Characterization of a Mesorhizobium loti {alpha}-Type Carbonic Anhydrase and Its Role in Symbiotic Nitrogen Fixation

Carbonic anhydrase (CA) (EC 4.2.1.1) is a widespread enzymecatalyzing the reversible hydration of CO₂ to bicarbonate, areaction that participates in many biochemical and physiologicalprocesses. Mesorhizobium loti, the microsymbiont of the modellegume Lotus japonicus, possesses on the symbiosis island agene (msi040) encoding an ${alpha}$ -type CA homologue, annotated as CAA1.In the present work, the CAA1 open reading frame from M. lotistrain R7A was cloned, expressed, and biochemically characterized,and it was proven to be an active ${alpha}$ -CA. The biochemical and physiologicalroles of the CAA1 gene in free-living and symbiotic rhizobiawere examined by using an M. loti R7A disruption mutant strain.Our analysis revealed that CAA1 is expressed in both nitrogen-fixingbacteroids and free-living bacteria during growth in batch cultures,where gene expression was induced by increased medium pH. L.japonicus plants inoculated with the CAA1 mutant strain showedno differences in top-plant traits and nutritional status butconsistently formed a higher number of nodules exhibiting higherfresh weight, N content, nitrogenase activity, and ${delta}$ ¹³C abundance.Based on these results, we propose that although CAA1 is notessential for nodule development and symbiotic nitrogen fixation,it may participate in an auxiliary mechanism that buffers thebacteroid periplasm, creating an environment favorable for NH₃protonation, thus facilitating its diffusion and transport tothe plant. In addition, changes in the nodule ${delta}$ ¹³C abundancesuggest the recycling of at least part of the HCO₃^– producedby CAA1.

INTRODUCTION

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INTRODUCTION

The interaction of the soil bacteria belonging to the generaMesorhizobium, Rhizobium, Bradyrhizobium, Sinorhizobium, andAzorhizobium with the root systems of leguminous plants resultsin the formation of a novel specialized plant organ, the rootnodule (27). Symbiotic nitrogen fixation (SNF), which takesplace inside nodules, represents a major mechanism for the enrichmentof agricultural and natural ecosystems in assimilated nitrogen(42). During this mutually beneficial plant-microbe interaction,the plant provides the microsymbiont with photosynthate, mainlyin the form of organic acids, together with other nutrients,in return for fixed nitrogen in the form of ammonium and aminoacids (28, 41). Thus, in mature nitrogen-fixing nodules, bothsymbionts must provide the proper chemical environment for boththe reduction of N₂ to ammonium by bacteroids and the efficientexchange of metabolites through the peribacteroid membrane andthe peribacteroid space (44). The establishment of this environmentis underpinned by the coordinated expression of specializedgenes in both organisms. Therefore, the elucidation of the exactphysiological and biochemical roles of these genes representsa major research goal for years to come. In the case of Mesorhizobiumloti, the microsymbiont of several Lotus species, includingthe model legume Lotus japonicus, many of the genes requiredfor nodulation, nitrogen fixation, and nutrient exchange resideon a symbiosis island, a chromosomally integrated element thatcan be transferred between mesorhizobia in the environment,converting them into strains able to nodulate Lotus species(18, 36).

Carbonic anhydrase (CA) (EC 4.2.1.1) is a zinc-containing metalloenzymethat catalyzes the reverse hydration of CO₂ to bicarbonate.CA is a ubiquitous enzyme, present in both prokaryotes and eukaryotes,and currently at least four distinct CA classes have been characterized,namely, ${alpha}$ , β, ${gamma}$ , and ${delta}$ (38). Interestingly, although the CAclasses show no significant sequence or structural homology,they share similar catalytic mechanisms employing a zinc atomthat is essential for catalysis (21, 22). Representatives ofall CA classes are widespread in prokaryotes, indicating theancient origins of these enzymes (33). In prokaryotes, ${alpha}$ -CAshave been characterized from several species, including thecyanobacteria Synechococcus and Anabaena (34) and the eubacteriaNeisseria gonorrhoeae (5), Helicobacter pylori (6), and Rhodopseudomonaspalustris (29).

CA catalyzes the reversible hydration of CO₂ to bicarbonate,a relatively simple but vital reaction, as it is involved inmany diverse biochemical and physiological processes, includingphotosynthesis, respiration, CO₂ and ion transport, and acid-basebalance. Similarly, in prokaryotes, various CA isoforms areessential for a wide range of cellular functions. Smith andFerry (32) summarized a variety of prokaryotic enzymes requiringor producing CO₂/HCO₃^–. In Escherichia coli, the expressionof the can gene, coding for a β-class CA, is required forgrowth in air, although it is dispensable when the partial pressureof CO₂ is high or during anaerobic growth (25). cynT, the onlycan paralog in E. coli, is transcribed as part of the cyanase(cyn) operon and serves to provide sufficient levels of cellularHCO₃^– for growth with cyanate as the nitrogen source (15).In R. palustris, a periplasmic ${alpha}$ -type CA has been proposed tobe essential for bicarbonate uptake (29). Finally, a periplasmic ${alpha}$ -type CA from H. pylori has been shown to be upregulated bylow environmental pH, being essential for buffering the periplasmicacidic pH. Recent studies suggest that this CA is an integralcomponent of the acid acclimation response that allows theseneutralophile bacteria to colonize the stomach (24, 43).

