Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Prevalence and phylogenetic relationship of two β-carbonic anhydrases in affiliates of Enterobacteriaceae

Prevalence and phylogenetic relationship of two β-carbonic anhydrases in affiliates of... Ann Microbiol (2013) 63:1275–1282 DOI 10.1007/s13213-012-0585-6 ORIGINAL ARTICLE Prevalence and phylogenetic relationship of two β-carbonic anhydrases in affiliates of Enterobacteriaceae Rishiram Ramanan & Krishnamurthi Kannan & Saravana Devi Sivanesan & Tapan Chakrabarti Received: 16 July 2012 /Accepted: 3 December 2012 /Published online: 30 December 2012 # Springer-Verlag Berlin Heidelberg and the University of Milan 2012 Abstract Enterobacteriaceae, one of the major families of Introduction microorganisms that inhabit the soil and gut, internally regulate constant fluctuations in soil and gut pH by buffer- Affiliates of Enterobacteriaceae inhabit the intestine and soil, ing these changes through the presence of carbonic anhy- performing a host of useful functions in a mutualistic–symbi- drase (CA). In our study, we prove the prevalence of β-CA, otic relationship. Both habitats are known to undergo constant derived from the can gene, in members of Enterobacteria- changes in intra-day pH and carbon dioxide (CO ) concentra- ceae by using a combination of experimental and bioinfor- tions. Consequently, members of Enterobacteriaceae are sub- matics approaches. Enzyme purification and western blot jected to extreme environments, such as low oxygen levels, analysis revealed the presence of β-CA in Enterobacter sp. low CO levels and pH changes, requiring the microorganisms RS1. Genetic studies confirmed the presence of β-CA in to possess carbonic anhydrase (CA) activity (Tashian 1989). both Enterobacter sp. RS1 and Citrobacter freundii SW3. Thezincmetallo-enzymeCAplays avital role in acid–base Our analysis of the divergence of cynT and can genes among homeostasis, buffering the pH in bacteria by catalyzing the harboring members indicated that the can gene was more reversible conversion of CO dioxide into bicarbonates. Micro- prominent in Enterobacteriaceae than cynT. Sequence anal- organisms express four forms of CA (α, β, γ and δ) which are ysis of the can gene revealed a >25 % similarity among all functionally convergent and evolutionarily independent (Smith sequences and a >50 % similarity among sequences from and Ferry 2000). The α-CA form is widely reported in eukar- the Enterobacteriaceae family. The β-CA from C. freundii yotes, especially mammals, the β-CA is reported in plants, SW3 and Enterobacter sp. RS1, isolated from soil and used animals and microorganisms, the, the γ-CA form is found in in this study, possessed a high similarity with the can gene. archea and bacteria and the δ-CA is restricted to diatoms and The close association among Enterobacteriaceae genera usu- dinoflagellates (Tripp et al. 2004). CA in photosynthetic micro- ally found in the soil and gut and the sequence similarity of organisms plays a major role alongside Rubisco in the carbon- β-CA in the different genera of Enterobacteriaceae suggest concentrating mechanism (Ghoshal et al. 2002). However, less the importance of the can gene in oscillating environmental is known about the physiological roles of the CAs in non- conditions. photosynthetic microorganisms. The β-CA is the most prevalent form of CA among the . . Keywords Carbonic anhydrase Enterobacteriaceae genera of the Enterobacteriaceae, although some members of . . Citrobacter freundii Enterobacter sp. Soil this family also possess γ-CA forms. However, the functions of these γ-CAs are still not well-defined. Escherichia coli possess three candidates for γ-CAs, YrdA, PaaY and CaiE, although these γ-CA class enzymes have not been found to have any activity (Eichler et al. 1994; Elssner et al. 2001; Ferrandez et al. 1998). There are multiple copies of both forms in some organ- : : : R. Ramanan K. Kannan (*) S. D. Sivanesan T. Chakrabarti isms. Four genes, cynT, can (also interchangeably denoted as Environmental Health Division, National Environmental yadF), cah and mig-5,encoding β-CA have been reported to be Engineering Research Institute (NEERI), Nehru Marg, expressed in Enterobacteriaceae. However, the cah and mig-5 Nagpur 440 020 Maharashtra, India e-mail: k_krishnamurthi@neeri.res.in genes have been found only sporadically among affiliates of 1276 Ann Microbiol (2013) 63:1275–1282 Enterobacteriaceae, with cynT and can being the most promi- isolates Enterobacter sp. RS1 (EU124384) and C. freundii nent members (Beach and Osuna 1998). The cynT gene was SW3 (EU124389), respectively. first identified in E. coli as a part of the cyn operon and was found to express β-CA, thus dispelling the fact that β-CA was Protein isolation and determination conserved among higher plants (Sung and Fuchs 1988). The indispensability and necessity of can gene expression under The intracellular protein extraction was carried out by first low CO concentration have been well established (Hashimoto washing the cell pellet with distilled water, then resuspend- and Kato 2003). Mutation analysis of the Ralstonia eutropha ing the pellet in lysis buffer containing 250 mM Tris–HCl can gene highlights the importance of the gene product, β-CA, buffer and Triton-X 100, followed by sonication for 10 min. for the survival of the organism at low CO concentrations The pellet was discarded after centrifugation at 12,000 g and (Kusian et al. 2002). The pH-dependent activity of the enzyme the supernatant stored for future analysis. The protein con- coded by can has also been studied, with the results suggesting centration was determined using a previously published a possible conformational change into a stable yet non-active method (Lowry et al. 1951) with bovine serum albumin form at lower pH and high activity at higher pH (Cronk et al. (Sigma, USA) as the standard, whereas the protein content 2001). However, numerous studies have established that CA is of the diluted chromatographic fractions was measured by essential for survival at low pH and low CO conditions monitoring the optical density at 280 nm. (Mitsuhashi et al. 2004; Sly and Hu 1995). Although the results of previous studies indicate the role of CA at low CO CA purification and assay and different pH levels, the preference of an organism for one gene over another has not been established in natural habitats, Carbonic anhydrase was purified from Enterobacter sp. strain such as the soil and gut. In these habitats, where intra-day pH RS1 using the method published in Ramanan et al. (2009b) fluctuations are common, it is also imperative to ascertain the with slight modifications. The crude protein obtained from role of CA in the survival of microorganisms. ammonium sulfate precipitation was loaded first onto a Studying the phylogenetic relationship of the can and cynT Sephadex-G-75 column, then onto a DEAE cellulose column. genes may provide information that will help explain the sim- The resulting purified enzyme was subsequently dialyzed ilarities and differences of these genes among members of the against Tris–HCl (pH 8.3) overnight. The hydration assay Enterobacteriaceae. In addition, studying the incidence of these (Wilbur–Anderson, W–A) for CA was performed using the genes among members of Enterobacteriaceae would further procedure of Khalifah (1971), and esterase activity was