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The Mycobacterium tuberculosis H37Ra gene MRA_1916 causes growth defects upon down-regulation.

Singh KS, Singh SK - Sci Rep (2015)

Bottom Line: The present study was taken-up to understand the role of DAO during different stages of growth and effect of its down-regulation on growth.Ethyl bromopyruvate (BrP) was used as an inhibitor of GlcB.Growth profile of WT under oxygen and nutritional stress showed changes in expression of DAO, GlcB, PckA (phosphoenolpyruvate carboxykinase: MRA_0219) and GlyA1 (serine hydroxymethyltransferase: MRA_1104).

View Article: PubMed Central - PubMed

Affiliation: Microbiology Division, CSIR-Central Drug Research Institute, B.S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow, U.P., India, Pin: 226031.

ABSTRACT
D-amino acid oxidases play an important role in converting D-amino acids to their corresponding α-keto acids. MRA_1916 of Mycobacterium tuberculosis H37Ra (Mtb-Ra) is annotated to be a D-amino acid oxidase (DAO). However, not much information is available about its physiological role during Mtb-Ra growth and survival. The present study was taken-up to understand the role of DAO during different stages of growth and effect of its down-regulation on growth. Recombinant Mtb-Ra strains with DAO and GlcB (malate synthase: MRA_1848) gene knockdown were developed and their growth was studied using Microtiter Alamar Blue Assay (MABA) with glycerol, acetate and glycine as a carbon source. Ethyl bromopyruvate (BrP) was used as an inhibitor of GlcB. MABA study showed inhibition of wild-type (WT) and knockdowns in the presence of BrP (2.5mM). However, growth inhibition of WT was less noticeable at lower concentrations of BrP. Mtb-Ra with DAO knockdown showed poor utilization of glycine in the presence of BrP. The DAO localization study showed its prominent distribution in cytosolic fraction and to some extent in cell wall and membrane fractions. Growth profile of WT under oxygen and nutritional stress showed changes in expression of DAO, GlcB, PckA (phosphoenolpyruvate carboxykinase: MRA_0219) and GlyA1 (serine hydroxymethyltransferase: MRA_1104).

No MeSH data available.


Related in: MedlinePlus

Effect of supplementation on growth: (a) Growth of DAO_KD and WT on different carbon sources with or without acetate or glyoxylate supplementation. (b) Growth of GlcB_KD and WT on different carbon sources with or without acetate or glyoxylate supplementation. Gly, Ac, Glo, Ser and Glyc refer to glycine, acetate, glyoxylate, serine and glycerol respectively. The data are mean ± SD of three independent experiments performed in triplicates, significance analysis was done by Student’s t-test, nsp > 0.05, *p < 0.05, ** < 0.01, ***p < 0.001.
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f3: Effect of supplementation on growth: (a) Growth of DAO_KD and WT on different carbon sources with or without acetate or glyoxylate supplementation. (b) Growth of GlcB_KD and WT on different carbon sources with or without acetate or glyoxylate supplementation. Gly, Ac, Glo, Ser and Glyc refer to glycine, acetate, glyoxylate, serine and glycerol respectively. The data are mean ± SD of three independent experiments performed in triplicates, significance analysis was done by Student’s t-test, nsp > 0.05, *p < 0.05, ** < 0.01, ***p < 0.001.

Mentions: However, comparatively higher inhibition of DAO_KD on glycine as a carbon source required further study. Hence, we performed supplementation studies using one of these (glycerol, glycine, serine or glyoxylate) as a source of carbon with or without additional acetate or glyoxylate supplementation (5 mM) and measured the growth by MABA (Fig. 3a). The growth retardation was more appreciable in DAO_KD in comparison of WT on media containing glycine alone (p = 0.0449) or glycine+glyoxylate (p = 0.0142). Similarly, significant growth retardation was seen in DAO_KD than in WT while using serine alone (p = 0.0015) or serine+glyoxylate (p = 0.026). The growth restoration was observed upon acetate supplementation to serine containing medium. Similarly, enhanced growth was observed in glyoxylate+acetate compared to glyoxylate alone with more restoration in WT than in DAO_KD. No difference in growth of WT and DAO_KD was noticed when glycerol was used as a primary carbon source and no effect of additional supplementation of glyoxylate or acetate was noticed (Fig. 3a).