In this work, we present the biochemical and physiological characterizationof an ${alpha}$ -class CA (CAA1) located in the symbiosis island of thenitrogen-fixing bacterium M. loti and showing high similarityto the periplasmic ${alpha}$ -CAs characterized in H. pylori and R. palustris(7, 29). Our goal was to test the functionality of the encodedpolypeptide and to assess the physiological role of the productof this gene during nodule development and function by usingan M. loti disruption mutant. Furthermore, we aimed at studyingthe effect of pH on the expression of this gene in free-livingM. loti. Based on preliminary findings, our hypothesis was thatCAA1-catalyzed CO₂ hydration might participate in a biochemicalmechanism for buffering of the bacteroid periplasm at a pH favorablefor NH₃ protonation.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Strains, culture conditions, and plant growth. M. loti strains R7A and R7A CAA1::pFUS2 were kindly providedby Clive Ronson (University of Otago, Otago, New Zealand). M.loti strain R7A CAA1::pFUS2 was constructed by insertion-duplicationmutagenesis. An internal fragment of the CAA1 gene was amplifiedfrom M. loti strain R7A genomic DNA and cloned into the HindIIIand BamHI sites of the pFUS2 suicide vector for CAA1 inactivationby homologous recombination and generation of transcriptionalfusion to lacZ (2). Gene disruption was performed by conjugationusing M. loti strain R7A to generate M. loti strain R7A CAA1::pFUS2.The gene interruption was confirmed by PCR analysis and sequencingof the insertion site.

For the complementation of the CAA1::pFUS2 mutant strain, theCAA1 open reading frame (ORF), together with 134 bp of the terminatorregion, was amplified using 5'-AAATCTAGATTGCGGCTGTGCCTTGACTG-3'and 5'-AAAGGTACCTTCCCCATGATCCGGTCA-3' primers. The amplificationproduct was cloned as an XbaI-KpnI fragment into the pFAJ1709vector, yielding the plasmid pFAJCAA1. pFAJ1709 carries thespsAB symbiotic plasmid stability loci from pNGR234 ${alpha}$ of Rhizobiumsp. strain NGR234, which confers plasmid stability in free-livingrhizobia and bacteroids (11). pFAJCAA1 was used for transformationof the CAA1::pFUS2 strain by electroporation at 12.5 kV/cm,25 µF, and 200 ${Omega}$ . Cells competent for electroporation wereprepared as previously described (17). Transformants were confirmedby retransformation of E. coli cells and restriction enzymedigestion. Plasmid stability in nodules was confirmed by plasmidrescue from antibiotic-resistant bacteroids.

All strains were grown at 28°C on yeast mannitol broth (YMB)or tryptone-yeast extract supplemented with the appropriateantibiotic (50 µg/ml gentamicin for CAA1::pFUS2; 4 µg/mltetracycline for the pFAJCAA1 plasmid). For the constructionof growth curves, 25-ml starter cultures were used to inoculate200 ml of YMB to an initial optical density at 600 nm (OD₆₀₀)of 0.05. The pH of the culture medium was adjusted to 5.5 withMES (morpholineethanesulfonic acid)/KOH (pK_a, 6.1), to 7 withHEPES/NaOH (pK_a, 7.5), and to 8 with Tris/H₂SO₄ (pK_a, 8.0).All buffer concentrations were 50 mM, except for Tris (75 mM).

L. japonicus (Gifu B-129) seeds were kindly provided by JensStougaard (University of Aarhus, Aarhus, Denmark). The seedswere scarified for 5 min with H₂SO₄, sterilized for 20 min in2% (vol/vol) NaOCl-0.02% (vol/vol) Tween 20, pregerminated at18°C in the dark for 72 h, and spot inoculated with a suspensionculture of the respective M. loti strain at an OD₆₀₀ of 0.1.The plants were grown with nitrogen-free Hoagland nutrient solutionin a controlled environment with an 18-h day/6-h night rotation,a 22°C day/18°C night temperature regime, and 70% airrelative humidity (16).

Expression and purification of the recombinant CAA1 polypeptide. The ORF of the CAA1 gene, excluding the stop codon, was amplifiedand cloned as an NcoI-XhoI fragment into the expression vectorpET28a(+). Two versions of the ORF, with and without the predictedsignal peptide, were amplified and cloned. The expression plasmidswere transformed into E. coli strain BL21(DE3). The cells weregrown at 23°C in LB broth with 50 µg/ml kanamycinto an OD₆₀₀ of 0.6. CAA1 expression was induced with 0.5 mMIPTG (isopropyl-β-D-thiogalactopyranoside) and 0.5 mM ZnSO₄for 6 h. The cells were harvested and resuspended in lysis buffer(50 mM NaH₂PO₄, pH 8, 300 mM NaCl, 10 mM imidazole) before celldisruption by sonication. The recombinant enzyme was purifiedfrom the resulting supernatant by nickel affinity chromatography(Qiagen, Hilden, Germany). The purified protein elutions wereevaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresisand Coomassie brilliant blue staining. Dialysis was performedagainst 25 mM MES, pH 6.3, or 20 mM HEPES, pH 7.5.

