Distribution and biological role of the oligopeptide-binding protein (OppA) in Xanthomonas species

In this study we investigated the prevalence of the oppA gene, encoding the oligopeptide binding protein (OppA) of the major bacterial oligopeptide uptake system (Opp), in different species of the genus Xanthomonas. The oppA gene was detected in two Xanthomonas axonopodis strains among eight tested Xanthomonas species. The generation of an isogenic oppA-knockout derivative of the Xac 306 strain, showed that the OppA protein neither plays a relevant role in oligopeptide uptake nor contributes to the infectivity and multiplication of the bacterial strain in leaves of sweet orange (Citrus sinensis) and Rangpur lime (Citrus limonia). Taken together these results suggest that the oppA gene has a recent evolutionary history in the genus and does not contribute in the physiology or pathogenesis of X. axonopodis.


Introduction
Oligopeptides play important roles in bacterial nutrition, representing important sources of nitrogen, carbon and other elements. They are also involved in intercellular gene signaling processes, such as those involved in chemotaxis, conjugation, spore formation and the development of the competence state (Detmers et al., 2001). Three distinct oligopeptide uptake systems, specifically committed to the transport of dipeptides (Dpp), tripeptides (Tpp) and oligopeptides (Opp), have been characterized in Escherichia coli, Salmonella enterica pv Typhimurum and Sinorhizobium meliloti (Hiles et al., 1987;Nogales et al., 2009).
In gram-negative bacteria, the Opp system belonging to the ATP-binding cassette (ABC) transporter family, comprises various functional and structural domains, viz., the substrate binding protein in the periplasm (OppA), two transmembrane pore-forming proteins (OppB and OppC), and the two membrane-associated ATPases (OppD and OppF) which generate energy from ATP hydrolysis re-quired for the transport process. The opp genes are frequently organized as a polycistronic operon (oppABCD/F), in which the binding component (OppA) is usually expressed at a higher stoichiometric ratio compared to the other components (Higgins and Hardie, 1983;Hiles et al., 1987;Monnet, 2003). Besides a role in nutrition, the Opp system participates in various physiological processes, such as the recycling of cell-wall peptides, quorum sensing and adhesion to host cells, genetic competence and sporulation (Rudner et al., 1991;Cundell et al., 1995;Alloing et al., 1998;Claverys et al., 2000;Detmers et al., 2001). Opp-encoding genes are found in approximately 50% of those bacterial species with available genomic sequences, although as yet, in only a few cases have their specific roles, either in physiology or pathogenesis, been investigated.
In the present study, we investigated the distribution of the oppA gene among different Xanthomonas species. In addition we evaluated the putative physiological role of OppA, namely that of the oligopeptide-binding component, in the Xac 306 strain, based on the generation of a specific knockout mutant. The present evidence demonstrated that the oppA gene is restricted to two X. axonopodis strains and does not play a specific role in the virulence of this phytopathogen.

Bacterial strains and growth conditions
All the bacterial strains used in this work are listed in Table 1. Bacterial strains were routinely cultivated in Circle Grow (CG) (Bio 101) broth at 30°C. For the oligopeptide uptake assays, the strains were cultivated overnight in M9 minimal medium supplemented with proline, methionine, histidine, tryptophan (100 mg mL -1 each), or in the same medium with proline replaced by the YPLG peptide (0.5 mg mL -1 ).

Gene screening and expression studies
Genomic DNA from all the Xanthomonas isolates was isolated, according to the procedure described by Llop et al. (1999). The oppA gene was amplified with genomic DNA, as template and the oligonucleotides Fw 5'-CGGC GCTCGGGTACCGTGGCGTTGGCGGTGCTG-3' and Rv 5'-GGCGGATCTA GATCAGTGGTGGTGGTGG TGTTTGCTCACCCAGGCGTC-3', based on the reported opp operon sequence (Silva et al., 2002). For Southern-blot analysis, genomic DNA from the tested samples was digested with Pst I, and then transferred to a nylon membrane (Hybond-N, Amersham Biosciences). A probe for the oppA gene was synthesized with [a 32 P]-dCTP, by random primer labeling, using the PCR generated DNA fragment described above. The labeled probe was hybridized with the membrane at 42°C for 16-20 h before exposure to autoradiography films (Kodak T-MAT G/RA film). Western-blot analyses were carried out with whole cell proteins sorted in 12.5% (w/v) acrylamide gels, followed by transference to nitrocellulose membranes (Millipore) and development of bands reacting with polyclonal monospecific anti-OppA antibody, as previously described (Moutran et al., 2004). The anti-OppA serum was raised in mice 342 Oshiro et al. parenterally immunized with a His-tagged protein produced in E. coli transformed with a recombinant pET-28a vector carrying the Xac oppA gene without the native signal peptide (Moutran et al., 2004).

