Structural and Mutational Analysis of Band 7 Proteins in the Cyanobacterium Synechocystis sp. Strain PCC 6803

Band 7 proteins, which encompass members of the stomatin, prohibitin, flotillin, and HflK/C protein families, are integral membrane proteins that play important physiological roles in eukaryotes but are poorly characterized in bacteria. We have studied the band 7 proteins encoded by the cyanobacterium Synechocystis sp. strain PCC 6803, with emphasis on their structure and proposed role in the assembly and maintenance of the photosynthetic apparatus. Mutagenesis revealed that none of the five band 7 proteins (Slr1106, Slr1128, Slr1768, Sll0815, and Sll1021) was essential for growth under a range of conditions (including high light, salt, oxidative, and temperature stresses), although motility was compromised in an Slr1768 inactivation mutant. Accumulation of the major photosynthetic complexes in the thylakoid membrane and repair of the photosystem II complex following light damage were similar in the wild type and a quadruple mutant. Cellular fractionation experiments indicated that three of the band 7 proteins (Slr1106, Slr1768, and Slr1128) were associated with the cytoplasmic membrane, whereas Slr1106, a prohibitin homologue, was also found in the thylakoid membrane fraction. Blue native gel electrophoresis indicated that these three proteins, plus Sll0815, formed large (>669-kDa) independent complexes. Slr1128, a stomatin homologue, has a ring-like structure with an approximate diameter of 16 nm when visualized by negative stain electron microscopy. No evidence for band 7/FtsH supercomplexes was found. Overall, our results indicate that the band 7 proteins form large homo-oligomeric complexes but do not play a crucial role in the biogenesis of the photosynthetic apparatus in Synechocystis sp. strain PCC 6803.Members of the band 7 superfamily of proteins are found throughout nature and are defined by a characteristic sequence motif, termed the SPFH domain, after the initials of the various subfamilies: the stomatins, the prohibitins, the flotillins (also known as “reggies”), and the HflK/C proteins (12, 49). The stomatins and prohibitins and to a lesser extent flotillins are highly conserved protein families and are found in a variety of organisms ranging from prokaryotes to higher eukaryotes (29, 34, 49), whereas HflK and HflC homologues are only present in bacteria.In eukaryotes band 7 proteins are linked with a variety of disease states consistent with important cellular functions (6). In general the eukaryotic band 7 proteins tend to be oligomeric and are involved in membrane-associated processes: for example, prohibitins are involved in modulating the activity of a membrane-bound FtsH protease (17, 46) and the assembly of mitochondrial respiratory complexes (30), stomatins are involved in ion channel function (47), and flotillins are involved in signal transduction and vesicle trafficking (25).In the case of prokaryotes, most work so far has focused on the roles of the HflK/C and YbbK (also known as QmcA, a stomatin homologue) band 7 proteins of Escherichia coli (7, 16, 17, 36) and the structure of a stomatin homologue in the archaeon Pyrococcus horikoshii (57). Much less is known about the structure, function, and physiological importance of band 7 proteins in other prokaryotes, especially the cyanobacteria (12).The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 is a widely used model organism for studying various aspects of cyanobacterial physiology and, in particular, oxygenic photosynthesis. One of the main areas of our research is to understand the mechanism by which the oxygen-evolving photosystem II (PSII) complex found in the thylakoid membrane of Synechocystis sp. strain PCC 6803 is repaired following light damage. Recent work has identified an important role for FtsH proteases in PSII repair (19, 41). Given that FtsH is known to form large supercomplexes with HflK/C in E. coli (36) and with prohibitins in Saccharomyces cerevisiae mitochondria (46), we hypothesized that one or more band 7 proteins might interact with FtsH in cyanobacteria and play a role in the selective turnover of the D1 reaction center polypeptide during PSII repair and so provide resistance to high light stress (40). This idea was given early support by the detection of both FtsH and Slr1106, a prohibitin homologue, in a His-tagged PSII preparation isolated from Synechocystis sp. strain PCC 6803 (40) and the detection of Slr1128 (a stomatin homologue), Sll1021 (a possible flotillin homologue), and FtsH in a His-tagged preparation of ScpD, a small chlorophyll a/b-like-binding protein that associates with PSII (56). Recent mutagenesis experiments have also suggested a role for Slr1128 in maintaining growth at high light intensities (53).In this paper we have used targeted gene disruption mutagenesis and various biochemical approaches to investigate the structure and function of band 7 proteins in Synechocystis sp. strain PCC 6803, with particular emphasis on PSII function. We provide evidence that four predicted band 7 proteins in Synechocystis sp. strain PCC 6803 (Slr1106, Slr1768, Slr1128, and Sll8015) form large independent complexes, which in the case of Slr1128 forms a ring-like structure. No evidence was found for the formation of supercomplexes with FtsH. Importantly, single and multiple insertion mutants lacking up to four of the five band 7 proteins are able to grow as well as the wild type (WT) under a range of growth conditions, including high light stress. Our results suggest that band 7 proteins are not essential in Synechocystis sp. strain PCC 6803 and are not required for efficient PSII repair. Possible functions of the cyanobacterial band 7 proteins are discussed in the light of recent results from other systems.     

The Biosynthetic Pathway for Myxol-2′ Fucoside (Myxoxanthophyll) in the Cyanobacterium Synechococcus sp. Strain PCC 7002

