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Gene encoding gamma-carbonic anhydrase is cotranscribed with argC and induced in response to stationary phase and high CO2 in Azospirillum brasilense Sp7.

Kaur S, Mishra MN, Tripathi AK - BMC Microbiol. (2010)

Bottom Line: Reverse transcription-PCR analysis indicated that gca1 in A. brasilense is co-transcribed with its upstream gene annotated as argC, which encodes a putative N-acetyl-gamma-glutamate-phosphate reductase. 5'-RACE also demonstrated that there was no transcription start site between argC and gca1, and the transcription start site located upstream of argC transcribed both the genes (argC-gca1).Using transcriptional fusions of argC-gca1 upstream region with promoterless lacZ, we further demonstrated that gca1 upstream region did not have any promoter and its transcription occurred from a promoter located in the argC upstream region.The organization and regulation of this gene along with the putative argC gene suggests its involvement in arginine biosynthetic pathway instead of the predicted CO2 hydration.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi-221005, India. tripathianil@rediffmail.com

ABSTRACT

Background: Carbonic anhydrase (CA) is a ubiquitous enzyme catalyzing the reversible hydration of CO2 to bicarbonate, a reaction underlying diverse biochemical and physiological processes. Gamma class carbonic anhydrases (gamma-CAs) are widespread in prokaryotes but their physiological roles remain elusive. At present, only gamma-CA of Methanosarcina thermophila (Cam) has been shown to have CA activity. Genome analysis of a rhizobacterium Azospirillum brasilense, revealed occurrence of ORFs encoding one beta-CA and two gamma-CAs.

Results: One of the putative gamma-CA encoding genes of A. brasilense was cloned and overexpressed in E. coli. Electrometric assays for CA activity of the whole cell extracts overexpressing recombinant GCA1 did not show CO2 hydration activity. Reverse transcription-PCR analysis indicated that gca1 in A. brasilense is co-transcribed with its upstream gene annotated as argC, which encodes a putative N-acetyl-gamma-glutamate-phosphate reductase. 5'-RACE also demonstrated that there was no transcription start site between argC and gca1, and the transcription start site located upstream of argC transcribed both the genes (argC-gca1). Using transcriptional fusions of argC-gca1 upstream region with promoterless lacZ, we further demonstrated that gca1 upstream region did not have any promoter and its transcription occurred from a promoter located in the argC upstream region. The transcription of argC-gca1 operon was upregulated in stationary phase and at elevated CO2 atmosphere.

Conclusions: This study shows lack of CO2 hydration activity in a recombinant protein expressed from a gene predicted to encode a gamma-carbonic anhydrase in A. brasilense although it cross reacts with anti-Cam antibody raised against a well characterized gamma-CA. The organization and regulation of this gene along with the putative argC gene suggests its involvement in arginine biosynthetic pathway instead of the predicted CO2 hydration.

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Heterologous overexpression, purification and western blot analysis of recombinant Gca1 of A. brasilense (A) SDS-PAGE gel electrophoresis (15%) of uninduced (lane 2) and induced (lane 3) cell lysates of transformants harboring pSK7. The Gca1 protein overproduced in E. coli pSK7 is encircled. Low range molecular weight marker, Bangalore Genei (lane 1). (B). Purification of recombinant Gca1 of A. brasilense under denaturing conditions SDS-PAGE gel (15%) showing induced crude extract of transformant harboring pSK7 (Lane 2); Ni-NTA purified His.Tag Gca1 (Lane 3); Low range molecular weight marker, Bangalore Genei (Lane 1). (C) Western blot analysis showing cross-reactivity of purified recombinant Gca1 with antisera raised against CAM.
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Figure 3: Heterologous overexpression, purification and western blot analysis of recombinant Gca1 of A. brasilense (A) SDS-PAGE gel electrophoresis (15%) of uninduced (lane 2) and induced (lane 3) cell lysates of transformants harboring pSK7. The Gca1 protein overproduced in E. coli pSK7 is encircled. Low range molecular weight marker, Bangalore Genei (lane 1). (B). Purification of recombinant Gca1 of A. brasilense under denaturing conditions SDS-PAGE gel (15%) showing induced crude extract of transformant harboring pSK7 (Lane 2); Ni-NTA purified His.Tag Gca1 (Lane 3); Low range molecular weight marker, Bangalore Genei (Lane 1). (C) Western blot analysis showing cross-reactivity of purified recombinant Gca1 with antisera raised against CAM.

Mentions: To examine whether gca1 gene encoded a functionally active protein, the gca1 ORF was amplified from the A. brasilense Sp7 genomic DNA and directionally cloned into the pET15b to construct an over-expression plasmid, pSK7 which, after confirmation by sequencing, was used for expression in E. coli and purification of the recombinant protein. SDS-PAGE analysis of extracts from uninduced versus induced cultures showed the presence of a protein of the expected size in the induced cells (Figure 3A). The size of the recombinant Gca1 (ca. 21 kDa) was larger than the predicted polypeptide size (19 kDa) due to the additional vector-encoded His-tag at the N-terminus of the protein. The recombinant protein was purified to homogeneity under denaturing conditions (Figure 3B). A clear band of purified protein in the position corresponding to the overexpressed protein in the crude lysate was visualized on the gel (Figure 3B). This band cross-reacted with anti-Cam antiserum (Figure 3C). The recognition of recombinant Gca1 with heterologous antibody indicates significant similarity between Gca1 and Cam.