Enzyme assays. CA activity was assayed by measuring changes in pH during thereaction by a dye indicator method (20). The assays were performedat 4°C. The buffer-indicator pairs used were (i) 50 mM MESplus 0.005% (wt/vol) bromothymol blue (pH 6.3; A₆₁₅) and (ii)50 mM HEPES plus 0.005% (wt/vol) phenol red (pH 7.5; A₅₅₇).Both solutions contained 50 mM sodium sulfate to maintain arelatively constant ionic strength of the reaction medium; 10µg of purified recombinant CAA1 protein was used in eachassay. Data were acquired using a Hitachi (Tokyo, Japan) U-2800spectrophotometer. The initial rate was measured during thefirst 5 seconds when the reaction was still at the linear phase.Unanalyzed rates, measured in the absence of the enzyme, wereused as negative controls and were subtracted from the observedrates. An ${alpha}$ -type bovine CA (ICN Biomedicals, Eschwege, Germany)was used as a positive control. Kinetic parameters were calculatedby fitting the corrected rate data to the Michaelis-Menten equationusing GraFit v3.5 (Erithacus Software, Surrey, United Kingdom).

Esterase activity was measured as p-nitrophenylacetate (4-NPA)hydrolysis at 25°C (3). The buffer used was 50 mM Tris/H₂SO₄,pH 7.6, and the substrate concentration was 3 mM. The reactionwas monitored for 5 min at 348 nm in a Hitachi U-2800 spectrophotometer.

β-Galactosidase activity was determined by o-nitrophenyl-β-D-galactopyranosidehydrolysis at 37°C according to the method of Miller (26).The substrate concentration was 0.4 M. The reaction was monitoredat 420 nm in a Hitachi U-2800 spectrophotometer.

Nitrogenase activity was estimated by acetylene reduction onwhole plants incubated at 25°C in 28-ml rubber cap tubescontaining 1/10 (vol/vol) acetylene. The ethylene produced atdifferent time points was quantified with a Perkin-Elmer 8500gas chromatograph (Perkin-Elmer Life and Analytical ScienceInc., Wellesley, MA) equipped with a 2-m Porapak R column anda flame ionization detector.

The histochemical staining of β-galactosidase activitywas a modification of the method described by Boivin et al.(4). The reaction buffer used was 0.1 M sodium-phosphate, pH7.0, and nodules were flash frozen in liquid nitrogen to aidthe penetration of glutaraldehyde. The nodules were then handsectioned and observed by bright-field microscopy.

Transcript analysis using real-time reverse transcription-qPCR. Total RNA was isolated from L. japonicus-inoculated roots andnodules and quantified by spectrophotometry and agarose gelelectrophoresis. RNA samples were treated with DNase I (Promega,Madison, WI) at 37°C for 45 min to eliminate contaminatinggenomic DNA. First-strand cDNA was reverse transcribed usingSuperScript II (Invitrogen, Paisley, United Kingdom) and randomhexanucleotides (Invitrogen). CAA1 transcripts were specificallyamplified using Ml0040-274F (5'-CAAATCAACATGCCGGAAGG-3') andMl0040-338R (5'-AACTGTGCCAGTTGGTAGACGC-3') primers. PCRs wereperformed on the Stratagene MX3005P using Power SYBR green mastermix (Applied Biosystems). PCR cycling started at 95°C for10 min, followed by 40 cycles of 95°C for 15 s and 60°Cfor 1 min. The primer specificity and the formation of primerdimers were monitored by dissociation curves and agarose gelelectrophoresis on a 4% (wt/vol) gel. The expression levelsof the M. loti polyribonucleotide nucleotidyltransferase gene(mlr5562), detected using mlr5562-499F (5'-ATCGACGAAATGCAGGAATCC-3')and mlr5562-576R (5'-TTCCGACTCAACCATCAGCAC-3') primers, wereused as internal standards. Relative transcript levels of thegene of interest (X) were calculated as a ratio to the mlr5562gene transcripts (S) as (1 + E)^–C^T, where ${Delta}$ C_T was calculatedas (C_T^X – C_T^S). The PCR efficiency (E) for each ampliconwas calculated by employing the linear regression method onthe log(fluorescence)-per-cycle-number data. All real-time quantitativePCRs (qPCRs) were performed on three biological repeats.

