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Journal of Bacteriology, September 2005, p. 6231-6237, Vol. 187, No. 17
0021-9193/05/$08.00+0     doi:10.1128/JB.187.17.6231-6237.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Xanthomonas campestris pv. campestris Possesses a Single Gluconeogenic Pathway That Is Required for Virulence{dagger}

Dong-Jie Tang,{ddagger} Yong-Qiang He,{ddagger} Jia-Xun Feng, Bao-Ren He, Bo-Le Jiang, Guang-Tao Lu, Baoshan Chen, and Ji-Liang Tang*

Guangxi Key Laboratory of Subtropical Bioresources Conservation and Utilization, The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering, and College of Life Science and Technology, Guangxi University, 100 Daxue Road, Nanning, Guangxi 530004, People's Republic of China

Received 17 January 2005/ Accepted 17 May 2005


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ABSTRACT
 
Disruption of ppsA, a key gene in gluconeogenesis, of Xanthomonas campestris pv. campestris resulted in the failure of the pathogen to grow in medium with pyruvate or C4-dicarboxylates as the sole carbon source and a significant reduction in virulence, indicating that X. campestris pv. campestris possesses only the malic enzyme-PpsA route in gluconeogenesis, which is required for virulence.


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TEXT
 
The ability to acquire nutrients from the host is essential for a pathogen to establish an infection. Among those nutrients, carbon is one of the most basic elements. Bacteria may use gluconeogenesis (Fig. 1) to synthesize glucose from nonsugar C2 or C3 compounds or the intermediates of the tricarboxylic acid (TCA) cycle when there is not sufficient hexose in their niches (17, 24, 26, 27). The first step of gluconeogenesis in bacteria is the synthesis of phosphoenolpyruvate (PEP) through the phosphoenolpyruvate carboxykinase (PckA) route and/or the malic enzyme-phosphoenolpyruvate synthase (PpsA) route (13, 17, 24, 25, 26). In some bacteria, the function of PpsA to synthesize PEP can be performed instead by pyruvate phosphate dikinase (PPDK) (10, 26). The PckA route is a one-step reaction in which PEP is synthesized from oxaloacetate catalyzed by PckA, and the malic enzyme-PpsA (or PPDK) route consists of two reactions: the first reaction is the conversion of malate to pyruvate catalyzed by the malic enzyme MaeA or MaeB and the second reaction is the synthesis of PEP from pyruvate catalyzed by PpsA using pyruvate, H2O, and ATP as substrates or by PPDK using pyruvate, phosphate, and ATP as substrates (24, 26). The PckA route and the malic enzyme-PpsA (or PPDK) route coexist in Escherichia coli (24), Rhodopseudomonas palustris (26), and Sinorhizobium meliloti (17). The synthesis of PEP from pyruvate is catalyzed by PpsA in E. coli and R. palustris and by PPDK in S. meliloti (17, 24, 26). Campylobacter jejuni, a bacterium that is unable to utilize glucose due to the absence of the key glycolytic enzyme 6-phosphofructokinase, possesses only the PckA route to synthesize PEP in gluconeogenesis (31). The gluconeogenic pathway has been shown to be required for virulence of the animal pathogen Mycobacterium bovis (5). The role of this pathway in the pathogenesis of a plant pathogen has not been reported.



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  		FIG. 1. Schematic drawing of the gluconeogenic pathway in microorganisms. The PckA route is shown as a blue arrow, the malic enzyme-PpsA route as red arrows, and the glyoxylate cycle as green arrows (modified from reference 24). CoA, coenzyme A.

The glyoxylate cycle is a truncated TCA cycle in which isocitrate is hydrolyzed to succinate and glyoxylate by isocitrate lyase (encoded by aceA, also named icl) and the glyoxylate is then condensed with acetyl coenzyme A to produce malate by malate synthase (encoded by mls). The primary function of the glyoxylate cycle is to permit C2 compounds to be converted to glucose through the gluconeogenic pathway (21). It has been reported that the glyoxylate cycle plays an important role in the pathogenesis of a number of pathogens (16, 20, 23, 32, 33).

