The sulfur/sulfonates transport systems in Xanthomonas citri pv. citri
© Pereira et al. 2015
Received: 28 August 2014
Accepted: 29 June 2015
Published: 14 July 2015
The Xanthomonas citri pv. citri (X. citri) is a phytopathogenic bacterium that infects different species of citrus plants where it causes canker disease. The adaptation to different habitats is related to the ability of the cells to metabolize and to assimilate diverse compounds, including sulfur, an essential element for all organisms. In Escherichia coli, the necessary sulfur can be obtained by a set of proteins whose genes belong to the cys regulon. Although the cys regulon proteins and their importance have been described in many other bacteria, there are no data related to these proteins in X. citri or in the Xanthomonas genus. The study of the relevance of these systems in these phytopathogenic bacteria that have distinct mechanisms of infection is one essential step toward understanding their physiology. In this work, we used bioinformatics, molecular modeling and transcription analysis (RT-PCR) to identify and characterize the putative cys regulon genes in X. citri.
We showed that the ATP Binding Cassette Transporter (ABC transporter) SbpCysUWA for sulfate uptake is conserved in X. citri and translated in presence of sulfate. On the other hand, differently from what is predicted in databases, according molecular modeling and phylogenetic analysis, X. citri does not show a proper taurine transporter, but two different ABC systems related to the alkanesulfonate/sulfonate transport that were recently acquired during evolution. RT-PCR analysis evidenced that these genes and their putative transcriptional regulator CysB are rather transcripted in XAM1, a medium with defined concentration of sulfate, than LB.
The presence of at least three distinct systems for sulfate and sulfonates assimilation in X. citri evidenced the importance of these compounds for the bacterium. The transcription of genes involved with alkanesulfonate/sulfur compounds in XAM1 along to CysB suggests that despite the differences in the transporters, the regulation of these systems might be similar to the described for E. coli. Altogether, these results will serve as a foundation for further studies aimed to understanding the relevance of sulfur in growth, virulence and pathogenesis of X. citri and related bacteria.
The plant-associated bacteria from the Xanthomonas genus exhibit a high degree of host plant specificity when invading diverse tissues and causing different types of diseases . Comparative genomic analysis has provided insights into the role of horizontal gene transfer and the understanding of the pathogenic adaptations in this genus . X. citri is one of the relevant species from the genus because it is the causative agent of the citrus canker, a disease that affects citrus plants and causes significant economic losses in Brazil and many other countries in the world. Although studies have demonstrated the importance of specific genes for biofilm formation , infection  and pathogenesis , there is a lack of information regarding the sulfur or sulfate assimilation pathways in this bacterium and the relevance of these compounds for infection and pathogenesis.
In Escherichia coli, the cys regulon genes encode a set of proteins that are associated with the acquisition of sulfate and organosulfur compounds, such as sulfonates (R-SO3-) and sulfate esters (RO-SO3-), as sulfur source for cysteine biosynthesis. The preferable source of sulfur is sulfate, which is transported by the ABC transporter SbpCysAUW. Once the sulfate assimilation is completed, CysNCDH proteins reduce it to sulfite, and the CysGIJ complex reduces sulfite to sulfide . In sulfate or cysteine starvation, the operons ssuABCDE and tauABCD are induced to constitute two ABC transporters that are required for uptake of alkanesulfonate (SsuABC) and taurine (TauABC), respectively, as well as the enzymes for the desulfonation of the organosulfonates (SsuDE and TauD, respectively) [9, 38]. The regulation of these genes is mediated by Cbl and CysB proteins, which consist of two LysR-type transcriptional activators [31, 37, 39]. CysB is the regulator for sulfur assimilation in E. coli, while the Cbl protein functions as an accessory element that is specific for the utilization of sulfur from organosulfur sources , activating the expression of the tau and ssu genes. Both regulators are closely related, sharing 41 % sequence identity and 60 % similarity .