Determination of nodule and top-plant C isotopic composition and total C and N content. Nodules were isolated from the plant root system and oven dried(3 days; 65°C). The same procedure was followed for thetop plant (leaf and stem). All plant material was then groundwith a mortar and pestle into a homogeneous fine powder, andsamples of ca. 0.5 mg were transferred into tin capsules (IVAAnalysentechnik, Meerbusch, Germany). Subsequently, the sampleswere injected into an elemental analyzer (NA 2500; CE Instruments,Milan, Italy) coupled to an isotope ratio mass spectrometer(Delta Plus; Finnigan MAT GmbH, Bremen, Germany) by a ConfloII interface (Finnigan MAT GmbH, Bremen, Germany). The carbonisotope composition ( ${delta}$ ¹³C abundance) of plant tissues is an information-richsignal providing useful insights into different plant functions(1). The ${delta}$ ¹³C abundance of plant tissues is described as follows: ${delta}$ ¹³C_plant = ${delta}$ ¹³C_atm – ${alpha}$ – (b – ${alpha}$ )C_i/C, where ${delta}$ ¹³Cis expressed in per mille ( ${per thousand}$ ), ${alpha}$ is the discrimination duringdiffusion (ca. 4.4 ${per thousand}$ ), b is the discrimination during carboxylationby ribulose-bisphosphate carboxylase (ca. 29 ${per thousand}$ ), C_i is the CO₂concentration inside the stomatal cavities, and C_atm is theatmospheric CO₂ concentration (12).

Electron microscopy. Nodules were harvested 26 days postinoculation (p.i.) and fixedwith 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2)for 2 h, postfixed with 1% osmium tetroxide for 2 h, dehydratedin a graded series of alcohol followed by propylenoxide, andembedded in Spurr resin. Ultrathin sections were cut and stainedwith uranyl acetate, and specimens were examined and photographedwith a Zeiss 9S transmission electron microscope. At least threenodules for each genotype (wild type and CAA1::pFUS2) were examinedby electron microscopy.

Data analysis. Statistical analysis was performed with SPSS 12.0 (SPSS, Inc.,Chicago, IL). Comparison of mean values between the two strains(Tables 1 and 2) was conducted with independent-sample t testsat a 95% level of significance.

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TABLE 1. Comparison of parameters for L. japonicus plants inoculated with M. loti strains R7A, R7A CAA1::pFUS2, and R7A CAA1::pFUS2-pFAJ1709/CA^a

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TABLE 2. C and N elemental and ¹³C isotopic ratio analysis of L. japonicus plant parts inoculated with M. loti strain R7A or R7A CAA1::pFUS2^a

RESULTS

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RESULTS

An ${alpha}$ -class CA isoform is present in the symbiosis island of M. loti. Previous work had demonstrated that genes encoding nodule-specificCA isoforms are present in various legume species, includingMedicago sativa (10), Glycine max (19), and L. japonicus (14).In contrast, to our knowledge, nothing is known about the presenceand the physiological roles of CA isoforms in the microsymbiont.To this end, in silico homology searches revealed the presenceof a gene coding for an ${alpha}$ -CA homologue in the symbiosis islandsof both the R7A (msi040) and MAFF303099 (mll6384) strains ofM. loti. In both strains, the encoded polypeptides consist of250 amino acids, with a predicted molecular mass of 27.2 kDa.Three amino acid substitutions were detected between msi040-and mll6384-encoded polypeptides, including Gly9Ala, Thr152Met,and Lys158Asn.

Comparative analysis of the M. loti R7A msi040 deduced aminoacid sequence, annotated as CAA1, revealed that it shares 62%,53% and 45% similarity with the ${alpha}$ -type CAs from R. palustris,N. gonorrhoeae, and H. pylori, respectively (Fig. 1). Furthermore,all amino acids participating in the formation of the ${alpha}$ -CA activesite were found to be conserved in the CAA1 polypeptide, includingHis114, His116, and His133, which correspond to the zinc ligands.In silico topology prediction revealed that, similarly to theother bacterial ${alpha}$ -CAs, CAA1 has an N-terminal sorting-signalpeptide, which possesses the Tat (for twin-arginine translocation)motif RRNLI starting at position 3. In addition, topology analysisof the CAA1 polypeptide using the HMMTOP tool (http://www.enzim.hu/hmmtop/)(39) revealed the presence of a transmembrane helix betweenamino acids 9 and 26, with the N terminus localized in the cytoplasmand the C terminus, including the active site, facing the periplasm.

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FIG. 1. Comparison of the deduced amino acid sequence of the M. loti ${alpha}$ -CA (CAA1) with those of previously characterized bacterial ${alpha}$ -CAs. The sequences included are as follows: CAA1, M. loti (strain R7A) ${alpha}$ -CA (CAD31445); Hp ${alpha}$ CA, H. pylori ${alpha}$ -CA (BAE66646); Ng ${alpha}$ CA, N. gonorrhoeae ${alpha}$ -CA (CAA72038); and Rp ${alpha}$ CA, R. palustris ${alpha}$ -CA (BAA82053). Identical residues are black, while conservative amino acid substitutions are gray shaded. Conserved amino acids important for catalysis are marked with asterisks, and the predicted signal peptide is overlined. The sequences were aligned using ClustalX (version 2).