Xanthomonas campestris pv. campestris, a yellow-pigmented {gamma}-proteobacterium, is the causal agent of the black rot disease of cruciferous crops worldwide, and it also infects the model plant Arabidopsis thaliana. It propagates and spreads in the apoplast of the host plant after infection (14, 29). X. campestris pv. campestris has been used as a model bacterium for studying microbe-plant interactions for over 2 decades, and a number of genes involved in pathogenicity have been identified (2, 4, 6, 9, 30). The whole genome of X. campestris pv. campestris strain ATCC 33913 was sequenced (8). Our group and collaborators have recently sequenced the whole genome of another X. campestris pv. campestris strain 8004 (our unpublished data). The genome annotation shows that homologs of all the appropriate genes for gluconeogenesis, except pckA and maeA, are present in both strains (8; our unpublished data).

Using a molecular genetic approach, we have demonstrated that X. campestris pv. campestris has only the malic enzyme-PpsA route to synthesize PEP in gluconeogenesis and the gluconeogenic pathway is required for its full virulence. We further found that the glyoxylate cycle is not required for the multiplication in planta and virulence of the pathogen. We speculate that X. campestris pv. campestris uses C4-dicarboxylates rather than C2 compounds as the primary carbon source through gluconeogenesis via the malic enzyme-PpsA route in planta.

The malic enzyme-PpsA route is the only route to synthesize PEP in gluconeogenesis of X. campestris pv. campestris. There exist two routes to synthesize PEP in gluconeogenesis in bacteria: the PckA route and the malic enzyme-PpsA route. The PckA route has been generally considered to be the main pathway of gluconeogenesis (19, 22). However, the genome annotation data show that there is no homolog of pckA in X. campestris pv. campestris (8; our unpublished data). To investigate whether the PckA route is functionally absent in X. campestris pv. campestris, ppsA was disrupted by using a method described previously (34). A 542-bp internal fragment of the ppsA coding region (from positions 12 to 553) was amplified by using chromosomal DNA of X. campestris pv. campestris wild-type strain 8004 (6) as the template and ppsAMF-ppsAMR mutation primer set (Table 1). After confirmation by sequencing, the amplified DNA fragment was cloned into the suicide plasmid pK18mob (28) to create recombinant plasmid pK1880 (Table 2). The plasmid pK1880 was introduced from E. coli JM109 (35) into X. campestris pv. campestris strain 8004 by triparental conjugation using pRK2073 as the helper plasmid (18). Transconjugants were screened on NYG medium (5 g of peptone, 3 g of yeast extract, and 20 g of glycerol per liter) (7) supplemented with rifampin and kanamycin to the final concentrations of 50 µg/ml and 25 µg/ml, and the obtained transconjugants with a mutation in the ppsA gene were confirmed by PCR using the chromosomal DNA of the transconjugants as the template and the p18conF-ppsAconR confirmation primer set (Table 1). The expected PCR product was further confirmed by sequencing. The resulting ppsA mutant was designated NK1880 (Table 2).


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  		TABLE 1. Primer sets used in this work


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  		TABLE 2. Bacterial strains and plasmids used in this work

For complementation of the ppsA mutant, the wild-type ppsA gene was amplified from strain 8004 by PCR using primer set ppsAF-ppsAR (Table 1) and cloned into pLAFR6 (15). The obtained recombinant plasmid pXC1880 (Table 2) was introduced into NK1880 by triparental conjugation. NK1880 carrying pXC1880 was screened on NYG supplemented with rifampin (50 µg/ml), kanamycin (25 µg/ml), and tetracycline (5 µg/ml) and confirmed by PCR with a specific primer set, ppsAF-ppsAR (Table 1). The resulting strain was designated CNK1880 (Table 2).

The abilities of X. campestris pv. campestris strains to utilize different carbon sources were determined by measuring the growth rates of the strains in noncarbohydrate minimal medium (NCM) [modified from MMX minimal medium (7), containing 2.0 g/liter (NH4)2SO4, 4.0 g/liter K2HPO4, 6.0 g/liter KH2PO4, and 0.2 g/liter MgSO4 · 7H2O] supplemented with different carbon sources (glucose, sucrose, malate, succinate, fumarate, and pyruvate at 0.5% [wt/vol] and acetate at 0.15% [wt/vol]).

