Functional analysis of two divalent metal-dependent regulatory genes dmdR1 and dmdR2 in Streptomyces coelicolor and proteome changes in deletion mutants.
In Gram-positive bacteria, the expression of iron-regulated genes is mediated by a class of divalent metal-dependent regulatory (DmdR) proteins. We cloned and characterized two dmdR genes of Streptomyces coelicolor that were located in two different nonoverlapping cosmids. Functional analysis of dmdR1 and dmdR2 was performed by deletion of each copy. Deletion of dmdR1 resulted in the derepression of at least eight proteins and in the repression of three others, as shown by 2D proteome analysis. These 11 proteins were characterized by MALDI-TOF peptide mass fingerprinting. The proteins that show an increased level in the mutant correspond to a DNA-binding hemoprotein, iron-metabolism proteins and several divalent metal-regulated enzymes. The levels of two other proteins--a superoxide dismutase and a specific glutamatic dehydrogenase--were found to decrease in this mutant. Complementation of the dmdR1-deletion mutant with the wild-type dmdR1 allele restored the normal proteome profile. By contrast, deletion of dmdR2 did not affect significantly the protein profile of S. coelicolor. One of the proteins (P1, a phosphatidylethanolamine-binding protein), overexpressed in the dmdR1-deleted mutant, is encoded by ORF3 located immediately upstream of dmdR2; expression of both ORF3 and dmdR2 is negatively controlled by DmdR1. Western blot analysis confirmed that dmdR2 is only expressed when dmdR1 is disrupted. Species of Streptomyces have evolved an elaborated regulatory mechanism mediated by the DmdR proteins to control the expression of divalent metal-regulated genes.
Flores
Francisco J
FJ
Area de Microbiología, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, Spain.
Barreiro
Carlos
C
Coque
Juan José R
JJ
Martín
Juan F
JF
D000595:Amino Acid Sequence; D001483:Base Sequence; D002413:Cations, Divalent / Q000378:metabolism*; D017931:DNA Primers; D017353:Gene Deletion; D005798:Genes, Bacterial; D005809:Genes, Regulator; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D020543:Proteome; D017386:Sequence Homology, Amino Acid; D048372:Streptomyces coelicolor / Q000235:genetics*
D002413:Cations, Divalent; D017931:DNA Primers; D020543:Proteome