CAA1 codes for an active ${alpha}$ -CA. To determine the catalytic properties of CAA1, the coding region,with and without the predicted signal peptide, was subclonedinto the pET-28a vector, which drives the expression of theencoded polypeptide as an N-terminal fusion to a six-His tag.When the signal peptide was included, we could not detect accumulationof the recombinant polypeptide in the soluble protein fractionof E. coli. Interestingly, low levels of the polypeptide accumulatedin the periplasmic protein fraction (Fig. 2A). In contrast,the recombinant polypeptide lacking the predicted signal peptidewas found to accumulate at high levels in the soluble cytoplasmicprotein fraction (Fig. 2B). The expressed polypeptide lackingthe signal peptide had a molecular mass of around 25 kDa onsodium dodecyl sulfate-polyacrylamide gel electrophoresis andwas purified over 90% by Ni affinity chromatography (Fig. 2B).

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FIG. 2. Heterologous expression, purification, and enzymatic assays for CA activity of the recombinant CAA1 polypeptide. (A) Accumulation of the recombinant CAA1⁺ polypeptide (arrow) containing the predicted N-terminal signal peptide in the E. coli periplasmic proteins (lane 2) in comparison to periplasmic proteins from E. coli cells transformed with the empty vector (lane 1). (B) Expression and purification of the recombinant CAA1^– polypeptide (arrow) lacking the N-terminal signal peptide. Lane 1, total soluble proteins from E. coli cells transformed with the empty vector; lane 2, total soluble proteins from E. coli cells transformed with the CAA1^– expression plasmid; lane 3, purified CAA1^– recombinant polypeptide. (C) Kinetic analysis of the CO₂ hydration activity was performed at pH 7.5 in HEPES/NaOH buffer. (D) Kinetic analysis of the bicarbonate dehydration activity was performed at pH 6.3 in MES/KOH buffer. Kinetic parameters were calculated by fitting the corrected rate data (change in absorbance per min [ ${Delta}$ Abs]) to the Michaelis-Menten equation using the program GraFit v3.5. The rates shown are means of three repeats (n = 3).

The CO₂ hydration activity of the CAA1 recombinant polypeptidewas measured at 4°C in a HEPES/NaOH, pH 7.5, buffer. Therecombinant enzyme exhibited strong CO₂ hydration activity (specificactivity, 19.14·10⁶ µmol/min/mg), while it followedMichaelis-Menten kinetics with a k_cat of (8.136 ± 1.296)·10⁶s^–1 and a K_m of 31.729 ± 8.33 mM (Fig. 2C). Thereverse reaction of bicarbonate dehydration was again assayedat 4°C in a MES/KOH pH 6.3 buffer (Fig. 2D). In this case,the recombinant enzyme exhibited a specific activity of 2.85·10⁶µmol/min/mg. Again, the recombinant CAA1 displayed Michaelis-Mentenkinetics, with the following respective kinetic parameters:k_cat = (1.215 ± 0.095)·10⁶ s^–1 and K_m =112.7 ± 19.5 mM. In contrast to previously characterized ${alpha}$ -CA isoforms, the recombinant CAA1 polypeptide did not possessany measurable esterase activity with 4-NPA as a substrate.

CAA1 expression is pH regulated in free-living M. loti. In order to gain insight into the possible physiological rolesof CAA1, an M. loti strain R7A disruption mutant was obtained.The mutant was created by homologous recombination using thesuicide vector pFUS2 (2), which also creates a transcriptionalfusion between the interrupted gene and a promoterless lacZgene. The disruption of CAA1 was confirmed by amplification,cloning, and sequencing of a 700-bp region including the 5'region of CAA1 and the pFUS2 region corresponding to the N terminusof the lacZ polypeptide. The analysis revealed that the CAA1ORF was disrupted after Val166 (Fig. 3), while pFUS2 inserteda stop codon after 19 amino acids. In a first effort, we evaluatedthe effect of CAA1 disruption on the free-living phenotype.Since the homologous periplasmic ${alpha}$ -CA of H. pylori is implicatedin the acid acclimation of the bacteria (24) and the gene appearsto be pH regulated (43), we tested the growth of the CAA1::pFUS2strain in batch cultures in YMB medium with various pH values(Fig. 4A). No difference in growth was observed between theCAA1::pFUS2 and the R7A control strains (Fig. 4A) at all pHvalues tested, while at pH 8, the growth of both strains wasstrongly restricted. As an indication of the CAA1 promoter activity,β-galactosidase activity was determined during the growthof the CAA1::pFUS2 strain at the different pH values (Fig. 4B).During the exponential growth phase, a twofold induction ofβ-galactosidase activity was detected at pH 8, while enzymeactivity remained high during the stationary phase. At pH 7,a moderate induction of β-galactosidase was detected duringthe exponential phase, reaching a maximum during the decelerating-growthand stationary phases. The lowest levels of β-galactosidaseactivity were observed at pH 5.5, where a slight increase wasobserved during the decelerating-growth phase.

