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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.

Show MeSH
Analysis of essential gene MTL50 values.Three curves from triplicate experiments of each gene were plotted so as to obtain three MTL50 values for statistical analyses. Significant difference (** = p<0.01, * = p<0.05) in MTL50 between genes was determined by a one-way ANOVA and Tukey HSD Test. Antisense PNA silencing (left panel) showed MTL50 of acpP to be significantly greater than the other three genes. Expressed antisense RNA silencing (right panel) showed MTL50 of acpP and ftsZ was significantly greater than that of murA only.
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pone-0006061-g006: Analysis of essential gene MTL50 values.Three curves from triplicate experiments of each gene were plotted so as to obtain three MTL50 values for statistical analyses. Significant difference (** = p<0.01, * = p<0.05) in MTL50 between genes was determined by a one-way ANOVA and Tukey HSD Test. Antisense PNA silencing (left panel) showed MTL50 of acpP to be significantly greater than the other three genes. Expressed antisense RNA silencing (right panel) showed MTL50 of acpP and ftsZ was significantly greater than that of murA only.

Mentions: Based on the above data, we propose minimum transcript level 50 (MTL50) as a measure of stringent requirement, somewhat similar to the IC50 measure of enzyme inhibition. MTL50 values directly reflect the relationship between the level of a particular mRNA and growth rate decline, thus indicating how stringently required an essential gene is for growth. By plotting the values of relative growth rate against relative mRNA level for each gene on the same graph, we are able to estimate the value of relative mRNA (MTL50) at 50% growth rate (Figure 5). We observed that a shift to the right indicates greater stringency as the minimum level of transcript needed to sustain 50% cell viability increases. Hence, a high MTL50 value indicates high gene stringency. This is because the value indicates the minimum required level of transcript for viability, not the minimum inhibition level of transcript, and hence differs from IC50 in this respect. Comparisons of the MTL50 values, determined by the two different gene-silencing methods, are shown in Figure 6. In general, MTL50 values were higher in the expressed antisense cultures compared to antisense PNA treated cultures. Both methods found that the most stringently required essential gene was acpP, followed by ftsZ. Antisense PNA silencing showed fabI to be the least stringent, whereas expressed antisense silencing found murA was significantly less stringently required, relative to the other four essential genes examined (Figures 5 and 6).


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)

Analysis of essential gene MTL50 values.Three curves from triplicate experiments of each gene were plotted so as to obtain three MTL50 values for statistical analyses. Significant difference (** = p<0.01, * = p<0.05) in MTL50 between genes was determined by a one-way ANOVA and Tukey HSD Test. Antisense PNA silencing (left panel) showed MTL50 of acpP to be significantly greater than the other three genes. Expressed antisense RNA silencing (right panel) showed MTL50 of acpP and ftsZ was significantly greater than that of murA only.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2698124&req=5

pone-0006061-g006: Analysis of essential gene MTL50 values.Three curves from triplicate experiments of each gene were plotted so as to obtain three MTL50 values for statistical analyses. Significant difference (** = p<0.01, * = p<0.05) in MTL50 between genes was determined by a one-way ANOVA and Tukey HSD Test. Antisense PNA silencing (left panel) showed MTL50 of acpP to be significantly greater than the other three genes. Expressed antisense RNA silencing (right panel) showed MTL50 of acpP and ftsZ was significantly greater than that of murA only.
Mentions: Based on the above data, we propose minimum transcript level 50 (MTL50) as a measure of stringent requirement, somewhat similar to the IC50 measure of enzyme inhibition. MTL50 values directly reflect the relationship between the level of a particular mRNA and growth rate decline, thus indicating how stringently required an essential gene is for growth. By plotting the values of relative growth rate against relative mRNA level for each gene on the same graph, we are able to estimate the value of relative mRNA (MTL50) at 50% growth rate (Figure 5). We observed that a shift to the right indicates greater stringency as the minimum level of transcript needed to sustain 50% cell viability increases. Hence, a high MTL50 value indicates high gene stringency. This is because the value indicates the minimum required level of transcript for viability, not the minimum inhibition level of transcript, and hence differs from IC50 in this respect. Comparisons of the MTL50 values, determined by the two different gene-silencing methods, are shown in Figure 6. In general, MTL50 values were higher in the expressed antisense cultures compared to antisense PNA treated cultures. Both methods found that the most stringently required essential gene was acpP, followed by ftsZ. Antisense PNA silencing showed fabI to be the least stringent, whereas expressed antisense silencing found murA was significantly less stringently required, relative to the other four essential genes examined (Figures 5 and 6).

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