The presence and importance of these systems have been shown for several bacterial species such as Salmonella typhimurium , Pseudomonas aeruginosa , Bacillus subtilis , and Acidithiobacillus ferrooxidans . In addition, sulfated metabolites have been implicated in the interactions between bacteria and their eukaryotic hosts, including species of the plant symbiont genus Rhizobium , Mycobacterium tuberculosis  and Xanthomonas oryzae . Recently, our group has expressed, purified and solved the three-dimensional structure of the alkanesulfonate-binding protein SsuA bound to three different alkanesulfonates. Through the monitoring of growth, infection and pathogenesis in Citrus sinensis leaves, we showed the importance of an alkanesulfonate binding protein for the growth, infection and production of xantham gum and the development of the canker citric phenotype .
Based on the previous information that has been described for E. coli and other microorganisms, we carried out bioinformatics and trancriptional analyses (RT-PCR) of X. citri to identify the putative cys regulon components. The genes belonging to the cys regulon in E. coli were used as template for a BlastP search against to X. citri genome. The protein sequences were compared and modeled, and the putative motifs and domains were characterized. Moreover, the transcription of genes belonging to the ABC transporters and some genes from cys pathway and ssu operons was evidenced by RT-PCR, suggesting they are required for the bacterial growth. Using this information, we were able to develop a model for sulfur assimilation in X. citri evidencing the expression of genes of the sulfate uptake and sulfur assimilation pathway. Moreover, the data show that X. citri presents significant differences related to the systems for uptake of aliphatic sulfonates and alkanesulfonates, as well as the ability to use sulfate- and sulfur-reduced compounds as sources of energy.
Search for cys regulon gene orthologs, sequence alignment and phylogenetic analysis
The genes belonging to the cys regulon in E. coli, as previously described [9, 14, 37–39], were obtained from the KEGG2 server (Bioinformatics Center Institute for Chemical Research Kyoto University, www.genome.jp) and used to perform a basic local alignment search BlastN (http://blast.ncbi.nlm.nih.gov) against the X. citri (TaxId: 346) genome database (Additional file 1: Table A1). All the default parameters of the program were used. The amino acid sequence alignments were carried out using ClustalX  and edited with GeneDoc . To build the phylogenetic tree of the periplasmic components from the ABC transporters related to sulfate or sulfonates uptake found in X. citri, the gene sequences of the periplasmic proteins [ssuA1 (GeneID: 1154920), ssuA2 (GeneID: 1157269) and sbp (GeneID: 1155088)] were submitted to the BlastP using the non-redundant sequence database. The criterion of choice of the proteins and microorganisms was based on the diversity of genus and similarity of function of the proteins. Description of the hosts and sequences is shown in Additional file 2: Table A2. Phylogenetic reconstruction and molecular evolutionary analyses were conducted with MEGA version 5 , using the neighbor-joining statistical method, p-distance to estimate the evolutionary distances, and 1000 Bootstrap Replications . All the gaps were treated as a complete deletion. The tree that shows the conservation of the cys regulon genes in different phylogenetic groups was built based on the 16S rDNA sequences from the microorganisms described in Additional file 3: Table A3. All the organisms with complete genome sequences were named in agreement with the codes of the KEGG Organisms Complete Genomes table. To name the microorganisms whose genomes were incomplete or absent, we used the capital first letter of the genus followed by two or three first letters from the species.
Molecular modeling of the proteins SsuA1, SflA and SsuD1 was performed using the Modeller 9v4 program  with basic (for one template) or advanced (for more than one template) scripts (http://salilab.org/modeller/tutorial/). A total of 10 models were generated for each target protein, and the best model was selected using the lowest value of the objective function curve. The templates used for the model building of each protein, as well as the Protein Data Bank (PDB) code and amino acid sequence identities, are presented in Additional file 4: Table A4. Consensus prediction of transmembrane domains was obtained with TOPCONS .
