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Isolation of a gene (pbsC) required for siderophore biosynthesis in fluorescent Pseudomonas sp. strain M114
C Adams1, D N Dowling, D J O'Sullivan
1Microbiology Department University College, Cork, Ireland.
Abstract:
An iron-regulated gene, pbsC, required for siderophore production in fluorescent Pseudomonas sp. strain M114 has been identified. A kanamycin-resistance cassette was inserted at specific restriction sites within a 7 kb genomic fragment of M114 DNA and by marker exchange two siderophore-negative mutants, designated M1 and M2, were isolated. The nucleotide sequence of approximately 4 kb of the region flanking the insertion sites was determined and a large open reading frame (ORF) extending for 2409 bp was identified. This gene was designated pbsC (pseudobactin synthesis C) and its putative protein product termed PbsC. PbsC was found to be homologous to a family of enzymes involved in the biosynthesis of secondary metabolites, including EntF of Escherichia coli. These enzymes are believed to act via ATP-dependent binding of AMP to their substrate. Several areas of high sequence homology between these proteins and PbsC were observed, including a conserved AMP-binding domain. The expression of pbsC is iron-regulated as revealed when a DNA fragment containing the upstream region was cloned in a promoter probe vector and conjugated into the wild-type strain, M114. The nucleotide sequence upstream of the putative translational start site contains a region homologous to previously defined -16 to -25 sequences of iron-regulated genes but did not contain an iron-box consensus sequence. It was noted that inactivation of the pbsC gene also affected other iron-regulated phenotypes of Pseudomonas M114.
Insights
Researchers identified the iron-regulated gene pbsC, essential for siderophore production in Pseudomonas. This gene
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Fluorescent Pseudomonas species produce siderophores for iron uptake.
- Iron regulation is crucial for bacterial adaptation and virulence.
Purpose of the Study:
- To identify and characterize the iron-regulated gene responsible for siderophore production in Pseudomonas sp. strain M114.
- To elucidate the function and regulation of the identified gene.
Main Methods:
- Genetic manipulation: insertion of a kanamycin-resistance cassette and marker exchange to create mutants.
- DNA sequencing: determination of the nucleotide sequence of the pbsC gene and its flanking regions.
- Bioinformatic analysis: comparison of the PbsC protein sequence with known enzymes.
- Gene expression analysis: cloning of the upstream region into a promoter probe vector to study iron regulation.
Main Results:
- Identification and characterization of the pbsC gene, encoding the PbsC protein involved in pseudobactin synthesis.
- PbsC shows homology to ATP-dependent enzymes involved in secondary metabolite biosynthesis, possessing a conserved AMP-binding domain.
- pbsC expression is iron-regulated, with its upstream region containing sequences similar to known iron-regulated promoters but lacking a canonical iron-box.
- Inactivation of pbsC impacted other iron-regulated phenotypes in Pseudomonas M114.
Conclusions:
- The pbsC gene is a key component of the siderophore synthesis pathway in Pseudomonas sp. strain M114.
- PbsC is a novel enzyme belonging to a family of secondary metabolite biosynthesis enzymes.
- The iron-regulation mechanism of pbsC involves unique promoter elements, suggesting a complex regulatory network.