Engineering orthogonal signaling pathways reveals the sparse distribtion of protein protein interactions in sequence space [RNA-seq]
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ABSTRACT:
ORGANISM(S): Escherichia coli str. K-12 substr. MG1655
PROVIDER: GSE128611 | GEO | 2019/09/20
REPOSITORIES: GEO
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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.
2019-09-20 | GSE120780 | GEO
Project description:The cross compatibility of functional PhoQ-PhoP mutants with altered specificity residues was analyzed by sort seq. We cloned and combinatorially combined 79 PhoQ* and 71 PhoP* variants, producing a library with a theoretical diversity of 5,609. The library was 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 (Fig. 2b). 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.
2019-07-27 | GSE120786 | GEO
Project description: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.
2008-07-16 | E-GEOD-12099 | biostudies-arrayexpress
Project description: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
2008-07-16 | GSE12099 | GEO
Project description:Background Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch. Methodology/Principal findings One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10-5 on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAOSCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexABoprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels. Conclusions By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system. We used microarrays to study changes in gene expression during early and late stationary phase of SCV and WT strains. SCV and WT cultures were grown in triplicate until early (24h) and late (48h) stationary phase. RNA was extracted, labelled and hybridised on Affymetrix P. aeruginosa expression microarrays.
2011-12-06 | E-GEOD-34141 | biostudies-arrayexpress
Project description:Background Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch. Methodology/Principal findings One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10-5 on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAOSCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexABoprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels. Conclusions By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system. We used microarrays to study changes in gene expression during early and late stationary phase of SCV and WT strains.
2011-12-06 | GSE34141 | GEO
Project description:We showed previously that insertion of Synechocystis 12-desaturase in Salmonellas membrane alters membrane physical state (MPS) followed by the expression of stress genes causing inability to survive within murine macrophages . Recently, we showed that expression of one membrane lipid domain (MLD) of 12-desaturase (ORF200) interferes with Salmonella MPS causing loss of virulence in mice and immunoprotection. We postulated that a putative -AMP intercalates specifically within phospholipids but, depending on its amino acid sequence, does so within particular key sensors of MLD. In this study we choose as target for a putative synthetic AMP, PhoP/PhoQ, a sensor that responds to low Mg2+ concentration during murine macrophages infection. We synthesized a modified DNA fragment coding for an amino acid sequence (NUF) similar to that fragment and expressed it in Salmonella LT2 strain. We show that the pattern of gene expression controlled by PhoP/PhoQ and other pathways involving phospholipids biosynthesis, stress proteins and genes coding for antigens are dysregulated. We also present RNAseq of strain expressing ORF200 and showed that the pattern of the same genes is altered. Accumulation of NUF conferred temporary immunoprotection suggesting that it is possible to address a synthetic peptide to a specific MLD. This represents a powerful procedure to synthesize -AMPs and generate live non virulent strains for vaccination.
2016-07-15 | E-MTAB-4544 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below.
2019-08-05 | GSE120789 | GEO
Project description:We developed a surface-displayed library of 117 human antimicrobial peptides (AMPs) and over 3000 human AMP variants in a laboratory E. coli strain expressing PhoP and PhoQ from Salmonella Typhimurium. We used sort-seq (fluorescence-activated cell sorting followed by next-generation sequencing) to characterize PhoPQ activation by these surface-displayed AMPs.
2022-11-09 | GSE174191 | GEO
Project description:Microarrays were used to identify Y. pestis genes that are differentially regulated under conditions of phoP overexpression. RNA samples were isolated from cultures of two isogenic Y. pestis strains, one overexpressing phoP (KIM10+phoP(delta)/PhoP) and the other not (KIM10+phoP(delta)/MMB67EH), and their gene expression profiles were compared.
2006-07-01 | E-MEXP-712 | biostudies-arrayexpress