Halotolerant cyanobacterium Aphanothece halophytica contains a betaine transporter active at alkaline pH and high salinity

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow in media of up to 3.0 M NaCl and pH 11. This cyanobacterium can synthesize betaine from glycine by three-step methylation using S-adenosylmethionine as a methyl donor. To unveil the mechanism of betaine uptake and efflux in this alkaliphile, we isolated and characterized a betaine transporter. A gene encoding a protein (BetT(A. halophytica)) that belongs to the betaine-choline-carnitine transporter (BCCT) family was isolated. Although the predicted isoelectric pH of a typical BCCT family transporter, OpuD of Bacillus subtilis, is basic, 9.54, that of BetT(A. halophytica) is acidic, 4.58. BetT(A. halophytica) specifically catalyzed the transport of betaine. Choline, gamma-aminobutyric acid, betaine aldehyde, sarcosine, dimethylglycine, and amino acids such as proline did not compete for the uptake of betaine by BetT(A. halophytica). Sodium markedly enhanced betaine uptake rates, whereas potassium and other cations showed no effect, suggesting that BetT(A. halophytica) is a Na(+)-betaine symporter. Betaine uptake activities of BetT(A. halophytica) were high at alkaline pH values, with the optimum pH around 9.0. Freshwater Synechococcus cells overexpressing BetT(A. halophytica) showed NaCl-activated betaine uptake activities with enhanced salt tolerance, allowing growth in seawater supplemented with betaine. Kinetic properties of betaine uptake in Synechococcus cells overexpressing BetT(A. halophytica) were similar to those in A. halophytica cells. These findings indicate that A. halophytica contains a Na(+)-betaine symporter that contributes to the salt stress tolerance at alkaline pH. BetT(A. halophytica) is the first identified transporter for compatible solutes in cyanobacteria.     

Tumor cytotoxicity and interleukin 1 production of blood monocytes of lung cancer patients

Summary The effects of lung cancer on the abilities of blood monocytes to produce interleukin-1 and to mediate antitumor activity were examined. The functional integrity of blood monocytes was determined by their capacity to respond in vitro to a variety of activating agents and become tumoricidal, as assessed by a radioactive release assay and ability to produce interleukin-1 in vitro. The results show that the presence of lung cancer significantly increased the number of harvested blood monocytes and that the spontaneous tumoricidal activity of these monocytes was slightly high as compared to monocytes obtained from healthy donors. The production of interleukin-1 by monocytes of healthy donors and lung cancer patients was similar. Blood monocytes obtained from lung cancer patients were less cytotoxic against allogeneic A375 melanoma cells as compared with those of healthy donors subsequent to incubation with a soluble muramyl dipeptide analog or lipopolysaccharide, but were as tumoricidal as those from healthy donors when activated with lipophilic muramyl tripeptide (MTP-PE) entrapped in multilamellar liposomes. The finding that monocytes of patients with lung cancer can respond to MTP-PE encapsulated in liposomes, recommends the use of these liposomes in therapy of human lung cancer.     

Halotolerant Cyanobacterium Aphanothece halophytica Contains NapA-Type Na+/H+ Antiporters with Novel Ion Specificity That Are Involved in Salt Tolerance at Alkaline pH

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow at NaCl concentrations up to 3.0 M and at pH values up to 11. The genome sequence revealed that the cyanobacterium Synechocystis sp. strain PCC 6803 contains five putative Na⁺/H⁺ antiporters, two of which are homologous to NhaP of Pseudomonas aeruginosa and three of which are homologous to NapA of Enterococcus hirae. The physiological and functional properties of NapA-type antiporters are largely unknown. One of NapA-type antiporters in Synechocystis sp. strain PCC 6803 has been proposed to be essential for the survival of this organism. In this study, we examined the isolation and characterization of the homologous gene in Aphanothece halophytica. Two genes encoding polypeptides of the same size, designated Ap-napA1-1 and Ap-napA1-2, were isolated. Ap-NapA1-1 exhibited a higher level of homology to the Synechocystis ortholog (Syn-NapA1) than Ap-NapA1-2 exhibited. Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 complemented the salt-sensitive phenotypes of an Escherichia coli mutant and exhibited strongly pH-dependent Na⁺/H⁺ and Li⁺/H⁺ exchange activities (the highest activities were at alkaline pH), although the activities of Ap-NapA1-2 were significantly lower than the activities of the other polypeptides. Only one these polypeptides, Ap-NapA1-2, complemented a K⁺ uptake-deficient E. coli mutant and exhibited K⁺ uptake activity. Mutagenesis experiments suggested the importance of Glu129, Asp225, and Asp226 in the putative transmembrane segment and Glu142 in the loop region for the activity. Overexpression of Ap-NapA1-1 in the freshwater cyanobacterium Synechococcus sp. strain PCC 7942 enhanced the salt tolerance of cells, especially at alkaline pH. These findings indicate that A. halophytica has two NapA1-type antiporters which exhibit different ion specificities and play an important role in salt tolerance at alkaline pH.     