Gene encoding gamma-carbonic anhydrase is cotranscribed with argC and induced in response to stationary phase and high CO2 in Azospirillum brasilense Sp7.

Kaur S, Mishra MN, Tripathi AK - BMC Microbiol. (2010)

Heterologous overexpression, purification and western blot analysis of recombinant Gca1 of A. brasilense (A) SDS-PAGE gel electrophoresis (15%) of uninduced (lane 2) and induced (lane 3) cell lysates of transformants harboring pSK7. The Gca1 protein overproduced in E. coli pSK7 is encircled. Low range molecular weight marker, Bangalore Genei (lane 1). (B). Purification of recombinant Gca1 of A. brasilense under denaturing conditions SDS-PAGE gel (15%) showing induced crude extract of transformant harboring pSK7 (Lane 2); Ni-NTA purified His.Tag Gca1 (Lane 3); Low range molecular weight marker, Bangalore Genei (Lane 1). (C) Western blot analysis showing cross-reactivity of purified recombinant Gca1 with antisera raised against CAM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2914000&req=5

Figure 3: Heterologous overexpression, purification and western blot analysis of recombinant Gca1 of A. brasilense (A) SDS-PAGE gel electrophoresis (15%) of uninduced (lane 2) and induced (lane 3) cell lysates of transformants harboring pSK7. The Gca1 protein overproduced in E. coli pSK7 is encircled. Low range molecular weight marker, Bangalore Genei (lane 1). (B). Purification of recombinant Gca1 of A. brasilense under denaturing conditions SDS-PAGE gel (15%) showing induced crude extract of transformant harboring pSK7 (Lane 2); Ni-NTA purified His.Tag Gca1 (Lane 3); Low range molecular weight marker, Bangalore Genei (Lane 1). (C) Western blot analysis showing cross-reactivity of purified recombinant Gca1 with antisera raised against CAM.
Mentions: To examine whether gca1 gene encoded a functionally active protein, the gca1 ORF was amplified from the A. brasilense Sp7 genomic DNA and directionally cloned into the pET15b to construct an over-expression plasmid, pSK7 which, after confirmation by sequencing, was used for expression in E. coli and purification of the recombinant protein. SDS-PAGE analysis of extracts from uninduced versus induced cultures showed the presence of a protein of the expected size in the induced cells (Figure 3A). The size of the recombinant Gca1 (ca. 21 kDa) was larger than the predicted polypeptide size (19 kDa) due to the additional vector-encoded His-tag at the N-terminus of the protein. The recombinant protein was purified to homogeneity under denaturing conditions (Figure 3B). A clear band of purified protein in the position corresponding to the overexpressed protein in the crude lysate was visualized on the gel (Figure 3B). This band cross-reacted with anti-Cam antiserum (Figure 3C). The recognition of recombinant Gca1 with heterologous antibody indicates significant similarity between Gca1 and Cam.

Bottom Line: Reverse transcription-PCR analysis indicated that gca1 in A. brasilense is co-transcribed with its upstream gene annotated as argC, which encodes a putative N-acetyl-gamma-glutamate-phosphate reductase. 5'-RACE also demonstrated that there was no transcription start site between argC and gca1, and the transcription start site located upstream of argC transcribed both the genes (argC-gca1).Using transcriptional fusions of argC-gca1 upstream region with promoterless lacZ, we further demonstrated that gca1 upstream region did not have any promoter and its transcription occurred from a promoter located in the argC upstream region.The organization and regulation of this gene along with the putative argC gene suggests its involvement in arginine biosynthetic pathway instead of the predicted CO2 hydration.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi-221005, India. tripathianil@rediffmail.com

ABSTRACT

Background: Carbonic anhydrase (CA) is a ubiquitous enzyme catalyzing the reversible hydration of CO2 to bicarbonate, a reaction underlying diverse biochemical and physiological processes. Gamma class carbonic anhydrases (gamma-CAs) are widespread in prokaryotes but their physiological roles remain elusive. At present, only gamma-CA of Methanosarcina thermophila (Cam) has been shown to have CA activity. Genome analysis of a rhizobacterium Azospirillum brasilense, revealed occurrence of ORFs encoding one beta-CA and two gamma-CAs.

Results: One of the putative gamma-CA encoding genes of A. brasilense was cloned and overexpressed in E. coli. Electrometric assays for CA activity of the whole cell extracts overexpressing recombinant GCA1 did not show CO2 hydration activity. Reverse transcription-PCR analysis indicated that gca1 in A. brasilense is co-transcribed with its upstream gene annotated as argC, which encodes a putative N-acetyl-gamma-glutamate-phosphate reductase. 5'-RACE also demonstrated that there was no transcription start site between argC and gca1, and the transcription start site located upstream of argC transcribed both the genes (argC-gca1). Using transcriptional fusions of argC-gca1 upstream region with promoterless lacZ, we further demonstrated that gca1 upstream region did not have any promoter and its transcription occurred from a promoter located in the argC upstream region. The transcription of argC-gca1 operon was upregulated in stationary phase and at elevated CO2 atmosphere.

Conclusions: This study shows lack of CO2 hydration activity in a recombinant protein expressed from a gene predicted to encode a gamma-carbonic anhydrase in A. brasilense although it cross reacts with anti-Cam antibody raised against a well characterized gamma-CA. The organization and regulation of this gene along with the putative argC gene suggests its involvement in arginine biosynthetic pathway instead of the predicted CO2 hydration.

Show MeSH
Related in: MedlinePlus