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RNA-Seq of Clostridium cellulovorans cultured in different carbon sources in exponential phase

ABSTRACT: RNA-Seq of Clostridium cellulovorans cultured in different carbon sources in exponential phase

PROVIDER: PRJEB57453 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR10482301_1.fastq.gz	Fastqsanger.gz
	ERR10482301_2.fastq.gz	Fastqsanger.gz
	ERR10482302_1.fastq.gz	Fastqsanger.gz
	ERR10482302_2.fastq.gz	Fastqsanger.gz
	ERR10482303_1.fastq.gz	Fastqsanger.gz

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Project description:Transcriptomics analysis of biopolymer (medium chain length polyhydroxyalkanoate) producing strain P.putida LS46 cultured with biodiesel derived waste carbon sources: studies of cellular adaptation to the industrial waste streams and metabolic profiling under the polymer producing conditions. We are reporting RNAseq analysis data here as part of our multi-level Omics study of medium chain length polyhydroxyalkanoate (mcl-PHA) producing strain P.putida LS46 culture with biodiesel derived waste glycerol and waste fatty acids. The data presented here will be used in two separate manuscripts. The objectives of this study are a): to evaluate cellular responses of P.putida LS46 under industrial waste stream. b): to study gene expression profile under two selected mcl-PHA producing conditions of P.putida LS46. Comparative multi-level Omics study: for objective a): Exponential P.putida LS46 cell from waste glycerol culture compared against reagent grade pure glycerol culture. For objective b): Two mcl-PHA producing conditions, namely stationary phase waste glycerol culture and exponential phase waste fatty acid culture of P.putida LS46, were compared against exponential phase waste glycerol culture of P.putida LS46. Major results from objective a): The waste glycerol substrate induced expression of a large number of genes putatively involved in heavy metal tolerance, including three gene clusters: a putative cusABC transcript unit and two copies of copAB, which are usually involved in copper resistance and tolerance to other monovalent heavy metals. A local gene relocation was observed in cluster 1 consisting cusABC and copAB relative to the KT2440 type strain according to the phylogenetic and gene neighbourhood analyses on various P. putida strains. P. putida LS46 also contains 11 putative MerR family regulators, which sense various environmental stimuli including heavy metals. MerR-1 is an ortholog of the copper response regulator of other gram-negative bacteria, and was highly up-regulated in waste glycerol cultures. Finally, a number of genes involved in cell responses to high extra-cellular Na+ concentrations, and genes of the fatty acid beta-oxidation pathway were up-regulated in waste glycerol cultures Major results from objective b): Regardless to the type of substrates, up-regulation of two mcl-PHA synthase (PhaC1 and PhaC2), and two phasin proteins (PhaF and PhaI) are the most common genotype under mcl-PHA production conditions. PhaG and possible PhaJ4 connect fatty acid de novo synthesis to mcl-PHA in waste glycerol culture. Interestingly, expression of gene, fabZ, in production of unsaturated fatty acid from fatty acid de novo synthesis was only observed in waste glycerol culture. On the other hand, PhaJ1 and PhaJ4 derived mcl-PHA production via fatty acid beta-oxidation was observed under waste fatty acid culture. These results would help to explain observed different production kinetics and monomer distribution of the polymer. Although under active mcl-PHA production condition, depression on the expression of glpF genes in glycerol transportation system prevent further channelling extra-cellular glycerol into the cell. Waste glycerol culture also triggers trahalose synthesis pathway, a potential competing pathway during mcl-PHA synthesizing. In waste fatty acid culture, the intermediates (acyl-CoA and 3-hydroxyacyl-CoA) of fatty acid beta-oxidation were used for mcl-PHA production and were also likely hydrolysed to their free acid forms via an up-regulated thioesteras coding gene, tesA. Acetyl-CoA cleaved from the pathway was clearly channeled into glyoxylate shut for C2 carbon assimilation over spillage as CO2 through TCA cycle or used in fatty acid biosynthesis pathway.

Project description:In consideration of the fact that BrpA expression is at its maximum during early-exponential phase as shown by Northern blotting and reporter gene fusion assays, we carried out a DNA microarray analysis using total RNA from early exponential phase cultures (OD600nm=0.3). It was found that 92 genes were up-regulated and 90 down-regulated in TW14D by a factor of at least 1.5-fold (p ≤ 0.001). At a level of P<0.01, 176 additional genes were found to be differentially expressed in TW14D, with 77 up- and 90 down-regulated. Based on the description and putative functions of the genes identified at the significance level of P<0.001, BrpA-deficiency affects almost every aspect of the cellular physiology as well as virulence properties, including amino acid biosynthesis, carbohydrates and energy metabolism, nucleic acids and DNA metabolism, transcriptional regulation, ABC transporters, molecular chaperones and other cellular processes, and hypothetical and conserved hypothetical proteins. The breadth of impact of BrpA-deficiency on the transcriptional profile of the deficient mutant is similar to what was observed previously with mid-exponential phase cells. However, comparison of the two transcriptional profiles revealed that only a small number of genes were consistently up- or down-regulated in both early- and mid-exponential phase cultures, which include recA (for recombinant protein RecA), gtfD (for glucosyltransferase D), wapA (for surface-associated protein WapA), groEL (for molecular chaperone GroEL) and sod (for Mn-dependent superoxide dismutase SOD). In addition, the magnitude of alterations in gene expression was also more dramatic in cells of the early-exponential phase, when compared to that in the mid-exponential phase cultures. Of the genes altered as a result of BrpA-deficiency in early-exponential phase cultures, several were found to encode proteins with potential roles in peptidoglycan biosynthesis. Among them are SMU.246 for a phospho-MurNAc-pentapeptide-transferase (RgpG), SMU.549 for an undecaprenyl-PP-MurNAc-pentapeptide-UDP-GlcNAc transferase (MurG), SMU.599 for a D-alanine-D-alanine ligase (DdlA), and SMU.1677 for a UDP-MurNAc-tripeptide synthetase (MurE). Besides, several genes encoding components of the Sec translocase were also found altered in TW14D. These included secA, secE and secY encoding the ATP-dependent motor of the translocation machinery, SecA, and the translocon pore components, SecE and SecY, respectively. Both secA and secE were down-regulated by more than 2-fold, while secY was up-regulated by more than 2-fold.

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RNA-Seq of Clostridium cellulovorans cultured in different carbon sources in exponential phase

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