Synechococcus sp. strain PCC 7002 produces a variety of carotenoids, which comprise predominantly dicylic β-carotene and two dicyclic xanthophylls, zeaxanthin and synechoxanthin. However, this cyanobacterium also produces a monocyclic myxoxanthophyll, which was identified as myxol-2′ fucoside. Compared to the carotenoid glycosides produced by diverse microorganisms, cyanobacterial myxoxanthophyll and closely related compounds are unusual because they are glycosylated on the 2′-OH rather than on the 1′-OH position of the ψ end of the molecule. In this study, the genes encoding two enzymes that modify the ψ end of myxoxanthophyll in Synechococcus sp. strain PCC 7002 were identified. Mutational and biochemical studies showed that open reading frame SynPCC7002_A2032, renamed cruF, encodes a 1′-hydroxylase and that open reading frame SynPCC7002_A2031, renamed cruG, encodes a 2′-O-glycosyltransferase. The enzymatic activity of CruF was verified by chemical characterization of the carotenoid products synthesized when cruF was expressed in a lycopene-producing strain of Escherichia coli. Database searches showed that homologs of cruF and cruG occur in the genomes of all sequenced cyanobacterial strains that are known to produce myxol or the acylic xanthophyll oscillaxanthin. The genomes of many other bacteria that produce hydroxylated carotenoids but do not contain crtC homologs also contain cruF orthologs. Based upon observable intermediates, a complete biosynthetic pathway for myxoxanthophyll is proposed. This study expands the suite of enzymes available for metabolic engineering of carotenoid biosynthetic pathways for biotechnological applications.A wide variety of organisms produce carotenoid glycosides, which act as natural surfactants, stabilize membranes, and possibly contribute to regulating the permeability of membranes to oxygen (4, 41, 51). The first carotenoid glycosides were isolated from saffron in 1818 (6). However, the structures of the glycosylated carotenoids phleixanthophyll and 4-keto-phleixanthophyll were the first to be completely determined, in 1967, after their isolation from Mycobacterium phlei (15, 34). Tertiary glycosides are relatively rare in nature but seem to be common in carotenoid biosynthesis (34). These include the glycosylated carotenoids of the myxobacteria, which have characteristic C-3′,4′ desaturation and C-1′ glycosylation (18, 19). Acylated carotenoid C-1′-glycosides are broadly distributed among bacteria, including Salinibacter ruber and members of the Chloroflexi and Chlorobi (24, 46, 47).The glycosylated carotenoids of cyanobacteria differ from the examples mentioned above in that glycosylation characteristically occurs on the C-2′-hydroxyl group rather than that at the C-1′ position of the ψ end of myxol (3′,4′-didehydro-1′,2′-dihydro-β,ψ-carotene-3,1′,2′-triol) or oscillol (3,4,3′,4′-tetradehydro-1,2,1′,2′-ψ,ψ-carotene-1,2,1′,2′-tetrol) to form myxoxanthophyll or oscillaxanthin, respectively (16, 17). Myxoxanthophyll is thus far found uniquely in members of the phylum Cyanobacteria, and this compound is named after the synonym for this group of organisms, i.e., Myxophyceae (14, 42). As carotenoids from increasingly diverse bacteria are characterized, the apparent uniqueness of myxoxanthophyll to cyanobacteria will probably not persist. For example, the aglycone myxol occurs in marine flavobacteria, along with saporaxanthin (38, 49). Oscillaxanthin, which was once thought to be unique to the Nostophyceae, was recently identified as the major pigment of three strains of Methylobacterium spp. (20). Moreover, oscillol appears to be a precursor of the glycosylated and acylated carotenoids of Thermomicrobium roseum (52).Several variations on the myxoxanthophyll pathway, which lead to a variety of possible compounds, are known to occur in cyanobacteria. A number of different sugars commonly occur in myxoxanthophyll derivatives found in different strains. Strains of Oscillatoria and Spirulina spp. produce compounds that are chinovosides, fucosides, or methylfucosides (1, 7). Derivatives containing fucose have been found in Nostoc punctiforme strain PCC 73102 and Nostoc sp. strain PCC 7120 (45), whereas myxoxanthophyll dimethylfucoside has been found in Synechocystis sp. strain PCC 6803 (42). Variations in the ring oxidation level of the basal compound, with the addition of a keto group at the C-4 position or of an additional hydroxyl group at the C-2 or C-4 position, may also lead to several related compounds.Myxol is presumably synthesized from lycopene, the acyclic precursor of all carotenoids in cyanobacteria (see Fig. ​Fig.1)1) (27). Because of the occurrence of a β-ionone ring in the final product, monocyclic γ-carotene is also presumed to be an intermediate in this pathway (21, 41). The question of what enzyme is responsible for the formation of the β-ionone ring from the linear ψ end of γ-carotene has been contentious but has recently been resolved. Although CrtL-type lycopene cyclases occur in some cyanobacteria, genes for lycopene cyclases of this family do not occur in the genomes of sequenced cyanobacteria that produce myxoxanthophyll (11, 26). Mohamed and Vermaas reported that the open reading frame (ORF) sll0254 encodes an enzyme thought to be involved simultaneously in the cyclization and ψ-end hydroxylation of lycopene in Synechocystis sp. strain PCC 6803 (31). However, subsequent studies of both Synechococcus sp. strain PCC 7002 and Synechocystis sp. strain PCC 6803 showed that this is not the case (11, 26). Like nearly all other cyanobacteria lacking CrtL homologs, both of these cyanobacteria contain two genes, cruA and cruP, which encode lycopene cyclases (26). A third class of organisms, which so far includes only Synechococcus sp. strains PCC 7942 and PCC 6301, have CrtL and CruP homologs.Open in a separate windowFIG. 1.HPLC elution profiles for pigments from two cyanobacteria. (A) HPLC elution profiles (obtained by the jegpsu method) for pigments extracted from the wild type (solid line) and the slr1293 mutant (dotted line) of Synechocystis sp. strain PCC 6803. (B) HPLC elution profiles (obtained by the jegpsu method) for pigments extracted from the wild type (solid line) and SynPCC7002_A1623 mutant (dotted line) of Synechococcus sp. strain PCC 7002. Peak identities: s, synechoxanthin; md, myxol-2′ dimethylfucoside; mf, myxol-2′ fucoside; z, zeaxanthin; c, cryptoxanthin; e, echinenone; and b, β-carotene.The well-conserved β-hydroxylase CrtR functions in the C-3 hydroxylation of myxoxanthophyll, and CrtR also functions in the synthesis of zeaxanthin, cryptoxanthin, and 3′-hydroxy-echinenone (11, 21, 27, 28). CrtR seems to be responsible for all C-3 hydroxylation reactions in Synechocystis sp. strain PCC 6803, and it is notable that CrtR seems to be extremely highly conserved among cyanobacteria (11, 27). Considerable confusion has existed concerning the remaining reactions of this biosynthetic pathway. Mohamed and Vermaas reported that ORF slr1293 encodes the 3′,4′ desaturase in Synechocystis sp. strain PCC 6803 (30). Furthermore, Mohamed and Vermaas additionally reported that the product of ORF sll0254 plays a dual role as a lycopene cyclase and a mediator of ψ-end hydroxylation during myxoxanthophyll biosynthesis in Synechocystis sp. strain PCC 6803 (31). However, subsequent studies have shown that the sll0254 product and its orthologs, renamed CruE, are carotene desaturases/methyltransferases that participate in the synthesis of the aromatic carotenoid synechoxanthin (12, 13).In this study, we identified two genes, cruF and cruG, which encode the C-1′-hydroxylase and 2′-O-glycosyltransferase, respectively, that are uniquely required for mxyoxanthophyll biosynthesis in Synechococcus sp. strain PCC 7002. Orthologs of these genes are found in all sequenced genomes of cyanobacteria that synthesize myxoxanthophyll. Additionally, in contrast to the data in a previous report (30), we show that ORF slr1293 of Synechocystis sp. strain PCC 6803 and its ortholog SynPCC7002_A2031 in Synechococcus sp. strain PCC 7002 do not play an essential role in myxoxanthophyll biosynthesis.     

Identification and Characterization of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase from Bacillus megaterium