The Mycobacterium tuberculosis H37Ra gene MRA_1916 causes growth defects upon down-regulation.

Singh KS, Singh SK - Sci Rep (2015)

Effect of supplementation on growth: (a) Growth of DAO_KD and WT on different carbon sources with or without acetate or glyoxylate supplementation. (b) Growth of GlcB_KD and WT on different carbon sources with or without acetate or glyoxylate supplementation. Gly, Ac, Glo, Ser and Glyc refer to glycine, acetate, glyoxylate, serine and glycerol respectively. The data are mean ± SD of three independent experiments performed in triplicates, significance analysis was done by Student’s t-test, nsp > 0.05, *p < 0.05, ** < 0.01, ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4632087&req=5

f3: Effect of supplementation on growth: (a) Growth of DAO_KD and WT on different carbon sources with or without acetate or glyoxylate supplementation. (b) Growth of GlcB_KD and WT on different carbon sources with or without acetate or glyoxylate supplementation. Gly, Ac, Glo, Ser and Glyc refer to glycine, acetate, glyoxylate, serine and glycerol respectively. The data are mean ± SD of three independent experiments performed in triplicates, significance analysis was done by Student’s t-test, nsp > 0.05, *p < 0.05, ** < 0.01, ***p < 0.001.
Mentions: However, comparatively higher inhibition of DAO_KD on glycine as a carbon source required further study. Hence, we performed supplementation studies using one of these (glycerol, glycine, serine or glyoxylate) as a source of carbon with or without additional acetate or glyoxylate supplementation (5 mM) and measured the growth by MABA (Fig. 3a). The growth retardation was more appreciable in DAO_KD in comparison of WT on media containing glycine alone (p = 0.0449) or glycine+glyoxylate (p = 0.0142). Similarly, significant growth retardation was seen in DAO_KD than in WT while using serine alone (p = 0.0015) or serine+glyoxylate (p = 0.026). The growth restoration was observed upon acetate supplementation to serine containing medium. Similarly, enhanced growth was observed in glyoxylate+acetate compared to glyoxylate alone with more restoration in WT than in DAO_KD. No difference in growth of WT and DAO_KD was noticed when glycerol was used as a primary carbon source and no effect of additional supplementation of glyoxylate or acetate was noticed (Fig. 3a).

Bottom Line: The present study was taken-up to understand the role of DAO during different stages of growth and effect of its down-regulation on growth.Ethyl bromopyruvate (BrP) was used as an inhibitor of GlcB.Growth profile of WT under oxygen and nutritional stress showed changes in expression of DAO, GlcB, PckA (phosphoenolpyruvate carboxykinase: MRA_0219) and GlyA1 (serine hydroxymethyltransferase: MRA_1104).

View Article: PubMed Central - PubMed

Affiliation: Microbiology Division, CSIR-Central Drug Research Institute, B.S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow, U.P., India, Pin: 226031.

ABSTRACT
D-amino acid oxidases play an important role in converting D-amino acids to their corresponding α-keto acids. MRA_1916 of Mycobacterium tuberculosis H37Ra (Mtb-Ra) is annotated to be a D-amino acid oxidase (DAO). However, not much information is available about its physiological role during Mtb-Ra growth and survival. The present study was taken-up to understand the role of DAO during different stages of growth and effect of its down-regulation on growth. Recombinant Mtb-Ra strains with DAO and GlcB (malate synthase: MRA_1848) gene knockdown were developed and their growth was studied using Microtiter Alamar Blue Assay (MABA) with glycerol, acetate and glycine as a carbon source. Ethyl bromopyruvate (BrP) was used as an inhibitor of GlcB. MABA study showed inhibition of wild-type (WT) and knockdowns in the presence of BrP (2.5mM). However, growth inhibition of WT was less noticeable at lower concentrations of BrP. Mtb-Ra with DAO knockdown showed poor utilization of glycine in the presence of BrP. The DAO localization study showed its prominent distribution in cytosolic fraction and to some extent in cell wall and membrane fractions. Growth profile of WT under oxygen and nutritional stress showed changes in expression of DAO, GlcB, PckA (phosphoenolpyruvate carboxykinase: MRA_0219) and GlyA1 (serine hydroxymethyltransferase: MRA_1104).

No MeSH data available.


Related in: MedlinePlus