Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of PhoP-regulon members under low Mg2+ Ca2+ condition of Xanthomonas oryzae pv. oryzae PXO99A

ABSTRACT: In bacteria, two-component regulatory system (TCSs) is generally characterized by a simple phosphotransfer scheme, composed of a sensor domain (a histidine kinase) and a response domain (a response regulator), which is responsible for detection of external stimuli. PhoP-PhoQ TCS of Xanthomonas oryzae pv. oryzae (Xoo), a causal agent of bacterial leaf blight disease in rice, was previously shown to be negatively regulated by RaxR-RaxH, another TCS that senses population cell density as well as modulates the activity of AvrXA21, a bacterial effector, recognized by a bacterial blight resistance gene, Xa21. In this work, whole-genome microarray was performed to analyze transcription profiling and identify member of PhoP regulon under Mg2+ and Ca2+ limited condition. This analysis revealed that PhoP governs broad cellular pathways including stress defense response, cation transportation, general metabolism, broad regulatory system, bacterial motility, and bacterial virulence. Array results provide a set of candidate genes, further biochemical pathway analysis, and signaling pathway crosstalks that need to be characterized to understand PhoP-dependent mechanisms and also suggest a putative regulatory loop between two phoP-phoQ and raxR-raxH TCSs. Implication of this analysis suggested that Xoo adopt PhoP-PhoQ system to perceive extracellular signals from certain environment such as low concentration of metal ions, and to regulate intracellular signals of other regulatory systems. Keywords: Comparative transcription profiling under limited Mg2+ Ca2+ condition Three biological and dye-swap replicates, total six samples for each dataset. Three datasets contained; (i) phoP knockout mutant vs. wildtype PXO99A in low concentration (10 µM) of MgCl2 and CaCl2, (ii) phoP knockout mutant in low (10 µM) vs. high (10mM) concentration of MgCl2 and CaCl2, and (iii) PXO99A in low (10 µM) vs. high (10mM) concentration of MgCl2 and CaCl2.

ORGANISM(S): Xanthomonas oryzae pv. oryzae PXO99A

SUBMITTER: Malinee Sriariyanun

PROVIDER: E-GEOD-12099 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The specificity of the kinase-regulator interaction is driven by a limited set of interfacial residues in each protein that strongly. To identify combinations of these interface residues that are functional and potentially insulated from existing two-component signaling pathways in E. coli, we constructed a dual library of mutants in which the key, coevolving interface residues of a canonical two-component system, PhoQ and PhoP, were randomized. We used NNS codons to randomize six residues in PhoQ and five residues in PhoP, all of which lie at the interface formed by the two proteins in complex and that are critical to determining partner specificity in all two-component signaling pathways. To identify functional combinations of residues, we first grew the library of PhoQ-PhoP variants overnight in medium with low Mg2+, which activates PhoQ. Because cells must phosphorylate PhoP to grow when extracellular Mg2+ is limiting, this step enriches for functional PhoQ-PhoP variants. Variants that survived selection in limiting Mg2+ were then subjected to Sort-Seq, using fluorescence-activated cell sorting (FACS) and deep sequencing to quantify the signal responsiveness of variants in the library. To gauge the phosphorylation of PhoP in vivo, we used a fluorescent transcriptional reporter, PmgrB-yfp. In the presence of low extracellular Mg2+, functional PhoQ promotes the phosphorylation of PhoP and the production of YFP, whereas in the presence of high concentrations of Mg2+, PhoQ drives the dephosphorylation of PhoP, limiting the accumulation of YFP). The library was grown in each condition for 6 hours before sorting and sequencing. To identify variants that are signal responsive and drive YFP production specifically in low Mg2+, we sorted cells from each condition into 8 separate bins and deep sequenced the randomized regions of variants collected in each bin. We then calculated the frequency of each variant in each bin to yield the distributions of individual variants in low and high Mg2+, which were fit to Gaussians. From these fits, we assessed the mean level of YFP in each condition and the fold-induction, or signal responsiveness, of each variant detected in the library. The 11 codons / amino acids listed in this dataset refer to codons 12, 14, 15, 18, and 19 in PhoP and codons 284, 288, 289, 292, 302, and 303 in PhoQ, in that order.

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Transcription profiling of PhoP-regulon members under low Mg2+ Ca2+ condition of Xanthomonas oryzae pv. oryzae PXO99A

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