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Concurrent growth rate and transcript analyses reveal essential gene stringency in Escherichia coli.

Goh S, Boberek JM, Nakashima N, Stach J, Good L - PLoS ONE (2009)

Bottom Line: The relationship between mRNA decline and growth rate decline reflects the degree of essentiality, or stringency, of an essential gene, which is here defined by the minimum transcript level for a 50% reduction in growth rate (MTL(50)).When applied to four growth essential genes, both RNA silencing methods resulted in MTL(50) values that reveal acpP as the most stringently required of the four genes examined, with ftsZ the next most stringently required.This method may be used to validate existing essential genes and to quantify drug target requirement.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden.

ABSTRACT

Background: Genes essential for bacterial growth are of particular scientific interest. Many putative essential genes have been identified or predicted in several species, however, little is known about gene expression requirement stringency, which may be an important aspect of bacterial physiology and likely a determining factor in drug target development.

Methodology/principal findings: Working from the premise that essential genes differ in absolute requirement for growth, we describe silencing of putative essential genes in E. coli to obtain a titration of declining growth rates and transcript levels by using antisense peptide nucleic acids (PNA) and expressed antisense RNA. The relationship between mRNA decline and growth rate decline reflects the degree of essentiality, or stringency, of an essential gene, which is here defined by the minimum transcript level for a 50% reduction in growth rate (MTL(50)). When applied to four growth essential genes, both RNA silencing methods resulted in MTL(50) values that reveal acpP as the most stringently required of the four genes examined, with ftsZ the next most stringently required. The established antibacterial targets murA and fabI were less stringently required.

Conclusions: RNA silencing can reveal stringent requirements for gene expression with respect to growth. This method may be used to validate existing essential genes and to quantify drug target requirement.

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Transcomplementation of antisense PNA and expressed antisense RNA effects on growth by target gene over-expression.Essential gene over-expression was induced by L-arabinose at indicated concentrations. PNAs were added to a final concentration of 4 µM. Concentrations of IPTG used for induction of acpP-, fabI-, ftsZ- and murA-antisense expression were 200 µM, 1 mM, 100 µM and 75 mM, respectively.
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pone-0006061-g001: Transcomplementation of antisense PNA and expressed antisense RNA effects on growth by target gene over-expression.Essential gene over-expression was induced by L-arabinose at indicated concentrations. PNAs were added to a final concentration of 4 µM. Concentrations of IPTG used for induction of acpP-, fabI-, ftsZ- and murA-antisense expression were 200 µM, 1 mM, 100 µM and 75 mM, respectively.

Mentions: To silence the four selected target genes using the two different silencing methods, we first needed to design, test and validate several new silencers. Fortunately, we were able to use previously developed silencers for acpP and fabI, and we used similar design guidelines for the additional silencers needed for this study. We found that all eight silencers are able to inhibit E. coli growth. Also, both PNA and expressed antisense RNA silencers displayed gene and sequence selective effects. Nevertheless, to ensure that all silence mainly the target gene, we carried out a set of rescue experiments where the RNA silencers were used under conditions that inhibited growth and we tested whether growth could be rescued by over-expression of the target gene open reading frame (ORF) from a plasmid. This transcomplementation strategy provides a more strict control of specificity relative to controls that involve sense, scrambled or irrelevant sequences, because it takes into account all transcript sequences present in the bacterial cell and the effector sequences remain unchanged. We designed antisense PNA specific for acpP (Ec108), fabI (Ec107), ftsZ (Ec326), and murA (Ec330) using optimal parameters for gene silencing [37], [38] (Table 1). To test PNA specificity, we constructed four DH5α strains containing either pBAD-acpP, pBAD-fabI, pBAD-ftsZ or pBAD-murA. Each strain was grown in the presence of antisense PNA specific to the target gene cloned into pBAD, and either with or without the addition of L-arabinose for target gene over-expression. Without L-arabinose, strains did not grow but upon induction by L-arabinose, were resistant to the growth inhibitory effects of the PNA (Figure 1). To test specificity of antisense expressed from plasmids, we constructed another four DH5α strains containing either pBAD-acpP+pHNA, pBAD-fabI+pHN682, pBAD-ftsZ+pHNZ or pBAD-murA+pHNM. Each strain was grown in the presence of IPTG for induction of expressed antisense, and either with or without the addition of L-arabinose to induce target gene over-expression. Without L-arabinose, growth of strains was inhibited, and L-arabinose addition complemented gene silencing and allowed strains to grow in the presence of expressed antisense (Figure 1). The results from the rescue experiments demonstrate that the eight silencers display gene selective growth inhibition.


