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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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Phenotype array results for peptide nitrogen sources.Normalized values for positive wells in PMs 6–8 are presented. Time course measurement data up to 138 hours is shown. Each row represents a compound and each column represents MAP-WT or leuD mutant at a particular time point. Expression range is indicated at top left side of the figure.
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pone-0037884-g003: Phenotype array results for peptide nitrogen sources.Normalized values for positive wells in PMs 6–8 are presented. Time course measurement data up to 138 hours is shown. Each row represents a compound and each column represents MAP-WT or leuD mutant at a particular time point. Expression range is indicated at top left side of the figure.

Mentions: Biolog phenotype microarray (PM) for microbial cells is a high-throughput assay for testing utilization of various nutrients. The assay chemistry uses a redox dye to measure respiration of cells [20]. When the bacterial cell is able to utilize a specific test nutrient in a given PM well, redox dye is reduced producing purple color. Since the dye reduction is mostly irreversible under physiological conditions, the color accumulates in the well over a period of hours, amplifying the signal and integrating the amount of respiration over time [20]. There are eight PM plates which test for the utilization of the following nutrient sources; PM1, 2- carbon sources, PM3- nitrogen sources, PM4- phosphorus and sulphur sources, PM5- nutrient supplements, PM6, 7 and 8- peptide nitrogen sources. In this study, we have utilized PMs 1–8 to establish the complete metabolic profile of MAP WT and MAPΔleuD. Of the total of 760 phenotypes tested, 182 tests were positive for MAP WT (Table S3).PM results show that the most preferred carbon source for MAP was Tween 80. Most preferred simple sugars were D-ribose, D-tagatose, D-fucose, D-trehalose and α-D-glucose respectively. Tween 40 and Tween 20 were utilized at the rate similar to D-ribose. Among amino acids, L-leucine was utilized most efficiently as carbon source. The most used nitrogen sources were alloxan and D-mannosamine. The overall results for positive phenotypes in PM plates 1–5 are given in Fig. 2. As shown in Fig. 2, when compared to MAP WT, the ability to utilize most of the nutrients was impaired in MAPΔleuD. A similar trend was observed for peptide nitrogen sources (Fig. 3). Therefore, the deletion of leuD gene resulted in the impairment of overall nutrient utilization capacity of MAP. However, one exception was the ability of MAPΔleuD to utilize phosphonoacetic acid while MAP WT did not use this chemical effectively.


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

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

Phenotype array results for peptide nitrogen sources.Normalized values for positive wells in PMs 6–8 are presented. Time course measurement data up to 138 hours is shown. Each row represents a compound and each column represents MAP-WT or leuD mutant at a particular time point. Expression range is indicated at top left side of the figure.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037884-g003: Phenotype array results for peptide nitrogen sources.Normalized values for positive wells in PMs 6–8 are presented. Time course measurement data up to 138 hours is shown. Each row represents a compound and each column represents MAP-WT or leuD mutant at a particular time point. Expression range is indicated at top left side of the figure.
Mentions: Biolog phenotype microarray (PM) for microbial cells is a high-throughput assay for testing utilization of various nutrients. The assay chemistry uses a redox dye to measure respiration of cells [20]. When the bacterial cell is able to utilize a specific test nutrient in a given PM well, redox dye is reduced producing purple color. Since the dye reduction is mostly irreversible under physiological conditions, the color accumulates in the well over a period of hours, amplifying the signal and integrating the amount of respiration over time [20]. There are eight PM plates which test for the utilization of the following nutrient sources; PM1, 2- carbon sources, PM3- nitrogen sources, PM4- phosphorus and sulphur sources, PM5- nutrient supplements, PM6, 7 and 8- peptide nitrogen sources. In this study, we have utilized PMs 1–8 to establish the complete metabolic profile of MAP WT and MAPΔleuD. Of the total of 760 phenotypes tested, 182 tests were positive for MAP WT (Table S3).PM results show that the most preferred carbon source for MAP was Tween 80. Most preferred simple sugars were D-ribose, D-tagatose, D-fucose, D-trehalose and α-D-glucose respectively. Tween 40 and Tween 20 were utilized at the rate similar to D-ribose. Among amino acids, L-leucine was utilized most efficiently as carbon source. The most used nitrogen sources were alloxan and D-mannosamine. The overall results for positive phenotypes in PM plates 1–5 are given in Fig. 2. As shown in Fig. 2, when compared to MAP WT, the ability to utilize most of the nutrients was impaired in MAPΔleuD. A similar trend was observed for peptide nitrogen sources (Fig. 3). Therefore, the deletion of leuD gene resulted in the impairment of overall nutrient utilization capacity of MAP. However, one exception was the ability of MAPΔleuD to utilize phosphonoacetic acid while MAP WT did not use this chemical effectively.

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