Oligopeptide uptake assay
Xac 306 and XoppA2 cells, grown overnight, were harvested by centrifugation, washed twice with saline and inoculated (1:100) in the M9 medium without proline and with the tetrapeptide YPLG (Sigma) (0.5 mg mL -1 ). After various incubation periods at 28°C, aliquots of the culture supernatant were harvested and dried by vacuum centrifugation. The peptides were extracted with cold methanol and submitted to RP-HPLC separation (LC 10A-VP binary Shimadzu HPLC system). Each fraction was analyzed in an ESI mass spectrometer (LCQDuo, ThermoFinnigan, USA), equipped with a nanospray source and connected to a nanoHPLC system (UltiMate HPLC System, LC Packings, Dionex, USA). The samples were introduced into the spectrometer by a flow rate at 1 mL/min, and then diluted in a solution of 5% acetonitrile and 0.2% formic acid. The spray voltage was kept at 1.8 kV, capillary voltage at 46 V, capillary temperature at 180°C and tube lens offset at -5 V. MS spectra were collected in centroid mode in the 50 to 2000 m/z range.

Protease assays
Cell-free culture medium aliquots of Xac 306 and XoppA2 strains prepared in M9 medium, were incubated with YPLG (0.5 mg mL -1 ) for different periods at 20-28°C. The presence of the tetrapeptide was monitored by RP-HPLC fractionation, and the samples analyzed by mass spectrometry, as described above. The same experiments were repeated in the presence of EDTA (ethylenediaminetetraacetic acid) added to the medium aliquots at a final concentration of 1 mM.

Plant infection experiments
Cells of the Xac 306 and XoppA2 strains were diluted in sterile destilled water to a final absorbance (OD 600 ) of 0.3. Aliquots (100 mL) of cell suspensions were inoculated into the leaves of two sensitive citrus hosts, namely sweet orange (Citrus sinensis) and Rangpur lime (Citrus limonia), through injuries in the leaf surface. Infiltrations were carried out at the lower part of the leaf using a needleless syringe, as previously reported by Laia et al. (2009).

Studies of in vivo growth kinetics
In vivo growth tests of Xac strains were carried out with sweet orange (C. sinensis). The whole procedure followed the experimental conditions described by Laia et al. (2009). Five discs of at least 6 different leaves were assayed for each time point and for each tested bacterial strain.

Results
Opp genes have only been discovered in the genome of the Xac 306 strain (Silva et al., 2002). Moreover, no orthologs have been detected in genomes of the Xcc ATCC33913, 8004 and B100 strains (Silva et al., 2002;Qian et al., 2005;Vorhölter et al., 2008), the Xcv 85-10 strain (Thieme et al., 2005), nor the Xoo strains KACC10331 (Lee et al., 2005), PX099 A (Salzberg et al., 2008) and MAF311018 (GenBank accession number AP008229). These results indicate that opp genes are restricted to Xac and may have a specific physiological role, such as host specificity, in this bacterial species. To further investigate the distribution of the Opp system in Xanthomonas species, the oppA gene was screened in eight additional Xanthomonas species without known genome sequences, including one additional Xcv strain, six isolates of different Xanthomonas species (X. bromi, X. codiaei, X. sacchari, X. pisi, X. theicola, and X. melonis, and two X. axonopodis pv aurantifolii (Xaa) strains (409 and 381). As shown in Figure 1, the oppA gene was detected only in the Xac 306 and Xaa 381 strains, previously classified within the citrus pathovar, but currently, and according to the low DNA homology with Xac strains, considered as belonging to a different pathovar altogether (Schaad et al., 2005). The oppA gene detected in both strains were actively transcribed and translated during in vitro growth. This was demonstrated by Western-blot analysis carried out with a specific anti-OppA polyclonal antibody generated in mice immunized with purified recombinant OppA protein from the Xac 306 strain. As shown in Figure 1C, the anti-OppA serum recognized a reactive protein band in the periplasm fraction of the two X. axonopodis strains (Xac 306 and Xaa 381), but not in extracts of any other Xanthomonas species The OppA protein in Xanthomonas 343 or strain. The expression of OppA protein was apparently constitutive, since no significant difference in its expression was detected in whole-cell extracts of cells kept under different culture medium conditions, supplemented or not with oligopeptides or leaf extracts of susceptible citrus hosts (data not shown).
The restricted distribution of the oppA gene in the Xanthomonas genus suggests that the Opp system plays a specific physiological role in these two strains, but not in the other tested species and strains. In order to investigate the putative role of OppA in the uptake of oligopeptides by X. axonopodis strains, we generated an isogenic oppA defective knockout mutant strain (XoppA2) by a gene replacement approach in which the native gene was replaced by non-functional knockout alleles.
The uptake of a synthetic tetrapeptide (YPLG) was monitored by mass spectrometry, using both the parental Xac 306 strain and the OppA-deficient XoppA2 strain based on the removal of the synthetic peptide from the culture medium during bacterial cell growth. The choice of the substrate peptide was based mainly on molecular modeling studies and docking analyses of different peptides to the Xac OppA structural model (Moutran et al., 2007). In order to validate the experimental approach, we employed two E. coli strains: one (the SS320 strain) proficient in the opp operon, and the other (the SS5013 strain) being an isogenic derivative deleted in the entire opp operon (Andrews and Short, 1985). As indicated in Figures 2A and 2B, the oligopeptides were efficiently removed from the culture supernatant by the E.coli SS320 strain, but not by the opp-defi- 344 Oshiro et al. cient SS5013 strain. On the other hand, when the same experiment was repeated with Xac 306 and its isogenic oppAderivative, XoppA2, no tetrapeptide was detected in the culture supernatants of either strain, after incubation periods ranging from 12 to 24 h at 28°C ( Figure 2C,D). This might be explained, either by the presence of an alternative Opp-independent oligopeptide uptake system in the Xac 306 strain, or by the production and secretion of proteases in the culture media.
In order to discover the reason for the rapid removal of tetrapeptide from Xac cultures, the oligopeptide was incubated with aliquots from the culture supernatant of both Xac strains. No tetrapeptide was detected after incubation with culture supernatants of both parent and mutant strains ( Figure 3B/D). In addition, EDTA prevented the in vitro peptide degradation following incubation with culture supernatants of both Xac strains ( Figure 3A,C). Such result indicated that the apparent uptake of the tetrapeptide was mainly attributed to the proteolytic attack by secreted proteases produced by the Xac 306 strain. Attempts to block protease activity in culture supernatants of actively growing Xac strains with EDTA failed, probably due to the large amount of secreted proteases (data not shown).
In order to evaluate the putative role of OppA in Xac pathogenesis in citrus hosts, we infected two susceptible citrus hosts, viz., sweet orange (C. sinensis) and Rangpur lime (C. limonia), with the Xac 306 and XoppA2 strains, and compared the induction of leaf lesions and multiplication in leaf tissues. Up to 10 days following infection, no difference was detected in the symptoms inflicted by the The OppA protein in Xanthomonas 345 two Xac strains in either citrus host (Figure 4). Furthermore, no significant difference in the growth curves of either of the two strains was detected during their multiplication in sweet orange leaf tissues (Figure 4). These results clearly indicated that deletion of the oppA gene did not impair the growth of Xac in susceptible citrus hosts.

