Elsevier

Journal of Proteomics

Volume 108, 28 August 2014, Pages 78-88
Journal of Proteomics

Phosphate regulated proteins of Xanthomonas citri subsp. citri: A proteomic approach

https://doi.org/10.1016/j.jprot.2014.05.005Get rights and content

Highlights

  • We show the presence and functionality of Pho regulon genes in Xanthomonas citri.

  • PstSCAB and the two-component system PhoR–PhoB are conserved in X. citri.

  • The proteins PstS and PhoX are highly induced during phosphate starvation.

  • ABC transporters, proteins related to pathogenesis are highly induced.

Abstract

Xanthomonas citri subsp. citri (X. citri) is the causative agent of the citrus canker, a disease that affects several citrus plants in Brazil and across the world. Although many studies have demonstrated the importance of genes for infection and pathogenesis in this bacterium, there are no data related to phosphate uptake and assimilation pathways. To identify the proteins that are involved in the phosphate response, we performed a proteomic analysis of X. citri extracts after growth in three culture media with different phosphate concentrations. Using mass spectrometry and bioinformatics analysis, we showed that X. citri conserved orthologous genes from Pho regulon in Escherichia coli, including the two-component system PhoR/PhoB, ATP binding cassette (ABC transporter) Pst for phosphate uptake, and the alkaline phosphatase PhoA. Analysis performed under phosphate starvation provided evidence of the relevance of the Pst system for phosphate uptake, as well as both periplasmic binding proteins, PhoX and PstS, which were formed in high abundance. The results from this study are the first evidence of the Pho regulon activation in X. citri and bring new insights for studies related to the bacterial metabolism and physiology.

Biological significance

Using proteomics and bioinformatics analysis we showed for the first time that the phytopathogenic bacterium X. citri conserves a set of proteins that belong to the Pho regulon, which are induced during phosphate starvation. The most relevant in terms of conservation and up-regulation were the periplasmic-binding proteins PstS and PhoX from the ABC transporter PstSBAC for phosphate, the two-component system composed by PhoR/PhoB and the alkaline phosphatase PhoA.

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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