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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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Effect of gene silencers on cell morphology.Cells were stained with DAPI before fluorescent microscopy. Left panel: AS19 untreated or treated with antisense PNA Ec108 (20 nM) targeting acpP, Ec107 (80 nM) targeting fabI, Ec326 (70 nM) targeting ftsZ or Ec330 (55 nM) targeting murA. Gross elongation of cells was observed only in cultures treated with ftsZ-specific PNA, Ec326. Right panel: TOP10 clones uninduced or induced with IPTG for antisense RNA expression. Concentrations of IPTG used for induction of acpP-, fabI-, ftsZ- and murA-antisense expression were 160 µM, 130 µM, 80 µM and 80 µM, respectively. Cells expressing ftsZ-antisense were grossly elongated compared to other cells.
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pone-0006061-g004: Effect of gene silencers on cell morphology.Cells were stained with DAPI before fluorescent microscopy. Left panel: AS19 untreated or treated with antisense PNA Ec108 (20 nM) targeting acpP, Ec107 (80 nM) targeting fabI, Ec326 (70 nM) targeting ftsZ or Ec330 (55 nM) targeting murA. Gross elongation of cells was observed only in cultures treated with ftsZ-specific PNA, Ec326. Right panel: TOP10 clones uninduced or induced with IPTG for antisense RNA expression. Concentrations of IPTG used for induction of acpP-, fabI-, ftsZ- and murA-antisense expression were 160 µM, 130 µM, 80 µM and 80 µM, respectively. Cells expressing ftsZ-antisense were grossly elongated compared to other cells.

Mentions: Both antisense PNA and expressed antisense specifically silenced mRNA of essential genes acpP, fabI, ftsZ or murA, as determined by analyses of transcript levels over an appropriate range of inhibitory but not lethal doses (Figure 3). Decreases in essential gene transcripts corresponded to decreases in bacterial growth rates (PNA data: R2 for acpP, fabI, ftsZ, murA = 0.87, 0.63, 0.89, 0.45), although the response was non-linear. It should be noted that the growth profiles observed for ftsZ-inhibited cultures, which displayed a rise and then a fall in culture turbidity, were consistent with a cell-division effect observed previously, where cells elongate without dividing [41], [42]. Phenotypic effects of ftsZ-inhibited cultures were confirmed by microscopy, revealing extremely elongated cells compared to untreated cells and cells treated with other PNAs. Cell phenotypes for antisense-expressing cells were similar to that of antisense PNA-treated cells (Figure 4).


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)

Effect of gene silencers on cell morphology.Cells were stained with DAPI before fluorescent microscopy. Left panel: AS19 untreated or treated with antisense PNA Ec108 (20 nM) targeting acpP, Ec107 (80 nM) targeting fabI, Ec326 (70 nM) targeting ftsZ or Ec330 (55 nM) targeting murA. Gross elongation of cells was observed only in cultures treated with ftsZ-specific PNA, Ec326. Right panel: TOP10 clones uninduced or induced with IPTG for antisense RNA expression. Concentrations of IPTG used for induction of acpP-, fabI-, ftsZ- and murA-antisense expression were 160 µM, 130 µM, 80 µM and 80 µM, respectively. Cells expressing ftsZ-antisense were grossly elongated compared to other cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2698124&req=5

pone-0006061-g004: Effect of gene silencers on cell morphology.Cells were stained with DAPI before fluorescent microscopy. Left panel: AS19 untreated or treated with antisense PNA Ec108 (20 nM) targeting acpP, Ec107 (80 nM) targeting fabI, Ec326 (70 nM) targeting ftsZ or Ec330 (55 nM) targeting murA. Gross elongation of cells was observed only in cultures treated with ftsZ-specific PNA, Ec326. Right panel: TOP10 clones uninduced or induced with IPTG for antisense RNA expression. Concentrations of IPTG used for induction of acpP-, fabI-, ftsZ- and murA-antisense expression were 160 µM, 130 µM, 80 µM and 80 µM, respectively. Cells expressing ftsZ-antisense were grossly elongated compared to other cells.
Mentions: Both antisense PNA and expressed antisense specifically silenced mRNA of essential genes acpP, fabI, ftsZ or murA, as determined by analyses of transcript levels over an appropriate range of inhibitory but not lethal doses (Figure 3). Decreases in essential gene transcripts corresponded to decreases in bacterial growth rates (PNA data: R2 for acpP, fabI, ftsZ, murA = 0.87, 0.63, 0.89, 0.45), although the response was non-linear. It should be noted that the growth profiles observed for ftsZ-inhibited cultures, which displayed a rise and then a fall in culture turbidity, were consistent with a cell-division effect observed previously, where cells elongate without dividing [41], [42]. Phenotypic effects of ftsZ-inhibited cultures were confirmed by microscopy, revealing extremely elongated cells compared to untreated cells and cells treated with other PNAs. Cell phenotypes for antisense-expressing cells were similar to that of antisense PNA-treated cells (Figure 4).

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