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Poly-L-glutamate/glutamine synthesis in the cell wall of Mycobacterium bovis is regulated in response to nitrogen availability.

Tripathi D, Chandra H, Bhatnagar R - BMC Microbiol. (2013)

Bottom Line: We observed that GS expression and activity decreased significantly in high nitrogen grown conditions.In high nitrogen conditions, the amount of PLG in cell wall was drastically reduced (below detectable limits) as compared to low nitrogen condition in M. bovis and in M. smegmatis strain complemented with M. bovis glnA1.Also, we have found that M. smegmatis complemented with M. bovis glnA1 was more efficient in biofilm formation than the wild type strain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biology and Genetic Engineering Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India. rakeshbhatnagar@jnu.ac.in.

ABSTRACT

Background: The cell wall of pathogenic mycobacteria is known to possess poly-L-glutamine (PLG) layer. PLG synthesis has been directly linked to glutamine synthetase (GS) enzyme. glnA1 gene encodes for GS enzyme in mycobacteria. PLG layer is absent in cell wall of avirulent Mycobacterium smegmatis, although M. smegmatis strain expressing GS enzyme of pathogenic mycobacteria can synthesize PLG layer in the cell wall. The role of GS enzyme has been extensively studied in Mycobacterium tuberculosis, however, little is known about GS enzyme in other mycobacterial species. Mycobacterium bovis, as an intracellular pathogen encounters nitrogen stress inside macrophages, thus it has developed nitrogen assimilatory pathways to survive in adverse conditions. We have investigated the expression and activity of M. bovis GS in response to nitrogen availability and effect on synthesis of PLG layer in the cell wall. M. smegmatis was used as a model to study the behaviour of glnA1 locus of M. bovis.

Results: We observed that GS expression and activity decreased significantly in high nitrogen grown conditions. In high nitrogen conditions, the amount of PLG in cell wall was drastically reduced (below detectable limits) as compared to low nitrogen condition in M. bovis and in M. smegmatis strain complemented with M. bovis glnA1. Additionally, biofilm formation by M. smegmatis strain complemented with M. bovis glnA1 was increased than the wild type M. smegmatis strain.

Conclusions: The physiological regulation of GS in M. bovis was found to be similar to that reported in other mycobacteria but this data revealed that PLG synthesis in the cell wall of pathogenic mycobacteria occurs only in nitrogen limiting conditions and on the contrary high nitrogen conditions inhibit PLG synthesis. This indicates that PLG synthesis may be a form of nitrogen assimilatory pathway during ammonium starvation in virulent mycobacteria. Also, we have found that M. smegmatis complemented with M. bovis glnA1 was more efficient in biofilm formation than the wild type strain. This indicates that PLG layer favors biofilm formation. This study demonstrate that the nitrogen availability not only regulates GS expression and activity in M. bovis but also affects cell surface properties by modulating synthesis of PLG.

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Immunogold localization of PLG in the cell wall of mycobacteria during nitrogen availability. Shown are transmission electron micrographs of the wild type M. bovis and recombinant M. smegmatis strain (MSFP, MSP1 and MSP2) in low and high nitrogen condition. The black arrows shown in the images marked the gold particle around the cell wall periphery in low nitrogen condition.
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Figure 6: Immunogold localization of PLG in the cell wall of mycobacteria during nitrogen availability. Shown are transmission electron micrographs of the wild type M. bovis and recombinant M. smegmatis strain (MSFP, MSP1 and MSP2) in low and high nitrogen condition. The black arrows shown in the images marked the gold particle around the cell wall periphery in low nitrogen condition.

Mentions: In order to validate the above observation, immunogold localization study was done to localize PLG in the cell wall. Exponential phase cultures were taken for localization studies. Gold particles were observed at the cell periphery of bacteria gown in nitrogen limiting conditions (FigureĀ 6). While no gold particles were seen at the cell periphery of mycobacterial strains grown in high nitrogen condition, as expected. Interestingly the less number of gold particles was found in the MSP2 strain as low amount of GS expression and PLG formation.


