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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

Confirmation of DAO and GlcB knockdowns: (a) PCR amplification using flanking region primer pair Primer 1/Primer 2, lane 1 is wild-type (WT), lane 2 is 1 kb DNA ladder (MBI Fermentas), lane 3 is pMV361-DAO plasmid construct, lane 4 is DAO_KD. (b) PCR using flanking region primer pair Primer 1/Primer 3: lane 1 is WT, lane 2 is 1 kb DNA ladder (MBI Fermentas), lane 3 is pMV361-DAO plasmid construct, lane 4 is DAO_KD. (c) Confirmation of DAO knockdown by expression analysis, WT and DAO_KD refer to wild-type and DAO knockdown strain. (d) Bar graph represents expression of DAO in WT and DAO_KD, Hsp65 was used as loading control, significance analysis was done by Student’s t-test, ***p < 0.001. (e) Confirmation of DAO knockdown by qRT-PCR, 16S rRNA was used as a reference for normalization. (f) PCR using flanking region primer pair Primer 1/Primer 2: lane 1 is 1 kb DNA ladder (MBI Fermentas), lane 2 is pMV361-GlcB plasmid construct, lane 3 is GlcB_KD, lane 4 is WT. (g) PCR using flanking region primer pair Primer 1/Primer 3, lane 1 1 kb DNA ladder (MBI Fermentas), lane 2 is pMV361-GlcB plasmid construct, lane 3 is GlcB_KD, lane 4 is WT. (h) Confirmation of GlcB knockdown by expression analysis, WT and GlcB_KD refer to wild-type and GlcB knockdown. (i) Bar graph represents expression of GlcB in WT and GlcB_KD, Hsp65 was used as loading control, significance analysis was done by Student’s t-test, ***p < 0.001. (j) Confirmation of GlcB knockdown by qRT-PCR, 16S rRNA was used as a reference for normalization. All immunoblots are representative of three independent experiments with similar observations. All transcript profiles are mean ± SD of three independent experiments performed in triplicates. (k) Diagram showing generalized knockdown construct map and its arrangement after integration into Mtb-Ra genome. The construction is based on the scheme proposed by Pena et al. (1997)30.
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f1: Confirmation of DAO and GlcB knockdowns: (a) PCR amplification using flanking region primer pair Primer 1/Primer 2, lane 1 is wild-type (WT), lane 2 is 1 kb DNA ladder (MBI Fermentas), lane 3 is pMV361-DAO plasmid construct, lane 4 is DAO_KD. (b) PCR using flanking region primer pair Primer 1/Primer 3: lane 1 is WT, lane 2 is 1 kb DNA ladder (MBI Fermentas), lane 3 is pMV361-DAO plasmid construct, lane 4 is DAO_KD. (c) Confirmation of DAO knockdown by expression analysis, WT and DAO_KD refer to wild-type and DAO knockdown strain. (d) Bar graph represents expression of DAO in WT and DAO_KD, Hsp65 was used as loading control, significance analysis was done by Student’s t-test, ***p < 0.001. (e) Confirmation of DAO knockdown by qRT-PCR, 16S rRNA was used as a reference for normalization. (f) PCR using flanking region primer pair Primer 1/Primer 2: lane 1 is 1 kb DNA ladder (MBI Fermentas), lane 2 is pMV361-GlcB plasmid construct, lane 3 is GlcB_KD, lane 4 is WT. (g) PCR using flanking region primer pair Primer 1/Primer 3, lane 1 1 kb DNA ladder (MBI Fermentas), lane 2 is pMV361-GlcB plasmid construct, lane 3 is GlcB_KD, lane 4 is WT. (h) Confirmation of GlcB knockdown by expression analysis, WT and GlcB_KD refer to wild-type and GlcB knockdown. (i) Bar graph represents expression of GlcB in WT and GlcB_KD, Hsp65 was used as loading control, significance analysis was done by Student’s t-test, ***p < 0.001. (j) Confirmation of GlcB knockdown by qRT-PCR, 16S rRNA was used as a reference for normalization. All immunoblots are representative of three independent experiments with similar observations. All transcript profiles are mean ± SD of three independent experiments performed in triplicates. (k) Diagram showing generalized knockdown construct map and its arrangement after integration into Mtb-Ra genome. The construction is based on the scheme proposed by Pena et al. (1997)30.