The wild-type strain 8004 was able to grow in the NCM medium supplemented with each of the following carbohydrates as the sole carbon source: glucose, sucrose, malate, succinate, fumarate, pyruvate, and acetate (Fig. 2). The ppsA mutant grew normally in NCM medium supplemented with glucose or sucrose but was unable to grow in NCM medium supplemented with malate, succinate, fumarate, pyruvate, or acetate as the sole carbon source (Fig. 2). Furthermore, the complemented strain CNK1880 behaved like the wild-type strain (Fig. 2). These results demonstrated that the PckA route is functionally absent and the malic enzyme-PpsA route is the only route to synthesize PEP from C4-dicarboxylate in gluconeogenesis in X. campestris pv. campestris.



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  		FIG. 2. Growth of X. campestris pv. campestris strains ({blacktriangleup}, wild-type strain 8004; {circ}, ppsA mutant NK1880; •, ppsA-complemented strain CNK1880) in media with different carbon sources. The NCM medium was supplemented with glucose (A), sucrose (B), fumarate (C), malate (D), succinate (E), or pyruvate (F) at 0.5% or sodium acetate (NaAC) at 0.15% (G). Data presented were from a representative experiment.

Of the 213 bacterial strains (eubacteria and archaea) with the whole genomes sequenced so far, 90 strains possess a single route for the synthesis of PEP in gluconeogenesis (36 possess the genes encoding the PckA route, and 54 possess the genes encoding the malic enzyme-PpsA route), 84 possess the genes encoding both the PckA and the malic enzyme-PpsA routes, and 39 have neither the pckA homolog nor the ppsA (or the ppd kinase gene) homolog (see Table S1 in the supplemental material). Theoretically, possession of both the PckA route and the malic enzyme-PpsA route for synthesizing PEP in gluconeogenesis may confer the bacterium advantages in acquiring carbon nutrients to survive in more diversified environments. For example, in E. coli, which possesses both the PckA route and the malic enzyme-PpsA route for the synthesis of PEP in gluconeogenesis, disruption of one of the two routes alone does not affect the normal function of gluconeogenesis of this bacterium (24). The limited genome data seem to show the trend that most free-living bacteria, including dominantly the saprophytic and opportunistic pathogens, possess both the PckA and the malic enzyme-PpsA routes, while the semiparasitic pathogens tend to have a single route, either PckA or malic enzyme-PpsA. Interestingly, Buchnera spp., the endocellular symbionts, and Mycoplasma spp., the obligate pathogens, have no homologous genes for PEP synthesis in gluconeogenesis (see Table S1 in the supplemental material). Thus, the pathway variation in gluconeogenesis seems to correlate with the living niches of the different bacteria to ensure the acquisition of sufficient carbon source.