Isolation and functional characterization of Ca2+/H+ antiporters from cyanobacteria

Genome sequences of cyanobacteria, Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and Thermosynechococcus elongatus BP-1 revealed the presence of a single Ca2+/H+ antiporter in these organisms. Here, we isolated the putative Ca2+/H+ antiporter gene from Synechocystis sp. PCC 6803 (synCAX) as well as a homologous gene from a halotolerant cyanobacterium Aphanothece halophytica (apCAX). In contrast to plant vacuolar CAXs, the full-length apCAX and synCAX genes complemented the Ca2+-sensitive phenotype of an Escherichia coli mutant. ApCAX and SynCAX proteins catalyzed specifically the Ca2+/H+ exchange reaction at alkaline pH. Immunological analysis suggested their localization in plasma membranes. The Synechocystis sp. PCC 6803 cells disrupted of synCAX exhibited lower Ca2+ efflux activity and a salt-sensitive phenotype. Overexpression of ApCAX and SynCAX enhanced the salt tolerance of Synechococcus sp. PCC 7942 cells. Mutagenesis analyses indicate the importance of two conserved acidic amino acid residues, Glu-74 and Glu-324, in the transmembrane segments for the exchange activity. These results clearly indicate that cyanobacteria contain a Ca2+/H+ antiporter in their plasma membranes, which plays an important role for salt tolerance.     

Overexpression of DnaK from a halotolerant cyanobacterium Aphanothece halophytica enhances growth rate as well as abiotic stress tolerance of poplar plants

The DnaK/Hsp70 family is a molecular chaperone that binds non-native states of other proteins, and concerns to various physiological processes in the bacterial, plant and animal cells. Previously, we showed that overexpression of DnaK from a halotolerant cyanobacterium Aphanothece halophytica (ApDnaK) enhances tolerance to abiotic stresses such as high salinity and high temperature in tobacco plants. Here, we tested the transformation of poplar (Populus alba) with ApDnaK for enhancing the growth of transformed poplar plants. Under control growth conditions, transgenic poplar plants exhibited similar growth rates with the wild-type plants during young seedlings under low light intensity, whereas they showed faster growth, larger plant size, and higher cellulose contents when poplar plants were grown under high light intensity. Transgenic young poplar plants exhibited more rapid recovery from the stresses of high salinity, drought, and low temperature compared with those of the wild type plants when poplar plants were grown under low light intensity. These results suggest that ApDnaK could be useful to enhance the growth rate as well as to increase the stress tolerance.     

Zymobacter palmae gen. nov., sp. nov., a new ethanol-fermenting peritrichous bacterium isolated from palm sap

Zymobacter palmae gen. nov., sp. nov. was proposed for a new ethanol-fermenting bacterium that was isolated from palm sap in Okinawa Prefecture, Japan. The bacterium is gram-negative, facultatively anaerobic, catalase-positive, oxidase-negative, nonspore-forming and peritrichously flagellated. It requires nicotinic acid for growth. It ferments hexoses, -linked di- and tri-saccarides, and sugar alcohols (fructose, galactose, glucose, mannose, maltose, melibiose, saccharose, raffinose, mannitol and sorbitol). Fifteen percent of maltose in broth medium is effectively fermented, whereas glucose with a concentration higher than 10% delayed growth initiation and decreased growth rates. Maltose is fermented to produce ethanol and CO₂ with a trace amount of acids. Approximately 2 mol of ethanol are produced from 1 mol moiety of hexose of maltose. The organism possesses ubiquinone-9. The G+C content of the DNA is 55.8+-0.4 mol%. Major cellular fatty acids were palmitic and oleic acids and cyclopropanic acid of C_19:0. Characteristic hydroxylated acid was 3-hydroxy dodecanoic acid. The bacterium is distinct from other ethanol-fermenting bacteria belonging to the genera Zymomonas Kluyver and van Niel 1936 and Saccharobacter Yaping et al. 1990 with respect to chemotaxonomic and other phenotypic characters to warrant to compose a new genus and a new species. The type strain is strain T109 (= IAM 14233).Abbreviation IAM IAM Culture Collection, Institute of Applied Microbiology. The University of Tokyo     

Interaction of plastocyanin with oligopeptides: effect of lysine distribution within the peptide

We synthesized and purified four oligopeptides containing four lysines (KKKK, GKKGGKK, KKGGGKK, and KGKGKGK) as models for the plastocyanin (PC) interacting site of cytochrome f. These peptides competitively inhibited electron transfer between cytochrome c and PC. The inhibitory effect increased as the peptide concentrations were increased. The association constants between PC and the peptides did not differ significantly (3500-5100 M(-1)), although the association constant of PC-KGKGKGK was a little larger than the constants between PC and other peptides. Changes in the absorption spectrum of PC were observed when the peptides were added to the PC solution: peaks and troughs were detected at about 460 and 630 nm and at about 560 and 700 nm, respectively, in the difference absorption spectra between the spectra with and without peptides. These changes were attributed to the structural change at the copper site of PC by interaction with the peptides. The structural change was most significant when tetralysine was used. These results show that binding of the oligopeptide to PC is slightly more efficient when lysines are distributed uniformly within the peptide, whereas the structural change of PC becomes larger when the lysines are close to each other within the peptide.     

