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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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Related in: MedlinePlus

27 Effect of growth phase and CO2 concentration on argC-gca1 promoter activity β-galactosidase assay was performed with A. brasilense Sp7 cells harbouring either pRKK200 (empty vector) or pSK8 and grown up to either exponential or stationary phase at ambient air, and exponential growing cells at high CO2 concentration. The assay was performed on two different occasions. The error bars indicate standard deviation from the three replicates.
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Figure 6: 27 Effect of growth phase and CO2 concentration on argC-gca1 promoter activity β-galactosidase assay was performed with A. brasilense Sp7 cells harbouring either pRKK200 (empty vector) or pSK8 and grown up to either exponential or stationary phase at ambient air, and exponential growing cells at high CO2 concentration. The assay was performed on two different occasions. The error bars indicate standard deviation from the three replicates.

Mentions: Comparison of β-galactosidase activity in the cells taken from exponential and stationary phase cultures (Figure 6) showed that PargC activity was significantly up-regulated (more than 2 fold) during stationary phase than in the exponential phase of growth. Similarly, β-galactosidase activity measured in exponentially growing cells of A. brasilense harboring pSK8 under 3% CO2 enriched atmosphere was ~3 fold higher than the cells grown in ambient atmosphere (Figure 6). These data suggested that the PargC is constitutively but weakly expressed in exponentially growing cells under optimal growth conditions but significantly induced in response to high CO2 or stationary phase.


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)

27 Effect of growth phase and CO2 concentration on argC-gca1 promoter activity β-galactosidase assay was performed with A. brasilense Sp7 cells harbouring either pRKK200 (empty vector) or pSK8 and grown up to either exponential or stationary phase at ambient air, and exponential growing cells at high CO2 concentration. The assay was performed on two different occasions. The error bars indicate standard deviation from the three replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: 27 Effect of growth phase and CO2 concentration on argC-gca1 promoter activity β-galactosidase assay was performed with A. brasilense Sp7 cells harbouring either pRKK200 (empty vector) or pSK8 and grown up to either exponential or stationary phase at ambient air, and exponential growing cells at high CO2 concentration. The assay was performed on two different occasions. The error bars indicate standard deviation from the three replicates.
Mentions: Comparison of β-galactosidase activity in the cells taken from exponential and stationary phase cultures (Figure 6) showed that PargC activity was significantly up-regulated (more than 2 fold) during stationary phase than in the exponential phase of growth. Similarly, β-galactosidase activity measured in exponentially growing cells of A. brasilense harboring pSK8 under 3% CO2 enriched atmosphere was ~3 fold higher than the cells grown in ambient atmosphere (Figure 6). These data suggested that the PargC is constitutively but weakly expressed in exponentially growing cells under optimal growth conditions but significantly induced in response to high CO2 or stationary phase.

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