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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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Determination of argC/gca1 transcription unit and transcription start site of argC/gca1 transcript. A. Agarose gel showing amplified products obtained by reverse transcriptasepolymerase chain reaction (RT-PCR) with total RNA isolated from Azospirillum brasilense Sp7 using argF/argR1 (Lane 3), gcaF1/gcaR1 (Lane 4) and argF1/gcaR3 (Lane 5) primer sets. Lane 1 and 2 shows the bands of 100 bp DNA ladder (NEB) and control without reverse transcriptase, respectively; B. Determination of argC/gca1 transcription start site by 5' RACE experiment. The electropherogram is representative of results from sequencing of several distinct clones obtained after 5'RACE. The first base (C) downstream of the dT tail-A corresponds to the first nucleotide transcribed or TSS; C. Schematic representation of the argC-gca1 chromosomal region of A. brasilense. Large arrows represent the ORFs, and their orientation shows the transcriptional direction. Small arrows indicate the location of primers used for RT-PCR and 5'RACE experiment. The nucleotides representing TSS (+1), putative -35 and -10 boxes, and SD are underlined. Start codon (ATG) of argC is italicized.
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Figure 5: Determination of argC/gca1 transcription unit and transcription start site of argC/gca1 transcript. A. Agarose gel showing amplified products obtained by reverse transcriptasepolymerase chain reaction (RT-PCR) with total RNA isolated from Azospirillum brasilense Sp7 using argF/argR1 (Lane 3), gcaF1/gcaR1 (Lane 4) and argF1/gcaR3 (Lane 5) primer sets. Lane 1 and 2 shows the bands of 100 bp DNA ladder (NEB) and control without reverse transcriptase, respectively; B. Determination of argC/gca1 transcription start site by 5' RACE experiment. The electropherogram is representative of results from sequencing of several distinct clones obtained after 5'RACE. The first base (C) downstream of the dT tail-A corresponds to the first nucleotide transcribed or TSS; C. Schematic representation of the argC-gca1 chromosomal region of A. brasilense. Large arrows represent the ORFs, and their orientation shows the transcriptional direction. Small arrows indicate the location of primers used for RT-PCR and 5'RACE experiment. The nucleotides representing TSS (+1), putative -35 and -10 boxes, and SD are underlined. Start codon (ATG) of argC is italicized.

Mentions: To determine if argC and gca1 genes are part of a single operon and transcribed as a single mRNA, reverse transcription-PCR (RT-PCR) experiments were performed using total RNA isolated from A. brasilense cultures using three different primer sets, (Table 1 and Figure 5C) gcaF1/gcaR1 to amplify gca1 ORF (519 bp), argF/argR1 for 687 bp portion of argC ORF and argF1/gcaR3 to amplify the transcript (625 bp) encompassing both argC and gca1 ORFs. Analysis of RT-PCR amplified product revealed that argF1/gcaR3 primer set produced a fragment of expected size (ca. 600) indicating that there was a single mRNA for these two genes. Amplicons of expected size, ca 700 bp and ca 500 bp, were also obtained with argC and gca1-specific primer sets, respectively (Figure 5A). RT-PCR analysis confirmed that these genes are, in fact, co-transcribed which suggests a new functional linkage between the two genes that may have interesting implications for A. brasilense physiology.


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)

Determination of argC/gca1 transcription unit and transcription start site of argC/gca1 transcript. A. Agarose gel showing amplified products obtained by reverse transcriptasepolymerase chain reaction (RT-PCR) with total RNA isolated from Azospirillum brasilense Sp7 using argF/argR1 (Lane 3), gcaF1/gcaR1 (Lane 4) and argF1/gcaR3 (Lane 5) primer sets. Lane 1 and 2 shows the bands of 100 bp DNA ladder (NEB) and control without reverse transcriptase, respectively; B. Determination of argC/gca1 transcription start site by 5' RACE experiment. The electropherogram is representative of results from sequencing of several distinct clones obtained after 5'RACE. The first base (C) downstream of the dT tail-A corresponds to the first nucleotide transcribed or TSS; C. Schematic representation of the argC-gca1 chromosomal region of A. brasilense. Large arrows represent the ORFs, and their orientation shows the transcriptional direction. Small arrows indicate the location of primers used for RT-PCR and 5'RACE experiment. The nucleotides representing TSS (+1), putative -35 and -10 boxes, and SD are underlined. Start codon (ATG) of argC is italicized.
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Figure 5: Determination of argC/gca1 transcription unit and transcription start site of argC/gca1 transcript. A. Agarose gel showing amplified products obtained by reverse transcriptasepolymerase chain reaction (RT-PCR) with total RNA isolated from Azospirillum brasilense Sp7 using argF/argR1 (Lane 3), gcaF1/gcaR1 (Lane 4) and argF1/gcaR3 (Lane 5) primer sets. Lane 1 and 2 shows the bands of 100 bp DNA ladder (NEB) and control without reverse transcriptase, respectively; B. Determination of argC/gca1 transcription start site by 5' RACE experiment. The electropherogram is representative of results from sequencing of several distinct clones obtained after 5'RACE. The first base (C) downstream of the dT tail-A corresponds to the first nucleotide transcribed or TSS; C. Schematic representation of the argC-gca1 chromosomal region of A. brasilense. Large arrows represent the ORFs, and their orientation shows the transcriptional direction. Small arrows indicate the location of primers used for RT-PCR and 5'RACE experiment. The nucleotides representing TSS (+1), putative -35 and -10 boxes, and SD are underlined. Start codon (ATG) of argC is italicized.
Mentions: To determine if argC and gca1 genes are part of a single operon and transcribed as a single mRNA, reverse transcription-PCR (RT-PCR) experiments were performed using total RNA isolated from A. brasilense cultures using three different primer sets, (Table 1 and Figure 5C) gcaF1/gcaR1 to amplify gca1 ORF (519 bp), argF/argR1 for 687 bp portion of argC ORF and argF1/gcaR3 to amplify the transcript (625 bp) encompassing both argC and gca1 ORFs. Analysis of RT-PCR amplified product revealed that argF1/gcaR3 primer set produced a fragment of expected size (ca. 600) indicating that there was a single mRNA for these two genes. Amplicons of expected size, ca 700 bp and ca 500 bp, were also obtained with argC and gca1-specific primer sets, respectively (Figure 5A). RT-PCR analysis confirmed that these genes are, in fact, co-transcribed which suggests a new functional linkage between the two genes that may have interesting implications for A. brasilense physiology.

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