A gene that codes for a novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase, designated PhaZ1, has been identified in the genome of Bacillus megaterium. A native PHB (nPHB) granule-binding assay showed that purified soluble PhaZ1 had strong affinity for nPHB granules. Turbidimetric analyses revealed that PhaZ1 could rapidly degrade nPHB granules in vitro without the need for protease pretreatment of the granules to remove surface proteins. Notably, almost all the final hydrolytic products produced from the in vitro degradation of nPHB granules by PhaZ1 were 3-hydroxybutyric acid (3HB) monomers. Unexpectedly, PhaZ1 could also hydrolyze denatured semicrystalline PHB, with the generation of 3HB monomers. The disruption of the phaZ1 gene significantly affected intracellular PHB mobilization during the PHB-degrading stage in B. megaterium, as demonstrated by transmission electron microscopy and the measurement of the PHB content. These results indicate that PhaZ1 is functional in intracellular PHB mobilization in vivo. Some of these features, which are in striking contrast with those of other known nPHB granule-degrading PhaZs, may provide an advantage for B. megaterium PhaZ1 in fermentative production of the biotechnologically valuable chiral compound (R)-3HB.Polyhydroxyalkanoates (PHAs) are a group of polyesters that are produced by numerous bacteria as carbon and energy storage materials in response to nutritional stress (13, 27, 29). Poly(3-hydroxybutyrate) (PHB) is the most common and intensively studied PHA. Intracellular native PHB (nPHB) granules are composed of a hydrophobic PHB core and a surface layer consisting of proteins and phospholipids (13). The PHB of intracellular nPHB granules is in an amorphous state. When intracellular nPHB granules are exposed to extracellular environments due to cell death and lysis, the amorphous PHB is transformed into a denatured semicrystalline state. nPHB granules subjected to physical damage or solvent extraction to remove the surface layer can also crystallize into denatured PHB (dPHB) (13, 15). Artificial PHB (aPHB) granules, in which PHB is in an amorphous state, can be prepared from semicrystalline dPHB and detergents (1, 11, 23, 31).Various extracellular PHB depolymerases (PhaZs) that are secreted by many PHB-degrading bacteria have been demonstrated to specifically degrade dPHB (13, 14, 37). One exception is that PhaZ7, an extracellular PHB depolymerase secreted by Paucimonas lemoignei, displays unusual substrate specificity for amorphous PHB, with 3-hydroxybutyrate (3HB) oligomers as the main products of enzymatic hydrolysis (7). PhaZ7 exhibits no enzymatic activity toward dPHB. So far, a growing number of intracellular PHB depolymerases have been characterized. The intracellular PHB depolymerase PhaZa1 of Ralstonia eutropha (also called Cupriavidus necator) H16 has recently been established to be especially important for the intracellular mobilization of accumulated PHB (42). The main in vitro hydrolytic products of PhaZa1 degradation of amorphous aPHB are 3HB oligomers (31). PhaZd1, another intracellular PHB depolymerase of R. eutropha H16, shows no significant amino acid similarity to PhaZa1. The in vitro hydrolytic products of PhaZd1 degradation of amorphous aPHB are also 3HB oligomers. A 3HB monomer is rarely detected as a hydrolytic product (1). The intracellular PHB depolymerase PhaZ of Paracoccus denitrificans was reported previously to degrade protease-treated nPHB granules in vitro, with the release of 3HB dimers and oligomers as the main hydrolytic products (6). Recently, we have identified a novel intracellular PHB depolymerase from Bacillus thuringiensis serovar “israelensis” (39). The B. thuringiensis PhaZ shows no significant amino acid similarity to any known PHB depolymerase. This PhaZ has strong amorphous PHB-hydrolyzing activity and can release a considerable amount of 3HB monomers by the hydrolysis of trypsin-treated nPHB granules (39). It is of note that purified PhaZd1 from R. eutropha, PhaZ from P. denitrificans, and PhaZ from B. thuringiensis need pretreatment of nPHB granules with protease to remove surface proteins for PHB degradation (1, 6, 39). They show only very little or no activity toward nPHB granules without trypsin pretreatment. It has been demonstrated previously that these intracellular PHB depolymerases cannot hydrolyze dPHB (1, 31, 39).(R)-3HB, a biotechnologically valuable chiral compound, has been widely used for syntheses of antibiotics, vitamins, and pheromones (3, 30, 38). One way to produce (R)-3HB is heterologous coexpression of a PHB synthetic operon and a gene encoding an amorphous PHB-degrading PhaZ in Escherichia coli (3, 18, 25, 33, 38). A common problem encountered by this method is that oligomeric and dimeric forms of 3HB often constitute a major portion of the products of enzymatic hydrolysis, thus requiring further hydrolysis by 3HB oligomer hydrolase or heating under alkaline conditions to generate 3HB monomers (3, 18, 25, 33).Bacillus megaterium genes involved in the biosynthesis of nPHB granules have been cloned from strain ATCC 11561 and characterized previously (19, 21, 22). A gene encoding the extracellular PHB depolymerase PhaZ from B. megaterium was recently cloned from strain N-18-25-9 (34). However, little is known about B. megaterium genes involved in the intracellular mobilization of PHB. In this study, we have identified in B. megaterium ATCC 11561 an intracellular PHB depolymerase that could rapidly degrade nPHB granules in vitro without the need for trypsin pretreatment of the nPHB granules. Moreover, almost all the in vitro hydrolytic products released from the degradation of amorphous PHB by this PhaZ were 3HB monomers. This PhaZ could also hydrolyze dPHB with the generation of 3HB monomers. Thus, it appears to be a novel intracellular PHB depolymerase and may have promising potential for biotechnological application in the production of enantiomerically pure (R)-3HB monomers.     

Characterization of an Alcohol Dehydrogenase from the Cyanobacterium Synechocystis sp. Strain PCC 6803 That Responds to Environmental Stress Conditions via the Hik34-Rre1 Two-Component System

The slr1192 (adhA) gene from Synechocystis sp. strain PCC 6803 encodes a member of the medium-chain alcohol dehydrogenase/reductase family. The gene product AdhA exhibits NADP-dependent alcohol dehydrogenase activity, acting on a broad variety of aromatic and aliphatic primary alcohols and aldehydes but not on secondary alcohols or ketones. It exhibits superior catalytic efficiency for aldehyde reduction compared to that for alcohol oxidation. The enzyme is a cytosolic protein present in photoautotrophically grown Synechocystis cells. The expression of AdhA is enhanced upon the exposure of cells to different environmental stresses, although it is not essential for survival even under such stress conditions. The induction of the expression of the adhA gene is dependent on the Hik34-Rre1 two-component system, as it is severely impaired in mutant strains lacking either the histidine kinase Hik34 or the response regulator Rre1. In vitro DNA-protein interaction analysis reveals that the response regulator Rre1 binds specifically to the promoter region of the adhA gene.Medium-chain dehydrogenases/reductases (MDR) constitute a superfamily of alcohol dehydrogenases that catalyze the reversible NAD(P)-dependent oxidation of alcohols to aldehydes or ketones. It includes a large number of structurally related proteins, which catalyze several types of enzymatic activity (23, 41, 44). Screening of complete genome sequences has revealed that this family is widespread, complex, and of ancient origin (22, 44). MDR alcohol dehydrogenases are found in mammals, plants, fungi, and bacteria (52). The alcohol dehydrogenases fulfill an astonishing variety of functions in cell metabolism (21), also being a key enzyme in ethanol generation by Saccharomyces cerevisiae (6) and bacteria (10). Furthermore, the generation of biofuels by photoautotrophic microorganisms is of great biotechnological interest (43). Complementation of a cyanobacterium''s enzyme machinery with a specific exogenous gene(s) can result in the ability to generate bioethanol from photosynthetically fixed CO₂ (11). Notwithstanding, current knowledge of cyanobacterial alcohol dehydrogenases is rather limited. In the cyanobacterium Synechocystis sp. strain PCC 6803 (referred here as Synechocystis), the slr1192 gene encodes a putative MDR alcohol dehydrogenase. According to in silico analyses (38, 44), the slr1192 protein has similarity with two subfamilies of MDRs: the yeast ADH family (Y-ADH) and the cinnamyl ADH family (CADH). Y-ADH-related enzymes have catabolic functions and are involved mainly in the metabolism of ethanol or short-chain alcohols for which they exhibit broad substrate specificity. CADH and related enzymes, on the other hand, perform anabolic functions and participate in biosynthetic pathways in plants and bacteria (5, 25, 44).In Synechocystis, the expression of slr1192 is induced by osmotic (35) or salt (48) stress. In higher plants, alcohol dehydrogenase activity appears to be involved in aerobic metabolism under certain stress conditions (26, 56) such as low temperature, water deficit, or ozone exposure, but its function remains unknown. A temperature decrease seems to induce the accumulation of alcohol dehydrogenase mRNA in Arabidopsis thaliana (20), corn, and rice (9).In general, cyanobacteria perceive and respond to environmental changes by means of two-component regulatory systems, a ubiquitous signal transduction pathway that represents a prevalent signaling mechanism in bacteria (8, 61). Two-component systems consist of a histidine kinase (Hik) and a response regulator (Rre) and generally induce or repress the expression of specific genes in response to environmental stimuli. The histidine kinase autophosphorylates a conserved histidine residue in response to the environmental signal and then transfers the phosphate group to a conserved aspartate residue of the response regulator, which mediates the transfer of the signal. In Synechocystis, different Hik-Rre systems have been identified as being regulators of the response to different environmental stresses (37). A membrane-bound histidine kinase, Hik33, is involved in the perception of cold, salt, and osmotic stress (33, 35, 39, 48, 54). A cytosolic histidine kinase, Hik34, has been shown to be involved in the perception of salt and hyperosmotic stress (33, 39, 48) as well as heat shock (53). Specifically, the couples Hik33-Rre31, Hik10-Rre13, Hik16 Hik41-Rre17, Hik34-Rre1, and a putative Hik2-Rre1 have been identified as being elements involved in the perception and transduction of signals promoted by hyperosmotic and salt stress (39, 48).In the present work, a biochemical characterization of the slr1192 protein (designated AdhA here) from Synechocystis has been performed, revealing that the tetrameric 140-kDa enzyme is active toward linear and aromatic primary alcohols and that it preferentially reduces aldehydes rather than oxidizing alcohols. In addition, an extensive analysis of the expression of the adhA gene has verified its induction in response to heat shock, hyperosmotic stress, salt stress, and the addition of benzyl alcohol (BA). The Hik34-Rre1 two-component system has been shown to play a relevant role in the regulation of the expression of the adhA gene under these stress conditions. A specific interaction of Rre1 with the promoter region of the adhA gene has also been demonstrated. In light of this finding and additional information presented here, the physiological role of AdhA in Synechocystis is discussed.     