Mentions: The recombinant strains were constructed by knocking down the expression of desired genes. This was achieved by gene silencing using antisense approach. For this amplicons coding for DAO and GlcB were cloned downstream of the hsp60 promoter in an antisense orientation in mycobacterial integrative vector pMV361. The hsp60 promoter is a constitutive promoter ensuring basal level of antisense transcripts under all conditions. The transcripts from antisense construct bind to the sense transcript being transcribed from the native gene and form a RNA duplex. This results in inhibition of native gene translation and overall decrease in cellular level of targeted gene product. Orientation of constructs in pMV361 was confirmed by PCR and pMV-DAO construct gave an amplicon of ~1.1 kb (Fig. 1a) with primer-1 and primer-2. This amplicon consisted of sequences coding for Hsp60 and DAO (Fig. 1k). Amplification with primer-1 and primer-3 flanking hsp60 and kanamycin resistance marker (aph) gave an amplicon of ~2.3 kb (Fig. 1b). No amplification was seen in WT. The immunoblotting studies with anti-DAO antibody (Fig. 1c) showed down-regulation of DAO expression in DAO_KD compared to WT (Fig. 1d). This was also confirmed by transcript analysis using real-time PCR (RT-PCR) (Fig. 1e). Similarly, primer pairs (primer-1 and primer-2) flanking hsp60 and glcB as well as hsp60 and aph (primer-1 and primer-3) were used to confirm the GlcB antisense constructs integration in Mtb-Ra genome (Fig. 1f,g). The immunoblotting studies with anti-GlcB antibody (Fig. 1h) showed down-regulation of GlcB expression in GlcB_KD (Fig. 1i). This was also confirmed by transcript analysis using RT-PCR (Fig. 1j). The genotype map shows the construction scheme for recombinant development and its arrangement after integration into Mtb-Ra genome (Fig. 1k).


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

Singh KS, Singh SK - Sci Rep (2015)

Confirmation of DAO and GlcB knockdowns: (a) PCR amplification using flanking region primer pair Primer 1/Primer 2, lane 1 is wild-type (WT), lane 2 is 1 kb DNA ladder (MBI Fermentas), lane 3 is pMV361-DAO plasmid construct, lane 4 is DAO_KD. (b) PCR using flanking region primer pair Primer 1/Primer 3: lane 1 is WT, lane 2 is 1 kb DNA ladder (MBI Fermentas), lane 3 is pMV361-DAO plasmid construct, lane 4 is DAO_KD. (c) Confirmation of DAO knockdown by expression analysis, WT and DAO_KD refer to wild-type and DAO knockdown strain. (d) Bar graph represents expression of DAO in WT and DAO_KD, Hsp65 was used as loading control, significance analysis was done by Student’s t-test, ***p < 0.001. (e) Confirmation of DAO knockdown by qRT-PCR, 16S rRNA was used as a reference for normalization. (f) PCR using flanking region primer pair Primer 1/Primer 2: lane 1 is 1 kb DNA ladder (MBI Fermentas), lane 2 is pMV361-GlcB plasmid construct, lane 3 is GlcB_KD, lane 4 is WT. (g) PCR using flanking region primer pair Primer 1/Primer 3, lane 1 1 kb DNA ladder (MBI Fermentas), lane 2 is pMV361-GlcB plasmid construct, lane 3 is GlcB_KD, lane 4 is WT. (h) Confirmation of GlcB knockdown by expression analysis, WT and GlcB_KD refer to wild-type and GlcB knockdown. (i) Bar graph represents expression of GlcB in WT and GlcB_KD, Hsp65 was used as loading control, significance analysis was done by Student’s t-test, ***p < 0.001. (j) Confirmation of GlcB knockdown by qRT-PCR, 16S rRNA was used as a reference for normalization. All immunoblots are representative of three independent experiments with similar observations. All transcript profiles are mean ± SD of three independent experiments performed in triplicates. (k) Diagram showing generalized knockdown construct map and its arrangement after integration into Mtb-Ra genome. The construction is based on the scheme proposed by Pena et al. (1997)30.
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Related In: Results  -  Collection