The gluconeogenic pathway is required for virulence and in planta growth of X. campestris pv. campestris. Gluconeogenesis has been shown or implied to be required for virulence of a number of animal pathogens such as Salmonella enterica serovar Typhimurium (1), M. bovis (5, 19), Mycobacterium tuberculosis (23), and Candida albicans (20). To investigate whether the virulence of the pathogen X. campestris pv. campestris is affected by a mutation in ppsA, the virulence of ppsA mutant NK1880 was tested by inoculating the mutant onto its host plant Chinese radish (Raphanus sativus L. cv. radiculus Pers.) grown in a greenhouse with a day/night cycle of 12 h/12 h illumination by fluorescent lamps at a temperature of 25 to 28°C. Two leaves per plant at the stage of four full-expanded leaves were inoculated by the leaf-clipping method (9) with bacterial cells grown in NYG medium at 28°C with shaking at 200 rpm for 15 h at a concentration of 108 cells/ml. Fifty leaves were inoculated for each strain in one assay that was repeated three times. After being kept at 100% humidity for 24 h, the inoculated plants were maintained in the growth conditions described above. Lesion lengths were measured 10 days postinoculation, and the data were statistically analyzed. As analyzed by t test, the mean length of the lesion caused by ppsA mutant NK1880 was significantly shorter than that of the lesion caused by the wild-type strain at a P of 0.01 (t test) (Fig. 3). Meanwhile, the lesion lengths of the complemented strain CNK1880 and the wild-type strain were not significantly different at a P of 0.05 (Fig. 3). To verify whether the reduced virulence was correlated to the reduced growth rate of the pathogen, we investigated the growth of bacteria in planta. A group of five inoculated radish leaves was homogenized in sterilized water and plated on NYG medium supplemented with rifampin (50 µg/ml) (for the wild-type strain), rifampin (50 µg/ml) plus kanamycin (25 µg/ml) (for the mutant strain), or rifampin (50 µg/ml) and kanamycin (25 µg/ml) plus tetracycline (5 µg/ml) (for the complemented strain). Bacterial CFU were counted after incubation at 28°C for 3 days. During the observation period, the in planta multiplication level of the ppsA mutant was significantly lower than that of the wild-type strain at each of the test points (P = 0.01), and the number of CFU of the ppsA mutant in the infected leaves was about 100-fold less than that of the wild-type strain from day 4 to day 10 after inoculation (Fig. 3C). These results demonstrated that an intact gluconeogenic pathway is required for full virulence and the reduced virulence is probably related to the reduced bacterial numbers of the mutant in planta.



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  		FIG. 3. ppsA is required for virulence and in planta growth of X. campestris pv. campestris. (A) Black rot disease symptoms caused by X. campestris pv. campestris strains on inoculated leaves of Chinese radish. Photographs were taken at day 10 postinoculation. (B) Average lesion lengths caused by X. campestris pv. campestris strains. Values are the means ± standard deviations (SD) from three repeats, using two leaves each from 25 plants. (C) Growth of bacteria in inoculated leaves. Data are the means ± SD from three repeats. {blacktriangleup}, wild-type strain 8004; {circ}, ppsA mutant NK1880; •, ppsA-complemented strain CNK1880.

The glyoxylate cycle is not required for virulence and in planta growth of X. campestris pv. campestris. The involvement of the glyoxylate cycle in microbial pathogenesis has been reported for both animal and plant pathogens (16, 20, 23, 32, 33). To investigate whether or not the glyoxylate cycle is required for the virulence of X. campestris pv. campestris, aceA and mls, genes encoding the glyoxylate cycle enzymes isocitrate lyase and malate synthase, respectively, were disrupted by using the same method for inactivating ppsA. The 439-bp fragment of the aceA coding region (from positions 6 to 444) and the 423-bp fragment of the mls coding region (from positions 9 to 431) were generated by PCR with specific primers (Table 1). The resulting aceA and mls mutants, NK0217 and NK0216 (Table 2), could grow normally in NCM medium supplemented with each of the following carbohydrates at a concentration of 0.5% (wt/vol) as the sole carbon source: glucose, sucrose, glycerol, succinate, fumarate, malate, and pyruvate (data not shown); however, they were unable to grow in NCM medium supplemented with acetate (0.15%) as the sole carbon source, in contrast to the wild-type strain (Fig. 4), indicating that the glyoxylate cycle had been specifically blocked in the mutants. Upon inoculation onto the plants by the leaf-clipping method at a bacterial concentration of 108 cells/ml, both mutants showed full virulence on Chinese radish and grew as well as the wild-type strain (Fig. 5), demonstrating that the glyoxylate cycle is not required for virulence and in planta multiplication of X. campestris pv. campestris.



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  		FIG. 4. The mls mutant and aceA mutant are unable to grow on minimal medium supplemented with acetate as the sole carbon source. {blacktriangleup}, wild-type strain 8004; {diamond}, mls mutant NK0216; {blacklozenge}, aceA mutant NK0217. OD600, optical density at 600 nm.