Characterization of the FtsZ-Interacting Septal Proteins SepF and Ftn6 in the Spherical-Celled Cyanobacterium Synechocystis Strain PCC 6803

Assembly of the tubulin-like cytoskeletal protein FtsZ into a ring structure at midcell establishes the location of the nascent division sites in prokaryotes. However, it is not yet known how the assembly and contraction of the Z ring are regulated, especially in cyanobacteria, the environmentally crucial organisms for which only one FtsZ partner protein, ZipN, has been described so far. Here, we characterized SepF and Ftn6, two novel septal proteins, in the spherical-celled strain Synechocystis PCC 6803. Both proteins were found to be indispensable to Synechocystis sp. strain PCC 6803. The depletion of both SepF and Ftn6 resulted in delayed cytokinesis and the generation of giant cells but did not prevent FtsZ polymerization, as shown by the visualization of green fluorescent protein (GFP)-tagged FtsZ polymers. These GFP-tagged Z-ring-like structures often appeared to be abnormal, because these reporter cells respond to the depletion of either SepF or Ftn6 with an increased abundance of total, natural, and GFP-tagged FtsZ proteins. In agreement with their septal localization, we found that both SepF and Ftn6 interact physically with FtsZ. Finally, we showed that SepF, but not Ftn6, stimulates the formation and/or stability of FtsZ polymers in vitro.Binary fission of a mother cell to form two daughter cells is a widely conserved cell proliferation mechanism. In nearly all bacteria, cell division is initiated by the polymerization into a ring-like structure at midcell of the tubulin homolog GTPase protein FtsZ, which is also found in some archae, as well as in plastids and some mitochondria (for reviews, see references 7, 21, and 33). The Z-ring is subsequently used as a scaffold for recruitment of downstream factors that execute the synthesis of the division septum. The assembly of this complex, also referred to as the divisome, has been thoroughly investigated in studies of the rod-shaped model organisms Escherichia coli and Bacillus subtilis) (for reviews, see references 3, 4, 7, 9, 11, 19, and 21). In E. coli, more than 10 different proteins are required for the progression and completion of cell division. They are designated Fts proteins because their depletion leads to filamentation of the bacteria, and they are recruited to the division site in the following sequential order: FtsZ→FtsA/ZipA/ZapB→FtsK→FtsQ and FtsL/FtsB→FtsW→FtsI and FtsN.The stability of the FtsZ protofilaments is thought to be important for assembly of the septal Z ring. Four FtsZ-interacting proteins have been shown to promote FtsZ polymerization and/or Z-ring stabilization, namely, ZapA and ZipA (found only in gammaproteobacteria), FtsA (an actin-like protein), and SepF (not found in gammaproteobacteria) (10, 31). Both FtsA and ZipA assemble at the Z-ring early and participate in its anchorage to the inner face of the cytoplasmic membrane of the cell. They also participate in the recruitment of the downstream cytokinetic factor FtsK. Subsequently, the recruitment of FtsQ and the FtsB/FtsL complex allow the progressive assembly of downstream factors (FtsW, FtsI, and FtsN) involved in synthesis of the septal cell wall (7).By contrast, the negative regulatory proteins MinCDE, DivIVA, EzrA, SulA, and Noc operate in the destabilization and positioning of the Z-ring at midcell (7, 21, 30), sometimes through a direct interaction with FtsZ (SulA, MinC, and ErzA).Little is known concerning cell division in cyanobacteria, in spite of their crucial importance to the biosphere (5, 27, 34) and their interest for biotechnologists (1, 6, 32). Cyanobacteria are also attractive because many species (such as E. coli and B. subtilis) exhibit a cylindrical morphology with a well-defined middle, whereas many others have a spherical shape (29) and thus possess an infinite number of potential division planes at the point of greatest cell diameter. Furthermore, as the progenitor of the chloroplasts (8), cyanobacteria can be of help for deciphering the stromal chloroplastic division machinery (33). Interestingly, several cell division factors occurring in E. coli and B. subtilis have been shown (FtsZ, MinCDE, and SulA) or proposed (FtsE, FtsI, FtsQ, and FtsW) to be conserved in cyanobacteria (23, 26) and chloroplasts (which lack MinC) (33). In contrast, ftsA, ftsB, zipA, ftsK, ftsL, ftsN, and zapA have not been detected in cyanobacteria.So far, cyanobacterial cytokinesis has mainly been investigated using the two unicellular species Synechococcus sp. strain PCC 7942 (rod shaped; hereafter S. elongatus) and Synechocystis sp. strain PCC 6803 (spherical-celled; hereafter Synechocystis sp.) and the filamentous strain Anabaena PCC 7120, all of which possess a fully sequenced genome (http://genome.kazusa.or.jp/cyanobase/) that is easily manipulated (16). Both FtsZ and ZipN/Ftn2/Arc6, a protein occurring only in cyanobacteria (ZipN [alternative name, Ftn2]) and plant chloroplasts (Arc6), were found to be crucial for cytokinesis (17, 23, 26) and to physically interact with each other (20, 23). We also reported that the MinCDE system participates in determining the correct positioning of the septal Z ring at midcell (23). In addition, it has recently been shown in studies of Synechococcus sp. that inactivation of both the cdv2 gene (an orthologue of the gene encoding B.subtilis sepF) and the ftn6 gene (present in only some cyanobacteria) promotes filamentation, though their role in cell division has yet to be characterized (16, 26).In a continuous effort to characterize the divisome machine of Synechocystis sp., we have used a combination of in vivo and in vitro techniques for thorough analysis of the SepF and Ftn6 proteins. We report here that both SepF and Ftn6 are crucial cytokinetic proteins that localize at the division site at midcell and whose depletion leads to the formation of giant cells that remain spherical. In agreement with their septal localization, both SepF and Ftn6 were found to interact physically with FtsZ; also, SepF, but not Ftn6, was found to stimulate the formation and/or stability of FtsZ polymers.     