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FIG. 3. Schematic representation of the CAA1::pFUS2 disruption. The positions in the CAA1 ORF of the histidine zinc ligands are represented as black lines, while the positions of other residues participating in the formation of the active site and the hydrophobic CO₂ binding pocket are represented as gray lines.

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FIG. 4. (A) Growth of the M. loti CAA1::pFUS (CAA1::lacZ transcriptional fusion) disruption mutant and R7A control strains in batch YMB cultures at pH 5.5, 7, and 8. (B) CAA1 promoter activity was estimated by β-galactosidase activity assays in samples taken at the indicated OD₆₀₀ values. The values represent the means of four assays (± standard deviations). prot., protein.

Symbiotic phenotypes of the CAA1 mutant. In order to study CAA1 expression during nodule development,we performed real-time reverse transcription-qPCR assays oncDNA templates reverse transcribed from total RNA isolated fromnodulated root segments at 7 days p.i. and nodules at 10, 14,21, and 28 days p.i.. This analysis revealed that CAA1 geneexpression was induced twofold in mature nitrogen-fixing nodulesat 21 days p.i. in comparison to the levels in nodulated rootsand young developing nodules at 10 days p.i. (Fig. 5). Nitrogenasetranscripts were first detectable at 14 days p.i., indicatingthe onset of the nitrogen fixation process (data not shown).

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FIG. 5. Accumulation of CAA1 gene transcripts during nodule development. Total RNA was isolated, reverse transcribed to cDNA, and subjected to real-time qPCR using gene-specific primers. Relative mRNA levels were calculated with respect to the level of the polynucleotide nucleotidyltransferase (mlr5562) transcripts. The bars show means plus standard deviations (n = 3). dpi, days p.i.

As CAA1 gene expression was found to be upregulated during SNF,we investigated the effects of CAA1 disruption on nodule developmentand SNF. To this end, L. japonicus seedlings were inoculatedwith the CAA1::pFUS2 mutant and with R7A as a control. Bothstrains were able to form nitrogen-fixing nodules on L. japonicusroots. To visualize the expression pattern of the CAA1::lacZfusion, the spatial distribution of the gene fusion activitywas detected with histochemical staining of nodules at 21 daysp.i. Strong expression of the CAA1::lacZ fusion was detectedthroughout the infected cells of the nodule central tissue (Fig.6A). In contrast, no histochemical staining was observed innodules of the same age occupied by the M. loti R7A controlstrain (Fig. 6B).

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FIG. 6. Histochemical localization of β-galactosidase activity in L. japonicus nodules formed by M. loti CAA1::pFUS and M. loti R7A. Nodules at 21 days p.i. were hand sectioned and stained with X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). (A) In CAA1::pFUS-induced nodules, β-galactosidase activity was evident in the bacteroid-containing cells in the nodule central tissue. (B) No histochemical staining was detected in the nodules formed by M. loti R7A control strain. Bars, 200 µm.

Interestingly, while nodule color and macroscopic structurewere similar between nodules formed by the mutant and the wild-typestrains, a significant increase in the number of mature nitrogen-fixingnodules formed by the CAA1::pFUS2 mutant was consistently observedat 28 days p.i. during several independent experiments (Table1). The increase in nodule number was accompanied by a significantincrease in nodule fresh weight. Although, an increase in dryweight was also observed in CAA1::pFUS2 nodules, it was statisticallyinsignificant. In contrast to the altered nodulation kinetics,no significant differences in the top-plant growth were observed,as depicted by the determination of total hypergeous organ freshand dry weights at both 14 and 28 days p.i. (Table 1). Nitrogenaseactivity in the nodules formed by both strains was estimatedby measuring their acetylene reduction capacities (Table 1).In young nodules at 14 days p.i., nitrogenase activity of theCAA1 mutant was 1.5-fold higher than that of the wild-type R7Astrain. In mature nodules at 28 days p.i., this difference wasparticularly pronounced, as in these fully developed nodules,nitrogenase activity levels were found to be almost twofoldhigher in the nodules occupied by the CAA1 mutant.

In an attempt to test whether the increased nodulation capacityand elevated nitrogenase activity of the CAA1 mutant had animmediate effect on the plant's nutritional status, we measuredthe total C and N contents of nodules and hypergeous organsfrom plants inoculated with the mutant and R7A wild-type strains,respectively. At 28 days p.i., the total nitrogen content ofnodules formed by the CAA1 mutant was significantly higher thanthat of nodules of the R7A wild type (Table 2). In contrast,the total carbon content was not significantly altered betweennodules formed by the two strains. This resulted in a significantdecrease in the C/N ratio of the nodules formed by the ${alpha}$ -CA mutant(Table 2). Interestingly, the excess nitrogen content foundin CAA1::pFUS2 nodules did not have any effect on top-plantnutritional status, as both nitrogen and carbon total contentsof the hypergeous organs were similar between plants nodulatedwith the CAA1 mutant and the R7A wild type, respectively (Table2). A recent study showed that reduced levels of CA affectedthe carbon isotope discrimination ( ${Delta}$ ¹³C) and thus the leaf organic ${delta}$ ¹³C abundance in C₄ plants (8). Therefore, we aimed to testwhether a similar effect on the nodulated L. japonicus plantscould be detected. Interestingly, CAA1 disruption resulted ina small but significant enrichment of the nodules in ¹³C (higher ${delta}$ ¹³C abundance), whereas no effect was detected on the top-plantorgans (Table 2).

