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Phenotypic and transcriptomic response of auxotrophic Mycobacterium avium subsp. paratuberculosis leuD mutant under environmental stress.

Chen JW, Scaria J, Chang YF - PLoS ONE (2012)

Bottom Line: Our results showed that deletion of leuD gene has a global effect on both MAP phenotypic and transcriptome response.The mutant strain had 30% less fatty acid content when compared to wildtype, thus supporting the results from transcriptional and computational analyses.Our results therefore reveal the intricate connection between the metabolism and virulence in MAP.

View Article: PubMed Central - PubMed

Affiliation: Department of Population Medicine & Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of severe gastroenteritis in cattle. To gain a better understanding of MAP virulence, we investigated the role of leuD gene in MAP metabolism and stress response. For this, we have constructed an auxotrophic strain of MAP by deleting the leuD gene using allelic exchange. The wildtype and mutant strains were then compared for metabolic phenotypic changes using Biolog phenotype microarrays. The responses of both strains to physiologically relevant stress conditions were assessed using DNA microarrays. Transcriptomic data was then analyzed in the context of cellular metabolic pathways and gene networks. Our results showed that deletion of leuD gene has a global effect on both MAP phenotypic and transcriptome response. At the metabolic level, the mutant strain lost the ability to utilize most of the carbon, nitrogen, sulphur, phosphorus and nutrient supplements as energy source. At the transcriptome level, more than 100 genes were differentially expressed in each of the stress condition tested. Systems level network analysis revealed that the differentially expressed genes were distributed throughout the gene network, thus explaining the global impact of leuD deletion in metabolic phenotype. Further, we find that leuD deletion impacted metabolic pathways associated with fatty acids. We verified this by experimentally estimating the total fatty acid content of both mutant and wildtype. The mutant strain had 30% less fatty acid content when compared to wildtype, thus supporting the results from transcriptional and computational analyses. Our results therefore reveal the intricate connection between the metabolism and virulence in MAP.

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Related in: MedlinePlus

Gene interaction network of 26 genes that were found to be changing at all stress conditions.7A. STRING database was searched using list of 26 genes that were found to be differentially expressed 1.5 fold or more at all stress conditions tested. Nodes (genes) are indicated by locus tag or gene name. Edges (interactions) are denoted by the color code for source of evidence and are indicated at the top side of Fig. 7B in the inset. 7B. List of predicted interaction partners for these 26 DEGs. List of predicted interaction partners are given as locus tag or gene name in the left most column. The evidence source for each interaction is represented as columns. Cumulative evidence score is given in the right most columns.
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pone-0037884-g007: Gene interaction network of 26 genes that were found to be changing at all stress conditions.7A. STRING database was searched using list of 26 genes that were found to be differentially expressed 1.5 fold or more at all stress conditions tested. Nodes (genes) are indicated by locus tag or gene name. Edges (interactions) are denoted by the color code for source of evidence and are indicated at the top side of Fig. 7B in the inset. 7B. List of predicted interaction partners for these 26 DEGs. List of predicted interaction partners are given as locus tag or gene name in the left most column. The evidence source for each interaction is represented as columns. Cumulative evidence score is given in the right most columns.

Mentions: We further attempted to elucidate the role of 26 DEGs that were found changing at least 1.5 fold in all stress conditions. We used manual search of STRING database to find the interaction partners for these genes (Fig. 7A). This analysis predicted the following interaction partners with very high confidence evidence scores; groL, MAP_3239, MAP_3058c, dnaK and MAP_2204c (Fig. 7B). Cumulative results of this analysis suggest that 26 DEGs found to be changing in all stress conditions are involved in heat shock response and maintenance of cell viability.


Phenotypic and transcriptomic response of auxotrophic Mycobacterium avium subsp. paratuberculosis leuD mutant under environmental stress.

Chen JW, Scaria J, Chang YF - PLoS ONE (2012)

Gene interaction network of 26 genes that were found to be changing at all stress conditions.7A. STRING database was searched using list of 26 genes that were found to be differentially expressed 1.5 fold or more at all stress conditions tested. Nodes (genes) are indicated by locus tag or gene name. Edges (interactions) are denoted by the color code for source of evidence and are indicated at the top side of Fig. 7B in the inset. 7B. List of predicted interaction partners for these 26 DEGs. List of predicted interaction partners are given as locus tag or gene name in the left most column. The evidence source for each interaction is represented as columns. Cumulative evidence score is given in the right most columns.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366959&req=5

pone-0037884-g007: Gene interaction network of 26 genes that were found to be changing at all stress conditions.7A. STRING database was searched using list of 26 genes that were found to be differentially expressed 1.5 fold or more at all stress conditions tested. Nodes (genes) are indicated by locus tag or gene name. Edges (interactions) are denoted by the color code for source of evidence and are indicated at the top side of Fig. 7B in the inset. 7B. List of predicted interaction partners for these 26 DEGs. List of predicted interaction partners are given as locus tag or gene name in the left most column. The evidence source for each interaction is represented as columns. Cumulative evidence score is given in the right most columns.
Mentions: We further attempted to elucidate the role of 26 DEGs that were found changing at least 1.5 fold in all stress conditions. We used manual search of STRING database to find the interaction partners for these genes (Fig. 7A). This analysis predicted the following interaction partners with very high confidence evidence scores; groL, MAP_3239, MAP_3058c, dnaK and MAP_2204c (Fig. 7B). Cumulative results of this analysis suggest that 26 DEGs found to be changing in all stress conditions are involved in heat shock response and maintenance of cell viability.

Bottom Line: Our results showed that deletion of leuD gene has a global effect on both MAP phenotypic and transcriptome response.The mutant strain had 30% less fatty acid content when compared to wildtype, thus supporting the results from transcriptional and computational analyses.Our results therefore reveal the intricate connection between the metabolism and virulence in MAP.

View Article: PubMed Central - PubMed

Affiliation: Department of Population Medicine & Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of severe gastroenteritis in cattle. To gain a better understanding of MAP virulence, we investigated the role of leuD gene in MAP metabolism and stress response. For this, we have constructed an auxotrophic strain of MAP by deleting the leuD gene using allelic exchange. The wildtype and mutant strains were then compared for metabolic phenotypic changes using Biolog phenotype microarrays. The responses of both strains to physiologically relevant stress conditions were assessed using DNA microarrays. Transcriptomic data was then analyzed in the context of cellular metabolic pathways and gene networks. Our results showed that deletion of leuD gene has a global effect on both MAP phenotypic and transcriptome response. At the metabolic level, the mutant strain lost the ability to utilize most of the carbon, nitrogen, sulphur, phosphorus and nutrient supplements as energy source. At the transcriptome level, more than 100 genes were differentially expressed in each of the stress condition tested. Systems level network analysis revealed that the differentially expressed genes were distributed throughout the gene network, thus explaining the global impact of leuD deletion in metabolic phenotype. Further, we find that leuD deletion impacted metabolic pathways associated with fatty acids. We verified this by experimentally estimating the total fatty acid content of both mutant and wildtype. The mutant strain had 30% less fatty acid content when compared to wildtype, thus supporting the results from transcriptional and computational analyses. Our results therefore reveal the intricate connection between the metabolism and virulence in MAP.

Show MeSH
Related in: MedlinePlus