Effects of Aeration on the Synthesis of Poly(3-Hydroxybutyrate) from Glycerol and Glucose in Recombinant Escherichia coli

Bioreactor cultures of Escherichia coli recombinants carrying phaBAC and phaP of Azotobacter sp. FA8 grown on glycerol under low-agitation conditions accumulated more poly(3-hydroxybutyrate) (PHB) and ethanol than at high agitation, while in glucose cultures, low agitation led to a decrease in PHB formation. Cells produced smaller amounts of acids from glycerol than from glucose. Glycerol batch cultures stirred at 125 rpm accumulated, in 24 h, 30.1% (wt/wt) PHB with a relative molecular mass of 1.9 MDa, close to that of PHB obtained using glucose.Polyhydroxyalkanoates (PHAs), accumulated as intracellular granules by many bacteria under unfavorable conditions (5, 8), are carbon and energy reserves and also act as electron sinks, enhancing the fitness of bacteria and contributing to redox balance (9, 11, 19). PHAs have thermoplastic properties, are totally biodegradable by microorganisms present in most environments, and can be produced from different renewable carbon sources (8).Poly(3-hydroxybutyrate) (PHB) is the best known PHA, and its accumulation in recombinant Escherichia coli from several carbon sources has been studied (1, 13). In the last few years, increasing production of biodiesel has caused a sharp fall in the cost of its main by-product, glycerol (22). Its use for microbial PHA synthesis has been analyzed for natural PHA producers, such as Methylobacterium rhodesianum, Cupriavidus necator (formerly called Ralstonia eutropha) (3), several Pseudomonas strains (22), the recently described bacterium Zobellella denitrificans (7), and a Bacillus sp. (18), among others. Glycerol has also been used for PHB synthesis in recombinant E. coli (12, 15). PHAs obtained from glycerol were reported to have a significantly lower molecular weight than polymer synthesized from other substrates, such as glucose or lactose (10, 23).Apart from the genes that catalyze polymer biosynthesis, natural PHA producers have several genes that are involved in granule formation and/or have regulatory functions, such as phasins, granule-associated proteins that have been shown to enhance polymer synthesis and the number and size of PHA granules (17, 24). The phasin PhaP has been shown to exert a beneficial effect on bacterial growth and PHB accumulation from glycerol in bioreactor cultures of strain K24KP, a recombinant E. coli that carries phaBAC and phaP of Azotobacter sp. FA8 (6).Because the redox state of the cells is known to affect the synthesis of PHB (1, 4, 14), the present study investigates the behavior of this recombinant strain under different aeration conditions, by using two substrates, glucose and glycerol, with different oxidation states.     

The N-Acetylmuramic Acid 6-Phosphate Etherase Gene Promotes Growth and Cell Differentiation of Cyanobacteria under Light-Limiting Conditions

Inactivation of sll0861 in Synechocystis sp. strain PCC 6803 or the homologous gene alr2432 in Anabaena sp. strain PCC 7120 had no effect on the growth of these organisms at a light intensity of 30 μmol photons m⁻² s⁻¹ but reduced their growth at a light intensity of 5 or 10 μmol photons m⁻² s⁻¹. In Anabaena, inactivation of the gene also significantly reduced the rate of heterocyst differentiation under low-light conditions. The predicted products of sll0861 and alr2432 and homologs of these genes showed similarity to N-acetylmuramic acid 6-phosphate etherase (MurQ), an enzyme involved in peptidoglycan recycling, in Escherichia coli. E. coli murQ and the cyanobacterial homologs could functionally substitute for each other. We hypothesize that murQ in cyanobacteria promotes low-light adaptation through reutilization of peptidoglycan degradation products.Cyanobacteria are procaryotes that perform oxygenic photosynthesis and have a Gram-negative cell wall structure (7). They are found in oceans, bodies of freshwater, and the soil surface and contribute significantly to global primary productivity (33). In many environments, light often is a limiting factor for their growth.The efficiency of light harvesting and the distribution of excitation energy in photosystems are important in low-light adaptation. In Prochlorococcus marinus, high- and low-light-adapted ecotypes differ in the number of pcb genes that encode light-harvesting antenna proteins (3, 11). In Synechocystis sp. PCC 6803, rpaC, a gene required for the transition state, can promote growth in white light at an intensity of 2 μmol photons m⁻² s⁻¹ (10, 22). On the other hand, reutilization of secreted substances or degradation products may promote growth under light-limiting conditions. For example, low-light conditions can stimulate the uptake of amino acids in the cyanobacterium Planktothrix rubescens (31).Bacteria can break down peptidoglycan (PG) and reutilize the degradation products to synthesize new PG. This process is called PG recycling. In cyanobacteria and other Gram-negative bacteria, PG forms a continuous layer completely surrounding the cell between the cytoplasmic membrane and the outer membrane (12). The net-like layer consists of glycan strands cross-linked by short peptides with GlcNAc-anhydro-N-acetylmuramic acid (anhMurNAc)-l-Ala-d-Glu-meso-diaminopimelic acid-d-Ala as the repeating unit (23). In Escherichia coli, PG is degraded to GlcNAc-anhMurNAc-peptides or GlcNAc-anhMurNAc and peptides in the periplasmic space, and the GlcNAc-anhMurNAc-peptides and GlcNAc-anhMurNAc are then imported into the cytoplasm by the permease AmpG (13). GlcNAc-anhMurNAc-peptides are processed into GlcNAc-anhMurNAc and tripeptides by AmpD (anhydro-N-acetylmuramyl-l-Ala amidase) and LdcA (LD-carboxypeptidase) in the cytoplasm and reutilized (13, 26). PG accounts for about 2% of the cell mass in Gram-negative bacteria. The reutilization of PG degradation products may promote growth under nutrient-limiting conditions. However, so far, no experimental evidence directly supports this hypothesis. For example, inactivation of ampG or other genes involved in PG recycling apparently does not affect the normal growth rate of E. coli (8, 13, 14, 27, 30), except that it results in autolysis during the stationary growth phase in an ldcA mutant (26).Cyanobacteria have a PG structure similar to that of Gram-negative bacteria, except for small differences, such as the thickness, degree of cross-linking, and covalent linkage of the polysaccharide (15, 16). In the present study, we found that a gene that is highly conserved in cyanobacteria has a function similar to that of murQ, a gene involved in reutilization of GlcNAc-anhMurNAc in E. coli. As shown in Fig. ​Fig.1,1, GlcNAc-anhMurNAc is processed into GlcNAc and anhMurNAc by NagZ (β-N-acetylglucosaminidase) (8), and then GlcNAc is phosphorylated by NagK (GlcNAc kinase), producing GlcNAc-6-P (24), while anhMurNAc is phosphorylated by AnmK (anhMurNAc kinase), producing MurNAc-6-P (28), and is converted by MurQ (MurNAc-6-P etherase) into GlcNAc-6-P (14, 29). GlcNAc-6-P deacetylase (NagA) further converts GlcNAc-6-P to GlcN-6-P, which can be used in synthesis of new PG or enter carbohydrate metabolism (24). We show here that murQ and its homologs in cyanobacteria can promote growth under light-limiting conditions. Also, in a filamentous N₂-fixing cyanobacterium, Anabaena sp. strain PCC 7120, the murQ homolog enhances heterocyst differentiation at a low light intensity.Open in a separate windowFIG. 1.Schematic diagram showing the PG recycling pathway described by Uehara et al. (29). anhMurNAC, anhydro-N-acetylmuramic acid; GlcN-6-P, glucosamine 6-phosphate; GlcNAc, N-acetylglucosamine; GlcNAc-6-P, N-acetylglucosamine 6-phosphate; MurNAC-6-P, N-acetylmuramic acid 6-phosphate.     

