Regulation of expression and biochemical characterization of a Î²-class carbonic anhydrase from the plant growth-promoting rhizobacterium,<i>Azospirillum brasilense</i>Sp7

Kaur, Savneet; Mishra, Mukti Nath; Tripathi, Anil Kumar

doi:10.1111/j.1574-6968.2009.01736.x

Cited by 16 publications

(14 citation statements)

References 34 publications

(52 reference statements)

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“…5). The expression of several heterotrophic b-CAs has been reported to be induced by low CO 2 (Aguilera et al, 2005;Amoroso et al, 2005;Kaur et al, 2009), whereas the expression of others is regulated by additional or alternative factors including growth rate, pH or stress (Kaur et al, 2009;Merlin et al, 2003). The differential expression of psCA proteins suggests their independent regulation and further supports the hypothesis that they play different physiological roles in P. aeruginosa.…”

Section: Psca1 Psca2 and Psca3 Are Functionally Active Casmentioning

confidence: 62%

Three functional β-carbonic anhydrases in Pseudomonas aeruginosa PAO1: role in survival in ambient air

et al. 2013

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Bacterial b-class carbonic anhydrases (CAs) are zinc metalloenzymes catalysing reversible hydration of CO 2. They maintain the intracellular balance of CO 2 /bicarbonate required for biosynthetic reactions and represent a new group of antimicrobial drug targets. Genome sequence analysis of Pseudomonas aeruginosa PAO1, an opportunistic human pathogen causing life threatening infections, identified three genes, PAO102, PA2053 and PA4676, encoding putative b-CAs that share 28-45 % amino acid sequence identity and belong to clades A and B. The genes are conserved among all sequenced pseudomonads. The CAs were cloned, heterologously expressed and purified. Metal and enzymic analyses confirmed that the proteins contain Zn 2+ and catalyse hydration of CO 2 to bicarbonate. PAO102 (psCA1) was 19-26-fold more active, and together with PA2053 (psCA2) showed CA activity at both pH 7.5 and 8.3, whereas PA4676 (psCA3) was active only at pH 8.3. Circular dichroism spectroscopy suggested that psCA2 and psCA3 undergo pH-dependent structural changes. Taken together, the data suggest that psCA1 may belong to type I and psCA3 to type II b-CAs. Immunoblot analysis showed that all three CAs are expressed in PAO1 cells when grown in ambient air and at 5 % CO 2 ; psCA1 appeared more abundant under both conditions. Growth studies of transposon mutants showed that the disruption of psCA1 impaired PAO1 growth in ambient air and caused a minor defect at high CO 2 . Thus, psCA1 contributes to the adaptation of P. aeruginosa to low CO 2 conditions and will be further studied for its role in virulence and as a potential antimicrobial drug target in this organism.

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Section: Psca1 Psca2 and Psca3 Are Functionally Active Casmentioning

confidence: 62%

Three functional β-carbonic anhydrases in Pseudomonas aeruginosa PAO1: role in survival in ambient air

et al. 2013

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“…Such convergent evolution among diverse groups of organisms suggests that CAs are fundamental to many life strategies (Smith et al, 1999). Indeed, these enzymes have been linked to a number of common and specialised biological processes, such as CO 2 concentration mechanisms required to maintain photosynthesis in plants, algae and cyanobacteria (Badger, 2003;Badger and Price, 1994); calcification to limit calcium toxicity in bacteria (Banks et al, 2010;Li et al, 2005b); maintenance of required CO 2 and bicarbonate levels for metabolic activity in both bacteria (Merlin et al, 2003) and fungi (Kaur et al, 2009) growing under CO 2 limited conditions; and metabolic flexibility in methanogenic archaea (Smith and Ferry, 2000). However, despite evidence of CA activity in soils, the variability and drivers of their expression by soil communities is poorly understood (Li et al, 2005a;Seibt et al, 2006;Wingate et al, 2009Wingate et al, , 2008.…”

Section: Introductionmentioning

confidence: 99%

Non-destructive estimates of soil carbonic anhydrase activity and associated soil water oxygen isotope composition

Jones

Ogée

Sauze

et al. 2017

Hydrol. Earth Syst. Sci.

