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lpxC and yafS are the most suitable internal controls to normalize real time RT-qPCR expression in the phytopathogenic bacteria Dickeya dadantii.

Hommais F, Zghidi-Abouzid O, Oger-Desfeux C, Pineau-Chapelle E, Van Gijsegem F, Nasser W, Reverchon S - PLoS ONE (2011)

Bottom Line: Out of these, we retained 10 genes representing different functional categories, different levels of expression (low, medium, and high) and with no systematic variation in expression correlating with growth conditions.The two most stable genes (ABF-0017965 (lpxC) and ABF-0020529 (yafS) were successfully used for normalization of RT-qPCR data.Finally, we demonstrated that the ortholog of lpxC and yafS in Pectobacterium atrosepticum also showed stable expression in diverse growth conditions.

View Article: PubMed Central - PubMed

Affiliation: Unité Microbiologie, Adaptation, Pathogénie CNRS-INSA-UCBL UMR 5240, Université Lyon 1, Villeurbanne, France. hommais@univ-lyon1.fr

ABSTRACT

Background: Quantitative RT-PCR is the method of choice for studying, with both sensitivity and accuracy, the expression of genes. A reliable normalization of the data, using several reference genes, is critical for an accurate quantification of gene expression. Here, we propose a set of reference genes, of the phytopathogenic bacteria Dickeya dadantii and Pectobacterium atrosepticum, which are stable in a wide range of growth conditions.

Results: We extracted, from a D. dadantii micro-array transcript profile dataset comprising thirty-two different growth conditions, an initial set of 49 expressed genes with very low variation in gene expression. Out of these, we retained 10 genes representing different functional categories, different levels of expression (low, medium, and high) and with no systematic variation in expression correlating with growth conditions. We measured the expression of these reference gene candidates using quantitative RT-PCR in 50 different experimental conditions, mimicking the environment encountered by the bacteria in their host and directly during the infection process in planta. The two most stable genes (ABF-0017965 (lpxC) and ABF-0020529 (yafS) were successfully used for normalization of RT-qPCR data. Finally, we demonstrated that the ortholog of lpxC and yafS in Pectobacterium atrosepticum also showed stable expression in diverse growth conditions.

Conclusions: We have identified at least two genes, lpxC (ABF-0017965) and yafS (ABF-0020509), whose expressions are stable in a wide range of growth conditions and during infection. Thus, these genes are considered suitable for use as reference genes for the normalization of real-time RT-qPCR data of the two main pectinolytic phytopathogenic bacteria D. dadantii and P. atrosepticum and, probably, of other Enterobacteriaceae. Moreover, we defined general criteria to select good reference genes in bacteria.

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Expression profiles of the 10 genes selected for the stability of their expression level and the synthetic RNA pAW.Gene expression profiles are measured from micro-arrays (A) and from quantitative real time RT-PCR (B). The first 32 conditions are those measured in exponential phase and the last 32 conditions are those measured in stationary phase. The order of conditions is M63 supplemented with saccharose (S), M63 supplemented with saccharose and Saintpaulia leaf extract (PL), M63 supplemented with saccharose and PGA (P), and M63 supplemented with saccharose, leaves and PGA (PLP). The order of stress is: no stress, oxidative stress, acid stress, and osmotic stress.
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pone-0020269-g002: Expression profiles of the 10 genes selected for the stability of their expression level and the synthetic RNA pAW.Gene expression profiles are measured from micro-arrays (A) and from quantitative real time RT-PCR (B). The first 32 conditions are those measured in exponential phase and the last 32 conditions are those measured in stationary phase. The order of conditions is M63 supplemented with saccharose (S), M63 supplemented with saccharose and Saintpaulia leaf extract (PL), M63 supplemented with saccharose and PGA (P), and M63 supplemented with saccharose, leaves and PGA (PLP). The order of stress is: no stress, oxidative stress, acid stress, and osmotic stress.

