Dataset Information

AraR, an l-Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions.

ABSTRACT:

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In Corynebacterium glutamicum ATCC 31831, a LacI-type transcriptional regulator AraR, represses the expression of l-arabinose catabolism (araBDA), uptake (araE), and the regulator (araR) genes clustered on the chromosome. AraR binds to three sites: one (BSB) between the divergent operons (araBDA and galM-araR) and two (BSE1 and BSE2) upstream of araE. L-Arabinose acts as an inducer of the AraR-mediated regulation. Here, we examined the roles of these AraR-binding sites in the expression of the AraR regulon. BSB mutation resulted in derepression of both araBDA and galM-araR operons. The effects of BSE1 and/or BSE2 mutation on araE expression revealed that the two sites independently function as the cis elements, but BSE1 plays the primary role. However, AraR was shown to bind to these sites with almost the same affinity in vitro. Taken together, the expression of araBDA and araE is strongly repressed by binding of AraR to a single site immediately downstream of the respective transcriptional start sites, whereas the binding site overlapping the -10 or -35 region of the galM-araR and araE promoters is less effective in repression. Furthermore, downregulation of araBDA and araE dependent on l-arabinose catabolism observed in the BSB mutant and the AraR-independent araR promoter identified within galM-araR add complexity to regulation of the AraR regulon derepressed by L-arabinose.

Importance

Corynebacterium glutamicum has a long history as an industrial workhorse for large-scale production of amino acids. An important aspect of industrial microorganisms is the utilization of the broad range of sugars for cell growth and production process. Most C. glutamicum strains are unable to use a pentose sugar L-arabinose as a carbon source. However, genes for L-arabinose utilization and its regulation have been recently identified in C. glutamicum ATCC 31831. This study elucidates the roles of the multiple binding sites of the transcriptional repressor AraR in the derepression by L-arabinose and thereby highlights the complex regulatory feedback loops in combination with l-arabinose catabolism-dependent repression of the AraR regulon in an AraR-independent manner.

SUBMITTER: Kuge T

PROVIDER: S-EPMC4652040 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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AraR, an l-Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions.

Kuge Takayuki T Teramoto Haruhiko H Inui Masayuki M

Journal of bacteriology 20150928 24

<h4>Unlabelled</h4>In Corynebacterium glutamicum ATCC 31831, a LacI-type transcriptional regulator AraR, represses the expression of l-arabinose catabolism (araBDA), uptake (araE), and the regulator (araR) genes clustered on the chromosome. AraR binds to three sites: one (BSB) between the divergent operons (araBDA and galM-araR) and two (BSE1 and BSE2) upstream of araE. L-Arabinose acts as an inducer of the AraR-mediated regulation. Here, we examined the roles of these AraR-binding sites in the ...[more]

PMID: 26416832

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Project description:BackgroundZinc is considered as an essential element for all living organisms, but it can be toxic at large concentrations. Bacteria therefore tightly regulate zinc metabolism. The Cg2502 protein of Corynebacterium glutamicum was a candidate to control zinc metabolism in this species, since it was classified as metalloregulator of the zinc uptake regulator (Zur) subgroup of the ferric uptake regulator (Fur) family of DNA-binding transcription regulators.ResultsThe cg2502 (zur) gene was deleted in the chromosome of C. glutamicum ATCC 13032 by an allelic exchange procedure to generate the zur-deficient mutant C. glutamicum JS2502. Whole-genome DNA microarray hybridizations and real-time RT-PCR assays comparing the gene expression in C. glutamicum JS2502 with that of the wild-type strain detected 18 genes with enhanced expression in the zur mutant. The expression data were combined with results from cross-genome comparisons of shared regulatory sites, revealing the presence of candidate Zur-binding sites in the mapped promoter regions of five transcription units encoding components of potential zinc ABC-type transporters (cg0041-cg0042/cg0043; cg2911-cg2912-cg2913), a putative secreted protein (cg0040), a putative oxidoreductase (cg0795), and a putative P-loop GTPase of the COG0523 protein family (cg0794). Enhanced transcript levels of the respective genes in C. glutamicum JS2502 were verified by real-time RT-PCR, and complementation of the mutant with a wild-type zur gene reversed the effect of differential gene expression. The zinc-dependent expression of the putative cg0042 and cg2911 operons was detected in vivo with a gfp reporter system. Moreover, the zinc-dependent binding of purified Zur protein to double-stranded 40-mer oligonucleotides containing candidate Zur-binding sites was demonstrated in vitro by DNA band shift assays.ConclusionWhole-genome expression profiling and DNA band shift assays demonstrated that Zur directly represses in a zinc-dependent manner the expression of nine genes organized in five transcription units. Accordingly, the Zur (Cg2502) protein is the key transcription regulator for genes involved in zinc homeostasis in C. glutamicum.

