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Analysis of transcript changes in a heme-deficient mutant of Escherichia coli in response to CORM-3 [Ru(CO)3Cl(glycinate)].

ABSTRACT: This article describes in extended detail the methodology applied for acquisition of transcriptomic data, and subsequent statistical data modelling, published by Wilson et al. (2015) in a study of the effects of carbon monoxide-releasing molecule-3 (CORM-3 [Ru(CO)3Cl(glycinate)]) on heme-deficient bacteria. The objective was to identify non-heme targets of CORM action. Carbon monoxide (CO) interacts with heme-containing proteins, in particular respiratory cytochromes; however, CORMs have been shown to elicit multifaceted effects in bacteria, suggesting that the compounds may have additional targets. We therefore sought to elucidate the activity of CORM-3, the first water-soluble CORM and one of the most characterised CORMs to date, in bacteria devoid of heme synthesis. Importantly, we also tested inactive CORM-3 (iCORM-3), a ruthenium co-ligand fragment that does not release CO, in order to differentiate between CO- and compound-related effects. A well-established hemA mutant of Escherichia coli was used for the study and, for comparison, parallel experiments were performed on the corresponding wild-type strain. Global transcriptomic changes induced by CORM-3 and iCORM-3 were evaluated using a Two-Color Microarray-Based Prokaryote Analysis (FairPlay III Labeling) by Agilent Technologies (Inc. 2009). Data acquisition was carried out using Agilent Feature Extraction software (v6.5) and data normalisation, as well as information about gene products and their function was obtained from GeneSpring GX v7.3 (Agilent Technologies). Functional category lists were created using KEGG (Kyoto Encyclopedia of Genes and Genomes). Relevant regulatory proteins for each gene were identified, where available, using regulonDB and EcoCyc (World Wide Web). Statistical data modelling was performed on the gene expression data to infer transcription factor activities. The transcriptomic data can be accessed through NCBI's Gene Expression Omnibus (GEO): series accession number GSE55097 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE55097).

SUBMITTER: Wilson JL

PROVIDER: S-EPMC4543538 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Photochemical CO2 reduction catalysed by trans(Cl)-Ru(bpy)(CO)2Cl2 (bpy = 2,2'-bipyridine) efficiently produces carbon monoxide (CO) and formate (HCOO-) in N,N-dimethylacetamide (DMA)/water containing [Ru(bpy)3]2+ as a photosensitizer and 1-benzyl-1,4-dihydronicotinamide (BNAH) as an electron donor. We have unexpectedly found catalyst concentration dependence of the product ratio (CO/HCOO-) in the photochemical CO2 reduction: the ratio of CO/HCOO- decreases with increasing catalyst concentration. The result has led us to propose a new mechanism in which HCOO- is selectively produced by the formation of a Ru(i)-Ru(i) dimer as the catalyst intermediate. This reaction mechanism predicts that the Ru-Ru bond dissociates in the reaction of the dimer with CO2, and that the insufficient electron supply to the catalyst results in the dominant formation of HCOO-. The proposed mechanism is supported by the result that the time-course profiles of CO and HCOO- in the photochemical CO2 reduction catalysed by [Ru(bpy)(CO)2Cl]2 (0.05 mM) are very similar to those of the reduction catalysed by trans(Cl)-Ru(bpy)(CO)2Cl2 (0.10 mM), and that HCOO- formation becomes dominant under low-intensity light. The kinetic analyses based on the proposed mechanism could excellently reproduce the unusual catalyst concentration effect on the product ratio. The catalyst concentration effect observed in the photochemical CO2 reduction using [Ru(4dmbpy)3]2+ (4dmbpy = 4,4'-dimethyl-2,2'-bipyridine) instead of [Ru(bpy)3]2+ as the photosensitizer is also explained with the kinetic analyses, reflecting the smaller quenching rate constant of excited [Ru(4dmbpy)3]2+ by BNAH than that of excited [Ru(bpy)3]2+. We have further synthesized trans(Cl)-Ru(6Mes-bpy)(CO)2Cl2 (6Mes-bpy = 6,6'-dimesityl-2,2'-bipyridine), which bears bulky substituents at the 6,6'-positions in the 2,2'-bipyridyl ligand, so that the ruthenium complex cannot form the dimer due to the steric hindrance. We have found that this ruthenium complex selectively produces CO, which strongly supports the catalytic mechanism proposed in this work.

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Analysis of transcript changes in a heme-deficient mutant of Escherichia coli in response to CORM-3 [Ru(CO)3Cl(glycinate)].

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