RNA extraction, cDNA synthesis and RT-PCR
The X. citri strain used in this study was grown at 28 °C overnight in Luria-Bertani (LB) modified broth (without NaCl) supplemented with ampicillin (100 μg/ml) at 28 °C at 200 rpm. After the growth period, the cultures were diluted 50 times, washed two times in sterile water and incubated in virulence induction medium to mimic the plant environment, XAM1 [10, 30], or LB until the mid end-exponential growth phase. Samples normalized by O.D. to contain about 1014 cells of each culture were transferred to a microcentrifuge tube and centrifuged for 2 min at 14,000 × g. We carefully remove the supernatant, leaving the pellet as dry as possible for suspension in 100 μl freshly prepared TE buffer containing lysozyme (10 mg/ml). The mixture was incubated at room temperature for 5 min. The following steps were performed according to the SV Total RNA Isolation System protocol (Promega, Madison, MA, USA). To check that there wasn’t DNA contamination we performed PCRs using the RNA samples and no amplifications were detected.
Reverse transcription was carried out on the day after RNA isolation using the GoScript™ Reverse Transcription System (Promega, Madison, MA, USA). A total mix of 50 ng RNA and 0.5 μg Random Primer per reaction was added, and the final volume was brought up to 5 μl with nuclease-free water. The RNA-primer mix was heated at 70 °C for 5 min followed by chilling in ice water for 5 min. Then, the RNA-primer mix was added to the reverse transcription reaction mix. For each cDNA reaction, the reverse transcription reaction mix was composed of the following: 4 μl GoScript™ 5x Reaction Buffer, 2 μl MgCl2, 1 μl PCR Nucleotide Mix, 1 μl GoScript™ Reverse Transcriptase and 7 μl nuclease-free water to a final volume of 15 μl. The RT-PCR temperature sequence was as follows: 25 °C for 5 min to assist annealing, incubation at 42 °C for one hour and incubation at 70 °C for 15 min to inactivate the reverse transcriptase. The cDNA was stored at -20 °C. The PCR was carried out in a 25 μl reaction mixture, using 1 μl of the RT (200 ng template for cDNAs and 100 ng for genomic DNA) reaction as template for 0,5 μl Taq DNA (5U/μl) and 20 pmol of each primer (Additional file 5: Table A5). As positive control of the reactions we used specific oligonucleotides to amplify a 271 bp 16S ribosomal RNA fragment (KEGG number XAC3896). The temperature of annealing for all genes was 51 °C. Amplification was performed in 40 cycles with a T1000 Thermo Cycler (BIO-RAD, Philadelphia, USA).
Results and discussion
X. citri conserves the Cys proteins for sulfate transport and desulfonation to sulfide
Systems for the transport of alkanesulfonate and aliphatic compounds in X. citri
Gene expression of the components from cys regulon and predicted ssu operons in X. citri
In this work, we showed for the first time, that the phytopatogenic bacterium X. citri has the ABC transporter for sulfate and two additional systems for the transport and oxidoreduction of alkanesulfonates or organosulfur compounds, respectively, SlfASsuDACB (Ssu1) and SsuDEACB (Ssu2). These systems were differentially induced in LB and XAM1. Comparative and phylogenetic analysis of the periplasmic binding proteins, as well the complete set of genes, showed that SlfASsuDACB differs from the classical taurine transporter and probably was originated from the β-proteobacteria group. The presence of more than one transporter for this kind of compound would give to the X. citri the advantageous capability to transport and assimilate distinct sources of sulfur, which is relevant for the bacterium maintenance and growth. The absence of these transporters in X. campestris, which has a different way of infection and pathogenesis, may also reflect the importance of these compounds for the bacterium. Since there is no previous data related to the sulfur assimilation in X. citri or Xanthomonas genus, the work presented here is an important step for understanding global sulfur metabolism in these bacteria and arises perspectives for further experimental investigations.
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, process number 2013/09172-9), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, process number: 474110/2009-5) and Laboratório Nacional de Biociências (LNBio), CNPEM, Brazil. A. M. and C.T.P. received FAPESP fellowships (proc. Number 2010/09812-0 and 2010/14207-8, respectively).
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