Teruo Ogawa

Distinct constitutive and low-CO2-induced CO2 uptake systems in cyanobacteria: genes involved and their phylogenetic relationship with homologous genes in other organisms.

Cyanobacteria possess a CO2-concentating mechanism that involves active CO2 uptake and HCO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} transport. For CO2 uptake, we have identified two systems in the cyanobacterium Synechocystis sp. strain PCC 6803, one induced at low CO2 and one constitutive. The low CO2-induced system showed higher maximal activity and higher affinity for CO2 than the constitutive system. On the basis of speculation that separate NAD(P)H dehydrogenase complexes were essential for each of these systems, we reasoned that inactivation of one system would allow selection of mutants defective in the other. Thus, mutants unable to grow at pH 7.0 in air were recovered after transformation of a ΔndhD3 mutant with a transposon-bearing library. Four of them had tags within slr1302 (designated cupB), a homologue of sll1734 (cupA), which is cotranscribed with ndhF3 and ndhD3. The ΔcupB, ΔndhD4, and ΔndhF4 mutants showed CO2-uptake characteristics of the low CO2induced system observed in wild type. In contrast, mutants ΔcupA, ΔndhD3, and ΔndhF3 showed characteristics of the constitutive CO2-uptake system. Double mutants impaired in one component of each of the systems were unable to take up CO2 and required high CO2 for growth. Phylogenetic analysis indicated that the ndhD3/ndhD4-, ndhF3/ndhF4-, and cupA/cupB-type genes are present only in cyanobacteria. Most of the cyanobacterial strains studied possess the ndhD3/ndhD4-, ndhF3/ndhF4-, and cupA/cupB-type genes in pairs. Thus, the two types of NAD(P)H dehydrogenase complexes essential for low CO2-induced and constitutive CO2-uptake systems associated with the NdhD3/NdhF3/CupA-homologues and NdhD4/NdhF4/CupB-homologues, respectively, appear to be present in these cyanobacterial strains but not in other organisms.

Expression and Functional Roles of the Two Distinct NDH-1 Complexes and the Carbon Acquisition Complex NdhD3/NdhF3/CupA/Sll1735 in Synechocystis sp PCC 6803

To investigate the (co)expression, interaction, and membrane location of multifunctional NAD(P)H dehydrogenase type 1 (NDH-1) complexes and their involvement in carbon acquisition, cyclic photosystem I, and respiration, we grew the wild type and specific ndh gene knockout mutants of Synechocystis sp PCC 6803 under different CO2 and pH conditions, followed by a proteome analysis of their membrane protein complexes. Typical NDH-1 complexes were represented by NDH-1L (large) and NDH-1M (medium size), located in the thylakoid membrane. The NDH-1L complex, missing from the ΔNdhD1/D2 mutant, was a prerequisite for photoheterotrophic growth and thus apparently involved in cellular respiration. The amount of NDH-1M and the rate of P700+ rereduction in darkness in the ΔNdhD1/D2 mutant grown at low CO2 were similar to those in the wild type, whereas in the M55 mutant (ΔNdhB), lacking both NDH-1L and NDH-1M, the rate of P700+ rereduction was very slow. The NDH-1S (small) complex, localized to the thylakoid membrane and composed of only NdhD3, NdhF3, CupA, and Sll1735, was strongly induced at low CO2 in the wild type as well as in ΔNdhD1/D2 and M55. In contrast with the wild type and ΔNdhD1/D2, which show normal CO2 uptake, M55 is unable to take up CO2 even when the NDH-1S complex is present. Conversely, the ΔNdhD3/D4 mutant, also unable to take up CO2, lacked NDH-1S but exhibited wild-type levels of NDH-1M at low CO2. These results demonstrate that both NDH-1S and NDH-1M are essential for CO2 uptake and that NDH-1M is a functional complex. We also show that the Na+/HCO3− transporter (SbtA complex) is located in the plasma membrane and is strongly induced in the wild type and mutants at low CO2.

Genes encoding A-type flavoproteins are essential for photoreduction of O2 in cyanobacteria.