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  		FIG. 5. mls and aceA are not required for virulence and in planta growth of X. campestris pv. campestris. (A) Black rot disease symptoms caused by X. campestris pv. campestris strains on inoculated leaves of Chinese radish. Photographs were taken at day 10 postinoculation. (B) Average lesion lengths caused by X. campestris pv. campestris strains. Values are the means ± standard deviations (SD) from three repeats, each with 50 leaves. (C) Growth of bacteria in inoculated leaves. Data are the means ± SD from three repeats. {blacktriangleup}, wild-type strain 8004; {diamond}, mls mutant NK0216; {blacklozenge}, aceA mutant NK0217.

C4-dicarboxylates could be the principal carbon sources for X. campestris pv. campestris during infection. X. campestris pv. campestris propagates and spreads in the apoplast of the host plant after infection (14, 29). Thus, the ability to acquire nutrients from the apoplast is critically important for it to cause disease. However, the nutritional requirements of X. campestris pv. campestris during infection and the molecular mechanism by which it acquires nutrients from the apoplast are still unclear. The observation that disruption of the gluconeogenic pathway resulted in significant reductions both in multiplication in planta and virulence of X. campestris pv. campestris suggested that the apoplast is lacking hexose but rich in gluconeogenic substrates (C2 or C3 compounds or the intermediates of the TCA cycle), and the gluconeogenic pathway is the only route to utilize these carbon sources to maintain the carbon and energy supplies for normal growth of X. campestris pv. campestris during infection. Furthermore, disruption of the glyoxylate cycle (mutation in aceA or mls) of X. campestris pv. campestris resulted in failure to grow in medium with C2 compounds as the sole carbon source (Fig. 4) but did not affect its virulence and growth in planta (Fig. 5), indicating that C2 compounds are not the major carbon source for X. campestris pv. campestris in planta. Combining these results with the fact that the wild-type strain grew well on medium with C4-dicarboxylic acids (Fig. 2) but poorly on medium with citrate (data not shown) as the sole carbon source, we suppose that C4-dicarboxylates could be the principal carbon sources for X. campestris pv. campestris during infection. This hypothesis is consistent with the findings with Rhizobium spp., that C4-dicarboxylic acids are the principal carbon sources for the bacteroid within nodules (3, 11, 12) but in contrast to the reports that nonsugar C2 compounds are the major carbon sources for M. tuberculosis and C. albicans during the infection of macrophage (20, 23). This information suggests that the carbon diet of a pathogen inside the host is correlated to the composition of the available carbon source in the infection site. From an evolutionary point of view, the nutritional requirements of a pathogen during infection and the molecular mechanism by which this pathogen acquires nutrients from the host may be the results of coevolution of the pathogens with their hosts.

The reduced virulence of the ppsA mutant may be due to its inability to convert C4-dicarboxylic acids to glucose for growth in planta. The gluconeogenic pathway may be required for fitness of X. campestris pv. campestris in planta and unlikely for the production of virulence factor(s). Although metabolic pathways are generally not considered to be virulence factors, the elucidation of the mechanism to acquire and metabolize nutrients during infection is critically important for fully understanding the pathogenesis of a pathogen.

Nucleotide and amino acid sequence accession numbers. The sequences of ppsA, aceA, and mls have been submitted to the GenBank database under accession numbers AY618213, AY724680, and AY724681, respectively.


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ACKNOWLEDGMENTS
 
We are grateful to David Hopwood and J. Maxwell Dow for their helpful discussions.

This work was supported by grants from the National Natural Science Foundation of China Key Program (30130010) and The National High Technology Research and Development Program of China (2001AA223051 and 2004AA223060).


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FOOTNOTES
 
* Corresponding author. Mailing address: College of Life Science and Technology, Guangxi University, 100 Daxue Road, Nanning, Guangxi 530004, People’s Republic of China. Phone: 86-771-3239566. Fax: 86-771-3239413. E-mail: jltang{at}gxu.edu.cn. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back

{ddagger} Dong-Jie Tang and Yong-Qiang He contributed equally to this work. Back


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Journal of Bacteriology, September 2005, p. 6231-6237, Vol. 187, No. 17
0021-9193/05/$08.00+0     doi:10.1128/JB.187.17.6231-6237.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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