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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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Schematic representation of the genomic organization of gene predicted to encode γ-CA in Azospirillum brasilense and other closely related α-proteobacteria sharing highest similarity for γ-CA sequences. Arrows indicate the positions and orientations of the potential ORFs predicted to encode γ-CA (black), N-acetyl-gamma-glutamyl phosphate reductase (gray), hypothetical proteins (lined) and other known proteins (white). 1. 50 S ribosomal protein; 2. 30 S ribosomal protein; 3. OmpA/MotB domain protein precursor; 4. Poly(3-hydroxyalkanoate) synthase; 5. phosphoribosyl AMP cyclohydrolase; 6. cystathionine beta lyase; 7. Acetyltransferase (GNAT family); 8. poly-beta hydroxybutyrate transferase; 9. Arylsulphatase regulator; 10. Aminotransferase; 11. ABC transporter component; 12. Binding protein dependent transport systems inner membrane component.
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Figure 4: Schematic representation of the genomic organization of gene predicted to encode γ-CA in Azospirillum brasilense and other closely related α-proteobacteria sharing highest similarity for γ-CA sequences. Arrows indicate the positions and orientations of the potential ORFs predicted to encode γ-CA (black), N-acetyl-gamma-glutamyl phosphate reductase (gray), hypothetical proteins (lined) and other known proteins (white). 1. 50 S ribosomal protein; 2. 30 S ribosomal protein; 3. OmpA/MotB domain protein precursor; 4. Poly(3-hydroxyalkanoate) synthase; 5. phosphoribosyl AMP cyclohydrolase; 6. cystathionine beta lyase; 7. Acetyltransferase (GNAT family); 8. poly-beta hydroxybutyrate transferase; 9. Arylsulphatase regulator; 10. Aminotransferase; 11. ABC transporter component; 12. Binding protein dependent transport systems inner membrane component.

Mentions: While analyzing the organization of gca1 chromosomal region of A. brasilense using genome database and NCBI database BLAST resources, a putative gene (annotated as argC) was identified that was located upstream of gca1 ORF in the same transcriptional orientation with an intergenic distance of 35 nucleotides (Figure 4). The argC gene product (351 amino acids) of A. brasilense shared high similarity with the ArgC protein of R. centenum, M. magneticum and R. rubrum. The N-acetyl-gamma-glutamate-phosphate reductase (EC 1.2.1.38) encoded by argC is involved in the arginine biosynthesis in prokaryotes [15]. The arginine biosynthetic pathway proceeds via N-acetylation of L-glutamate by N-acetylglutamate synthase (ArgA) yielding N-acetylglutamate which is converted into N-acetylglutamyl-phosphate by N-acetylglutamate 5-phosphotransferase encoded by argB. N-acetylglutamyl-phosphate is subsequently reduced to N-acetylglutamic semialdehyde by N-acetylglutamyl-phosphate reductase, encoded by the argC gene. Thus the ArgC protein catalyses the third step in the pathway of biosynthesis of arginine from glutamate [15].


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)

Schematic representation of the genomic organization of gene predicted to encode γ-CA in Azospirillum brasilense and other closely related α-proteobacteria sharing highest similarity for γ-CA sequences. Arrows indicate the positions and orientations of the potential ORFs predicted to encode γ-CA (black), N-acetyl-gamma-glutamyl phosphate reductase (gray), hypothetical proteins (lined) and other known proteins (white). 1. 50 S ribosomal protein; 2. 30 S ribosomal protein; 3. OmpA/MotB domain protein precursor; 4. Poly(3-hydroxyalkanoate) synthase; 5. phosphoribosyl AMP cyclohydrolase; 6. cystathionine beta lyase; 7. Acetyltransferase (GNAT family); 8. poly-beta hydroxybutyrate transferase; 9. Arylsulphatase regulator; 10. Aminotransferase; 11. ABC transporter component; 12. Binding protein dependent transport systems inner membrane component.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Schematic representation of the genomic organization of gene predicted to encode γ-CA in Azospirillum brasilense and other closely related α-proteobacteria sharing highest similarity for γ-CA sequences. Arrows indicate the positions and orientations of the potential ORFs predicted to encode γ-CA (black), N-acetyl-gamma-glutamyl phosphate reductase (gray), hypothetical proteins (lined) and other known proteins (white). 1. 50 S ribosomal protein; 2. 30 S ribosomal protein; 3. OmpA/MotB domain protein precursor; 4. Poly(3-hydroxyalkanoate) synthase; 5. phosphoribosyl AMP cyclohydrolase; 6. cystathionine beta lyase; 7. Acetyltransferase (GNAT family); 8. poly-beta hydroxybutyrate transferase; 9. Arylsulphatase regulator; 10. Aminotransferase; 11. ABC transporter component; 12. Binding protein dependent transport systems inner membrane component.
Mentions: While analyzing the organization of gca1 chromosomal region of A. brasilense using genome database and NCBI database BLAST resources, a putative gene (annotated as argC) was identified that was located upstream of gca1 ORF in the same transcriptional orientation with an intergenic distance of 35 nucleotides (Figure 4). The argC gene product (351 amino acids) of A. brasilense shared high similarity with the ArgC protein of R. centenum, M. magneticum and R. rubrum. The N-acetyl-gamma-glutamate-phosphate reductase (EC 1.2.1.38) encoded by argC is involved in the arginine biosynthesis in prokaryotes [15]. The arginine biosynthetic pathway proceeds via N-acetylation of L-glutamate by N-acetylglutamate synthase (ArgA) yielding N-acetylglutamate which is converted into N-acetylglutamyl-phosphate by N-acetylglutamate 5-phosphotransferase encoded by argB. N-acetylglutamyl-phosphate is subsequently reduced to N-acetylglutamic semialdehyde by N-acetylglutamyl-phosphate reductase, encoded by the argC gene. Thus the ArgC protein catalyses the third step in the pathway of biosynthesis of arginine from glutamate [15].

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