Characterization of the Synechocystis Strain PCC 6803 Penicillin-Binding Proteins and Cytokinetic Proteins FtsQ and FtsW and Their Network of Interactions with ZipN

Because very little is known about cell division in noncylindrical bacteria and cyanobacteria, we investigated 10 putative cytokinetic proteins in the unicellular spherical cyanobacterium Synechocystis strain PCC 6803. Concerning the eight penicillin-binding proteins (PBPs), which define three classes, we found that Synechocystis can survive in the absence of one but not two PBPs of either class A or class C, whereas the unique class B PBP (also termed FtsI) is indispensable. Furthermore, we showed that all three classes of PBPs are required for normal cell size. Similarly, the putative FtsQ and FtsW proteins appeared to be required for viability and normal cell size. We also used a suitable bacterial two-hybrid system to characterize the interaction web among the eight PBPs, FtsQ, and FtsW, as well as ZipN, the crucial FtsZ partner that occurs only in cyanobacteria and plant chloroplasts. We showed that FtsI, FtsQ, and ZipN are self-interacting proteins and that both FtsI and FtsQ interact with class A PBPs, as well as with ZipN. Collectively, these findings indicate that ZipN, in interacting with FtsZ and both FtsI and FtQ, plays a similar role to the Escherichia coli FtsA protein, which is missing in cyanobacteria and chloroplasts.The peptidoglycan layer (PG) of bacterial cell wall is a major determinant of cell shape, and the target of our best antibiotics. It is built from long glycan strands of repeating disaccharides cross-linked by short peptides (38). The resultant meshwork structure forms a strong and elastic exoskeleton essential for maintaining shape and withstanding intracellular pressure. Cell morphogenesis and division have been essentially studied in the rod-shaped organisms Escherichia coli and Bacillus subtilis, which divide through a single medial plane (8, 10, 21, 23). These organisms have two modes of cell wall synthesis: one involved in cell elongation and the second operating in septation (2). Each mode of synthesis is ensured by specific protein complexes involving factors implicated in the last step of PG synthesis (2). The complete assembly of PG requires a glycosyl transferase that polymerizes the glycan strands and a transpeptidase that cross-links them via their peptide side chains (35). Both activities are catalyzed by penicillin-binding proteins (PBPs), which can be divided into three classes: class A and class B high-molecular-weight (HMW) PBPs and class C low-molecular-weight (LMW) PBPs (35).Class A PBPs exhibit both transglycosylase and transpeptidase activities. In E. coli, they seem to be nonspecialized (2), as they operate in the synthesis of both cylindrical wall (cell elongation) and septal PG (cytokinesis). In B. subtilis, PBP1 (class A) is partially localized to septal sites and its depletion leads to cell division defects (31).Class B PBPs, which comprise two proteins in most bacteria, are monofunctional transpeptidases (35), each involved in longitudinal and septal growth of cell wall, respectively (36). In E. coli, this protein, PBP3, is also termed FtsI, because it belongs to the Fts group of cell division factors whose depletion leads to the filamentation phenotype (11). These at least 10 Fts proteins are recruited to the division site at mid-cell in the following sequential order: FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsL/FtsB, FtsW, FtsI, and FtsN (11). The cytoplasmic protein FtsZ is the first recruited to the division site, where it polymerizes in a ring-like structure (1), which serves as a scaffold for the recruitment of the other Fts proteins and has been proposed to drive the division process (6). Together the Fts proteins form a complex machine coordinating nucleoid segregation, membrane constriction, septal PG synthesis, and possibly membrane fusion.Unlike the other PBPs, class C PBPs do not operate in PG synthesis but rather in maturation or recycling of PG during cell septation (35). They are subdivided into four types. Class C type 5 PBP removes the terminal d-alanine residue from pentapeptide side-chains (dd-carboxypeptidase activity). Types 4 and 7 are able to cleave the peptide cross-links (endopeptidase activity). Finally, type AmpH, which does not have a defined enzymatic activity, is believed to play a role in the normal course of PG synthesis, remodeling or recycling (for a review, see reference 35).In contrast to rod-shaped bacteria, less is known concerning PG synthesis, morphogenesis, and cytokinesis, and their relationships, in spherical-celled bacteria, even though a wealth of them have a strong impact on the environment and/or human health. Furthermore, unlike rod-shaped bacteria spherical-celled bacteria possess an infinite number of potential division planes at the point of greater cell diameter, and they divide through alternative perpendicular planes (26, 36, 37, 39). The spherical cells of Staphylococcus aureus seem to insert new PG strands only at the septum, and accordingly the unique class A PBP localizes at the septum during cell division (36). In contrast, the rugby-ball-shaped cells of Streptococcus pneumoniae synthesize cell wall at both the septum and the neighboring region called “equatorial rings” (36). Accordingly, class A PBP2a and PBP1a were found to operate in elongation and septation, respectively (29).In cyanobacteria, which are crucial to the biosphere in using solar energy to renew the oxygenic atmosphere and which make up the biomass for the food chain (7, 30, 40), cell division is currently investigated in two unicellular models with different morphologies: the rod-shaped Synechococcus elongatus strain PCC 7942 (19, 28) and the spherical-celled Synechocystis strain PCC 6803 (26), which both possess a small fully sequenced genome (http://genome.kazusa.or.jp/cyanobase/) that is easily manipulable (18). In both organisms FtsZ and ZipN/Arc6, a protein occurring only in cyanobacteria (ZipN) and plant chloroplasts (Arc6), were found to be crucial for cytokinesis (19, 26, 28) and to physically interact with each other (25, 26). Also, interestingly, recent studies of cell division in the filamentous cyanobacterium Anabaena (Nostoc) strain PCC 7120, showed that this process is connected with the differentiation of heterocysts, the cells dedicated to nitrogen fixation (34).In a continuous effort to study the cell division machine of the unicellular spherical cyanobacterium Synechocystis, we have presently characterized its eight presumptive PBPs (22) that define three classes and the putative cytokinetic proteins FtsQ and FtsW, as well as their network of interactions between each other and ZipN. Both FtsI and FtsQ were found to be key players in cell division in interacting with ZipN and class A PBPs. Consequently, ZipN in interacting with FtsZ (26), FtsI, and FtQ, like the FtsA protein of E. coli, could play a role similar to FtsA, which is absent in cyanobacteria and chloroplasts.     