deter- help researchers to arrive at a conclusion on this predilection mined using the procedure reported by Armstrong et al. (1966). factor. The results of our study contribute information on the presence of cynT and can gene in Enterobacteriaceae genera Western blot analysis and the possible implications of the presence of these genes. Crude protein samples were separated using sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and Materials and methods were transferred to a nitrocellulose membrane (Bio-Rad, Her- cules, CA) using a wet transfer unit for 2 h at 4 °C and 80 V. Bacterial strains and growth conditions All blocking and antiserum incubations were performed in TBST (20 mM Tris–HCl, pH 7.5, 0.5 M NaCl, 0.05 % Tween Pure cultures were isolated from soil samples, grown in Luria– 20). Themembranewas blocked2hin 5 %skim milk–Tris Bertani broth at 37 °C in a rotary shaker for 24 h at 100 rpm buffer solution–Tween 20 (TBST) at room temperature. The and processed for protein analysis. Genomic DNA was membrane was incubated overnight at 4 °C with β-CA anti- extracted and purified from cell pellets, and its purity was body diluted 1:1,000. E. coli CA was used as a positive assessed using a spectrophotometric method at A /A standard and the molecular weight marker for CA. The mem- 260 280 (Sambrook and Russell 2001 ). The culture was studied for brane was then rinsed three times in TBST and incubated for morphological, physiological and biochemical characteristics 2 h at room temperature with a 1:3,000 dilution of pig anti- following standard procedures. Universal 16S rDNA PCR rabbit horseradish peroxidase-conjugated immunoglobulin G, forward (5′-AGAGTTTGATCMTGGCTCAG) and reverse which resulted in an antigen–antibody complex (Alber and (5′-AAGGAGGTGWTCCARCC) primers were used in the Ferry 1996; Ramanan et al. 2009a). amplification of 16S rDNA genes. The amplification products were purified by using a DNA extraction kit (Genei, India) and Cloning and sequencing of can and cynT genes the DNA was sequenced (Bangalore Genei, India). The obtained 16S rDNA sequence has been deposited in GenBank, The primer sets were self-designed with the sequence details of the E. coli can gene. The forward and reverse primer sequences National Center for Biotechnology Information (NCBI) for Ann Microbiol (2013) 63:1275–1282 1277 used in our study to amplify the cynT gene were 5′- sufficient for purification of the enzyme to homogeneity GACGTGTTTTTTTGTCAGG and 5′-AGATACACTCAT- (Table 1). SDS-PAGE of the purified CA with the high CAGCAAC, respectively. The forward and reverse primer activity fraction revealed that the molecular weight of the sequences for the can gene were 5′-TTATTTGTGG enzyme was approximately 24 kDa protein, which was TTGGCGTGT and 5′-AGATACACTCATCAGCAAC, re- supported by the western blot results (Fig. 1). The western spectively. A 650-bp DNA fragment was amplified from both blot analysis of the protein sample showed a cross-reaction Enterobacter sp. RS1 and C. freundii strain SW3byPCR in a for Enterobacter sp. RS1 with the E. coli β-CA used as the Applied Biosystems (USA) thermal cycler (model GeneAmp positive control and molecular weight marker (Fig. 2). The 9,700) in a total reaction volume of 50 μl containing 50 ng CA enzyme from Enterobacter sp. RS1 possessed a similar genomic DNA, 50 pmol of each primer, 0.2 mM of each molecular weight to that of the E. coli CA (approx. 24 kDa). dNTPs, 1× polymerase buffer and 1 U Taq DNA polymerase This is the first study to confirm the presence of β-CA in an (New England Biolabs, USA). The program used consisted of a Enterobacter genus of Enterobacteriaceae. Earlier studies denaturation step at 94 °C for 4 min, 30 cycles at 94 °C for 30 s, have confirmed the presence of β-CA by western blotting 55 °C for 45 s and 72 °C for 45 s and a final extension at 72 °C in E. coli, C. freundii and Salmonella typhimurium among for 5 min. The PCR product was purified and cloned into the T- the affiliates of Enterobacteriaceae (Ramanan et al. 2009a; vector (Bangalore Genei, India), which was further sequenced. Smith et al. 1999). Phylogenetic analysis Sequencing and phylogenetic analysis of the β-CA gene The deduced amino acid sequences of the can gene obtained Sequencing of the approximately 650-bp amplicon con- were compared against database sequences using tBLASTx firmed the presence of the β-CA gene in Enterobacter sp. provided by NCBI (http://www.ncbi.nlm.nih.gov) and were RS1 and C. freundii SW3. The BLAST program for the aligned and clustered using Clustal-X. Phylogenetic trees sequenced and cloned amplicons yielded similar sequences were constructed using the PHYLIP programs and were visu- of β-CA, with most belonging to those of the Enterobacter- alized using TREEVIEW software, ver. 1.6.6 (Thompson et iaceae family. The phylogenetic tree suggests that both al. 1997). The phylogenetic tree based upon the neighbor- Enterobacter sp. RS1 and C. freundii SW3 β-CA genes joining (NJ) algorithm was performed with 1,000 bootstrap- are closely related to the can gene of many Enterobacter- ping replicates. The COG (Clusters of Orthologous Groups) iaceae affiliates (Fig. 3). Hence, we constructed a multiple database together with the COGNITOR program (http:// alignment of deduced amino acids among homologs from www.ncbi.nlm.nih.gov/COG/old/xognitor.html)wereused to the Enterobacteriaceae family, a total of 18 protein sequen- fit experimental sequences into the COGs to unearth paralogs ces, which yielded 21 identical amino acids, 39 conserved within complete genomes. All sequences for comparisons sequences and 42 semi-conserved sequences (data not were retrieved from protein databases using the Entrez pro- shown). It should be noted that cloned amplicons obtained gram (NCBI, National Institutes of Health, USA). from both the microorganisms used in this study shared a high similarity with the can gene in terms of phylogenetic relationship and sequence identity. Results Phylogenetic analysis of the can and cynT genes Purification and western blotting of CA from Enterobacter sp The ENTREZ program was searched for genera harboring The purification of CA from Enterobacter sp. was carried the can gene encoding β-CA. One representative species out by ammonium sulfate precipitation, gel filtration and ion from each genus was selected for multiple alignment of exchange chromatography, in that order. These steps were deduced amino acid sequences of the can gene (data not Table 1 Sequential purification of carbonic anhydrase from the ammonium sulfate-precipitated protein extract of Enterobacter sp. RS1 assayed by esterase activity Purification stages Total protein (mg/ml) Total activity (U/ml) Specific activity (U/mg) Relative yield (%) Purification fold Concentrated protein extract RS1 153 303 1.980 100 1 Gel filtration RS1 37 219 5.92 72 2.99 DEAE cellulose RS1 3.53 154 43.62 50.8 22.03 One enzyme unit is defined as formation of μmol p-nitro phenol (p-NP) per minute 1278 Ann Microbiol (2013) 63:1275–1282 Fig. 1 Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS- PAGE) of purified beta-carbonic anhydrase (β-CA) from Enterobacter sp. RS1 following three consecutive chromatographic steps. Lanes: 1 purified β-CA with a molecular weight of approx. 