Finally, a comparison of the ultrastructure of nodules initiatedby the CAA1 mutant in comparison to the wild-type nodules failedto reveal any significant differences in bacteroid differentiationand morphology. In addition, no significant changes in bacteroiddensity were observed (data not shown).

In order to verify that the observed differences in nodule number,weight, and nitrogenase activity could be unambiguously attributedto the CAA1 disruption, the CAA1 coding and terminator sequenceswere cloned into the pFAJ1709 vector and reinserted into theCAA1::pFUS2 mutant strain. L. japonicus plants nodulated bythe resulting strain did not exhibit any significant differencesin nodule number, fresh and dry weight, or nitrogenase activityin comparison to the plants nodulated with the R7A control strain(Table 1).

DISCUSSION

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DISCUSSION

In this report, we present biochemical and physiological studiesof CAA1, an M. loti ${alpha}$ -type CA encoded by a single gene presentin the symbiosis island (18, 36). In contrast to the widespreaddistribution of CAs, only a few prokaryotes have been shownso far to possess active ${alpha}$ -type CA isoforms. They include H.pylori (6), the CO₂-requiring pathogen N. gonorrhoeae (5), thepurple non-sulfur phototrophic bacterium R. palustris (29),and the cyanobacteria Anabaena and Synechococcus (34). Similarly,in silico homology searches revealed that ${alpha}$ -type CA isoformsare not present in all genera of rhizobia. In addition to M.loti, ${alpha}$ -type CA homologues are present only in the Sinorhizobiummeliloti pSymA plasmid (gene SMa0045) and the Bradyrhizobiumjaponicum chromosome (gene bll1137 in strain USDA110).

A common feature of all bacterial ${alpha}$ -type CAs is the presenceof an N-terminal signal peptide, responsible for the periplasmiclocalization of the enzyme. In CAA1, the respective N-terminalsignal peptide possesses the characteristic motif found in theproteins exported through the Tat pathway. Although, we do notyet have experimental confirmation for the periplasmic localizationof CAA1 in M. loti, the N-terminal signal peptide resulted inthe periplasmic localization of the recombinant CAA1 in E. coli(Fig. 2A).

Comparison of the CAA1 polypeptide with characterized bacterialand mammalian ${alpha}$ -type CAs revealed that the identified zinc ligandsare invariant in all ${alpha}$ -CAs. In addition, all the hydrophobicresidues forming the CO₂ binding pocket near the enzyme activesite, namely, Val135, Val146, Leu199, Val208, and Trp210, areidentical in all bacterial ${alpha}$ -CAs, as well as the human CAII.Our functional assays confirmed that CAA1 codes for an active ${alpha}$ -type CA that is able to catalyze both the hydration of CO₂and the dehydration of bicarbonate. In contrast to the mammalianisoforms, we failed to detect any CAA1 esterase activity. Similarly,low 4-NPA hydrolase activity was also reported for the H. pylori ${alpha}$ -CA (6).

The possible biochemical and physiological roles of CAA1 duringsymbiosis were studied by the use of a CAA1::pFUS2 disruptionmutant strain. These experiments revealed that the CAA1::pFUS2strain was able to form nitrogen-fixing nodules on L. japonicus,although inactivation of the gene caused various symbiotic effects.The mutant strain formed a higher number of nodules with increasedfresh weight and nitrogenase activity (Table 1), although nodifferences were observed in nodule ultrastructure and bacterialdistribution in infected cells. The observed effects did notimprove the growth and nutritional status of plants nodulatedby the mutant strain (Table 2). The only difference observedin the nodules of these plants was an increase in their nitrogencontent. These findings indicate that any increase in nitrogenfixation due to increased nitrogenase activity is not beneficialfor the nonsymbiotic plant organs.

