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Iron-responsive gene expression in Pseudomonas fluorescens M114: cloning and characterization of a
R Sexton1, P R Gill, M J Callanan
1Department of Microbiology, University College Cork, Ireland.
Abstract:
In response to iron limitation. Pseudomonas fluorescens M114 induces a number of genes including an iron-scavenging siderophore termed pseudobactin M114, its cognate receptor, PbuA, and a casein protease. A Tn5lacZ-induced mutant (M114FA1) was isolated that exhibits a pleiotropic phenotype and lacks the ability to express these iron-regulated genes. A cosmid clone was identified which complements this mutation. This clone is capable of activating a number of iron-regulated promoter fusion constructs from P. fluorescens M114 and Pseudomonas putida WCS358 and can also promote expression of these fusions in Escherichia coli. A series of insertion mutants was constructed by homologous recombination which were unable to transcribe the promoter fusions. DNA sequence analysis of the complementing region identified one open reading frame (ORF) termed pbrA (pseudobactin regulation activation) and the deduced amino acid sequence shows domains with significant homology to a number of ECF (extracytoplasmic function) transcriptional regulators of the sigma 70 sigma factor family, including fecl required for expression of the ferric dicitrate outer-membrane receptor protein of E. coli. Sequences upstream of the pbrA gene suggest that transcription of pbrA may also be iron regulated.
Insights
Pseudomonas fluorescens M114 regulates iron uptake genes via PbrA, an ECF sigma factor. This regulator activates siderophore and protease gene expression under iron limitation, crucial for bacterial survival.
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Genetics
Background:
- Pseudomonas fluorescens M114 produces siderophores like pseudobactin M114, PbuA receptor, and casein protease in response to iron deficiency.
- Iron regulation is critical for bacterial adaptation and virulence.
- A mutant (M114FA1) lacking these iron-regulated genes was identified.
Purpose of the Study:
- To identify and characterize the genetic regulator controlling iron-responsive gene expression in Pseudomonas fluorescens M114.
- To understand the mechanism of pseudobactin M114 biosynthesis and uptake regulation.
Main Methods:
- Isolation and complementation of a Tn5lacZ-induced mutant.
- Construction of promoter-fusion constructs and insertion mutants.
- DNA sequencing and bioinformatic analysis of the identified open reading frame (ORF).
Main Results:
- A cosmid clone complemented the mutant, restoring expression of iron-regulated genes.
- DNA sequencing revealed an ORF, pbrA, encoding a putative extracytopmatic function (ECF) sigma factor.
- PbrA shares homology with known ECF transcriptional regulators, suggesting a conserved regulatory mechanism.
Conclusions:
- PbrA is a novel transcriptional regulator essential for activating iron-scavenging and iron-regulated genes in Pseudomonas fluorescens.
- The findings elucidate a key regulatory pathway for iron acquisition in Pseudomonas species.
- PbrA represents a potential target for modulating bacterial iron metabolism.