24 kDa, 2 molecular weight marker Fig. 3 Phylogenetic analysis of sequences similar to the experimental sequences of Citrobacter freundii SW3 and Enterobacter sp.RS1.The scale bar is given in fixed nucleotide substitutions per sequence position. GenBank shown), leading to the identification of the can gene in 15 accession numbers of sequences used in this analysis are given in parenthesis different genera. Nonetheless, 13 of the 19 genera identified belong to the Enterobacteriaceae family, indicating that the can gene was not diversely distributed among the bacterial domain studied and that the sequences of the can gene were conserved (Figs. 3, 4) (Merlin et al. 2003). The sequence alignment of the can gene also suggested a >25 % similarity among all sequences and a >50 % similarity among sequen- ces from the Enterobacteriaceae family. The analysis of Fig. 4 Phylogenetic tree of can gene sequences from 15 genera of Fig. 2 Western blot analysis of β-CA validated the presence of β-CA Enterobacteriaceae. The scale bar is given in fixed nucleotide substi- in Enterobacter sp. RS1 (lane 2) with Escherichia coli CA used as the tutions per sequence position. GenBank accession numbers of sequen- positive control and molecular weight marker (lane 1) ces used in this analysis are given in parenthesis Ann Microbiol (2013) 63:1275–1282 1279 enzyme characteristics suggested that the β-CA encoded by blot analysis and purification of CA from Enterobacter sp. the gene is usually a protein with 215–220 amino acids and RS1 suggest that this gene is expressed in most members of an approximate molecular weight of 24–25 kDa (Table 2). Enterobacteriaceae (Figs. 2 and 4). The β-CA from Enterobacter sp. RS1 and C. freundii SW3 One representative species from each genus was selected also seem to be homologs of the can genes reported. The for phylogenetic analysis and multiple sequence alignment phylogenetic data coupled with the results of the western of the cynT gene was performed (data not shown). The Table 2 Beta-carbonic anhydrase-encoding genes present among members of the Enterobacteriaceae family Genus Microorganism Gene type Characteristics Reference or Common natural habitats accession number (Janda and Abbott 2006) Molecular Number of weight (Da) amino acids Citrobacter Citrobacter koseri ATCC can 28,681 258 YP_001456157 Soil, feces, water, sewage, food BAA-895 Citrobacter freundii SW3 can 24,000 186 EU919747 (this study) Soil, water, sewage, food, and the intestinal tracts of animals and humans Enterobacter Enterobacter sp. 638 can 24,959 220 YP_001175412 Soil, fresh water, plants, sewage, animal feces Enterobacter sakazakii ± cynT 23,055 207 YP_001439024 Soil, water, sewage, plants Enterobacter sp. RS1 can 24,000 202 EU919748 (this study) Soil Erwinia Erwinia tasmaniensis can 25,027 220 YP_001906796 Soil and plants Erwinia carotovora cah 26,533 244 YP_051416 Soil and plants Escherichia Escherichia coli can 25,024 220 NP_285822 Gastrointestinal tract of humans O157:H7 EDL933 and many of the warm blooded animals, soil, water, and plants Escherichia albertii can 24,993 220 ZP_02902992 Soil, water, and plants Escherichia coli str. K-12 can 24,966 220 YP_001729083 Gastrointestinal tract of humans cynT 23,633 219 NP_414873 and many of the warm blooded animals, soil, water, and plants Klebsiella Klebsiella pneumoniae ± cynT 23,113 211 YP_001335672 Soil, water, plants cah 27,145 246 Ref. [8] can 23,244 204 YP_001333824 Pectobacterium Pectobacterium carotovorum cah 26,645 244 AAC77891 Soil and plants Photorhabdus Photorhabdus luminescens cynT 23,679 219 NP_927481 Gastrointestinal lumen of Photorhabdus luminescens ± can 24,629 217 NP_928206 entomopathogenic nematodes and soil Providencia Providencia stuartii ± can 25,827 217 EDU60433 Soil, water, sewage Salmonella Salmonella enterica can 24,690 220 YP_215158 Gastrointestinal tract of humans mig 5 26,513 246 YP_209302 and animals, soil, and water Salmonella typhimurium can 24,690 220 NP_459176 Gastrointestinal tract of humans and animals, and soil Serratia Serratia proteamaculans ± can 24,604 218 YP_001480220 Soil, water, plants Shigella Shigella flexneri can 24,966 220 YP_687707 Soil and water Shigella boydii can 24,947 220 YP_406676 Soil and water Shigella dysenteriae can 24,992 220 YP_401762 Soil and water Shigella sonnei can 24,966 220 YP_309163 Soil and water Sodalis Sbodalis glossinidius ± can 24,760 218 YP_454163 Gastrointestinal tract of insects Yersinia Yersinia pseudotuberculosis can 24,739 220 YP_069265 Soil and water Yersinia pestis can 24,739 220 NP_406869 Soil and water cynT 26,681 251 NP_994143 Yersinia enterocolitica can 24,709 220 YP_001005059 Soil and water ± represents putative presence of the gene Sodalis glossinidius is the only microorganism among Enterobacteriaceae which primarily does not inhabit soil 1280 Ann Microbiol (2013) 63:1275–1282 sequence identity analysis using Clustal X revealed that the Lin et al. 1995; Taneva et al. 2006). Members of the Enter- selected sequences were highly diverse, with only four obacteriaceae family, especially the genera used in this study, amino acid sequences being semi-conserved and no identi- Citrobacter and Enterobacter, are major soil residents. As cal amino acid among the sequences. The cynT gene was indicated in Table 2, the primary natural habitat of all micro- distributed among 37 genera tested, of which only three organisms belonging to Enterobacteriaceae is the soil, with the genera belong to Enterobacteriaceae family, including E. exception of Sodalis glossinidius (Janda and Abbott 2006). coli (Fig. 5; Table 2). The results of the phylogenetic diver- CA plays a crucial role in soil microorganisms and intestinal sity and sequence identity analysis suggest that the cynT microflora of higher animals and invertebrates, where extreme gene may have independently evolved among the genera, pH changes are common occurrences (König and Varma which is plausible considering the evolutionary indepen- 2005; Winfield and Groisman 2003). It has been proven that dence of all classes of CA. E. coli encodes proteins that mediate resistance to pH, espe- cially acidic pH (Winfield and Groisman 2003). The role of CA anhydrase in pH homeostasis has also been noted (Sly and Discussion Hu 1995). Although these studies have established the role of CA at low CO and pH changes, the results of our study Soil and the intestines of mammals and humans are subjected establish the prevalence of CA among soil microorganisms, to varying pH ranges and CO concentrations, and genera of especially Enterobacteriaceae, which experience these condi- the Enterobacteriaceae are known to adapt well to such tions. There is a strong possibility that the can gene present in changeable conditions (Epron et al. 1999; Leyer et al. 1995; C. freundii and Enterobacter sp. as well as the can gene Fig. 5 Phylogenetic tree of cynT sequences from 37 genera. CynT gene was found to be widespread among the bacterial domain. Haloquadratum walsbyi (YP_657802), a square halophilic archaeon, is the only member of the domain archaea to be reported to possess cynT. The scale bar is given in fixed nucleotide substitutions per sequence position. GenBank accession numbers of sequences used in this analysis are given in parenthesis Ann Microbiol (2013) 63:1275–1282 1281 identified in other Enterobacteriaceae affiliates are quite sim- Armstrong J, Myers D, Verpoorte J, Edsall J (1966) Purification and properties of human erythrocyte carbonic anhydrases. J Biol ilar in