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Overexpression of cloned RhsA sequences perturbs the cellular translational machinery in Escherichia coli.

ABSTRACT: RhsA is a member of the multigene Rhs family and consists of a complex genetic sequence. This sequence consists of several distinct components, including a GC-rich core (core open reading frame [ORF]), an AT-rich extension (ext-a1) of the core ORF and an AT-rich region following the core extension (dsORF-a1). The functions of RhsA and the different distinct components, which can include open reading frames, are not well understood. Here, we study the effect of overexpression of the ext-a1 sequence and the ext-a1 3' region, which includes a partial sequence of dsORF-a1, on Escherichia coli cells. Cells expressing these sequences show reduced cell growth and cell viability. The expression of these sequences dramatically affects different components of the transcription and translation machinery. Transcriptomic analysis reveals an increase in the expression of genes involved in transcription, RNA processing, and nucleotide biosynthesis and metabolism and a decrease in the expression of amino acid biosynthesis genes and transfer RNAs. Further, expression of the above-mentioned RhsA components increases ribosomal gene expression, as well as rRNA and ribosome abundance. Proteomic analysis reveals an overall reduction of protein expression at the genome-wide level in cells expressing the above-mentioned RhsA components. Based on these observations, we suspect a translation product of ext-a1 affects different regulatory mechanisms that control rRNA synthesis.

SUBMITTER: Aggarwal K

PROVIDER: S-EPMC3165670 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Overexpression of cloned RhsA sequences perturbs the cellular translational machinery in Escherichia coli.

Aggarwal Kunal K Lee Kelvin H KH

Journal of bacteriology 20110715 18

RhsA is a member of the multigene Rhs family and consists of a complex genetic sequence. This sequence consists of several distinct components, including a GC-rich core (core open reading frame [ORF]), an AT-rich extension (ext-a1) of the core ORF and an AT-rich region following the core extension (dsORF-a1). The functions of RhsA and the different distinct components, which can include open reading frames, are not well understood. Here, we study the effect of overexpression of the ext-a1 sequen ...[more]

PMID: 21764923

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Project description:Diacylglycerol acyltransferase (DGAT) is the rate-limiting enzyme in triacylglycerol biosynthesis in eukaryotic organisms. Triacylglycerols are important energy-storage oils in plants such as peanuts, soybeans and rape. In this study, Arachis hypogaea type 2 DGAT (AhDGAT2) genes were cloned from the peanut cultivar 'Luhua 14' using a homologous gene sequence method and rapid amplification of cDNA ends. To understand the role of AhDGAT2 in triacylglycerol biosynthesis, two AhDGAT2 nucleotide sequences that differed by three amino acids were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli Rosetta (DE3). Following IPTG induction, the isozymes (AhDGAT2a and AhDGAT2b) were expressed as 64.5 kDa GST fusion proteins. Both AhDGAT2a and AhDGAT2b occurred in the host cell cytoplasm and inclusion bodies, with larger amounts in the inclusion bodies. Overexpression of AhDGATs depressed the host cell growth rates relative to non-transformed cells, but cells harboring empty-vector, AhDGAT2a-GST, or AhDGAT2b-GST exhibited no obvious growth rate differences. Interestingly, induction of AhDGAT2a-GST and AhDGAT2b-GST proteins increased the sizes of the host cells by 2.4-2.5 times that of the controls (post-IPTG induction). The total fatty acid (FA) levels of the AhDGAT2a-GST and AhDGAT2a-GST transformants, as well as levels of C12:0, C14:0, C16:0, C16:1, C18:1n9c and C18:3n3 FAs, increased markedly, whereas C15:0 and C21:0 levels were lower than in non-transformed cells or those containing empty-vectors. In addition, the levels of some FAs differed between the two transformant strains, indicating that the two isozymes might have different functions in peanuts. This is the first time that a full-length recombinant peanut DGAT2 has been produced in a bacterial expression system and the first analysis of its effects on the content and composition of fatty acids in E. coli. Our results indicate that AhDGAT2 is a strong candidate gene for efficient FA production in E. coli.

Dataset Information

Overexpression of cloned RhsA sequences perturbs the cellular translational machinery in Escherichia coli.

Publications

Overexpression of cloned RhsA sequences perturbs the cellular translational machinery in Escherichia coli.

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