Mentions: Next, we required that the selected genes satisfied the following criteria: (i) they are associated, as far as possible, with different functional categories to minimize the risk of choosing co-regulated genes; (ii) they belong to different transcription units; (iii) they show no sign of correlation between the expression profiles and growth conditions (supplementary data). Among the 49 genes, 22 encode metabolic enzymes, 9 genes encode membrane proteins involved in membrane traffic and 6 are predicted to encode transcriptional regulators, the others encoding hypothetical proteins or proteins with unknown function. No functional category is overrepresented, with the exception of three genes which encode proteins involved in heme biosynthesis: hemF and hemXY. One of the most stable genes is lpxC (ABF-0017965). This gene encodes for a UDP-3-O-acyl N-acetylglucosamine deacetylase that catalyzes the second step of the lipid A-precursor biosynthetic pathway [11]. Seven enzymes are involved in this pathway and most of the encoding genes are significantly down-regulated in oxidative stress when grown at the exponential phase, suggesting that the expression of lpxC (ABF-0017965) is stable whereas that of the other genes from the same pathway is under regulation. This means that enzymes from the same metabolic pathway may be differentially regulated. Among the 49 selected genes, only hemY, hemX and yadH, yadG are organized in operons. hemXY are transcribed with two other genes, hemC and hemD. Remarkably, expression of hemCD is induced by osmotic stress (1.5 log), suggesting a difference in the regulation mechanisms between hemXY and hemCD. This is in accordance with a previous study, using E. coli, that demonstrates the presence of two transcription initiation sites in this operon [12]. The expression of the two other genes organized in operons, yadH and yadG, is very similar (supplementary data) and seems to be correlated to growth phases and stresses. These genes were discarded for the rest of the study. We examined the expression profiles of the 47 genes (supplementary data) and checked for systematic variation and correlations between expression and growth conditions. In spite of the weak variation in the expression levels of the selected genes, some correlations were found between the expression levels of several genes and the growth conditions tested. For example, the expression level of ABF-0020153 is weakly, but significantly, induced by oxidative or osmotic stresses (supplementary data). Such genes were discarded for the remainder of the study. Eventually we retained, for further investigation, ten genes satisfying the criteria above. Three of these genes display a low expression level (mean of log expression level below 9): ABF-0018436, ABF-0018449 and ABF-20529 (yafS). Three others have high expression levels (mean of log expression level above 11): ABF-0016832 (yqcD), ABF-0017965 (lpxC) and ABF-0015677 (yhbN). Finally, four genes have an intermediate expression level (mean of log expression level between 9 and 11): ABF-0020403 (rraA), ABF-0015073, ABF-0016418 (ddlA) and ABF-0018748. Their expression level profiles, measured from micro-arrays, are presented in Figure 2A.


lpxC and yafS are the most suitable internal controls to normalize real time RT-qPCR expression in the phytopathogenic bacteria Dickeya dadantii.

Hommais F, Zghidi-Abouzid O, Oger-Desfeux C, Pineau-Chapelle E, Van Gijsegem F, Nasser W, Reverchon S - PLoS ONE (2011)

Expression profiles of the 10 genes selected for the stability of their expression level and the synthetic RNA pAW.Gene expression profiles are measured from micro-arrays (A) and from quantitative real time RT-PCR (B). The first 32 conditions are those measured in exponential phase and the last 32 conditions are those measured in stationary phase. The order of conditions is M63 supplemented with saccharose (S), M63 supplemented with saccharose and Saintpaulia leaf extract (PL), M63 supplemented with saccharose and PGA (P), and M63 supplemented with saccharose, leaves and PGA (PLP). The order of stress is: no stress, oxidative stress, acid stress, and osmotic stress.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102694&req=5