properties—i.e. expressed only in a low CO environ- Chem 241:5137–5149 ment and at pH-defining conditions. The experimental Bach S, Almeida A, Carniel E (2000) The Yersinia high-pathogenicity evidence and results of the bioinformatic analysis presented island is present in different members of the family Enterobacter- in this study indicate that CA coded by the can gene play a iaceae. FEMS Microbiol Lett 183:289–294 Beach M, Osuna R (1998) Identification and characterization of the fis definite role in Enterobacteriaceae. The scenario of can gene operon in enteric bacteria. J Bacteriol 180:5932–5946 prevalence may possibly be similar to that of the Yersinia Cronk J, Endrizzi J, Cronk M, O’neill J, Zhang K (2001) Crystal high-pathogenicity island, which has been demonstrated to structure of E. coli β-carbonic anhydrase, an enzyme with an unusual pH-dependent activity. Protein Sci 10:911–922 be a part of the genome in all Enterobacteriaceae affiliates Eichler K, Bourgis F, Buchet A, Kleber H, Mandrand-Berthelot M through horizontal gene transfers (Bach et al. 2000). (1994) Molecular characterization of the cai operon necessary for Based on the results of their study of the cynT gene and carnitine metabolism in Escherichia coli. Mol Microbiol 13:775– its metabolism, Sung and Fuchs (1988) reported that the cynT gene is usually in repressed form and expressed only Elssner T, Engemann C, Baumgart K, Kleber H (2001) Involve- ment of coenzyme A esters and two new enzymes, an enoyl- for cyanate metabolism. Its role is to prevent the depletion of CoA hydratase and a CoA-transferase, in the hydration of intracellular bicarbonate, which accompanies the cyanase- crotonobetaine to L-carnitine by Escherichia coli. Biochem- catalyzed bicarbonate-dependent hydrolysis of cyanate. This istry 40:11140–11148 study led to breakthroughs in studies on the other paralog, Epron D, Farque L, Lucot E, Badot P-M (1999) Soil CO efflux in a beech forest: dependence on soil temperature and soil water the can gene, which encodes β-CA, and it was subsequently content. Ann For Sci 56:221–226 proven that to survive under atmospheric CO concentra- Ferrandez A, Minambres B, Garcia B, Olivera E, Luengo J, Garcia J, tions and at varying pH conditions, the expression of this Diaz E (1998) Catabolism of phenylacetic acid in Escherichia gene is mandatory (Hashimoto and Kato 2003; Kusian et al. coli: characterization of a new aerobic hybrid pathway. J Biol Chem 73:25974–25986 2002; Mitsuhashi et al. 2004). Ghoshal D, Husic H, Goyal A (2002) Dissolved inorganic carbon concentration mechanism in Chlamydomonas moewusii.Plant Physiol Biochem 40:299–305 Conclusion Hashimoto M, Kato J-I (2003) Indispensability of the Escherichia coli carbonic anhydrases yadF and cynT in cell proliferation at low CO partial pressure. Biosci Biotechnol Biochem 67:919–922 Based on our results, we conclude that the can gene is Janda J, Abbott S (2006) The Enterobacter. ASM Press, Washington preferred over the cynT gene, considering its predomi- D.C. nance in Enterobacteriaceae. The abundance of the can König H, Varma A (2005) Intestinal microorganisms of termites and other invertebrates, 1st edn. Springer, Berlin gene among the Enterobacteriaceae family and its un- Khalifah R (1971) The carbon dioxide hydration activity of carbonic usual properties as revealed by earlier reports (Cronk et anhydrase. J Biol Chem 246:2561–2573 al. 2001;Merlinetal. 2003; Ramanan et al. 2009a) Kusian B, Sultemeyer D, Bowien B (2002) Carbonic anhydrase is provide a valid insight into this predilection factor. essential for growth of Ralstonia eutropha at ambient CO .J Bacteriol 184:5018–5026 Thepresenceofthe can gene among C. freundii and Leyer G, Wang L-L, Johnson E (1995) Acid Adaptation of Escherichia Enterobacter sp., its prevalence in almost all of the coli O157:H7 increases survival in acidic roods. Appl Environ members of the Enterobacteriaceae family and the in- Microbiol 61:3752–3755 dispensability of the can gene for the survival of organ- Lin J, Lee I, Frey J, Slonczewski J, Foster J (1995) Comparative analysis of extreme acid survival in Salmonella typhimurium, isms at varying pH and low CO concentrations, which Shigella flexneri,and Escherichia coli. J Bacteriol 177:4097– are conditions that usually prevail in the soil, as proven by other studies, highlight its value. Lowry O, Rosebrough N, Farr A, Randall R (1951) Protein measure- ment with the Folin phenol reagent. J Biol Chem 193:265–275 Merlin C, Masters M, McAteer S, Coulson A (2003) Why is carbonic Acknowledgments Rishiram Ramanan thanks the University Grants anhydrase essential to Escherichia coli. J Bacteriol 185:6415– Commission, New Delhi for the award of Senior Research Fellowship and for providing financial support. The authors thank the Department Mitsuhashi S, Ohnishi J, Hayashi M, Ikeda M (2004) A gene homol- of Biotechnology, Government of India for the financial support pro- ogous to β-type carbonic anhydrase is essential for the growth of vided for this project. Corynebacterium glutamicum under atmospheric conditions. Appl Microbiol Biotechnol 63:592–601 Ramanan R, Kannan K, Sivanesan S, Mudliar S, Kaur S, Tripathi A, Chakrabarti T (2009a) Bio-sequestration of carbon dioxide using carbonic anhydrase enzyme purified from Citrobacter freundii. References World J Microbiol Biotechnol 25:981–987 Ramanan R, Kannan K, Vinayagamoorthy N, Ramkumar K, Sivanesan Alber B, Ferry J (1996) Characterization of heterologously produced S, Chakrabarti T (2009b) Purification and characterization of a novel plant-type carbonic anhydrase from Bacillus subtilis. Bio- carbonic anhydrase from Methanosarcina thermophila. J Bacter- technol Bioprocess Eng 14:32–37 iol 178:3270–3274 1282 Ann Microbiol (2013) 63:1275–1282 Sambrook J, Russell R (2001) Molecular cloning: a laboratory manual, respiration in a temperate forest exposed to elevated CO concen- 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring tration. Glob Chang Biol 12:983–994 Sly W, Hu P (1995) Human carbonic anhydrases and carbonic anhy- Tashian R (1989) The carbonic anhydrases: widening perspectives on drase deficiencies. Annu Rev Biochem 64:375–401 their evolution, expression and function. BioEssays 10:186–192 Smith K, Ferry J (2000) Prokaryotic carbonic anhydrases. FEMS Thompson J, Gibson T, Plewniak F, Jeanmougin F, Higgins D (1997) Microbiol Rev 24:335–366 The Clustal X Windows interface: flexible strategies for multiple Smith K, Jakubzick C, Whittam T, Ferry J (1999) Carbonic anhydrase sequence alignment aided by quality analysis tools. Nucleic Acids is an ancient enzyme widespread in prokaryotes. Proc Natl Acad Res 25:4876–4882 Sci USA 96:15184–15189 Tripp B, Bell C, Cruz F, Krebs C, Ferry J (2004) A role for iron in an Sung Y-C, Fuchs J (1988) Characterization of the cyn Operon in ancient carbonic anhydrase. J Biol Chem 279:6683–6687 Escherichia coli K12. J Biol Chem 263:14769–14775 Winfield M, Groisman E (2003) Role of nonhost environments in the Taneva L, Pippen J, Schlesinger W, Gonzalez-Meler M (2006) The lifestyles of Salmonella and Escherichia coli. Appl Environ turnover of carbon pools contributing to soil CO and soil Microbiol 69:3687–3694 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Prevalence and phylogenetic relationship of two β-carbonic anhydrases in affiliates of Enterobacteriaceae

Download PDF
8 pages

Loading next page...
