . Mol. Plant Pathol. 10, 749–766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Widenhorn, K.A. , Somers, J.M. and Kay, W.W. (1989) Genetic regulation of the tricarboxylate transport operon (tctI) of Salmonella typhimurium . J. Bacteriol. 171, 4436–4441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yamamoto, H. , Murata, M. and Sekiguchi, J. (2000) The CitST two‐component system regulates the expression of the Mg‐citrate transporter in Bacillus subtilis . Mol. Microbiol. 37, 898–912. [DOI] [PubMed] [Google Scholar]
  47. Yanisch‐Perron, C. , Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene, 33, 103–119. [DOI] [PubMed] [Google Scholar]
  48. Yu, Z.H. , Wang, J.F. , Stall, R.E. and Vallejos, C.E. (1995) Genomic localization of tomato genes that control a hypersensitive reaction to Xanthomonas campestris pv. vesicatoria . Genetics, 141, 675–682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zhao, Y. , Blumer, S.E. and Sundin, G.W. (2005) Identification of Erwinia amylovora genes induced during infection of immature pear tissue. J. Bacteriol. 187, 8088–8103. [DOI] [PMC free article] [PubMed] [Google Scholar]

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