Identification of the Polyhydroxyalkanoate (PHA)-Specific Acetoacetyl Coenzyme A Reductase among Multiple FabG Paralogs in Haloarcula hispanica and Reconstruction of the PHA Biosynthetic Pathway in Haloferax volcanii

Genome-wide analysis has revealed abundant FabG (β-ketoacyl-ACP reductase) paralogs, with uncharacterized biological functions, in several halophilic archaea. In this study, we identified for the first time that the fabG1 gene, but not the other five fabG paralogs, encodes the polyhydroxyalkanoate (PHA)-specific acetoacetyl coenzyme A (acetoacetyl-CoA) reductase in Haloarcula hispanica. Although all of the paralogous fabG genes were actively transcribed, only disruption or knockout of fabG1 abolished PHA synthesis, and complementation of the ΔfabG1 mutant with the fabG1 gene restored both PHA synthesis capability and the NADPH-dependent acetoacetyl-CoA reductase activity. In addition, heterologous coexpression of the PHA synthase genes (phaEC) together with fabG1, but not its five paralogs, reconstructed the PHA biosynthetic pathway in Haloferax volcanii, a PHA-defective haloarchaeon. Taken together, our results indicate that FabG1 in H. hispanica, and possibly its counterpart in Haloarcula marismortui, has evolved the distinct function of supplying precursors for PHA biosynthesis, like PhaB in bacteria. Hence, we suggest the renaming of FabG1 in both genomes as PhaB, the PHA-specific acetoacetyl-CoA reductase of halophilic archaea.Several haloarchaeal species belonging to the genera Haloferax, Haloarcula, Natrialba, and Haloquadratum are capable of synthesizing short-chain-length polyhydroxyalkanoates (SCL-PHAs) (6, 8, 14, 16), a large family of biopolymers with desirable biodegradability, biocompatibility, and thermoplastic features (31). Although the metabolic pathways of PHAs in bacteria have been characterized in detail (10, 15, 20, 25, 26, 37), the genes involved in PHA biosynthesis in haloarchaea were not recognized until recently, when the PHA synthase genes were identified and characterized for Haloarcula marismortui, Haloarcula hispanica, and Haloferax mediterranei (6, 19). These archaeal PHA synthases are all composed of two subunits, PhaE and PhaC. They are homologous to the class III PHA synthases from bacteria but have a longer C-terminal extension in the PhaC subunit. Nevertheless, the pathway of supplying the PHA precursors has not yet been clarified for any haloarchaeal strain.Both H. mediterranei and H. hispanica are able to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from unrelated carbon sources, despite the content of the (R)-3-hydroxyvalerate (3-HV) monomer of PHBV in H. mediterranei (10 to 13 mol%) (4, 19) being much higher than that in H. hispanica (∼3 mol%) (19). Conversely, the bacteria Ralstonia eutropha and Synechocystis sp. strain PCC6803, which possess class I and III PHA synthases, respectively, accumulate just poly(3-hydroxybutyrate) (PHB) when the 3-HV-related carbon sources (i.e., propionate and valerate) are not supplied (30). In these two bacteria, the biosynthesis of the (R)-3-hydroxybutyrate coenzyme A [(R)-3-HB-CoA] precursor is conducted by two steps. First, two acetyl-CoA molecules are condensed into one acetoacetyl-CoA molecule by the enzyme β-ketothiolase (PhaA). The acetoacetyl-CoA is then reduced to (R)-3-HB-CoA by a PHA-specific acetoacetyl-CoA reductase (PhaB). The resulting (R)-3-HB-CoA is subsequently incorporated into PHB, catalyzed by PHA synthases (26, 36).Both PhaB and FabG belong to the short-chain dehydrogenase/reductase (SDR) superfamily, whose members are homologous in sequence and have several conserved motifs (27, 29). Interestingly, although FabGs naturally reduce 3-ketoacyl-ACP to form (R)-3-hydroxyacyl-ACP in fatty acid biosynthesis, a few FabGs also recognize 3-ketoacyl-CoA and hence function in PHA biosynthesis. For example, the FabG proteins of Escherichia coli and Pseudomonas aeruginosa have been demonstrated to supply precursors for PHA biosynthesis in recombinant E. coli cells (21, 22, 32, 35). In addition, several FabG paralogs may have evolved a distinct function, to be responsible only for PHA accumulation. This situation was observed in Synechocystis sp. strain PCC6803, where the originally annotated FabG (12) was renamed PhaB after an understanding of its function in PHA biosynthesis (36).Genome-wide analysis of H. marismortui ATCC 43049 (1) revealed eight FabG paralogs in this haloarchaeon. Similarly, multiple fabG paralog genes (fabG1 to fabG6) were also observed in the newly sequenced genome of H. hispanica (our unpublished data). In this study, we demonstrate that fabG1, but not the other five fabG paralogs, encodes the PHA-specific acetoacetyl-CoA reductase in H. hispanica. It is responsible for providing (R)-3-HB-CoA for PHA biosynthesis in Haloarcula species, and interestingly, this enzyme also functions well in Haloferax volcanii, endowing this PHA-defective strain with the ability to accumulate PHA when cotransformed with PHA synthase genes.     

A TRAP Transporter for Pyruvate and Other Monocarboxylate 2-Oxoacids in the Cyanobacterium Anabaena sp. Strain PCC 7120

In the cyanobacterium Anabaena sp. strain PCC 7120, open reading frames (ORFs) alr3026, alr3027, and all3028 encode a tripartite ATP-independent periplasmic transporter (TRAP-T). Wild-type filaments showed significant uptake of [¹⁴C]pyruvate, which was impaired in the alr3027 and all3028 mutants and was inhibited by several monocarboxylate 2-oxoacids, identifying this TRAP-T system as a pyruvate/monocarboxylate 2-oxoacid transporter.The tripartite ATP-independent periplasmic transporter (TRAP-T) family of proteins (family 2.A.56 in the transporter classification database [19]) comprises transporters that consist of three components: a small membrane protein usually bearing 4 transmembrane segments (TMSs), a large membrane protein usually bearing 12 TMSs that is the membrane translocator, and a periplasmic substrate binding protein (10). The TRAP transporters use the energy of an electrochemical ion gradient to drive uphill substrate transport (7, 14). TRAP-T family members are widely present in bacteria and archaea, but only a few substrates, including different types of carboxylates, have been identified for them (20). In vitro binding analyses with the periplasmic solute binding proteins RRC01191 from Rhodobacter capsulatus (20) and TakP from Rhodobacter sphaeroides (8) have shown that they bind monocarboxylate 2-oxoacids, including pyruvate. Additionally, pyruvate induces the TRAP-T periplasmic solute binding protein {"type":"entrez-protein","attrs":{"text":"SMb21353","term_id":"1320617611","term_text":"SMB21353"}}SMb21353 in Sinorhizobium meliloti strain 1021 (13). We are not aware, however, of any study showing a direct role of any of these proteins in pyruvate transport in vivo.Cyanobacteria are a morphologically diverse group of photoautotrophic bacteria that includes unicellular and multicellular (filamentous) organisms (18). Most cyanobacteria can use ammonium or nitrate ions as nitrogen sources, and some can also assimilate urea or fix atmospheric N₂ (5). Some filamentous cyanobacteria fix N₂ in differentiated cells called heterocysts that are formed under combined nitrogen deprivation (6). A TRAP transporter is involved in sodium-dependent glutamate uptake in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 (17). It is composed of proteins GtrA and GtrB (small and large membrane subunits, respectively) and GtrC (periplasmic substrate binding protein). A cluster of open reading frames (ORFs), alr3026, alr3027, and all3028, encoding proteins similar to TRAP-T proteins, is found in the genome of the filamentous, heterocyst-forming Anabaena sp. strain PCC 7120 (9). The proteins are Alr3026, with 4 predicted TMSs; Alr3027, with 13 predicted TMSs (however, the N-terminal TMS is a predicted signal peptide that could be removed, producing a mature protein of 12 TMSs); and All3028, a predicted periplasmic solute binding protein. Whereas the two membrane proteins are most similar to proteins of the Synechocystis Gtr glutamate transporter (Alr3026 shares 63% identity with GtrA, and Alr3027, 77% identity with GtrB), the periplasmic solute binding protein, All3028, is more similar to Rhodobacter capsulatus RRC01191 (47% identity) and Rhodobacter sphaeroides TakP (49% identity) than to Synechocystis GtrC (about 18% identity in a 300-amino-acid overlap). It was of interest, therefore, to determine the substrate(s) for this Anabaena transporter, which we approached by mutation and transport analysis.     