 
/lp/springer-journals/prevalence-and-phylogenetic-relationship-of-two-carbonic-anhydrases-in-UNbYL90y4c

References (37)

Publisher
Springer Journals
Copyright
Copyright © 2012 by Springer-Verlag Berlin Heidelberg and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
eISSN
1869-2044
DOI
10.1007/s13213-012-0585-6
Publisher site
See Article on Publisher Site

Abstract

Ann Microbiol (2013) 63:1275–1282 DOI 10.1007/s13213-012-0585-6 ORIGINAL ARTICLE Prevalence and phylogenetic relationship of two β-carbonic anhydrases in affiliates of Enterobacteriaceae Rishiram Ramanan & Krishnamurthi Kannan & Saravana Devi Sivanesan & Tapan Chakrabarti Received: 16 July 2012 /Accepted: 3 December 2012 /Published online: 30 December 2012 # Springer-Verlag Berlin Heidelberg and the University of Milan 2012 Abstract Enterobacteriaceae, one of the major families of Introduction microorganisms that inhabit the soil and gut, internally regulate constant fluctuations in soil and gut pH by buffer- Affiliates of Enterobacteriaceae inhabit the intestine and soil, ing these changes through the presence of carbonic anhy- performing a host of useful functions in a mutualistic–symbi- drase (CA). In our study, we prove the prevalence of β-CA, otic relationship. Both habitats are known to undergo constant derived from the can gene, in members of Enterobacteria- changes in intra-day pH and carbon dioxide (CO ) concentra- ceae by using a combination of experimental and bioinfor- tions. Consequently, members of Enterobacteriaceae are sub- matics approaches. Enzyme purification and western blot jected to extreme environments, such as low oxygen levels, analysis revealed the presence of β-CA in Enterobacter sp. low CO levels and pH changes, requiring the microorganisms RS1. Genetic studies confirmed the presence of β-CA in to possess carbonic anhydrase (CA) activity (Tashian 1989). both Enterobacter sp. RS1 and Citrobacter freundii SW3. Thezincmetallo-enzymeCAplays avital role in acid–base Our analysis of the divergence of cynT and can genes among homeostasis, buffering the pH in bacteria by catalyzing the harboring members indicated that the can gene was more reversible conversion of CO dioxide into bicarbonates. Micro- prominent in Enterobacteriaceae than cynT. Sequence anal- organisms express four forms of CA (α, β, γ and δ) which are ysis of the can gene revealed a >25 % similarity among all functionally convergent and evolutionarily independent (Smith sequences and a >50 % similarity among sequences from and Ferry 2000). The α-CA form is widely reported in eukar- the Enterobacteriaceae family. The β-CA from C. freundii yotes, especially mammals, the β-CA is reported in plants, SW3 and Enterobacter sp. RS1, isolated from soil and used animals and microorganisms, the, the γ-CA form is found in in this study, possessed a high similarity with the can gene. archea and bacteria and the δ-CA is restricted to diatoms and The close association among Enterobacteriaceae genera usu- dinoflagellates (Tripp et al. 2004). CA in photosynthetic micro- ally found in the soil and gut and the sequence similarity of organisms plays a major role alongside Rubisco in the carbon- β-CA in the different genera of Enterobacteriaceae suggest concentrating mechanism (Ghoshal et al. 2002). However, less the importance of the can gene in oscillating environmental is known about the physiological roles of the CAs in non- conditions. photosynthetic microorganisms. The β-CA is the most prevalent form of CA among the . . Keywords Carbonic anhydrase Enterobacteriaceae genera of the Enterobacteriaceae, although some members of . . Citrobacter freundii Enterobacter sp. Soil this family also possess γ-CA forms. However, the functions of these γ-CAs are still not well-defined. Escherichia coli possess three candidates for γ-CAs, YrdA, PaaY and CaiE, although these γ-CA class enzymes have not been found to have any activity (Eichler et al. 1994; Elssner et al. 2001; Ferrandez et al. 1998). There are multiple copies of both forms in some organ- : : : R. Ramanan K. Kannan (*) S. D. Sivanesan T. Chakrabarti isms. Four genes, cynT, can (also interchangeably denoted as Environmental Health Division, National Environmental yadF), cah and mig-5,encoding β-CA have been reported to be Engineering Research Institute (NEERI), Nehru Marg, expressed in Enterobacteriaceae. However, the cah and mig-5 Nagpur 440 020 Maharashtra, India e-mail: k_krishnamurthi@neeri.res.in genes have been found only sporadically among affiliates of 1276 Ann Microbiol (2013) 63:1275–1282 Enterobacteriaceae, with cynT and can being the most promi- isolates Enterobacter sp. RS1 (EU124384) and C. freundii nent members (Beach and Osuna 1998). The cynT gene was SW3 (EU124389), respectively. first identified in E. coli as a part of the cyn operon and was found to express β-CA, thus dispelling the fact that β-CA was Protein isolation and determination conserved among higher plants (Sung and Fuchs 1988). The indispensability and necessity of can gene expression under The intracellular protein extraction was carried out by first low CO concentration have been well established (Hashimoto washing the cell pellet with distilled water, then resuspend- and Kato 2003). Mutation analysis of the Ralstonia eutropha ing the pellet in lysis buffer containing 250 mM Tris–HCl can gene highlights the importance of the gene product, β-CA, buffer and Triton-X 100, followed by sonication for 10 min. for the survival of the organism at low CO concentrations The pellet was discarded after centrifugation at 12,000 g and (Kusian et al. 2002). The pH-dependent activity of the enzyme the supernatant stored for future analysis. The protein con- coded by can has also been studied, with the results suggesting centration was determined using a previously published a possible conformational change into a stable yet non-active method (Lowry et al. 1951) with bovine serum albumin form at lower pH and high activity at higher pH (Cronk et al. (Sigma, USA) as the standard, whereas the protein content 2001). However, numerous studies have established that CA is of the diluted chromatographic fractions was measured by essential for survival at low pH and low CO conditions monitoring the optical density at 280 nm. (Mitsuhashi et al. 2004; Sly and Hu 1995). Although the results of previous studies indicate the role of CA at low CO CA purification and assay and different pH levels, the preference of an organism for one gene over another has not been established in natural habitats, Carbonic anhydrase was purified from Enterobacter sp. strain such as the soil and gut. In these habitats, where intra-day pH RS1 using the method published in Ramanan et al. (2009b) fluctuations are common, it is also imperative to ascertain the with slight modifications. The crude protein obtained from role of CA in the survival of microorganisms. ammonium sulfate precipitation was loaded first onto a Studying the phylogenetic relationship of the can and cynT Sephadex-G-75 column, then onto a DEAE cellulose column. genes may provide information that will help explain the sim- The resulting purified enzyme was subsequently dialyzed ilarities and differences of these genes among members of the against Tris–HCl (pH 8.3) overnight. The hydration assay Enterobacteriaceae. In addition, studying the incidence of these (Wilbur–Anderson, W–A) for CA was performed using