Poly-L-glutamate/glutamine synthesis in the cell wall of Mycobacterium bovis is regulated in response to nitrogen availability.

Tripathi D, Chandra H, Bhatnagar R - BMC Microbiol. (2013)

Immunogold localization of PLG in the cell wall of mycobacteria during nitrogen availability. Shown are transmission electron micrographs of the wild type M. bovis and recombinant M. smegmatis strain (MSFP, MSP1 and MSP2) in low and high nitrogen condition. The black arrows shown in the images marked the gold particle around the cell wall periphery in low nitrogen condition.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852478&req=5

Figure 6: Immunogold localization of PLG in the cell wall of mycobacteria during nitrogen availability. Shown are transmission electron micrographs of the wild type M. bovis and recombinant M. smegmatis strain (MSFP, MSP1 and MSP2) in low and high nitrogen condition. The black arrows shown in the images marked the gold particle around the cell wall periphery in low nitrogen condition.
Mentions: In order to validate the above observation, immunogold localization study was done to localize PLG in the cell wall. Exponential phase cultures were taken for localization studies. Gold particles were observed at the cell periphery of bacteria gown in nitrogen limiting conditions (FigureĀ 6). While no gold particles were seen at the cell periphery of mycobacterial strains grown in high nitrogen condition, as expected. Interestingly the less number of gold particles was found in the MSP2 strain as low amount of GS expression and PLG formation.

Bottom Line: We observed that GS expression and activity decreased significantly in high nitrogen grown conditions.In high nitrogen conditions, the amount of PLG in cell wall was drastically reduced (below detectable limits) as compared to low nitrogen condition in M. bovis and in M. smegmatis strain complemented with M. bovis glnA1.Also, we have found that M. smegmatis complemented with M. bovis glnA1 was more efficient in biofilm formation than the wild type strain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biology and Genetic Engineering Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India. rakeshbhatnagar@jnu.ac.in.

ABSTRACT

Background: The cell wall of pathogenic mycobacteria is known to possess poly-L-glutamine (PLG) layer. PLG synthesis has been directly linked to glutamine synthetase (GS) enzyme. glnA1 gene encodes for GS enzyme in mycobacteria. PLG layer is absent in cell wall of avirulent Mycobacterium smegmatis, although M. smegmatis strain expressing GS enzyme of pathogenic mycobacteria can synthesize PLG layer in the cell wall. The role of GS enzyme has been extensively studied in Mycobacterium tuberculosis, however, little is known about GS enzyme in other mycobacterial species. Mycobacterium bovis, as an intracellular pathogen encounters nitrogen stress inside macrophages, thus it has developed nitrogen assimilatory pathways to survive in adverse conditions. We have investigated the expression and activity of M. bovis GS in response to nitrogen availability and effect on synthesis of PLG layer in the cell wall. M. smegmatis was used as a model to study the behaviour of glnA1 locus of M. bovis.

Results: We observed that GS expression and activity decreased significantly in high nitrogen grown conditions. In high nitrogen conditions, the amount of PLG in cell wall was drastically reduced (below detectable limits) as compared to low nitrogen condition in M. bovis and in M. smegmatis strain complemented with M. bovis glnA1. Additionally, biofilm formation by M. smegmatis strain complemented with M. bovis glnA1 was increased than the wild type M. smegmatis strain.

Conclusions: The physiological regulation of GS in M. bovis was found to be similar to that reported in other mycobacteria but this data revealed that PLG synthesis in the cell wall of pathogenic mycobacteria occurs only in nitrogen limiting conditions and on the contrary high nitrogen conditions inhibit PLG synthesis. This indicates that PLG synthesis may be a form of nitrogen assimilatory pathway during ammonium starvation in virulent mycobacteria. Also, we have found that M. smegmatis complemented with M. bovis glnA1 was more efficient in biofilm formation than the wild type strain. This indicates that PLG layer favors biofilm formation. This study demonstrate that the nitrogen availability not only regulates GS expression and activity in M. bovis but also affects cell surface properties by modulating synthesis of PLG.

Show MeSH
Related in: MedlinePlus