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Abstract. The contribution of photosynthesis and soil respiration to net land-atmosphere carbon dioxide (CO 2 ) exchange can be estimated based on the differential influence of leaves and soils on budgets of the oxygen isotope composition (δ 18 O) of atmospheric CO 2 . To do so, the activity of carbonic anhydrases (CAs), a group of enzymes that catalyse the hydration of CO 2 in soils and plants, needs to be understood. Measurements of soil CA activity typically involve the inversion of models describing the δ 18 O of CO 2 fluxes to solve for the apparent, potentially catalysed, rate of CO 2 hydration. This requires information about the δ 18 O of CO 2 in isotopic equilibrium with soil water, typically obtained from destructive, depth-resolved sampling and extraction of soil water. In doing so, an assumption is made about the soil water pool that CO 2 interacts with, which may bias estimates of CA activity if incorrect. Furthermore, this can represent a significant challenge in data collection given the potential for spatial and temporal variability in the δ 18 O of soil water and limited a priori information with respect to the appropriate sampling resolution and depth. We investigated whether we could circumvent this requirement by inferring the rate of CO 2 hydration and the δ 18 O of soil water from the relationship between the δ 18 O of CO 2 fluxes and the δ 18 O of CO 2 at the soil surface measured at different ambient CO 2 conditions. This approach was tested through laboratory incubations of air-dried soils that were re-wetted with three waters of different δ 18 O. Gas exchange measurements were made on these soils to estimate the rate of hydration and the δ 18 O of soil water, followed by soil water extraction to allow for comparison. Estimated rates of CO 2 hydration were 6.8-14.6 times greater than the theoretical uncatalysed rate of hydration, indicating that CA were active in these soils. Importantly, these estimates were not significantly different among water treatments, suggesting that this represents a robust approach to assay the activity of CA in soil. As expected, estimates of the δ 18 O of the soil water that equilibrates with CO 2 varied in response to alteration to the δ 18 O of soil water. However, these estimates were consistently more negative than the composition of the soil water extracted by cryogenic vacuum distillation at the end of the gas measurements with differences of up to −3.94 ‰ VSMOW-SLAP. These offsets suggest that, at least at lower water contents, CO 2 -H 2 O isotope equilibration primarily occurs with water pools that are bound to particle surfaces and are depleted in 18 O compared to bulk soil water.

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“…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.…”

Section: Resultsmentioning

confidence: 99%

“…A. brasilense genome revealed the presence of one β-CA and two putative γ-CA encoding genes. Recently, we have shown that β-CA gene in A. brasilense encoded a functionally active protein, and its expression was regulated by growth phase, CO 2 concentration and pH [13]. In this work, one of the putative ORFs whose amino acid sequence shared significant identity with other members of the γ-CA family was characterized.…”

Section: Discussionmentioning

confidence: 98%

“…Earlier, we have cloned the gene encoding β-CA from A. brasilense , overexpresed, purified and characterized β-CA. We also showed that the transcription of bca gene was down regulated by stationary phase, elevated CO 2 and acidic pH [13]. The objective of this study was to study the role of a putative gene encoding γ-carbonic anhydrase in A. brasilense Sp7.…”

Section: Introductionmentioning

confidence: 99%

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

2010

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BackgroundCarbonic 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 (γ-CAs) are widespread in prokaryotes but their physiological roles remain elusive. At present, only γ-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 β-CA and two γ-CAs.ResultsOne of the putative γ-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-γ-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.ConclusionsThis study shows lack of CO2 hydration activity in a recombinant protein expressed from a gene predicted to encode a γ-carbonic anhydrase in A. brasilense although it cross reacts with anti-Cam antibody raised against a well characterized γ-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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Regulation of expression and biochemical characterization of a Î²-class carbonic anhydrase from the plant growth-promoting rhizobacterium,Azospirillum brasilenseSp7

Cited by 16 publications

References 34 publications

Three functional β-carbonic anhydrases in Pseudomonas aeruginosa PAO1: role in survival in ambient air

Three functional β-carbonic anhydrases in Pseudomonas aeruginosa PAO1: role in survival in ambient air

Non-destructive estimates of soil carbonic anhydrase activity and associated soil water oxygen isotope composition

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

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