Cloning, nucleotide sequence, and mutagenesis of a gene (irpA) involved in iron-deficient growth of the cyanobacterium Synechococcus sp. strain PCC7942.
We describe the cloning and sequencing of a gene from the cyanobacterium Synechococcus sp. strain PCC7942, designated irpA (iron-regulated protein A), that encodes for a protein involved in iron acquisition or storage. Polyclonal antibodies raised against proteins which accumulate during iron-deficient growth were used as probes to isolate immunopositive clones from a lambda gt11 genomic expression library. The clone, designated lambda gtAN26, carried a 1.7-kilobase (kb) chromosomal DNA insert and was detected by cross-reactivity with antibody against a 36-kilodalton protein. It was possible to map a 20-kb portion of the chromosome with various DNA probes from lambda gt11 and lambda EMBL-3 clones, and Southern blot analysis revealed that the irpA gene was present in a single copy and localized within a 1.7-kb PstI fragment. DNA sequencing revealed an open reading frame of 1,068 nucleotides capable of encoding 356 amino acids which yields a protein with a molecular weight of 38,584. The hydropathy profile of the polypeptide indicated a putative N-terminal signal sequence of 44 amino acid residues. IrpA is a cytoplasmic membrane protein as determined by biochemistry and electron microscopy immunocytochemistry. The upstream region of the irpA gene contained a consensus sequence similar to the aerobactin operator in Escherichia coli. This fact, plus a mutant with a mutation in irpA that is unable to grow under iron-deficient conditions, led us to suggest that irpA is regulated by iron and that the gene product is involved in iron acquisition or storage.
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000235:genetics* / Q000254:growth & development; D004252:DNA Mutational Analysis; D005786:Gene Expression Regulation; D005796:Genes; D007150:Immunohistochemistry; D007501:Iron / Q000378:metabolism*; D000090463:Iron Deficiencies; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012995:Solubility; D013057:Spectrum Analysis
D008565:Membrane Proteins; D007501:Iron