Dataset Information

Repeated Phenotypic Evolution by Different Genetic Routes in Pseudomonas fluorescens SBW25.

ABSTRACT: Repeated evolution of functionally similar phenotypes is observed throughout the tree of life. The extent to which the underlying genetics are conserved remains an area of considerable interest. Previously, we reported the evolution of colony switching in two independent lineages of Pseudomonas fluorescens SBW25. The phenotypic and genotypic bases of colony switching in the first lineage (Line 1) have been described elsewhere. Here, we deconstruct the evolution of colony switching in the second lineage (Line 6). We show that, as for Line 1, Line 6 colony switching results from an increase in the expression of a colanic acid-like polymer (CAP). At the genetic level, nine mutations occur in Line 6. Only one of these-a nonsynonymous point mutation in the housekeeping sigma factor rpoD-is required for colony switching. In contrast, the genetic basis of colony switching in Line 1 is a mutation in the metabolic gene carB. A molecular model has recently been proposed whereby the carB mutation increases capsulation by redressing the intracellular balance of positive (ribosomes) and negative (RsmAE/CsrA) regulators of a positive feedback loop in capsule expression. We show that Line 6 colony switching is consistent with this model; the rpoD mutation generates an increase in ribosomal gene expression, and ultimately an increase in CAP expression.

SUBMITTER: Gallie J

PROVIDER: S-EPMC6519391 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Repeated Phenotypic Evolution by Different Genetic Routes in Pseudomonas fluorescens SBW25.

Gallie Jenna J Bertels Frederic F Remigi Philippe P Ferguson Gayle C GC Nestmann Sylke S Rainey Paul B PB

Molecular biology and evolution 20190501 5

Repeated evolution of functionally similar phenotypes is observed throughout the tree of life. The extent to which the underlying genetics are conserved remains an area of considerable interest. Previously, we reported the evolution of colony switching in two independent lineages of Pseudomonas fluorescens SBW25. The phenotypic and genotypic bases of colony switching in the first lineage (Line 1) have been described elsewhere. Here, we deconstruct the evolution of colony switching in the second ...[more]

PMID: 30835268

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Project description:BACKGROUND: Pyoverdines (PVDs) are high affinity siderophores, for which the molecular mechanisms of biosynthesis, uptake and regulation have been extensively studied in Pseudomonas aeruginosa PAO1. However, the extent to which this regulatory model applies to other pseudomonads is unknown. Here, we describe the results of a genomic, genetic and structural analysis of pyoverdine-mediated iron uptake by the plant growth-promoting bacterium P. fluorescens SBW25. RESULTS: In silico analysis of the complete, but un-annotated, SBW25 genome sequence identified 31 genes putatively involved in PVD biosynthesis, transport or regulation, which are distributed across seven different regions of the genome. PVD gene iron-responsiveness was tested using 'lacZ fusions to five PVD loci, representative of structural and regulatory genes. Transcription of all fusions increased in response to iron starvation. In silico analyses suggested that regulation of fpvR (which is predicted to encode a cytoplasmic membrane-spanning anti-sigma factor) may be unique. Transcriptional assays using gene expression constructs showed that fpvR is positively regulated by FpvI (an extracytoplasmic family (ECF) sigma factor), and not directly by the ferric uptake regulator (Fur) as for PAO1. Deletion of pvdL, encoding a predicted non-ribosomal peptide synthetase (NRPS) involved in PVD chromophore biosynthesis confirmed the necessity of PvdL for PVD production and for normal growth in iron-limited media. Structural analysis of the SBW25 PVD shows a partly cyclic seven residue peptide backbone, identical to that of P. fluorescens ATCC13525. At least 24 putative siderophore receptor genes are present in the SBW25 genome enabling the bacterium to utilize 19 structurally distinct PVDs from 25 different Pseudomonas isolates. CONCLUSION: The genome of P. fluorescens SBW25 contains an extensively dispersed set of PVD genes in comparison to other sequenced Pseudomonas strains. The PAO1 PVD regulatory model, which involves a branched Fpv signaling pathway, is generally conserved in SBW25, however there is a significant difference in fpvR regulation. SBW25 produces PVD with a partly cyclic seven amino acid residue backbone, and is able to utilize a wide variety of exogenous PVDs.

Project description:HutC is known as a transcriptional repressor specific for histidine utilization (hut) genes in Gram-negative bacteria, including Pseudomonas fluorescens SBW25. However, its precise mode of protein-DNA interactions hasn't been examined with purified HutC proteins. Here, we performed electrophoretic mobility shift assay (EMSA) and DNase I footprinting using His6-tagged HutC and biotin-labeled probe of the hut promoter (PhutU). Results revealed a complex pattern of HutC oligomerization, and the specific protein-DNA interaction is disrupted by urocanate, a histidine derivative, in a concentration-dependent manner. Next, we searched for putative HutC-binding sites in the SBW25 genome. This led to the identification of 143 candidate targets with a P value less than 10-4 HutC interaction with eight selected candidate sites was subsequently confirmed by EMSA analysis, including the type IV pilus assembly protein PilZ, phospholipase C (PlcC) for phosphatidylcholine hydrolyzation, and key regulators of cellular nitrogen metabolism (NtrBC and GlnE). Finally, an isogenic hutC deletion mutant was subjected to transcriptome sequencing (RNA-seq) analysis and phenotypic characterization. When bacteria were grown on succinate and histidine, hutC deletion caused upregulation of 794 genes and downregulation of 525 genes at a P value of <0.05 with a fold change cutoff of 2.0. The hutC mutant displayed an enhanced spreading motility and pyoverdine production in laboratory media, in addition to the previously reported growth defect on the surfaces of plants. Together, our data indicate that HutC plays global regulatory roles beyond histidine catabolism through low-affinity binding with operator sites located outside the hut locus.IMPORTANCE HutC in Pseudomonas is a representative member of the GntR/HutC family of transcriptional regulators, which possess a N-terminal winged helix-turn-helix (wHTH) DNA-binding domain and a C-terminal substrate-binding domain. HutC is generally known to repress expression of histidine utilization (hut) genes through binding to the PhutU promoter with urocanate (the first intermediate of the histidine degradation pathway) as the direct inducer. Here, we first describe the detailed molecular interactions between HutC and its PhutU target site in a plant growth-promoting bacterium, P. fluorescens SBW25, and further show that HutC possesses specific DNA-binding activities with many targets in the SBW25 genome. Subsequent RNA-seq analysis and phenotypic assays revealed an unexpected global regulatory role of HutC for successful bacterial colonization in planta.

Dataset Information

Repeated Phenotypic Evolution by Different Genetic Routes in Pseudomonas fluorescens SBW25.

Publications

Repeated Phenotypic Evolution by Different Genetic Routes in Pseudomonas fluorescens SBW25.

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