Unknown,Transcriptomics,Genomics,Proteomics

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IpsA, a novel transcriptional regulator required for inositol derived lipid formation in Corynebacteria and Mycobacteria

ABSTRACT: The development of new drugs against tuberculosis and diphtheria is focused on disrupting the biogenesis of the cell wall, the unique architecture of which confers resistance against current therapies. The enzymatic pathways involved in the synthesis of the cell wall by these pathogens are well understood but the underlying regulatory mechanisms are largely unknown. Here, we characterize IpsA, a LacI-type transcriptional regulator conserved among Mycobacteria and Corynebacteria that plays a role in the regulation of cell wall biogenesis. IpsA triggers myo-inositol formation by activating ino1, which encodes inositol phosphate synthase. An IpsA deletion mutant of Corynebacterium glutamicum cultured on glucose displayed significantly impaired growth and presented an elongated cell morphology. Analysis of the polar lipid fraction of the cell wall revealed the absence of inositol-derived lipids. The phenotype of the C. glutamicum M-NM-^TipsA mutant was complemented by homologues from Corynebacterium diphtheriae (dip1969) and Mycobacterium tuberculosis (rv3575), indicating the conserved function of IpsA in the pathogenic species. Additional targets of IpsA with putative functions in cell wall biogenesis were identified and IpsA was shown to bind to a conserved palindromic motif within the corresponding promoter regions. myo-inositol was identified as an effector of IpsA, causing the dissociation of the IpsA-DNA complex in vitro. This characterization of IpsA function and of its regulon sheds light on the complex transcriptional control of cell wall biogenesis in the mycolata taxon and generates novel targets for drug development. To identify genes which were regulated by IpsA (Cg2910), we performed DNA microarray analyses of ATCC 13032 M-NM-^TipsA against wild type. For this purpose RNA was isolated from cells cultivated in CGXII minimal medium with 2% glucose (w v-1) and harvested in the exponential growth phase at an OD600 of 1. Three biological replicates were performed.

ORGANISM(S): Corynebacterium glutamicum ATCC 13032

SUBMITTER: Tino Polen

PROVIDER: E-GEOD-50210 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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IpsA, a novel LacI-type regulator, is required for inositol-derived lipid formation in Corynebacteria and Mycobacteria.

Baumgart Meike M Luder Kerstin K Grover Shipra S Gätgens Cornelia C Besra Gurdyal S GS Frunzke Julia J

BMC biology 20131230

<h4>Background</h4>The development of new drugs against tuberculosis and diphtheria is focused on disrupting the biogenesis of the cell wall, the unique architecture of which confers resistance against current therapies. The enzymatic pathways involved in the synthesis of the cell wall by these pathogens are well understood, but the underlying regulatory mechanisms are largely unknown.<h4>Results</h4>Here, we characterize IpsA, a LacI-type transcriptional regulator conserved among Mycobacteria a ...[more]

PMID: 24377418

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Project description:DNA affinity chromatography with the promoter region of the Corynebacterium glutamicum pck gene, encoding phosphoenolpyruvate carboxykinase (PEPCk), led to the isolation of four transcriptional regulators, i.e., RamA, GntR1, GntR2 and IolR. Determination of the PEPCk activity of the deletion mutants M-NM-^TramA, M-NM-^TgntR1M-NM-^TgntR2, and M-NM-^TiolR indicated that RamA represses pck during growth on glucose about twofold, whereas GntR1, GntR2, and IolR activate pck expression about twofold, irrespective whether glucose or acetate served as carbon source. The DNA binding sites of the four regulators in the pck promoter region were identified and their positions correlated with the predicted functions as repressor or activators. The iolR gene is located upstream and in divergent orientation to a iol gene cluster, encoding proteins involved in myo-inositol uptake and degradation. Comparative DNA microarray analysis of the M-NM-^TiolR mutant and the parental wild-type revealed strongly elevated (>100-fold) mRNA levels of the iol genes in the mutant, indicating that the primary function of IolR is the repression of the iol genes. IolR binding sites were identified in the promoter regions of iolC, iolT and iolR itself, which presumably is subject to negative autoregulation. A consensus DNA-binding motif was identified (5M-bM-^@M-^Y-KGWCHTRACA-3M-bM-^@M-^Y) which corresponds well to those of other GntR-type regulators of the HutC family. Taken together, our results disclose a complex regulation of the pck gene in C. glutamicum and identify IolR as an efficient repressor of genes involved in myo-inositol catabolism of this organism. To identify genes that are potentially regulated by IolR, the transcriptome profile of the iolR deletion strain was compared to that of the WT using DNA microarray analysis. The strains were grown in CGXII minimal medium with 4% (wt/vol) glucose as sole carbon source and RNA was isolated from cells harvested in the early exponential growth phase (OD600 of about 5). The transcriptome comparison was performed in triplicate starting from independent cultures. The second replicate included a dye-swap.