An interesting hint about the possible physiological role ofCAA1 during symbiosis comes from the physiological role of theH. pylori periplasmic ${alpha}$ -CA homologue. H. pylori and other Helicobacterspp. are able to colonize the acidic mammalian stomach, althoughthey are neutralophiles. This phenotype is mainly dependenton urease activity and urea influx through the UreI proton-gatedchannel (31). The products of urease activity in the cytoplasmare 2NH₃ plus CO₂, which diffuse very rapidly across the innermembrane into the periplasmic space. There, the ${alpha}$ -CA-catalyzedhydration of CO₂ provides both protons for NH₄⁺ formation andbicarbonate, which can buffer the periplasm at a pH value closeto its pK_a of 6.1, within the range of bacterial growth ability(24). Interestingly, a very similar situation exists in thesymbiotic nitrogen-fixing bacteroids, as large amounts of NH₃and CO₂ diffuse across the inner membrane into an acidifiedsymbiosome space, where NH₃ is protonated to NH₄⁺ for transportacross the symbiosome membrane (9, 44). The acidification ofthe symbiosome space is facilitated by a proton pumping P-typeH⁺-ATPase (13, 37, 40), although proteomic studies of the symbiosomemembrane fraction of L. japonicus and pea also revealed thepresence of V-type H⁺-ATPases (30, 45). The presence of an active ${alpha}$ -CA enzyme in the periplasm of the nitrogen-fixing bacteroidscould provide a complementary source of protons for the acidificationof the periplasm and the protonation of NH₃. In the presenceof the periplasmic CA, the hydration of the CO₂ produced bybacteroid respiration could be accelerated several thousandfold.The protons produced during the CA-catalyzed formation of HCO₃^–could subsequently be used for the protonation of NH₃. The rapidconversion of NH₃ to NH₄⁺ could serve as a trap, which in turnfacilitates the further diffusion of NH₃ from the bacteroidacross a steep concentration gradient. In addition, similarlyto H. pylori, the bicarbonate produced can buffer the periplasmat a slightly acidic pH, favoring NH₃ protonation. The absenceof a suitable buffering system, for maintaining the acidic externalpH could result in a less stable and unpredictable periplasmicpH (NH₄⁺/NH₃ pK_a, 9.2). The possible role of CAA1 in maintainingan acidic periplasm during SNF is supported by the observationthat the expression of the respective gene is upregulated bothupon the onset of nitrogen fixation and at alkaline pH valuesin free-living bacteria. Similarly, the expression of the H.pylori ${alpha}$ -CA was shown to be pH regulated by the ArsRS two-componentsystem (43). In the CAA1::pFUS2 nodules, the formation of NH₄⁺outside the bacteroid inner membrane could result in a localizedpH increase, which in turn would reduce the rate of NH₃ protonation,diffusion from the bacteroid cytosol, and transport to the plantcytosol, resulting in the observed increase in nodule N content.The increase in nodule number observed at later stages afterthe onset of SNF (28 days p.i.), in size, and in nitrogenaseactivity could represent a response to compensate for the reducednitrogen supplied by the microsymbiont. The increased nodulationkinetics observed after the onset of nitrogen fixation is atypical feature found in ineffective rhizobium-legume symbiosis(23, 35). In our plants, the increased nodulation kinetics wasaccompanied by an increase in nitrogenase activity, indicatingthat CAA1 disruption may limit the supply of symbiotically fixednitrogen to the plant at the step of transport and assimilation,rather than N₂ reduction. It should be pointed out that if sucha CA-based mechanism for facilitated NH₃ protonation and bufferingof the bacteroid periplasm exists, this mechanism would probablyhave an auxiliary role, as is indicated by the ability of theCAA1::pFUS2 mutant to form nitrogen-fixing nodules and the absenceof homologous genes from the genomes of certain rhizobium species.

An interesting clue to the possible physiological roles of CAA1comes from the altered ${delta}$ ¹³C of nodules occupied by the CAA1::pFUS2mutant strain. A recent study indicated that CA is involvedin carbon isotope discrimination ( ${Delta}$ ¹³C) in the leaves of theC₄ plant Flaveria bidentis (8). In particular, it was demonstratedthat reduced CA activity results in increased ${Delta}$ ¹³C and thus ¹³C-depletedleaves. In the present study, nodules lacking the M. loti ${alpha}$ -typeCA were ¹³C enriched compared to the wild-type leaves, possiblyindicating that at least part of the inorganic carbon channeledthrough CAA1 could be used as a substrate for carboxylationreactions from either the rhizobia or the plant. Although furtherresearch is needed in this direction, it indicates that CAA1is involved in carbon isotope discrimination during dark CO₂fixation in nodulated roots.

In this report, we have studied the biochemical properties andthe possible physiological roles of the CAA1 ${alpha}$ -class CA locatedin the symbiosis island of M. loti. Although plant CA isoformsare upregulated in nodules, to our knowledge, this is the firstreport addressing the role of the bacterial CA isoforms duringSNF. Nevertheless, further work is needed in order to provideadditional support for the proposed model of CA-facilitatedNH₃ protonation. In addition, the study of CAA1 involvementin nodule carbon economy would be greatly facilitated by L.japonicus lines with the plant CAs silenced, currently underconstruction in our laboratory.

ACKNOWLEDGMENTS

The M. loti R7A CAA1::pFUS2 disruption mutant strain was kindlyprovided by Clive Ronson (University of Otago, Otago, New Zealand).

FOOTNOTES

* Corresponding author. Mailing address: Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece. Phone: (30) 210 5294343. Fax: (30) 210 5294314. E-mail: mflem{at}aua.gr

Published ahead of print on 13 February 2009.

REFERENCES

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