The KtrA and KtrE Subunits Are Required for Na+-Dependent K+ Uptake by KtrB across the Plasma Membrane in Synechocystis sp. Strain PCC 6803

The Na⁺-dependent K⁺ uptake KtrABE system is essential for the adaptation of Synechocystis to salinity stress and high osmolality. While KtrB forms the K⁺-translocating pore, the role of the subunits KtrA and KtrE for Ktr function remains elusive. Here, we characterized the role of KtrA and KtrE in Ktr-mediated K⁺ uptake and in modulating Na⁺ dependency. Expression of KtrB alone in a K⁺ uptake-deficient Escherichia coli strain conferred low K⁺ uptake activity that was not stimulated by Na⁺. Coexpression of both KtrA and KtrE with KtrB increased the K⁺ transport activity in a Na⁺-dependent manner. KtrA and KtrE were found to be localized to the plasma membrane in Synechocystis. Site-directed mutagenesis was used to analyze the role of single charged residues in KtrB for Ktr function. Replacing negatively charged residues facing the extracellular space with residues of the opposite charge increased the apparent K_m for K⁺ in all cases. However, none of the mutations eliminated the Na⁺ dependency of Ktr-mediated K⁺ transport. Mutations of residues on the cytoplasmic side had larger effects on K⁺ uptake activity than those of residues on the extracellular side. Further analysis revealed that replacement of R262, which is well conserved among Ktr/Trk/HKT transporters in the third extracellular loop, by Glu abolished transport activity. The atomic-scale homology model indicated that R262 might interact with E247 and D261. Based on these data, interaction of KtrA and KtrE with KtrB increased the K⁺ uptake rate and conferred Na⁺ dependency.Cyanobacterium Synechocystis sp. strain PCC 6803 contains a number of different K⁺ uptake systems that may contribute to satisfying its requirement of K⁺ (3, 19, 36). Among these systems, Ktr has been shown to have a major role not only in K⁺ uptake but also in adaptation against high-osmolarity stress (3, 19). Inactivation of the ktr gene renders the cells hypersensitive to high concentrations of NaCl and the nonionic compound sorbitol. Ktr-mediated K⁺ uptake depends on the presence of Na⁺ in the medium, which is likely to be an adaptation to salinity stress. A requirement of Na⁺ for K⁺ transport activity has also been found in the homologous protein from Vibrio alginolyticus (21). This dependency on Na⁺ is a unique property of Ktr-type transporters and has not been found in other types of K⁺ transporters or channels (32). The structure and function of Ktr-type transporters have been studied in a number of organisms (3, 6, 7, 9, 11-14, 18-20, 30, 32-34). The Ktr system from Synechocystis consists of three subunits, KtrA, KtrB, and KtrE (19). The KtrE gene and the KtrB gene form a cistron, whereas the KtrA gene resides at a site distant from the KtrEB genes in the Synechocystis genome (19). KtrB, the K⁺-translocating subunit, is a member of the Ktr/Trk/HKT family of K⁺ transporters. These transporters have been proposed to have evolved from two membrane-spanning K⁺ channels (6, 7). According to the model, this type of transporter contains eight transmembrane domains, which consist of a 4-fold-repeated membrane-pore-membrane (M1-P-M2) motif (6, 7, 13, 18). An intramolecular electrostatic interaction of Synechocystis KtrB has been proposed to stabilize the protein in its active configuration (12). In addition, a conserved His in the external region in Synechocystis KtrB has been shown to be crucial for KtrB function (39). The region of the Vibrio Ktr protein responsible for gating of ion permeation has been identified (9). However, not much is known about the mechanism of Na⁺ binding to KtrB in Synechocystis.The KtrA subunit belongs to the family of KTR (K⁺-transport nucleotide binding)/RCK (regulating the conductance of K⁺ channels) proteins, which contain a Rossmann-fold sequence encoding β-α protein structure for NAD⁺/NADH binding (17). Accordingly KtrA has been proposed to regulate the K⁺ transport activity of KtrB by changing its binding from NAD⁺ to NADH through a ligand-mediated conformational switch mechanism (25). It has also been shown that ATP promotes complex formation between KtrA and KtrB and that KtrAB from V. alginolyticus when expressed in Escherichia coli cells requires both ATP and the membrane potential for its activity (17).KtrE is a unique subunit found only in Synechocystis; it is not involved in KtrB-mediated K⁺ transport in V. alginolyticus and Bacillus subtilis (11, 32). The termination codon of ktrE overlaps the initiation codon of ktrB in the same cistron, which has not been found in other bacterial ktrB-related genes. Coexpression of KtrA with KtrB alone does not complement the growth defect of an E. coli K⁺ uptake mutant. However, introduction of KtrE into the same mutant background in addition to KtrA and KtrB complements the mutation of the K⁺ uptake system (19). Interestingly, the KtrE protein has been shown to function as a digalactosyldiacylglycerol (DGDG) synthase (EC 2.4.6.241), an enzyme that produces DGDG from monogalactosyldiacylglycerol (MGDG). KtrE has therefore also been designated DgdA (1). Under nonstress conditions, DGDG is found in the thylakoid membranes, which helps stabilize the photosystem II complex in Synechocystis (29). Under phosphate-limited conditions, DGDG is synthesized instead of phospholipids in Synechocystis (1). However, KtrB functions as a major K⁺-conducting transport pore in the Synechocystis plasma membrane. The subcellular localization of KtrE has not been identified directly. Inactivation of ktrE (also called dgdA) in Synechocystis does not result in sensitivity to osmotic stress imposed by 300 mM sorbitol (1). This may be inconsistent with the requirement of KtrE for KtrB-mediated K⁺ uptake in the presence of KtrA in the E. coli expression system (19).Because of these uncertainties about the roles of the KtrA and KtrE subunits in K⁺ uptake by KtrB in Synechocystis and about the identity of the Na⁺ binding site in KtrB, we examined the subcellular localization and membrane association of KtrA and KtrE, the requirement of these subunits for KtrB-mediated K⁺ uptake, and the primary target for Na⁺ binding in KtrB.