Concurrent growth rate and transcript analyses reveal essential gene stringency in Escherichia coli.

Goh S, Boberek JM, Nakashima N, Stach J, Good L - PLoS ONE (2009)

Transcomplementation of antisense PNA and expressed antisense RNA effects on growth by target gene over-expression.Essential gene over-expression was induced by L-arabinose at indicated concentrations. PNAs were added to a final concentration of 4 µM. Concentrations of IPTG used for induction of acpP-, fabI-, ftsZ- and murA-antisense expression were 200 µM, 1 mM, 100 µM and 75 mM, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2698124&req=5

pone-0006061-g001: Transcomplementation of antisense PNA and expressed antisense RNA effects on growth by target gene over-expression.Essential gene over-expression was induced by L-arabinose at indicated concentrations. PNAs were added to a final concentration of 4 µM. Concentrations of IPTG used for induction of acpP-, fabI-, ftsZ- and murA-antisense expression were 200 µM, 1 mM, 100 µM and 75 mM, respectively.
Mentions: To silence the four selected target genes using the two different silencing methods, we first needed to design, test and validate several new silencers. Fortunately, we were able to use previously developed silencers for acpP and fabI, and we used similar design guidelines for the additional silencers needed for this study. We found that all eight silencers are able to inhibit E. coli growth. Also, both PNA and expressed antisense RNA silencers displayed gene and sequence selective effects. Nevertheless, to ensure that all silence mainly the target gene, we carried out a set of rescue experiments where the RNA silencers were used under conditions that inhibited growth and we tested whether growth could be rescued by over-expression of the target gene open reading frame (ORF) from a plasmid. This transcomplementation strategy provides a more strict control of specificity relative to controls that involve sense, scrambled or irrelevant sequences, because it takes into account all transcript sequences present in the bacterial cell and the effector sequences remain unchanged. We designed antisense PNA specific for acpP (Ec108), fabI (Ec107), ftsZ (Ec326), and murA (Ec330) using optimal parameters for gene silencing [37], [38] (Table 1). To test PNA specificity, we constructed four DH5α strains containing either pBAD-acpP, pBAD-fabI, pBAD-ftsZ or pBAD-murA. Each strain was grown in the presence of antisense PNA specific to the target gene cloned into pBAD, and either with or without the addition of L-arabinose for target gene over-expression. Without L-arabinose, strains did not grow but upon induction by L-arabinose, were resistant to the growth inhibitory effects of the PNA (Figure 1). To test specificity of antisense expressed from plasmids, we constructed another four DH5α strains containing either pBAD-acpP+pHNA, pBAD-fabI+pHN682, pBAD-ftsZ+pHNZ or pBAD-murA+pHNM. Each strain was grown in the presence of IPTG for induction of expressed antisense, and either with or without the addition of L-arabinose to induce target gene over-expression. Without L-arabinose, growth of strains was inhibited, and L-arabinose addition complemented gene silencing and allowed strains to grow in the presence of expressed antisense (Figure 1). The results from the rescue experiments demonstrate that the eight silencers display gene selective growth inhibition.

Bottom Line: The relationship between mRNA decline and growth rate decline reflects the degree of essentiality, or stringency, of an essential gene, which is here defined by the minimum transcript level for a 50% reduction in growth rate (MTL(50)).When applied to four growth essential genes, both RNA silencing methods resulted in MTL(50) values that reveal acpP as the most stringently required of the four genes examined, with ftsZ the next most stringently required.This method may be used to validate existing essential genes and to quantify drug target requirement.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden.

ABSTRACT

Background: Genes essential for bacterial growth are of particular scientific interest. Many putative essential genes have been identified or predicted in several species, however, little is known about gene expression requirement stringency, which may be an important aspect of bacterial physiology and likely a determining factor in drug target development.

Methodology/principal findings: Working from the premise that essential genes differ in absolute requirement for growth, we describe silencing of putative essential genes in E. coli to obtain a titration of declining growth rates and transcript levels by using antisense peptide nucleic acids (PNA) and expressed antisense RNA. The relationship between mRNA decline and growth rate decline reflects the degree of essentiality, or stringency, of an essential gene, which is here defined by the minimum transcript level for a 50% reduction in growth rate (MTL(50)). When applied to four growth essential genes, both RNA silencing methods resulted in MTL(50) values that reveal acpP as the most stringently required of the four genes examined, with ftsZ the next most stringently required. The established antibacterial targets murA and fabI were less stringently required.

Conclusions: RNA silencing can reveal stringent requirements for gene expression with respect to growth. This method may be used to validate existing essential genes and to quantify drug target requirement.

Show MeSH