Discussion
In the present study we investigated the prevalence of the oppA gene, and the corresponding OppA protein, in different Xanthomonas species, an important group of phytopathogens inflicting heavy losses in several economically relevant crops. The present results demonstrated that, in contrast to other bacterial groups, such as enterobacterial species and lactic acid bacteria, the oppA gene was detected in only two out of the three tested X. axonopodis strains. Furthermore, studies screening revealed that oppA is also absent in the genomes of the Xccs 8004 and Xcv 85-10 strains, as well as two Xoo strains with reported genome sequences (Lee et al., 2005;Thieme et al., 2005). We also demonstrated that early-branching Xanthomonas species, including the ancestral X. sacchari and X. theicola, do not carry the oppA gene. Similarly, as recently defined by Parkinson and colleagues (2007), other three established Xanthomonas phylogenetic groups, represented by X. bromi, X. melonis, X. pisi and X. codiaei, also do not carry the oppA gene. In spite of our previous observations that the opp operon does not present biased codon usage, distinct GC content or the presence of adjacent insertion sequences and transposase-encoding genes (Moutran et al., 2004), the present results indicate that the opp genes have been acquired in a recent evolutionary event in the Xanthomonas genus and remained restricted to some X. axonopodis strains.
Generation of an oppA-deficient strain led us to conclude that the Opp system does not play a significant role in the uptake of a tetrapeptide, the most likely substrate of OppA encoded by the Xac 306 strain, as previously determined by molecular modeling tools (Moutran et al., 2007). Monitoring of the peptide uptake by mass spectrometry showed that, in contrast to E. coli, the tetrapeptide is quickly degraded by secreted proteases produced by both Xac 306 and XoppA2 strains. Hence, it may be deduced that the abundant production of extracellular proteases produced by Xac, as well as other Xanthomonas species, could constitute an abundant source of amino acids derived from the proteolytic degradation of host proteins. Under such conditions, the function of an oligopeptide uptake system would be dispensable, since free amino acid residues are actively transported by different dedicated uptake systems. This conclusion is supported by the lack of the oppA gene in most Xanthomonas species and the presence of a stop codon located 129 bp downstream of the first structural codon in the Xac 306 oppD/F cistron, this encoding the ATPase component required for the generation of energy to the transport process (Silva et al., 2002;Moutran et al., 2004). The finding that eight other Xanthomonas species, besides one Xaa strain, which do not carry opp genes, lends further support to the notion that, in contrast to other bacterial species, Xanthomonas oppA genes really represent pseudo genes on the way to disappearing from the genomes of these strains.
In accordance with this idea, no measurable difference in colonization, infection and generation of leaf lesions was observed in two susceptible citrus hosts infected with either the Xac 306 or the isogenic oppA-deficient strains. The absence of any significant pathogenic impact involving the Xac 306 strain in various citric hosts, lends further support to the conception that the Opp system is not functional in this strain and does not contribute to the pathogenesis of this bacterial strain in different citrus hosts. Collectively, the present evidence indicates that the Xac OppA, and consequently the Opp system, in contrast to other bacterial species, does not play a relevant physiological role.