pone-0020269-g002: Expression profiles of the 10 genes selected for the stability of their expression level and the synthetic RNA pAW.Gene expression profiles are measured from micro-arrays (A) and from quantitative real time RT-PCR (B). The first 32 conditions are those measured in exponential phase and the last 32 conditions are those measured in stationary phase. The order of conditions is M63 supplemented with saccharose (S), M63 supplemented with saccharose and Saintpaulia leaf extract (PL), M63 supplemented with saccharose and PGA (P), and M63 supplemented with saccharose, leaves and PGA (PLP). The order of stress is: no stress, oxidative stress, acid stress, and osmotic stress.
Mentions: Next, we required that the selected genes satisfied the following criteria: (i) they are associated, as far as possible, with different functional categories to minimize the risk of choosing co-regulated genes; (ii) they belong to different transcription units; (iii) they show no sign of correlation between the expression profiles and growth conditions (supplementary data). Among the 49 genes, 22 encode metabolic enzymes, 9 genes encode membrane proteins involved in membrane traffic and 6 are predicted to encode transcriptional regulators, the others encoding hypothetical proteins or proteins with unknown function. No functional category is overrepresented, with the exception of three genes which encode proteins involved in heme biosynthesis: hemF and hemXY. One of the most stable genes is lpxC (ABF-0017965). This gene encodes for a UDP-3-O-acyl N-acetylglucosamine deacetylase that catalyzes the second step of the lipid A-precursor biosynthetic pathway [11]. Seven enzymes are involved in this pathway and most of the encoding genes are significantly down-regulated in oxidative stress when grown at the exponential phase, suggesting that the expression of lpxC (ABF-0017965) is stable whereas that of the other genes from the same pathway is under regulation. This means that enzymes from the same metabolic pathway may be differentially regulated. Among the 49 selected genes, only hemY, hemX and yadH, yadG are organized in operons. hemXY are transcribed with two other genes, hemC and hemD. Remarkably, expression of hemCD is induced by osmotic stress (1.5 log), suggesting a difference in the regulation mechanisms between hemXY and hemCD. This is in accordance with a previous study, using E. coli, that demonstrates the presence of two transcription initiation sites in this operon [12]. The expression of the two other genes organized in operons, yadH and yadG, is very similar (supplementary data) and seems to be correlated to growth phases and stresses. These genes were discarded for the rest of the study. We examined the expression profiles of the 47 genes (supplementary data) and checked for systematic variation and correlations between expression and growth conditions. In spite of the weak variation in the expression levels of the selected genes, some correlations were found between the expression levels of several genes and the growth conditions tested. For example, the expression level of ABF-0020153 is weakly, but significantly, induced by oxidative or osmotic stresses (supplementary data). Such genes were discarded for the remainder of the study. Eventually we retained, for further investigation, ten genes satisfying the criteria above. Three of these genes display a low expression level (mean of log expression level below 9): ABF-0018436, ABF-0018449 and ABF-20529 (yafS). Three others have high expression levels (mean of log expression level above 11): ABF-0016832 (yqcD), ABF-0017965 (lpxC) and ABF-0015677 (yhbN). Finally, four genes have an intermediate expression level (mean of log expression level between 9 and 11): ABF-0020403 (rraA), ABF-0015073, ABF-0016418 (ddlA) and ABF-0018748. Their expression level profiles, measured from micro-arrays, are presented in Figure 2A.

Bottom Line: Out of these, we retained 10 genes representing different functional categories, different levels of expression (low, medium, and high) and with no systematic variation in expression correlating with growth conditions.The two most stable genes (ABF-0017965 (lpxC) and ABF-0020529 (yafS) were successfully used for normalization of RT-qPCR data.Finally, we demonstrated that the ortholog of lpxC and yafS in Pectobacterium atrosepticum also showed stable expression in diverse growth conditions.

View Article: PubMed Central - PubMed

Affiliation: Unité Microbiologie, Adaptation, Pathogénie CNRS-INSA-UCBL UMR 5240, Université Lyon 1, Villeurbanne, France. hommais@univ-lyon1.fr

ABSTRACT

Background: Quantitative RT-PCR is the method of choice for studying, with both sensitivity and accuracy, the expression of genes. A reliable normalization of the data, using several reference genes, is critical for an accurate quantification of gene expression. Here, we propose a set of reference genes, of the phytopathogenic bacteria Dickeya dadantii and Pectobacterium atrosepticum, which are stable in a wide range of growth conditions.

Results: We extracted, from a D. dadantii micro-array transcript profile dataset comprising thirty-two different growth conditions, an initial set of 49 expressed genes with very low variation in gene expression. Out of these, we retained 10 genes representing different functional categories, different levels of expression (low, medium, and high) and with no systematic variation in expression correlating with growth conditions. We measured the expression of these reference gene candidates using quantitative RT-PCR in 50 different experimental conditions, mimicking the environment encountered by the bacteria in their host and directly during the infection process in planta. The two most stable genes (ABF-0017965 (lpxC) and ABF-0020529 (yafS) were successfully used for normalization of RT-qPCR data. Finally, we demonstrated that the ortholog of lpxC and yafS in Pectobacterium atrosepticum also showed stable expression in diverse growth conditions.

Conclusions: We have identified at least two genes, lpxC (ABF-0017965) and yafS (ABF-0020509), whose expressions are stable in a wide range of growth conditions and during infection. Thus, these genes are considered suitable for use as reference genes for the normalization of real-time RT-qPCR data of the two main pectinolytic phytopathogenic bacteria D. dadantii and P. atrosepticum and, probably, of other Enterobacteriaceae. Moreover, we defined general criteria to select good reference genes in bacteria.

Show MeSH
Related in: MedlinePlus