Distinct Constitutive and Low-CO2-Induced CO2 Uptake

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Distinct Constitutive and Low-CO2-Induced CO2 Uptake Distinct constitutive and low-CO2-induced CO2 uptake systems in cyanobacteria: Genes involved and their phylogenetic relationship with homologous genes in other organisms Mari Shibata*, Hiroshi Ohkawa*, Takakazu Kaneko†, Hideya Fukuzawa‡, Satoshi Tabata†, Aaron Kaplan§, and Teruo Ogawa*¶ *Bioscience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan; †Kazusa DNA Research Institute, Kisarazu, Chiba 292-0814, Japan; ‡Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan; and §Department of Plant Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel Edited by William L. Ogren, U.S. Department of Agriculture, Hilton Head Island, SC, and approved July 24, 2001 (received for review May 23, 2001) Cyanobacteria possess a CO2-concentating mechanism that in- unable to grow under an air level of CO2 (7). The single ؊ volves active CO2 uptake and HCO3 transport. For CO2 uptake, we mutants ⌬ndhD3 and ⌬ndhD4, on the other hand, possess have identified two systems in the cyanobacterium Synechocystis CO2-uptake activity and can grow under low CO2 conditions sp. strain PCC 6803, one induced at low CO2 and one constitutive. (7). These results raised the possibility of multiple systems for The low CO2-induced system showed higher maximal activity and CO2 uptake. In this report, we bring evidence for the presence higher affinity for CO2 than the constitutive system. On the basis of two CO2-uptake systems, one constitutive and one induc- of speculation that separate NAD(P)H dehydrogenase complexes ible, in Synechocystis sp. PCC 6803, and further identify two were essential for each of these systems, we reasoned that inac- genes, sll1734 and slr1302 (designated cupA and cupB for their tivation of one system would allow selection of mutants defective involvement in CO2 uptake) as essential components of the in the other. Thus, mutants unable to grow at pH 7.0 in air were inducible and constitutive systems, respectively. recovered after transformation of a ⌬ndhD3 mutant with a trans- To assess the presence of homologous genes encoding con- poson-bearing library. Four of them had tags within slr1302 (des- stitutive and inducible CO2 uptake systems in other organisms, ignated cupB), a homologue of sll1734 (cupA), which is cotrans- we used databases available in web sites (ref. 8; http:͞͞www. cribed with ndhF3 and ndhD3. The ⌬cupB, ⌬ndhD4, and ⌬ndhF4 kazusa.or.jp͞cyano͞; http:͞͞www.jgi.doe.gov͞tempweb͞JGI࿝ ͞ ͞ mutants showed CO2-uptake characteristics of the low CO2- microbial html index.html). We also made use of the genome induced system observed in wild type. In contrast, mutants ⌬cupA, sequence of two cyanobacterial strains, Thermosynechococcus ⌬ndhD3, and ⌬ndhF3 showed characteristics of the constitutive elongatus and Gloeobacter violaceus PCC 7421, recently com- CO2-uptake system. Double mutants impaired in one component of pleted at Kazusa DNA Research Institute. These sequences each of the systems were unable to take up CO2 and required high enabled us to construct phylogenetic trees for genes essential to CO2 for growth. Phylogenetic analysis indicated that the ndhD3͞ CO2 uptake in Synechocystis sp. PCC 6803. We show that most ndhD4-, ndhF3͞ndhF4-, and cupA͞cupB-type genes are present of the cyanobacterial strains investigated possess sets of genes only in cyanobacteria. Most of the cyanobacterial strains studied encoding components of NDH-1 complexes involved in both the possess the ndhD3͞ndhD4-, ndhF3͞ndhF4-, and cupA͞cupB-type constitutive and the inducible CO2-uptake systems, but that genes in pairs. Thus, the two types of NAD(P)H dehydrogenase these genes apparently are missing in green algae. complexes essential for low CO2-induced and constitutive CO2- Materials and Methods uptake systems associated with the NdhD3͞NdhF3͞CupA-homo- Growth Conditions. logues and NdhD4͞NdhF4͞CupB-homologues, respectively, appear Wild-type (WT) and mutant cells of Synecho- to be present in these cyanobacterial strains but not in other cystis sp. strain PCC 6803 (hereafter Synechocystis 6803) were grown at 30°C in BG11 medium (9), buffered at pH 8.0, and organisms. ͞ bubbled with either 3% (vol vol) CO2 in air or air alone, as NAD(P)H dehydrogenase ͉ constitutive CO uptake ͉ affinity to CO ͉ described (6). Solid medium was BG11 buffered at pH 7.0, 2 2 supplemented with 1.5% agar and 5 mM sodium thiosulfate. CO -concentrating mechanism 2 Continuous illumination was provided by fluorescent lamps at 50 ␮mol photons mϪ2⅐sϪ1. n cyanobacteria, NAD(P)H dehydrogenase (NDH-1) is es- Isential for both CO2 uptake (1–3) and photosystem-1 (PSI) Construction and Isolation of Mutants. Construction of the ⌬ndhD3, cyclic electron transport (4). It has been postulated that uptake ⌬ndhD4, ⌬ndhF3, ⌬ndhF4, and ⌬cupA mutants has been of CO2 is energized by NDH-1-dependent