Phosphate regulated proteins of Xanthomonas citri subsp. citri: A proteomic approach
Graphical abstract
Introduction
Phosphate (Pi) is an essential element for microorganisms because it is involved in energy metabolism and signal transduction, virulence, pathogenicity, adherence and infection [1], [2], [3]. In depletion of Pi Escherichia coli activates adaptive, pathogenic [4] and survival [5] responses, which involve the up and down regulation of many genes, nearly 10% of the genome [6], [7]. Many genes that are regulated during the adaptive response are members of the Pho (phosphate) regulon [7] defined by the involvement of a two-component regulatory system that comprises the PhoR and PhoB proteins [2]. PhoR encodes a histidine kinase, and PhoB encodes a response regulator that responds when the extracellular phosphate concentration falls below 4 μM. Activation or repression mediated by PhoB involves its phosphorylation and binding to the Pho-dependent promoter regions known as Pho-boxes [2]. One of these systems is the phosphate starvation system (Pst), a specific ATP-binding cassette transporter (ABC transporter) that is responsible for Pi uptake, signal transduction and transport with high affinity [2], [7]. Mutations on genes of the Pst system lead to the constitutive expression of the Pho regulon evidencing the function of this transporter also as a regulator [8]. Although many genes have been identified as Pi-regulated in E. coli only 31 genes were shown to be directly controlled by the PhoR/PhoB proteins, which include mostly genes involved with uptake and assimilation of other sources of phosphate, the genes of the Pst system and others [8]. Compared to iron and other relevant ions that absence or starvation can stimulate pathogenic processes, phosphate is also important for survival and proliferation by virtue of its relevance for the constitution of nucleic acids and phosphorylation. A number of reports have shown the relationship between pho genes and virulence [2], adhesion [9], biofilm formation [10], toxin production [11] and stress response [3], [2]. However, despite the importance of this ion and its relationship with pathogenesis, there is scant information in the literature regarding if protein expression is controlled by phosphate starvation and the expression of the Pho regulon proteins in the phytopathogenic bacterium Xanthomonas citri. X. citri is the causative agent of the citrus canker, a disease that affects citrus plants and the economy of many countries, including Brazil, which is one of the largest exporters of orange fruit and juice [12].
In this work, we analysed the proteome of X. citri after growing it in culture media with different phosphate concentrations in order to identify the putative Pho regulon members and to compare the proteins expressed during the adaptive responses. Using bioinformatics to support our results, we demonstrated that a set of proteins is regulated by phosphate, which includes a two-component system, the PstSCAB transporter and PhoA, an alkaline phosphatase that is up regulated when the Pho regulon is activated [7]. The proteomic and associated bioinformatics analyses indicate that the majority of the Pho regulon genes in X. citri are conserved and reveal, for the first time, the importance of the ABC transporter PstSCAB for phosphate assimilation in X. citri.
Section snippets
Search for Pho regulon orthologues in X. citri genome
The X. citri used in this work was Xanthomonas axonopodis pv. citri strain 306 [13]. Amino acid sequences of the E. coli Pho regulon proteins described by Hsieh and Wanner [8] were obtained from KEGG2 server (Bioinformatics Center Institute for Chemical Research Kyoto University, www.genome.jp) and used to perform a basic local alignment search BlastP (http://blast.ncbi.nlm.nih.gov) against the Xanthomonas axonopodis pv. citri (taxid:346) database using non-redundant protein sequence (nr) as
Orthologues of Pho regulon present in X. citri subsp. citri
Although the phosphate levels in the environment affect the expression of many genes, at least 36 genes were characterised in E. coli with altered expression levels due to limited phosphate availability (Pho regulon genes) [2], [7], [8]. Using the sequences of the corresponding proteins and the BlastP tool, we examined the X. citri database to identify orthologues of these 36 proteins. The results revealed that X. citri conserved at least 22 putative orthologues from the 36 Pho regulon genes
Discussion
The Pho regulon has been largely described in E. coli as well as the relationship between Pho regulon genes and virulence and pathogenesis [1], [2], but there are no available data reporting the phosphate regulation in phytopathogenic bacteria from Xanthomonas genus, including X. citri. In this work, we performed a simple, rapid and inexpensive method to characterise the X. citri proteome based on 1D SDS-PAGE gel fractionation. A total of 272 proteins were identified and classified in 19
Conclusions
Altogether, our data showed the first set of evidences of the presence of Pho regulon genes in X. citri, the bacterium responsible for the citrus canker disease. Firstly, we showed that X. citri conserves the phosphate ABC transporter PstSCAB and the two-component system PhoR/PhoB and that both systems are highly induced during phosphate starvation. The presence and induction of the alkaline phosphatase PhoA also corroborates the Pho regulon activation. On the other hand, although we did not
Conflict of interest
The authors declare no conflict interest.
Acknowledgements
This work was supported by the Coordenação de Aperfeiçoamento Pessoal de Nível Superior — CAPES, Brazil and the Laboratório Nacional de Biociências, LNBio, Campinas, Brazil.
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