Unknown,Transcriptomics,Genomics,Proteomics

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Expanding the Pathways of Manganese Homeostasis: Role of a Small Manganese Chaperone Protein, MntS

ABSTRACT: Escherichia coli possesses >65 small proteins of <50 amino acids, many of which are uncharacterized. We have identified a new small protein, MntS, involved in manganese homeostasis. Manganese is a critical micronutrient, serving as an enzyme cofactor and protecting against oxidative stress. Yet manganese is toxic in excess and little is known about its function in cells. Bacteria carefully control intracellular manganese levels using the transcription regulator MntR. Before this work, mntH, which encodes a manganese importer, was the only gene known to respond to manganese via MntR repression in E. coli K12. We demonstrated that mntS is another member of the MntR manganese regulon. We also identified yebN, which encodes a putative manganese efflux pump, as the first gene positively regulated by MntR in Enterobacteria. Since MntS is expressed when manganese levels are low, causes manganese sensitivity when overexpressed, and binds manganese, we propose that MntS may be a manganese chaperone. This study reveals new factors involved in manganese regulation and metabolism and expands our knowledge of how small proteins function. Two E. coli strains, MG1655 (wild type) and GSO458 (Delta-mntR) were grown to OD600 ~ 0.5 in M9 glucose media at 37 M-BM-:C and treated with 10 microM MnCl2. In the first experiment, this incubation with 10 microM MnCl2 was for 60 min and in the second experiment, it was for 10 min. RNA was extracted using the hot phenol method and cDNA prepared and hybridized according the manufacturer's instructions (Affymetrix).

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

SUBMITTER: Lauren Waters

PROVIDER: E-GEOD-25318 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

The Escherichia coli MntR miniregulon includes genes encoding a small protein and an efflux pump required for manganese homeostasis.

Waters Lauren S LS Sandoval Melissa M Storz Gisela G

Journal of bacteriology 20110909 21

Manganese is a critical micronutrient for cells, serving as an enzyme cofactor and protecting against oxidative stress. Yet, manganese is toxic in excess and little is known about its distribution in cells. Bacteria control intracellular manganese levels by the transcription regulator MntR. When this work began, the only Escherichia coli K-12 gene known to respond to manganese via MntR repression was mntH, which encodes a manganese importer. We show that mntS (formerly the small RNA gene rybA) i ...[more]

PMID: 21908668

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Project description:The SOS response to DNA damage in bacteria is a well-known component of the complex transcriptional responses to genotoxic environmental stresses such as exposure to reactive oxygen species, alkylating agents, and many of the antibiotics targeting DNA replication. However, bacteria such as Bacillus subtilis also respond to conditions that perturb DNA replication via a transcriptional response mediated by the replication initiation protein DnaA. In addition to regulating the initiation of DNA replication, DnaA directly regulates the transcription of specific genes. Conditions that perturb DNA replication can trigger the accumulation of active DnaA, activating or repressing transcription of genes in the DnaA regulon. We report here that simply growing B. subtilis in LB medium altered DnaA-dependent gene expression in a manner consistent with the accumulation of active DnaA, and that this was part of a general transcriptional response to manganese limitation. The SOS response to DNA damage was not induced under these conditions. One of the genes positively regulated by DnaA in Bacillus subtilis encodes a protein that inhibits the initiation of sporulation, Sda. Sda expression was induced as cells entered stationary phase in LB but not in LB supplemented with manganese, and induction of Sda inhibited sporulation-specific gene expression and the onset of spore morphogenesis. In the absence of Sda, manganese-limited cells initiated spore development but failed to form mature spores. These data highlight that DnaA-dependent gene expression may influence the response of bacteria to a range of environmental conditions, including conditions that are not obviously associated with genotoxic stress. Cultures of Bacillus subtilis strains with the genetic background of sda+ or delta-sda were grown in LB and LB+100 micro-M MnCl2 at 37degC until 3 hours after the onset of stationary phase. At 3 timepoints, aliqots were removed and RNA isolated for each culture: 1) Mid exponential, 2) T0 (onset of stationary phase), and 3) T3 (3 hours after onset of stationary phase). cDNA was prepared from each sample using 10 micro-g of RNA. Reference cDNA was prepared from pooled RNA from all samples (common reference for all arrays).

Dataset Information

Expanding the Pathways of Manganese Homeostasis: Role of a Small Manganese Chaperone Protein, MntS

Publications

The Escherichia coli MntR miniregulon includes genes encoding a small protein and an efflux pump required for manganese homeostasis.

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