the genes among members of Enterobacteriaceae would further procedure of Khalifah (1971), and esterase activity was deter- help researchers to arrive at a conclusion on this predilection mined using the procedure reported by Armstrong et al. (1966). factor. The results of our study contribute information on the presence of cynT and can gene in Enterobacteriaceae genera Western blot analysis and the possible implications of the presence of these genes. Crude protein samples were separated using sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and Materials and methods were transferred to a nitrocellulose membrane (Bio-Rad, Her- cules, CA) using a wet transfer unit for 2 h at 4 °C and 80 V. Bacterial strains and growth conditions All blocking and antiserum incubations were performed in TBST (20 mM Tris–HCl, pH 7.5, 0.5 M NaCl, 0.05 % Tween Pure cultures were isolated from soil samples, grown in Luria– 20). Themembranewas blocked2hin 5 %skim milk–Tris Bertani broth at 37 °C in a rotary shaker for 24 h at 100 rpm buffer solution–Tween 20 (TBST) at room temperature. The and processed for protein analysis. Genomic DNA was membrane was incubated overnight at 4 °C with β-CA anti- extracted and purified from cell pellets, and its purity was body diluted 1:1,000. E. coli CA was used as a positive assessed using a spectrophotometric method at A /A standard and the molecular weight marker for CA. The mem- 260 280 (Sambrook and Russell 2001 ). The culture was studied for brane was then rinsed three times in TBST and incubated for morphological, physiological and biochemical characteristics 2 h at room temperature with a 1:3,000 dilution of pig anti- following standard procedures. Universal 16S rDNA PCR rabbit horseradish peroxidase-conjugated immunoglobulin G, forward (5′-AGAGTTTGATCMTGGCTCAG) and reverse which resulted in an antigen–antibody complex (Alber and (5′-AAGGAGGTGWTCCARCC) primers were used in the Ferry 1996; Ramanan et al. 2009a). amplification of 16S rDNA genes. The amplification products were purified by using a DNA extraction kit (Genei, India) and Cloning and sequencing of can and cynT genes the DNA was sequenced (Bangalore Genei, India). The obtained 16S rDNA sequence has been deposited in GenBank, The primer sets were self-designed with the sequence details of the E. coli can gene. The forward and reverse primer sequences National Center for Biotechnology Information (NCBI) for Ann Microbiol (2013) 63:1275–1282 1277 used in our study to amplify the cynT gene were 5′- sufficient for purification of the enzyme to homogeneity GACGTGTTTTTTTGTCAGG and 5′-AGATACACTCAT- (Table 1). SDS-PAGE of the purified CA with the high CAGCAAC, respectively. The forward and reverse primer activity fraction revealed that the molecular weight of the sequences for the can gene were 5′-TTATTTGTGG enzyme was approximately 24 kDa protein, which was TTGGCGTGT and 5′-AGATACACTCATCAGCAAC, re- supported by the western blot results (Fig. 1). The western spectively. A 650-bp DNA fragment was amplified from both blot analysis of the protein sample showed a cross-reaction Enterobacter sp. RS1 and C. freundii strain SW3byPCR in a for Enterobacter sp. RS1 with the E. coli β-CA used as the Applied Biosystems (USA) thermal cycler (model GeneAmp positive control and molecular weight marker (Fig. 2). The 9,700) in a total reaction volume of 50 μl containing 50 ng CA enzyme from Enterobacter sp. RS1 possessed a similar genomic DNA, 50 pmol of each primer, 0.2 mM of each molecular weight to that of the E. coli CA (approx. 24 kDa). dNTPs, 1× polymerase buffer and 1 U Taq DNA polymerase This is the first study to confirm the presence of β-CA in an (New England Biolabs, USA). The program used consisted of a Enterobacter genus of Enterobacteriaceae. Earlier studies denaturation step at 94 °C for 4 min, 30 cycles at 94 °C for 30 s, have confirmed the presence of β-CA by western blotting 55 °C for 45 s and 72 °C for 45 s and a final extension at 72 °C in E. coli, C. freundii and Salmonella typhimurium among for 5 min. The PCR product was purified and cloned into the T- the affiliates of Enterobacteriaceae (Ramanan et al. 2009a; vector (Bangalore Genei, India), which was further sequenced. Smith et al. 1999). Phylogenetic analysis Sequencing and phylogenetic analysis of the β-CA gene The deduced amino acid sequences of the can gene obtained Sequencing of the approximately 650-bp amplicon con- were compared against database sequences using tBLASTx firmed the presence of the β-CA gene in Enterobacter sp. provided by NCBI (http://www.ncbi.nlm.nih.gov) and were RS1 and C. freundii SW3. The BLAST program for the aligned and clustered using Clustal-X. Phylogenetic trees sequenced and cloned amplicons yielded similar sequences were constructed using the PHYLIP programs and were visu- of β-CA, with most belonging to those of the Enterobacter- alized using TREEVIEW software, ver. 1.6.6 (Thompson et iaceae family. The phylogenetic tree suggests that both al. 1997). The phylogenetic tree based upon the neighbor- Enterobacter sp. RS1 and C. freundii SW3 β-CA genes joining (NJ) algorithm was performed with 1,000 bootstrap- are closely related to the can gene of many Enterobacter- ping replicates. The COG (Clusters of Orthologous Groups) iaceae affiliates (Fig. 3). Hence, we constructed a multiple database together with the COGNITOR program (http:// alignment of deduced amino acids among homologs from www.ncbi.nlm.nih.gov/COG/old/xognitor.html)wereused to the Enterobacteriaceae family, a total of 18 protein sequen- fit experimental sequences into the COGs to unearth paralogs ces, which yielded 21 identical amino acids, 39 conserved within complete genomes. All sequences for comparisons sequences and 42 semi-conserved sequences (data not were retrieved from protein databases using the Entrez pro- shown). It should be noted that cloned amplicons obtained gram (NCBI, National Institutes of Health, USA). from both the microorganisms used in this study shared a high similarity with the can gene in terms of phylogenetic relationship and sequence identity. Results Phylogenetic analysis of the can and cynT genes Purification and western blotting of CA from Enterobacter sp The ENTREZ program was searched for genera harboring The purification of CA from Enterobacter sp. was carried the can gene encoding β-CA. One representative species out by ammonium sulfate precipitation, gel filtration and ion from each genus was selected for multiple alignment of exchange chromatography, in that order. These steps were deduced amino acid sequences of the can gene (data not Table 1 Sequential purification of carbonic anhydrase from the ammonium sulfate-precipitated protein extract of Enterobacter sp. RS1 assayed by esterase activity Purification stages Total protein (mg/ml) Total activity (U/ml) Specific activity (U/mg) Relative yield (%) Purification fold Concentrated protein extract RS1 153 303 1.980 100 1 Gel filtration RS1 37 219 5.92 72 2.99 DEAE cellulose RS1 3.53 154 43.62 50.8 22.03 One enzyme unit is defined as formation of μmol p-nitro phenol (p-NP) per minute 1278 Ann Microbiol (2013) 63:1275–1282 Fig. 1 Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS- PAGE) of purified beta-carbonic anhydrase (β-CA) from Enterobacter sp. RS1 following three consecutive chromatographic steps. Lanes: 1 purified β-CA with a molecular weight of approx. 