Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Differential expression of genes in the WT and hupB KO strain of Mycobacterium tuberculosis H37Rv upon iron limitation

ABSTRACT: HupB is a 28 kDa in Mycobacterium tuberculosis that is co-expressed with the siderophores mycobactin and carboxymycobactin upon iron limitation. High levels of all the three components are seen in low iron (LI; 0.02 M-BM-5g Fe / mL) organisms, with negligible expression in high iron organisms (HI; 8 M-BM-5g Fe / mL). We generated a hupB knock out mutant of M. tuberculosis (H37Rv M-bM-^HM-^F hupB) and studied the differential expression of genes upon iron limitation in the WT H37Rv and the mutant. The RNA transcripts of the WT H37Rv, grown under high and low iron conditions of growth were isolated and subjected to microarray analysis to identify the iron-regulated genes and second, the differential expression of genes in iron-limited H37Rv M-bM-^HM-^F hupB vs iron-limited WT H37Rv was analysed. Microarray analysis was done commercially by Genotypic Technology (Bangalore, India), an authorised service provider for Agilent Technologies. The study revealed the up-regulation of all the mbt genes of the mycobactin biosynthetic machinery in LI - H37Rv and several other reported iron-regulated genes. The salient feature of this study is the failure of LI - H37Rv M-bM-^HM-^F hupB to show any up-regulation of the mbt genes as compared to LI - H37Rv. Among several other genes influenced by HupB, the mutant strain showed low levels of mmpL5 and mmpS5 transcripts, whose expressed products are reported to be associated with siderophore transport and biosynthesis. One-color experiment,Organism: Mycobacterium tuberculosis ,Custom Mycobacterium tuberculosis 8x15k Array designed byGenotypic Technology Private Limited (AMADID: 20181), Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)

ORGANISM(S): Mycobacterium tuberculosis

SUBMITTER: Manjula Sritharan

PROVIDER: E-GEOD-53254 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Iron-regulated protein HupB of Mycobacterium tuberculosis positively regulates siderophore biosynthesis and is essential for growth in macrophages.

Pandey Satya Deo SD Choudhury Mitali M Yousuf Suhail S Wheeler Paul R PR Gordon Stephen V SV Ranjan Akash A Sritharan Manjula M

Journal of bacteriology 20140307 10

Mycobacterium tuberculosis expresses the 28-kDa protein HupB (Rv2986c) and the Fe(3+)-specific high-affinity siderophores mycobactin and carboxymycobactin upon iron limitation. The objective of this study was to understand the functional role of HupB in iron acquisition. A hupB mutant strain of M. tuberculosis, subjected to growth in low-iron medium (0.02 μg Fe ml(-1)), showed a marked reduction of both siderophores with low transcript levels of the mbt genes encoding the MB biosynthetic machine ...[more]

PMID: 24610707

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Project description:Because iron toxicity and deficiency are equally life threatening, maintaining intracellular iron levels within a narrow optimal range is critical for nearly all known organisms. However, regulatory mechanisms that establish homeostasis are not well understood in organisms that dwell in environments at the extremes of pH, temperature, and salinity. Under conditions of limited iron, the extremophile Halobacterium salinarum, a salt-loving archaeon, mounts a specific response to scavenge iron for growth. We have identified and characterized the role of two transcription factors (TFs), Idr1 and Idr2, in regulating this important response. An integrated systems analysis of TF knockout gene expression profiles and genome-wide binding locations in the presence and absence of iron has revealed that these TFs operate collaboratively to maintain iron homeostasis. In the presence of iron, Idr1 and Idr2 bind near each other at 24 loci in the genome, where they are both required to repress some genes. In contrast, Idr1 and Idr2 are both necessary to activate other genes in a putative a feed forward loop. Even at loci bound independently, the two TFs target different genes with similar functions in iron homeostasis. We discuss conserved and unique features of the Idr1-Idr2 system in the context of similar systems in organisms from other domains of life. Data in this GEO archive are linked to the publication: Schmid AK, Pan M, Sharma K, Baliga NS.2011. Two transcription factors are necessary for iron homeostasis in a salt-dwelling archaeon.Nucleic Acids Res.39(7):2519-33. The Δura3 parent, Δidr2 and Δidr1, and Δ idr1Δidr2 mutant strains were grown to mid-logarithmic phase (OD600 ~0.4 – 0.8) in CDM with all trace metals except iron. Cultures were split in half and FeSO4 was added to one half, while the other was continued under iron limitation. 8-mL samples were collected from each culture every 20 minutes for 60 minutes (see also experimental design, Supplementary Figure 1, Schmid et al., 2011). RNA from two biological replicate time courses were prepared, averages of these replicates are reported in the published study, whereas data from each replicate are reported here. The zero time point was harvested immediately before the addition of iron. Each Sample is based on two arrrays (one with dye-swap).

Project description:Pelagic aggregates function as hotspots for microbial activity and biological carbon pumps for exporting OM fixed by photoautotrophs to sediments in lakes and oceans. In iron-rich (ferruginous) lakes, photoferrotrophic or chemolithoautotrophic bacteria appear to contribute to CO2 fixation by oxidizing reduced iron which leads to the formation of iron-rich pelagic aggregates called iron-snow. In acidic lakes, iron snow is colonized mainly by acidophilic iron-cycling microbes that can trigger interspecies aggregation mechanisms. However, the significance of iron oxidizers in carbon fixation, their general role in iron snow functioning, and the flow of carbon within iron snow is still unclear. Here, we combined a two-year metatranscriptome analysis with a 13CO2 metabolic labeling approach to determine general metabolic activities. Protein-based stable isotope probing (protein-SIP) was used to trace the 13CO2 incorporation in iron snow microcosms over time under both oxic and anoxic conditions. Analysis of our mRNA-derived metatranscriptome data identified four key players (Leptospirillum, Ferrovum, Acidithrix, Acidiphilium) with relative abundances (59.6%-85.7%) in iron snow encoding a variety of ecologically relevant pathways, including carbon fixation, polysaccharide biosynthesis, and flagellar-based motility. We did not detect transcriptional activity for carbon fixation from archaea or eukaryotes. The largest numbers of expressed genes (3008, 2991, 2936) matched to the genomes of our previously obtained iron snow isolates (Acidithrix sp. C25, Acidiphilium sp. C61, Acidocella sp. C78) separately. 13CO2 incorporation studies identified Leptospirillum and Ferrovum, as the main active chemolithoautotrophs under oxic conditions, and Ferrovum was the main active organism under anoxic conditions as well. Small amounts of labeled 13C (Relative isotope abundance: 1.0%-5.3%) were found in the heterotrophic Acidiphilium and Acidocella. Overall, our data show that iron oxidizers play an important role in the formation of iron minerals and CO2 fixation, but the majority of fixed C apparently did not reach other iron snow microbes. This finding suggests that most of the fixed C will be directly exported to the sediment without feeding heterotrophs in the water column in acidic ferruginous lakes.

Dataset Information

Differential expression of genes in the WT and hupB KO strain of Mycobacterium tuberculosis H37Rv upon iron limitation

Publications

Iron-regulated protein HupB of Mycobacterium tuberculosis positively regulates siderophore biosynthesis and is essential for growth in macrophages.

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