Metalloregulation in Bacillus subtilis: isolation and characterization of two genes differentially repressed by metal ions

Abstract

We have cloned two metal-regulated genes (mrgA and mrgC) from BaciUus subtilis by using transposon Tn917-lacZ. Both were isolated as iron-repressible gene fusions, but the metal specificity and sensitivity of gene repression are distinct. Transcription of mrgA-lacZ is induced at the end of logarithmic-phase growth in minimal medium, and this induction is prevented by excess manganese, iron, cobalt, or copper. Limitation for metal ions is sufficient for mrgA-lacZ induction, since resuspension in medium lacking both manganese and iron rapidly induces transcription. Transcription of mrgC-lacZ is also induced by iron deprivation but is not repressed by added manganese or other metal ions. Expression of mrgC-lacZ and a 2,3-dihydroxybenzoic acid-based siderophore is repressed in parallel by iron, and in both cases, only iron effects repression. We have cloned and sequenced the promoter and regulatory regions of both mrgA and mrgC. Both genes are preceded by a predicted crA-dependent promoter element with overlapping sequences similar to the iron box consensus element for recognition by the Escherichia coli ferric uptake regulator protein (Fur). Mutation of the putative iron box for gene mrgC leads to partial derepression in iron-replete medium.The regulation of gene expression in response to changing levels of metal ions requires a sensor function, to monitor metal ion levels, and an effector function, to mediate a transcriptional or translational response (12). Two of the best-studied metalloregulatory proteins, the mercury-sensing MerR protein and the iron-sensing Fur protein, serve both as direct metal ion sensors and as transcription factors (2,33). The importance of iron as an essential nutrient and enzyme cofactor has led to sophisticated regulatory mechanisms in both bacteria and eukaryotes to control intracellular iron levels. We are interested in the mechanisms by which iron regulates gene expression in the gram-positive organism Bacillus subtilis.Iron is an important nutrient for virtually all microorganisms, yet it is largely insoluble at neutral pH under aerobic conditions. Like many other organisms, B. subtilis synthesizes and excretes a Fe(III)-specific chelator (siderophore) under conditions of iron starvation. Early studies identified 2,3-dihydroxybenzoic acid (DHBA) and 2,3-dihydroxybenzoylglycine in cell culture supernatants of B. subtilis grown under iron limitation (14). Recent studies indicate that some B. subtilis strains have inducible uptake systems for hydroxamate-type siderophores as well (30). The synthesis of siderophore and subsequent uptake of ferri-siderophore complexes are inducible processes, suggesting that B. subtilis has one or more iron-sensing metalloregulatory proteins (24,25,30,39). We report the cloning and characterization of two metalloregulated promoter regions from B. subtilis that are affected by iron availability.We have determined the growth phase and metal ion dependence of gene expression for two strains containing transcriptional fusions to lacZ. Expression of mrgC-la...

Molecular characterization of the ferric-uptake regulator, Fur, from Staphylococcus aureus The GenBank accession numbers for the S. aureus fur gene and fhu operon reported in this paper are AF118839 and AF132117, respectively.

“…A 20 base oligonucleotide primer specific to the coding region was The production of siderophores by B. subtilis HB1000, HB6543, HB6543/pCU1-fur and HB6543/pCU1 strains were assayed as described by Chen et al (1993). The white bars represent the ratios of OD 510 /OD 600 in the iron-limiting minimal media and the black bars indicate the ratios in iron-rich minimal media.…”

The Bacillus subtilis iron-sparing response is mediated by a Fur-regulated small RNA and three small, basic proteins

“…An mrgC-lacZ transcriptional fusion is induced by the iron chelator EDDHA or by growth under iron-limiting conditions (27). With the completion of the B. subtilis genome sequence in 1997 (28), we noted that the mrgC ORF terminates after only 29 codons (Fig.…”

“…The third Fur-regulated basic protein, FbpC, is encoded by the first identified member of the Fur regulon, metal-regulated gene C (mrgC) (27). An mrgC-lacZ transcriptional fusion is induced by the iron chelator EDDHA or by growth under iron-limiting conditions (27).…”

An archaeal antioxidant: Characterization of a Dps-like protein from Sulfolobus solfataricus

“…Iron metabolism and oxidative stress are thus intimately interwoven, with the toxicity of one being dependent on the other. The significance of these selective pressures remains evident in the genomes of modern organisms where complex and overlapping defense mechanisms protect against oxygen toxicity and iron stress (1)(2)(3).…”