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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

Agarose-gel showing amplified products obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) with total RNA isolated from Azospirillum brasilense Sp7 grown in minimal (lane 1) and rich medium (lane 2). Lower strip is showing the amplification of 16 S RNA from the same amount of RNA sample as a control.
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Figure 2: Agarose-gel showing amplified products obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) with total RNA isolated from Azospirillum brasilense Sp7 grown in minimal (lane 1) and rich medium (lane 2). Lower strip is showing the amplification of 16 S RNA from the same amount of RNA sample as a control.

Mentions: Before extending the study on functional analysis of gca1 in A. brasilense, the expression of gca1 gene in A. brasilense cells was examined. Cell extracts of A. brasilense showed very low level of carbonic anhydrase activity of 0.3 ± 0.1 U/mg. Since A. brasilense genome also encodes a functional β-CA [13], it was not clear if the observed CA activity was due to β-CA or also due to γ-CA. To determine whether gca1 is expressed in A. brasilense under ambient conditions, RT-PCR with RNA samples isolated from the mid-log phase cultures grown in minimal (MMAB) or rich (LB) medium was performed. The ~500 bp gca1 transcripts was produced from both the RNA samples (Figure 2) which was confirmed by sequencing the cDNA amplicons. These results indicated that A. brasilense gca1 is constitutively expressed in cells grown in minimal or rich medium under ambient atmospheric conditions.


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)

Agarose-gel showing amplified products obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) with total RNA isolated from Azospirillum brasilense Sp7 grown in minimal (lane 1) and rich medium (lane 2). Lower strip is showing the amplification of 16 S RNA from the same amount of RNA sample as a control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Agarose-gel showing amplified products obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) with total RNA isolated from Azospirillum brasilense Sp7 grown in minimal (lane 1) and rich medium (lane 2). Lower strip is showing the amplification of 16 S RNA from the same amount of RNA sample as a control.
Mentions: Before extending the study on functional analysis of gca1 in A. brasilense, the expression of gca1 gene in A. brasilense cells was examined. Cell extracts of A. brasilense showed very low level of carbonic anhydrase activity of 0.3 ± 0.1 U/mg. Since A. brasilense genome also encodes a functional β-CA [13], it was not clear if the observed CA activity was due to β-CA or also due to γ-CA. To determine whether gca1 is expressed in A. brasilense under ambient conditions, RT-PCR with RNA samples isolated from the mid-log phase cultures grown in minimal (MMAB) or rich (LB) medium was performed. The ~500 bp gca1 transcripts was produced from both the RNA samples (Figure 2) which was confirmed by sequencing the cDNA amplicons. These results indicated that A. brasilense gca1 is constitutively expressed in cells grown in minimal or rich medium under ambient atmospheric conditions.

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