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Next Generation Sequencing Facilitates Quantitative Analysis of Mycobacterium smegmatis C2 155 Transcriptomes treated with overexpression strains MS_Vec and MS_Rv2872

ABSTRACT: To gain molecular insight into the role of Rv2872 in increased vancomycin resistance, change of the intracellular amino acids content and cell wall structure, the transcriptome of Rv2872 overexpression M. smegmatis was assessed through RNA-Seq analysis. The transcriptome data showed that the transcription of four genes involved in peptidoglycan synthesis was significantly lower in the MS_Rv2872 overexpression strain . The decreased vancomycin targets might underlie the observed vancomycin resistance. Consistent with altered intracellular amino acids levels in the recombinant M. smegmatis MS_Rv2872, the transcription level of relevant genes was also altered. This is also seen in the altered fatty acids levels. The data showed that 38 fatty acid metabolism genes were down regulated, and 15 were up regulated in the recombinant M.smegmatis MS_Rv2872.

ORGANISM(S): Mycolicibacterium smegmatis MC2 155

PROVIDER: GSE109134 | GEO | 2021/01/12

REPOSITORIES: GEO

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Project description:For over 30 years, serine hydroxamate has been used to chemically stimulate a stringent response in Escherichia coli and other bacteria. These studies have elucidated numerous characteristics of the classical stringent response beyond the simple cellular response to an amino acid shortage, including phospholipid synthesis and protease up-regulation. In this study, the effects of a serine hydroxamate addition on high cell density recombinant E. coli were examined and compared to the effects of recombinant protein production to determine overlaps, as recombinant protein production stress has often been attributed to amino acid shortages. Both the transcriptome and growth characteristics were evaluated and compared. The serine hydroxamate addition profoundly decreased the culture growth rate, whereas, recombinant protein production did not. Conversely, the transcriptome profile of the recombinant E. coli cultures were relatively unaffected by the serine hydroxamate addition, yet recombinant protein production dramatically changed the transcriptome profile. A subset of the classical stringent response genes were effected by the serine hydroxamate addition, whereas, recombinant protein production regulated numerous classical stringent response genes; however, not all. The genes that were regulated by the serine hydroxamate addition include numerous fatty acid synthesis genes, in agreement with altered phospholipids synthesis reports. These results indicate that recombinant protein production and the stringent response have many overlapping responses; however, are far from identical. It was hypothesized that recombinant protein production leads to a stringent response due to the high amino acid synthesis demands related to recombinant protein synthesis. A comparison of the transcriptomes during recombinant protein production and a chemical imposed stringent response would assist with determining what portion of the “metabolic burden” associated with recombinant protein production is due to amino acid shortages. In this study, the transcriptome profiles of recombinant E. coli were examined and compared for the three culture conditions: 1) Normal growth, no external stress; 2) L-serine hydroxamate addition (to mediate a stringent response); and 3) IPTG-induction to produce the recombinant protein chloramphenicol acetyltransferase (CAT). The transcriptome profiles from these three conditions were analyzed using Affymetrix Anti-sense E. coli GeneChip® microarrays.

Dataset Information

Next Generation Sequencing Facilitates Quantitative Analysis of Mycobacterium smegmatis C2 155 Transcriptomes treated with overexpression strains MS_Vec and MS_Rv2872

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