O(2) photoreduction by photosynthetic electron transfer, the Mehler reaction, was observed in all groups of oxygenic photosynthetic organisms, but the electron transport chain mediating this reaction remains unidentified. We provide the first evidence for the involvement of A-type flavoproteins that reduce O(2) directly to water in vitro. Synechocystis sp. strain PCC 6803 mutants defective in flv1 and flv3, encoding A-type flavoproteins, failed to exhibit O(2) photoreduction but performed normal photosynthesis and respiration. We show that the light-enhanced O(2) uptake was not due to respiration or photorespiration. After dark acclimation, photooxidation of P(700) was severely depressed in mutants Deltaflv1 and Deltaflv3 but recovered after light activation of CO(2) fixation, which gives P(700) an additional electron acceptor. Inhibition of CO(2) fixation prevented recovery but scarcely affected P(700) oxidation in the wild-type, where the Mehler reaction provides an alternative route for electrons. We conclude that the source of electrons for O(2) photoreduction is PSI and that the highly conserved A-type flavoproteins Flv1 and Flv3 are essential for this process in vivo. We propose that in cyanobacteria, contrary to eukaryotes, the Mehler reaction produces no reactive oxygen species and may be evolutionarily related to the response of anaerobic bacteria to O(2).

Two types of functionally distinct NAD(P)H dehydrogenases in Synechocystis sp. strain PCC6803

The ndhD gene encodes a membrane protein component of NAD(P)H dehydrogenase. The genome ofSynechocystis sp. PCC6803 contains 6 ndhDgenes. Three mutants were constructed by disrupting highly homologousndhD genes in pairs. Only theΔndhD1/ΔndhD2(ΔndhD1/D2) mutant was unable to grow under photoheterotrophic conditions and exhibited low respiration rate, although the mutant grew normally under photoautotrophic conditions in air. The ΔndhD3/ΔndhD4(ΔndhD3/D4) mutant grew very slowly in air and did not take up CO2. The results demonstrated the presence of two types of functionally distinct NAD(P)H dehydrogenases in Synechocystis PCC6803 cells. TheΔndhD5/ΔndhD6(ΔndhD5/D6) mutant grew like the wild-type strain. Under far-red light (>710 nm), the level of P700+was high in ΔndhD1/D2 and M55 (ndhB-less mutant) at low intensities. The capacity ofQ A (tightly bound plastoquinone) reduction by plastoquinone pool, as measured by the fluorescence increase in darkness upon addition of KCN, was much less inΔndhD1/D2 and M55 than inΔndhD3/D4 andΔndhD5/D6. We conclude that electrons from NADPH are transferred to the plastoquinone pool mainly by the NdhD1·NdhD2 type of NAD(P)H dehydrogenases.

Genes essential to iron transport in the cyanobacterium Synechocystis sp. strain PCC 6803.

Genes encoding polypeptides of an ATP binding cassette (ABC)-type ferric iron transporter that plays a major role in iron acquisition in Synechocystis sp. strain PCC 6803 were identified. These genes are slr1295, slr0513, slr0327, and recently reported sll1878 (Katoh et al., J. Bacteriol. 182:6523-6524, 2000) and were designated futA1, futA2, futB, and futC, respectively, for their involvement in ferric iron uptake. Inactivation of these genes individually or futA1 and futA2 together greatly reduced the activity of ferric iron uptake in cells grown in complete medium or iron-deprived medium. All the fut genes are expressed in cells grown in complete medium, and expression was enhanced by iron starvation. The futA1 and futA2 genes appear to encode periplasmic proteins that play a redundant role in iron binding. The deduced products of futB and futC genes contain nucleotide-binding motifs and belong to the ABC transporter family of inner-membrane-bound and membrane-associated proteins, respectively. These results and sequence similarities among the four genes suggest that the Fut system is related to the Sfu/Fbp family of iron transporters. Inactivation of slr1392, a homologue of feoB in Escherichia coli, greatly reduced the activity of ferrous iron transport. This system is induced by intracellular low iron concentrations that are achieved in cells exposed to iron-free medium or in the fut-less mutants grown in complete medium.

Synergistic action of red and blue light and action spectra for malate formation in guard cells of Vicia faba L.

Malate formation in guard cells of Vicia faba leaves is enhanced by light. The action spectrum for this effect was determined for epidermal strips of Vicia faba, and two different spectra were obtained under different light conditions and with and without background irradiation with high-irradiance red light (>600 nm, 3.0 mW cm−2) superimposed on monochromatic light of other wavelengths. The spectrum obtained at quantum fluxes of 1.7–2.2 nE cm−2 s−1 of monochromatic light without background red light showed a sharp peak at 433 nm with a shoulder around 475 nm and a lower peak at 670–680 nm; the spectrum obtained at much lower quantum fluxes of 0.05–0.07 nE cm−2 s−1 of monochromatic light with red-light background had two peaks of comoparable heights at 380 and 460 nm. The formation of malate with 430-nm blue light was saturated at a quantum flux of 3 nE cm−2 s−1 without the background red light but at a much lower quantum flux of 0.2 nE cm−2 s−1 with the background red light. At this low intensity, blue light was practically ineffective without background red light. A synergistic action of red light presumably absorbed by the chlorophylls, and blue light absorbed by a yellow pigment is thus demonstrated by these experiments. The action maxima at 380 and 460 nm for the blue-light effect in the presence of background red light agree with the absorption maxima of flavins.