Suppressed expression of the apoplastic ascorbate oxidase gene increases salt tolerance in tobacco and Arabidopsis plants

Transgenic tobacco plants expressing the ascorbate oxidase (AAO) gene in sense and antisense orientations, and an Arabidopsis mutant in which the T-DNA was inserted into a putative AAO gene, were used to examine the potential roles of AAO for salt-stress tolerance in plants. AAO activities in the transgenic tobacco plants expressing the gene in sense and antisense orientations were, respectively, about 16-fold and 0.2-fold of those in the wild type. Under normal growth conditions, no significant differences in phenotypes were observed, except for a delay in flowering time in the antisense plants. However, at high salinity, the percentage germination, photosynthetic activity, and seed yields were higher in antisense plants, with progressively lower levels in the wild type and the sense plants. The redox state of apoplastic ascorbate in sense plants was very low even under normal growth conditions. Upon salt stress, the redox state of symplastic and apoplastic ascorbate decreased among the three types of plants, but was lowest in the sense plants. The hydrogen peroxide contents in the symplastic and apoplastic spaces were higher in sense plants, progressively lower in the wild type, followed by the antisense plants. The Arabidopsis T-DNA inserted mutant exhibited very low ascorbate oxidase activity, and its phenotype was similar to that of antisense tobacco plants. These results suggest that the suppressed expression of apoplastic AAO under salt-stress conditions leads to a relatively low level of hydrogen peroxide accumulation and a high redox state of symplastic and apoplastic ascorbate which, in turn, permits a higher seed yield.     

Functional Differences Between Cyanobacterial DnaK1 Chaperones from the Halophyte Aphanothece halophytica and the Freshwater Species Synechococcus elongatus Expressed in Escherichia coli

DnaK chaperones participate in essential cellular processes including the assistance of the folding, structural maintenance, trafficking, and degradation of proteins, the control of stress responses, and so on. In contrast to the situation found in most other bacterial groups, the cyanobacteria contain multiple dnaK homolog genes whose cellular roles remain ambiguous. We compared in this work the in vivo chaperone capabilities of the DnaK1 members from the halophyte Aphanothece halophytica and the freshwater species Synechococcus elongatus. The corresponding dnaK1 genes were expressed in Escherichia coli, and the abilities of the encoded chaperones to provide for both general and specific functions conducted by E. coli DnaK were analyzed. Synechococcus DnaK1 was far more effective than A. halophytica DnaK1 in replacing E. coli DnaK in all activities tested in vivo, including changes in cell morphology and downregulation of the heat shock response, prevention of the aggregation of misfolded proteins, and restoration of thermotolerance to dnaK-deficient mutants. Thus, regardless of an extensive sequence similarity and comparable in vitro chaperone capabilities, the two cyanobacterial DnaK1 chaperones functionally differed under in vivo conditions. The overall results reinforce the notion that A. halophytica DnaK1 and Synechococcus DnaK1 evolved different substrate specificity since they separated from a common ancestor.     

Electron Flow from NAD(P)H Dehydrogenase to Photosystem I is Required for Adaptation to Salt Shock in the Cyanobacterium Synechocystis sp. PCC 6803

The role of the NAD(P)H-dehydrogenase complex in adaptationto salt stress was examined in an ndhB-inacti-vated mutant ofthe cyanobacterium Synechocystis sp. PCC 6803. Wild-type cellsand ndhB-inactivated mutant cells grew at similar rates underconditions of low salinity (<0.6M NaCl) and high CO² (3%).However, when the concentration of NaCl in the culture mediumwas higher than 0.6 M, the mutant cells grew much more slowlythan the wild-type cells. Upon addition of high concentrationsof NaCl, the oxygen-evolving activity was rapidly inhibitedbut then it recovered, with the rate of recovery depending onthe concentration of NaCl. The recovery of the mutant cellswas significantly delayed when the concentration of NaCl wasabove 0.3 M. At 0.9 M NaCl, wild-type cells recovered with ahalf time of about 40 min, while mutant cells did not recover.The kinetics of changes in Chi fluorescence confirmed theseresults. In wild-type cells, input of electrons from the cytosolto PSI via the NAD(P)H-dehy-drogenase complex increased uponsalt shock. It appears, therefore, that the electron flow fromthe cytosol to PSI via NAD(P)H-dehydrogenase is essential forthe adaptation of cyanobacteria to salt shock. (Received June 11, 1997; Accepted September 24, 1997)