Project description:We recently showed that the two-component system (TCS) HrrSA plays a central role in the control of heme homeostasis in the Gram-positive soil bacterium Corynebacterium glutamicum. Here, we characterized the function of another TCS of this organism, ChrSA, which exhibits significant sequence similarity to HrrSA, and provide evidence for cross-regulation of the two systems. In this study ChrSA was shown to be crucial for heme resistance of C. glutamicum by activation of the putative heme-detoxifying ABC-transporter HrtBA in the presence of heme. Deletion of either hrtBA or chrSA resulted in a strongly increased sensitivity towards heme. DNA microarray analysis and gel retardation assays with the purified response regulator ChrA provided evidence for ChrA being a repressor of the heme biosynthesis operon hemAC and hemH and an activator of the ctaE-qcrCAB operon, encoding subunits of the cytochrome bc1-aa3 supercomplex of the respiratory chain. The heme oxygenase gene, hmuO, showed a strongly decreased mRNA level in the M-NM-^TchrSA mutant, but no significant binding of ChrA was observed in vitro. Promoter fusion studies of PchrSA with eyfp indicated positive autoregulation of the chrSA operon in the presence of heme. Interestingly, ChrA was also shown to bind to the hrrA promoter and to repress transcription of the paralog response regulator, suggesting a close link between HrrSA and ChrSA. Mutational analysis and in silico prediction resulted in the deduction of a 16-bp weakly conserved inverted repeat as consensus DNA-binding motif of ChrA. Altogether, the present study emphasizes ChrSA as a second TCS, besides HrrSA, involved in heme-dependent gene regulation in C. glutamicum. To identify the influence of ChrSA on global gene expression , DNA microarray analyses were performed with the M-NM-^TchrSA mutant compared to C. glutamicum wild type. For this purpose RNA was isolated from exponentially growing cells cultivated in CgXII minimal medium with 4% glucose and either 2.5 M-BM-5M FeSO4 or 2.5 M-BM-5M hemin as iron source. Three biological replicates were performed.

Project description:The Gram-positive soil bacterium Corynebacterium glutamicum is widely used in industrial fermentative processes for the production of amino acids. The world production of L-lysine has surpassed 2 million tons per year. Glucose is taken up into the C. glutamicum cell by the phosphotransferase system PTS which can be replaced and/or enhanced by a permease and a glucokinase. Heterologous expression of the gene for the high-affinity glucose permease from Streptomyces coelicolor and of the Bacillus subitilis glucokinase gene fully compensated for the absence of the PTS in ï??hpr strains and strains grew as fast with glucose as C. glutamicum wild type. Growth of PTS-positive strains with glucose was accelerated when the endogenous inositol permease IolT2 and the glucokinase from Bacillus subtilis were overproduced using plasmid pEKEx3-IolTBest. When the genome-reduced C. glutamicum strain GRLys1 carrying additional in-frame deletions of sugR and ldhA to derepress glycolytic and PTS genes and to circumvent formation of L-lactate as by-product was transformed with this plasmid, a 40% higher L-lysine titer and a 30% higher volumetric productivity as compared to GRLys1(pEKEx3) resulted. The non-proteinogenic amino acid pipecolic acid (L-PA), a precursor of immunosuppressants, peptide antibiotics or piperidine alkaloids, can be derived from L-lysine. To enable production of L-PA by the L-lysine producing strain, the L-Lysine dehydrogenase gene lysDH from Silicibacter pomeroyi and the endogenous pyrroline 5-carboxylate reductase gene proC were expressed as synthetic operon. This enabled C. glutamicum to L-PA with a yield of 0.49 Â± 0.03 gg-1 and a volumetric productivity of 0.04 Â± 0.00 gL-1h-1.To the best of our knowledge, this is the first fermentative process for the production of L-PA. Two conditions tested, 200 mM NaCl Vs 200 mM pipecolic supplemented in the culture medium, control experiments done with the addition of 200mM of NaCl. Four technical replicates.