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

f1: Confirmation of DAO and GlcB knockdowns: (a) PCR amplification using flanking region primer pair Primer 1/Primer 2, lane 1 is wild-type (WT), lane 2 is 1 kb DNA ladder (MBI Fermentas), lane 3 is pMV361-DAO plasmid construct, lane 4 is DAO_KD. (b) PCR using flanking region primer pair Primer 1/Primer 3: lane 1 is WT, lane 2 is 1 kb DNA ladder (MBI Fermentas), lane 3 is pMV361-DAO plasmid construct, lane 4 is DAO_KD. (c) Confirmation of DAO knockdown by expression analysis, WT and DAO_KD refer to wild-type and DAO knockdown strain. (d) Bar graph represents expression of DAO in WT and DAO_KD, Hsp65 was used as loading control, significance analysis was done by Student’s t-test, ***p < 0.001. (e) Confirmation of DAO knockdown by qRT-PCR, 16S rRNA was used as a reference for normalization. (f) PCR using flanking region primer pair Primer 1/Primer 2: lane 1 is 1 kb DNA ladder (MBI Fermentas), lane 2 is pMV361-GlcB plasmid construct, lane 3 is GlcB_KD, lane 4 is WT. (g) PCR using flanking region primer pair Primer 1/Primer 3, lane 1 1 kb DNA ladder (MBI Fermentas), lane 2 is pMV361-GlcB plasmid construct, lane 3 is GlcB_KD, lane 4 is WT. (h) Confirmation of GlcB knockdown by expression analysis, WT and GlcB_KD refer to wild-type and GlcB knockdown. (i) Bar graph represents expression of GlcB in WT and GlcB_KD, Hsp65 was used as loading control, significance analysis was done by Student’s t-test, ***p < 0.001. (j) Confirmation of GlcB knockdown by qRT-PCR, 16S rRNA was used as a reference for normalization. All immunoblots are representative of three independent experiments with similar observations. All transcript profiles are mean ± SD of three independent experiments performed in triplicates. (k) Diagram showing generalized knockdown construct map and its arrangement after integration into Mtb-Ra genome. The construction is based on the scheme proposed by Pena et al. (1997)30.
Mentions: The recombinant strains were constructed by knocking down the expression of desired genes. This was achieved by gene silencing using antisense approach. For this amplicons coding for DAO and GlcB were cloned downstream of the hsp60 promoter in an antisense orientation in mycobacterial integrative vector pMV361. The hsp60 promoter is a constitutive promoter ensuring basal level of antisense transcripts under all conditions. The transcripts from antisense construct bind to the sense transcript being transcribed from the native gene and form a RNA duplex. This results in inhibition of native gene translation and overall decrease in cellular level of targeted gene product. Orientation of constructs in pMV361 was confirmed by PCR and pMV-DAO construct gave an amplicon of ~1.1 kb (Fig. 1a) with primer-1 and primer-2. This amplicon consisted of sequences coding for Hsp60 and DAO (Fig. 1k). Amplification with primer-1 and primer-3 flanking hsp60 and kanamycin resistance marker (aph) gave an amplicon of ~2.3 kb (Fig. 1b). No amplification was seen in WT. The immunoblotting studies with anti-DAO antibody (Fig. 1c) showed down-regulation of DAO expression in DAO_KD compared to WT (Fig. 1d). This was also confirmed by transcript analysis using real-time PCR (RT-PCR) (Fig. 1e). Similarly, primer pairs (primer-1 and primer-2) flanking hsp60 and glcB as well as hsp60 and aph (primer-1 and primer-3) were used to confirm the GlcB antisense constructs integration in Mtb-Ra genome (Fig. 1f,g). The immunoblotting studies with anti-GlcB antibody (Fig. 1h) showed down-regulation of GlcB expression in GlcB_KD (Fig. 1i). This was also confirmed by transcript analysis using RT-PCR (Fig. 1j). The genotype map shows the construction scheme for recombinant development and its arrangement after integration into Mtb-Ra genome (Fig. 1k).

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