The Plant-Like C2 Glycolate Cycle and the Bacterial-Like Glycerate Pathway Cooperate in Phosphoglycolate Metabolism in Cyanobacteria

The occurrence of a photorespiratory 2-phosphoglycolate metabolism in cyanobacteria is not clear. In the genome of the cyanobacterium Synechocystis sp. strain PCC 6803, we have identified open reading frames encoding enzymes homologous to those forming the plant-like C2 cycle and the bacterial-type glycerate pathway. To study the route and importance of 2-phosphoglycolate metabolism, the identified genes were systematically inactivated by mutagenesis. With a few exceptions, most of these genes could be inactivated without leading to a high-CO2-requiring phenotype. Biochemical characterization of recombinant proteins verified that Synechocystis harbors an active serine hydroxymethyltransferase, and, contrary to higher plants, expresses a glycolate dehydrogenase instead of an oxidase to convert glycolate to glyoxylate. The mutation of this enzymatic step, located prior to the branching of phosphoglycolate metabolism into the plant-like C2 cycle and the bacterial-like glycerate pathway, resulted in glycolate accumulation and a growth depression already at high CO2. Similar growth inhibitions were found for a single mutant in the plant-type C2 cycle and more pronounced for a double mutant affected in both the C2 cycle and the glycerate pathway after cultivation at low CO2. These results suggested that cyanobacteria metabolize phosphoglycolate by the cooperative action of the C2 cycle and the glycerate pathway. When exposed to low CO2, glycine decarboxylase knockout mutants accumulated far more glycine and lysine than wild-type cells or mutants with inactivated glycerate pathway. This finding and the growth data imply a dominant, although not exclusive, role of the C2 route in cyanobacterial phosphoglycolate metabolism.

Mutation of ndh Genes Leads to Inhibition of CO2 Uptake Rather than HCO3− Uptake in Synechocystis sp. Strain PCC 6803

Six mutants (B1 to B6) that grew poorly in air on BG11 agar plates buffered at pH 8.0 were rescued after mutations were introduced into ndhB of wild-type (WT) Synechocystis sp. strain PCC 6803. In these mutants and a mutant (M55) lacking ndhB, CO(2) uptake was much more strongly inhibited than HCO(3)(-) uptake, i.e., the activities of CO(2) and HCO(3)(-) uptake in B1 were 9 and 85% of those in the WT, respectively. Most of the mutants grew very slowly or did not grow at all at pH 6.5 or 7.0 in air, and their ability to grow under these conditions was correlated with CO(2) uptake capacity. Detailed studies of B1 and M55 indicated that the mutants grew as fast as the WT in liquid at pH 8.0 under air, although they grew poorly on agar plates. The contribution of CO(2) uptake appears to be larger on solid medium. Five mutants were constructed by inactivating each of the five ndhD genes in Synechocystis sp. strain PCC 6803. The mutant lacking ndhD3 grew much more slowly than the WT at pH 6.5 under 50 ppm CO(2), although other ndhD mutants grew like the WT under these conditions and showed low affinity for CO(2) uptake. These results indicated the presence of multiple NAD(P)H dehydrogenase type I complexes with specific roles.

Cyanobacterial NADPH dehydrogenase complexes

Cyanobacteria possess functionally distinct multiple NADPH dehydrogenase (NDH-1) complexes that are essential to CO2 uptake, photosystem-1 cyclic electron transport and respiration. The unique nature of cyanobacterial NDH-1 complexes is the presence of subunits involved in CO2 uptake. Other than CO2 uptake, chloroplastic NDH-1 complex has a similar role as cyanobacterial NDH-1 complexes in photosystem-1 cyclic electron transport and respiration (chlororespiration). In this mini-review we focus on the structure and function of cyanobacterial NDH-1 complexes and their phylogeny. The function of chloroplastic NDH-1 complex and characteristics of plants defective in NDH-1 are also described for comparison.

Inorganic carbon acquisition systems in cyanobacteria

This minireview focuses on the mechanism of inorganic carbon uptake in cyanobacteria and in particular the two CO2-uptake systems and two bicarbonate transporters recently identified in Synechocycstis PCC 6803, and their presence in other cyanobacterial strains.