24 kDa, 2 molecular weight marker Fig. 3 Phylogenetic analysis of sequences similar to the experimental sequences of Citrobacter freundii SW3 and Enterobacter sp.RS1.The scale bar is given in fixed nucleotide substitutions per sequence position. GenBank shown), leading to the identification of the can gene in 15 accession numbers of sequences used in this analysis are given in parenthesis different genera. Nonetheless, 13 of the 19 genera identified belong to the Enterobacteriaceae family, indicating that the can gene was not diversely distributed among the bacterial domain studied and that the sequences of the can gene were conserved (Figs. 3, 4) (Merlin et al. 2003). The sequence alignment of the can gene also suggested a >25 % similarity among all sequences and a >50 % similarity among sequen- ces from the Enterobacteriaceae family. The analysis of Fig. 4 Phylogenetic tree of can gene sequences from 15 genera of Fig. 2 Western blot analysis of β-CA validated the presence of β-CA Enterobacteriaceae. The scale bar is given in fixed nucleotide substi- in Enterobacter sp. RS1 (lane 2) with Escherichia coli CA used as the tutions per sequence position. GenBank accession numbers of sequen- positive control and molecular weight marker (lane 1) ces used in this analysis are given in parenthesis Ann Microbiol (2013) 63:1275–1282 1279 enzyme characteristics suggested that the β-CA encoded by blot analysis and purification of CA from Enterobacter sp. the gene is usually a protein with 215–220 amino acids and RS1 suggest that this gene is expressed in most members of an approximate molecular weight of 24–25 kDa (Table 2). Enterobacteriaceae (Figs. 2 and 4). The β-CA from Enterobacter sp. RS1 and C. freundii SW3 One representative species from each genus was selected also seem to be homologs of the can genes reported. The for phylogenetic analysis and multiple sequence alignment phylogenetic data coupled with the results of the western of the cynT gene was performed (data not shown). The Table 2 Beta-carbonic anhydrase-encoding genes present among members of the Enterobacteriaceae family Genus Microorganism Gene type Characteristics Reference or Common natural habitats accession number (Janda and Abbott 2006) Molecular Number of weight (Da) amino acids Citrobacter Citrobacter koseri ATCC can 28,681 258 YP_001456157 Soil, feces, water, sewage, food BAA-895 Citrobacter freundii SW3 can 24,000 186 EU919747 (this study) Soil, water, sewage, food, and the intestinal tracts of animals and humans Enterobacter Enterobacter sp. 638 can 24,959 220 YP_001175412 Soil, fresh water, plants, sewage, animal feces Enterobacter sakazakii ± cynT 23,055 207 YP_001439024 Soil, water, sewage, plants Enterobacter sp. RS1 can 24,000 202 EU919748 (this study) Soil Erwinia Erwinia tasmaniensis can 25,027 220 YP_001906796 Soil and plants Erwinia carotovora cah 26,533 244 YP_051416 Soil and plants Escherichia Escherichia coli can 25,024 220 NP_285822 Gastrointestinal tract of humans O157:H7 EDL933 and many of the warm blooded animals, soil, water, and plants Escherichia albertii can 24,993 220 ZP_02902992 Soil, water, and plants Escherichia coli str. K-12 can 24,966 220 YP_001729083 Gastrointestinal tract of humans cynT 23,633 219 NP_414873 and many of the warm blooded animals, soil, water, and plants Klebsiella Klebsiella pneumoniae ± cynT 23,113 211 YP_001335672 Soil, water, plants cah 27,145 246 Ref. [8] can 23,244 204 YP_001333824 Pectobacterium Pectobacterium carotovorum cah 26,645 244 AAC77891 Soil and plants Photorhabdus Photorhabdus luminescens cynT 23,679 219 NP_927481 Gastrointestinal lumen of Photorhabdus luminescens ± can 24,629 217 NP_928206 entomopathogenic nematodes and soil Providencia Providencia stuartii ± can 25,827 217 EDU60433 Soil, water, sewage Salmonella Salmonella enterica can 24,690 220 YP_215158 Gastrointestinal tract of humans mig 5 26,513 246 YP_209302 and animals, soil, and water Salmonella typhimurium can 24,690 220 NP_459176 Gastrointestinal tract of humans and animals, and soil Serratia Serratia proteamaculans ± can 24,604 218 YP_001480220 Soil, water, plants Shigella Shigella flexneri can 24,966 220 YP_687707 Soil and water Shigella boydii can 24,947 220 YP_406676 Soil and water Shigella dysenteriae can 24,992 220 YP_401762 Soil and water Shigella sonnei can 24,966 220 YP_309163 Soil and water Sodalis Sbodalis glossinidius ± can 24,760 218 YP_454163 Gastrointestinal tract of insects Yersinia Yersinia pseudotuberculosis can 24,739 220 YP_069265 Soil and water Yersinia pestis can 24,739 220 NP_406869 Soil and water cynT 26,681 251 NP_994143 Yersinia enterocolitica can 24,709 220 YP_001005059 Soil and water ± represents putative presence of the gene Sodalis glossinidius is the only microorganism among Enterobacteriaceae which primarily does not inhabit soil 1280 Ann Microbiol (2013) 63:1275–1282 sequence identity analysis using Clustal X revealed that the Lin et al. 1995; Taneva et al. 2006). Members of the Enter- selected sequences were highly diverse, with only four obacteriaceae family, especially the genera used in this study, amino acid sequences being semi-conserved and no identi- Citrobacter and Enterobacter, are major soil residents. As cal amino acid among the sequences. The cynT gene was indicated in Table 2, the primary natural habitat of all micro- distributed among 37 genera tested, of which only three organisms belonging to Enterobacteriaceae is the soil, with the genera belong to Enterobacteriaceae family, including E. exception of Sodalis glossinidius (Janda and Abbott 2006). coli (Fig. 5; Table 2). The results of the phylogenetic diver- CA plays a crucial role in soil microorganisms and intestinal sity and sequence identity analysis suggest that the cynT microflora of higher animals and invertebrates, where extreme gene may have independently evolved among the genera, pH changes are common occurrences (König and Varma which is plausible considering the evolutionary indepen- 2005; Winfield and Groisman 2003). It has been proven that dence of all classes of CA. E. coli encodes proteins that mediate resistance to pH, espe- cially acidic pH (Winfield and Groisman 2003). The role of CA anhydrase in pH homeostasis has also been noted (Sly and Discussion Hu 1995). Although these studies have established the role of CA at low CO and pH changes, the results of our study Soil and the intestines of mammals and humans are subjected establish the prevalence of CA among soil microorganisms, to varying pH ranges and CO concentrations, and genera of especially Enterobacteriaceae, which experience these condi- the Enterobacteriaceae are known to adapt well to such tions. There is a strong possibility that the can gene present in changeable conditions (Epron et al. 1999; Leyer et al. 1995; C. freundii and Enterobacter sp. as well as the can gene Fig. 5 Phylogenetic tree of cynT sequences from 37 genera. CynT gene was found to be widespread among the bacterial domain. Haloquadratum walsbyi (YP_657802), a square halophilic archaeon, is the only member of the domain archaea to be reported to possess cynT. The scale bar is given in fixed nucleotide substitutions per sequence position. GenBank accession numbers of sequences used in this analysis are given in parenthesis Ann Microbiol (2013) 63:1275–1282 1281 identified in other Enterobacteriaceae affiliates are quite sim- Armstrong J, Myers D, Verpoorte J, Edsall J (1966) Purification and properties of human erythrocyte carbonic anhydrases. J Biol ilar in properties—i.e. expressed only in a low CO environ- Chem 241:5137–5149 ment and at pH-defining conditions. The experimental Bach S, Almeida A, Carniel E (2000) The Yersinia high-pathogenicity evidence and results of the bioinformatic analysis presented island is present in different members of the family Enterobacter- in this study indicate that CA coded by the can gene play a iaceae. FEMS Microbiol Lett 183:289–294 Beach M, Osuna R (1998) Identification and characterization of the fis definite role in Enterobacteriaceae. The scenario of can gene operon in enteric bacteria. J Bacteriol 180:5932–5946 prevalence may possibly be similar to that of the Yersinia Cronk J, Endrizzi J, Cronk M, O’neill J, Zhang K (2001) Crystal high-pathogenicity island, which has been demonstrated to structure of E. coli β-carbonic anhydrase, an enzyme with an unusual pH-dependent activity. Protein Sci 10:911–922 be a part of the genome in all Enterobacteriaceae affiliates Eichler K, Bourgis F, Buchet A, Kleber H, Mandrand-Berthelot M through horizontal gene transfers (Bach et al. 2000). (1994) Molecular characterization of the cai operon necessary for Based on the results of their study of the cynT gene and carnitine metabolism in Escherichia coli. Mol Microbiol 13:775– its metabolism, Sung and Fuchs (1988) reported that the cynT gene is usually in repressed form and expressed only Elssner T, Engemann C, Baumgart K, Kleber H (2001) Involve- ment of coenzyme A esters and two new enzymes, an enoyl- for cyanate metabolism. Its role is to prevent the depletion of CoA hydratase and a CoA-transferase, in the hydration of intracellular bicarbonate, which accompanies the cyanase- crotonobetaine to L-carnitine by Escherichia coli. Biochem- catalyzed bicarbonate-dependent hydrolysis of cyanate. This istry 40:11140–11148 study led to breakthroughs in studies on the other paralog, Epron D, Farque L, Lucot E, Badot P-M (1999) Soil CO efflux in a beech forest: dependence on soil temperature and soil water the can gene, which encodes β-CA, and it was subsequently content. Ann For Sci 56:221–226 proven that to survive under atmospheric CO concentra- Ferrandez A, Minambres B, Garcia B, Olivera E, Luengo J, Garcia J, tions and at varying pH conditions, the expression of this Diaz E (1998) Catabolism of phenylacetic acid in Escherichia gene is mandatory (Hashimoto and Kato 2003; Kusian et al. coli: characterization of a new aerobic hybrid pathway. J Biol Chem 73:25974–25986 2002; Mitsuhashi et al. 2004). Ghoshal D, Husic H, Goyal A (2002) Dissolved inorganic carbon concentration mechanism in Chlamydomonas moewusii.Plant Physiol Biochem 40:299–305 Conclusion Hashimoto M, Kato J-I (2003) Indispensability of the Escherichia coli carbonic anhydrases yadF and cynT in cell proliferation at low CO partial pressure. Biosci Biotechnol Biochem 67:919–922 Based on our results, we conclude that the can gene is Janda J, Abbott S (2006) The Enterobacter. ASM Press, Washington preferred over the cynT gene, considering its predomi- D.C. nance in Enterobacteriaceae. The abundance of the can König H, Varma A (2005) Intestinal microorganisms of termites and other invertebrates, 1st edn. Springer, Berlin gene among the Enterobacteriaceae family and its un- Khalifah R (1971) The carbon dioxide hydration activity of carbonic usual properties as revealed by earlier reports (Cronk et anhydrase. J Biol Chem 246:2561–2573 al. 2001;Merlinetal. 2003; Ramanan et al. 2009a) Kusian B, Sultemeyer D, Bowien B (2002) Carbonic anhydrase is provide a valid insight into this predilection factor. essential for growth of Ralstonia eutropha at ambient CO .J Bacteriol 184:5018–5026 Thepresenceofthe can gene among C. freundii and Leyer G, Wang L-L, Johnson E (1995) Acid Adaptation of Escherichia Enterobacter sp., its prevalence in almost all of the coli O157:H7 increases survival in acidic roods. Appl Environ members of the Enterobacteriaceae family and the in- Microbiol 61:3752–3755 dispensability of the can gene for the survival of organ- Lin J, Lee I, Frey J, Slonczewski J, Foster J (1995) Comparative analysis of extreme acid survival in Salmonella typhimurium, isms at varying pH and low CO concentrations, which Shigella flexneri,and Escherichia coli. J Bacteriol 177:4097– are conditions that usually prevail in the soil, as proven by other studies, highlight its value. Lowry O, Rosebrough N, Farr A, Randall R (1951) Protein measure- ment with the Folin phenol reagent. J Biol Chem 193:265–275 Merlin C, Masters M, McAteer S, Coulson A (2003) Why is carbonic Acknowledgments Rishiram Ramanan thanks the University Grants anhydrase essential to Escherichia coli. J Bacteriol 185:6415– Commission, New Delhi for the award of Senior Research Fellowship and for providing financial support. The authors thank the Department Mitsuhashi S, Ohnishi J, Hayashi M, Ikeda M (2004) A gene homol- of Biotechnology, Government of India for the financial support pro- ogous to β-type carbonic anhydrase is essential for the growth of vided for this project. Corynebacterium glutamicum under atmospheric conditions. Appl Microbiol Biotechnol 63:592–601 Ramanan R, Kannan K, Sivanesan S, Mudliar S, Kaur S, Tripathi A, Chakrabarti T (2009a) Bio-sequestration of carbon dioxide using carbonic anhydrase enzyme purified from Citrobacter freundii. References World J Microbiol Biotechnol 25:981–987 Ramanan R, Kannan K, Vinayagamoorthy N, Ramkumar K, Sivanesan Alber B, Ferry J (1996) Characterization of heterologously produced S, Chakrabarti T (2009b) Purification and characterization of a novel plant-type carbonic anhydrase from Bacillus subtilis. Bio- carbonic anhydrase from Methanosarcina thermophila. J Bacter- technol Bioprocess Eng 14:32–37 iol 178:3270–3274 1282 Ann Microbiol (2013) 63:1275–1282 Sambrook J, Russell R (2001) Molecular cloning: a laboratory manual, respiration in a temperate forest exposed to elevated CO concen- 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring tration. Glob Chang Biol 12:983–994 Sly W, Hu P (1995) Human carbonic anhydrases and carbonic anhy- Tashian R (1989) The carbonic anhydrases: widening perspectives on drase deficiencies. Annu Rev Biochem 64:375–401 their evolution, expression and function. BioEssays 10:186–192 Smith K, Ferry J (2000) Prokaryotic carbonic anhydrases. FEMS Thompson J, Gibson T, Plewniak F, Jeanmougin F, Higgins D (1997) Microbiol Rev 24:335–366 The Clustal X Windows interface: flexible strategies for multiple Smith K, Jakubzick C, Whittam T, Ferry J (1999) Carbonic anhydrase sequence alignment aided by quality analysis tools. Nucleic Acids is an ancient enzyme widespread in prokaryotes. Proc Natl Acad Res 25:4876–4882 Sci USA 96:15184–15189 Tripp B, Bell C, Cruz F, Krebs C, Ferry J (2004) A role for iron in an Sung Y-C, Fuchs J (1988) Characterization of the cyn Operon in ancient carbonic anhydrase. J Biol Chem 279:6683–6687 Escherichia coli K12. J Biol Chem 263:14769–14775 Winfield M, Groisman E (2003) Role of nonhost environments in the Taneva L, Pippen J, Schlesinger W, Gonzalez-Meler M (2006) The lifestyles of Salmonella and Escherichia coli. Appl Environ turnover of carbon pools contributing to soil CO and soil Microbiol 69:3687–3694

Journal

Annals of Microbiology – Springer Journals

Published: Dec 30, 2012

There are no references for this article.