Project description:In a manner similar to ubiquitin, the prokaryotic ubiquitin-like protein (Pup) has been shown to target proteins for degradation via the proteasome in mycobacteria. However, not all actinobacteria possessing the Pup protein also harbor a proteasome, suggesting fates for pupylated proteins other than degradation via a proteasome or degradation at all. In the present study we set out to study pupylation in the proteasome-lacking non-pathogenic model microorganism and biotechnological workhorse Corynebacterium glutamicum. A defined pup deletion mutant of C. glutamicum ATCC 13032 grew as the control indicating that pupylation seems to be dispensable under the conditions tested. By expression of homologous Pup carrying a poly-histidine tag in C. glutamicum ATCC 13032 we purified the first pupylome of a microorganism lacking a proteasome. Multidimensional Protein Identification Technology (MudPIT) unraveled 54 proteins being pupylated in this organism. Similar to mycobacteria, the majority of pupylated proteins in C. glutamicum can be classified as enzymes of the metabolism or as involved in translation. These results help to elucidate the common target pathways of pupylation in bacteria. Sample 1: For growth in CGXII minimal media, a preculture 1 was grown in 5 ml BHI medium inoculated with a single colony from a fresh BHI agar plate and incubated at 170 rpm for 8 hours. Then 500 M-BM-5l of preculture 1 were used to inoculate preculture 2 in 100 ml shake flasks with 20 ml CGXII minimal medium containing 4 % (w/v) glucose and incubated over night (140 rpm , 30 M-BM-0C ). Subsequent main cultures (50 ml CGXII medium with 4 % glucose) were inoculated with cells from preculture 2 to an OD600 of about 0.4. For DNA microarray analysis, cells were harvested in the exponential growth phase at an OD600 of 4 to 5. After extraction of total RNA 25M-BM-5g of total RNA from C. glutamicum Delta-pup and C. glutamicum WT were compared in two-color microarray analysis. Overall three biological replicates were done. Sample 2-4: For growth in CGXII minimal media, a preculture 1 was grown in 5 ml BHI medium inoculated with a single colony from a fresh BHI agar plate and incubated at 170 rpm for 8 hours. Then 500 M-BM-5l of preculture 1 were used to inoculate preculture 2 in 100 ml shake flasks with 20 ml CGXII minimal medium containing 4 % (w/v) glucose and incubated over night (140 rpm , 30 M-BM-0C ). Subsequent main cultures (50 ml CGXII medium with 4 % glucose) were inoculated with cells from preculture 2 to an OD600 of about 0.4. For growth experiments in complex BHI medium, 20 ml brain heart infusion (BHI) medium (Difco Laboratories, Detroit, USA) were inoculated with a single colony from a fresh BHI agar plate and incubated at 140 rpm as overnight cultures. Subsequent main cultures (50 ml BHI medium) were inoculated with cells from overnight cultures to an optical density at 600 nm (OD600) of about 1. For DNA microarray analysis, cells were harvested in the exponential growth phase at an OD600 of 4 to 5. After extraction of total RNA 25M-BM-5g of total RNA from C. glutamicum Delta-pup and C. glutamicum WT were compared in two-color microarray analysis. Overall three biological replicates were done for each growth medium. Sample 5-6: For growth experiments in complex BHI medium, 20 ml brain heart infusion (BHI) medium (Difco Laboratories, Detroit, USA) were inoculated with a single colony from a fresh BHI agar plate and incubated at 140 rpm as overnight cultures. Subsequent main cultures (50 ml BHI medium) were inoculated with cells from overnight cultures to an optical density at 600 nm (OD600) of about 1. For DNA microarray analysis, cells were harvested in the exponential growth phase at an OD600 of 4 to 5. After extraction of total RNA 25M-BM-5g of total RNA from C. glutamicum Delta-pup and C. glutamicum WT were compared in two-color microarray analysis. Overall three biological replicates were done.

Project description:The response regulator HrrA belonging to the HrrSA two-component system (previously named CgtSR11) is known to be repressed by the global iron-dependent regulator DtxR in Corynebacterium glutamicum. Sequence analysis indicated an involvement of the HrrSA system in heme-dependent gene expression. Growth experiments revealed that the non-pathogenic soil bacterium C. glutamicum is able to use hemin or hemoglobin as sole iron source. In DNA microarray analyses a putative operon encoding the hemin-binding protein HtaA and the putative hemin ABC transporter HmuTUV showed a strong upregulation in heme-grown cells. Deletion of the hmu operon clearly affects heme utilization, but does not completely abolish growth on heme or hemoglobin. As a central part of this study, we investigated the regulon of the response regulator HrrA via comparative transcriptome analysis of a hrrA deletion mutant and C. glutamicum wild type in combination with DNA-protein interaction studies with purified HrrA protein. Our data provide evidence for a heme-dependent transcriptional activation of heme oxygenase (hmuO), an enzyme involved in the utilization of heme as iron source. Besides hmuO, HrrA was shown to activate the expression of heme-containing components of the respiratory chain, namely ctaD and the ctaE-qcrCAB operon encoding subunits I and III of cytochrome aa3 oxidase and three subunits of the cytochrome bc1 complex. Furthermore, HrrA represses almost all genes involved in heme biosynthesis, including glutamyl-tRNA reductase (hemA), uroporphyrinogen decarboxylase (hemE), and ferrochelatase (hemH). Thus, our data clearly emphasize a central role of the HrrSA system in the control of heme homeostasis in C. glutamicum. Three biological replicates of each experiment were performed. Experiment 1: Transcriptome comparison of wild type grown und FeSO4 or heme as iron source; Exp. 2: WT vs. hrrA deletion mutant grown on FeSO4; Exp. 3: WT vs. hrrA mutant grown on heme. For analysis via DNA microarraysose RNA was isolated from exponentially growing cells cultivated in CgXII medium containing glucose as carbon source and either 2.5 uM FeSO4 or 2.5 uM heme as iron source.

Dataset Information

IpsA, a novel transcriptional regulator required for inositol derived lipid formation in Corynebacteria and Mycobacteria

Publications

IpsA, a novel LacI-type regulator, is required for inositol-derived lipid formation in Corynebacteria and Mycobacteria.

Similar Datasets

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