PSI-cyclic electron described in a previous paper (6) and͞or deposited in the web transport (1). However, observations that mutants defective in site ‘‘CyanoMutants’’ (http:͞͞www.kazusa.or.jp͞cyano͞ ndhD3 display normal cyclic electron transport but are unable mutants͞). The constructs used to generate the single mutants to induce high-affinity CO2 uptake suggest the presence of PLANT BIOLOGY multiple NDH-1 complexes (5–7). Two types of functionally distinct NDH-1 complexes were recently recognized in Syn- This paper was submitted directly (Track II) to the PNAS office. ͞ echocystis sp. strain PCC 6803 with the aid of mutants impaired Abbreviations: H cells, cells grown under 3% (vol vol) CO2 in air; L cells, cells acclimated in one or more subunits of NDH-1 (7). One complex, con- to air for 18 h in the light; NDH-1, NAD(P)H dehydrogenase; WT, wild type; PSI, photosystem-1; CmR, chloramphenicol resistance. taining NdhD1 or NdhD2, plays a major role in PSI-cyclic ¶To whom reprint requests should be addressed. E-mail: [email protected] electron flow but is not involved in CO2 uptake (7). When the u.ac.jp. second type of NDH-1 complex is inactivated (in the double ⌬ ͞⌬ The publication costs of this article were defrayed in part by page charge payment. This mutant ndhD3 ndhD4), nearly normal PSI-cyclic electron article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. flow is observed, but the mutant does not take up CO2 and is §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.191258298 PNAS ͉ September 25, 2001 ͉ vol. 98 ͉ no. 20 ͉ 11789–11794 Downloaded by guest on September 28, 2021 were also used to transform appropriate mutants of Synechocystis Table 1. Mutants unable to grow at pH 7.0 in air isolated after 6803 to introduce multiple mutations. The sll1732 (designated transformation of a ⌬ndhD3 mutant with a ndhF3), sll1733 (ndhD3), and sll1734 (cupA) genes are expressed transposon-bearing library as an operon, and the mutants constructed by inactivating any of Mutant Tagged Positions Gene these genes grew much more slowly than the WT cells under 50 names genes interrupted* Directions† products‡ ppm CO2 (5, 6). This suggested that these genes are essential to the induced high-affinity CO2-uptake system. If so, isolation and NB-29 slr1302 305854 F CupB analysis of high CO2-requiring mutants after inactivation of NB-30 slr1302 306595 R CupB ⌬ndhD3 mutant also should enable us to identify the genes NB-31 slr1302 306299 R CupB involved in the constitutive low-affinity CO2-uptake system. NB-32 slr1302 306580 F CupB By using a Genomic Priming System (New England Biolabs), NB-33 sll0522 3267274 F NdhE a transposon containing a gene that confers chloramphenicol NB-34 slr1347 1742934 R CA resistance (CmR) was randomly inserted into the DNA in each NB-35 slr1347 1743132 F CA insert of 110 cosmids, which contained DNA fragments of NB-38 sll0247 1518083 R LHC Synechocystis 6803 previously used for genome sequencing (8). NB-39 slr0364 2354056 F H.P. The ⌬ndhD3 strain of Synechocystis 6803 was transformed with NB-41 slr0684 429962 F H.P. this transposon inactivation library, and CmR mutants unable to NB-42 sll1488 3379184 F H.P. grow at pH 7.0 in air were isolated. Genomic DNA isolated from NB-44 slr0687 431822 F PleD each mutant was digested with HhaI and, after self ligation, was NB-45 slr1521 1613061 R H.P. used as a template for inverse PCR with primers complementary ͞͞ R *Numbers represent those of nucleotide sequences in the Cyanobase (http: to the N- and C-terminal regions of the Cm cassette. The exact www.kazusa.or.jp͞cyano͞). position of the cassette in the mutant genome was determined by †F, forward; R, reverse. sequencing the PCR product. ‡CA, carboxysome-localized CA; LHC, light-harvesting chlorophyll induced under iron stress conditions; H.P., hypothetical protein. ͞ CO2 Exchange Measurements. Cells grown under 3% (vol vol) CO2 in air (H cells) or acclimated to air for 18 h in the light (L cells) were harvested by centrifugation, resuspended in 25 ml of 20 mM had interruptions in slr1347, a homologue of icfA in Synecho- N-Tris(hydroxymethyl)methyl-2-amino-ethanesulfonic acid coccus sp. strain PCC 7942, which encodes a carboxysome- (TES)-KOH buffer, pH 7.0, containing 15 mM NaCl to a cell localized carbonic anhydrase essential for the growth of cells in ␮ Ϫ1 density corresponding to 4.3 g chlorophyll ml and placed in an air level of CO2 (11), and four mutants had interruptions a reaction vessel (10). CO2 exchange of the cell suspension was in the genes encoding hypothetical proteins. NB-33 had the tag measured at 30°C by using an open infrared gas-analysis system in ndhE, which is present as a single copy in Synechocystis 6803 that records the rate of CO2 exchange as a function of time. N2, and encodes a component essential to all types of NDH-1 O2, and CO2 were mixed (using a standard gas generator model complexes.
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