Project description:BACKGROUND: Recent discoveries on bacterial transcriptomes gave evidence that small RNAs (sRNAs) have important regulatory roles in prokaryotic cells. Modern high-throughput sequencing approaches (RNA-Seq) enable the most detailed view on transcriptomes offering an unmatched comprehensiveness and single-base resolution. Whole transcriptome data obtained by RNA-Seq can be used to detect and characterize all transcript species, including small RNAs. Here, we describe an RNA-Seq approach for comprehensive detection and characterization of small RNAs from Corynebacterium glutamicum, an actinobacterium of high industrial relevance and model organism for medically important Corynebacterianeae, such as C. diphtheriae and Mycobacterium tuberculosis. RESULTS: In our RNA-Seq approach, total RNA from C. glutamicum ATCC 13032 was prepared from cultures grown in minimal medium at exponential growth or challenged by physical (heat shock, cold shock) or by chemical stresses (diamide, H2O2, NaCl) at this time point. Total RNA samples were pooled and sequencing libraries were prepared from the isolated small RNA fraction. High throughput short read sequencing and mapping yielded over 800 sRNA genes. By determining their 5'- and 3'-ends and inspection of their locations, these potential sRNA genes were classified into UTRs of mRNAs (316), cis-antisense sRNAs (543), and trans-encoded sRNAs (262). For 77 of trans-encoded sRNAs significant sequence and secondary structure conservation was found by a computational approach using a whole genome alignment with the closely related species C. efficiens YS-314 and C. diphtheriae NCTC 13129. Three selected trans-encoded sRNAs were characterized by Northern blot analysis and stress-specific transcript patterns were found. CONCLUSIONS: The study showed comparable numbers of sRNAs known from genome-wide surveys in other bacteria. In detail, our results give deep insight into the comprehensive equipment of sRNAs in C. glutamicum and provide a sound basis for further studies concerning the functions of these sRNAs.

Project description:Corynebacterium glutamicum belongs to the microbes of enormous biotechnological relevance. In particular, its strain ATCC 13032 is a widely used producer of L-amino acids at an industrial scale. Its apparent robustness also turns it into a favorable platform host for a wide range of further compounds, mainly because of emerging bio-based economies. A deep understanding of the biochemical processes in C. glutamicum is essential for a sustainable enhancement of the microbe's productivity. Computational systems biology has the potential to provide a valuable basis for driving metabolic engineering and biotechnological advances, such as increased yields of healthy producer strains based on genome-scale metabolic models (GEMs). Advanced reconstruction pipelines are now available that facilitate the reconstruction of GEMs and support their manual curation. This article presents iCGB21FR, an updated and unified GEM of C. glutamicum ATCC 13032 with high quality regarding comprehensiveness and data standards, built with the latest modeling techniques and advanced reconstruction pipelines. It comprises 1042 metabolites, 1539 reactions, and 805 genes with detailed annotations and database cross-references. The model validation took place using different media and resulted in realistic growth rate predictions under aerobic and anaerobic conditions. The new GEM produces all canonical amino acids, and its phenotypic predictions are consistent with laboratory data. The in silico model proved fruitful in adding knowledge to the metabolism of C. glutamicum: iCGB21FR still produces L-glutamate with the knock-out of the enzyme pyruvate carboxylase, despite the common belief to be relevant for the amino acid's production. We conclude that integrating high standards into the reconstruction of GEMs facilitates replicating validated knowledge, closing knowledge gaps, and making it a useful basis for metabolic engineering. The model is freely available from BioModels Database under identifier MODEL2102050001.

Dataset Information

AraR, an l-Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions.

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Importance

Publications

AraR, an l-Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions.

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