Molecular and biochemical characterization of a plant-like iota-class glutathione S-transferase from the halotolerant cyanobacterium Halothece sp. PCC7418.

AIMS: This study identifies a unique glutathione S-transferase (GST) in extremophiles using genome, phylogeny, bioinformatics, functional characterization, and RNA sequencing analysis. METHODS AND RESULTS: Five putative GSTs (H0647, H0729, H1478, H3557, and H3594) were identified in Halothece sp. PCC7418. Phylogenetic analysis suggested that H0647, H1478, H0729, H3557, and H3594 are distinct GST classes. Of these, H0729 was classified as an iota-class GST, encoding a high molecular mass GST protein with remarkable features. The protein secondary structure of H0729 revealed the presence of a glutaredoxin (Grx) Cys-Pro-Tyr-Cys (C-P-Y-C) motif that overlaps with the N-terminal domain and harbors a topology similar to the thioredoxin (Trx) fold. Interestingly, recombinant H0729 exhibited a high catalytic efficiency for both glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB), with catalytic efficiencies that were 155- and 32-fold higher, respectively, compared to recombinant H3557. Lastly, the Halothece gene expression profiles suggested that antioxidant and phase II detoxification encoding genes are crucial in response to salt stress. CONCLUSION: Iota-class GST was identified in cyanobacteria. This GST exhibited a high catalytic efficiency toward xenobiotic substrates. Our findings shed light on a diversified evolution of GST in cyanobacteria and provide functional dynamics of the genes encoding the enzymatic antioxidant and detoxification systems under abiotic stresses.

Active role of the protein translation machinery in protecting against stress tolerance in Synechococcus elongatus PCC7942.

In vivo protein synthesis is crucial for all domains of life. It is accomplished through translational machinery, and a key step is the translocation of tRNA-mRNA by elongation factor G (EF-G). Genome-based analysis revealed two EF-G encoding genes (S0885 and S2082) in the freshwater cyanobacterium model Synechococcus elongatus PCC7942. S0885 is the essential EF-G gene for photosynthesis. We generated a strain of S. elongatus PCC7942 that overexpressed S0885 (OX-S0885) to identify EF-G functionality. RT-PCR and Western blot analyses revealed increased transcriptional and translational levels in OX-S0885 at 10.5-13.5 and 2.0-3.0 fold, respectively. Overexpression of S0885 led to an increase in specific growth rate. Additionally, polysome-to-monosome ratio (P/M) and RNA-to-protein ratio (R/P) were elevated in OX-S0885 compared with the empty vector. Interestingly, R/P in OX-S0885 was retained at more than 70% under oxidative stress while R/P in the empty vector was severely depleted, suggesting the maintenance of translation. Thus, S0885 appeared to be the important target of oxidative stress because it was protected by the stress response system to maintain its function. These results suggest that cyanobacterial EF-G has a primary function in translation and an unrelated activity during stress conditions. These findings support the substantial role of EF-G in the formation and maintenance of cellular protein formation, and in the protection of the global translational mechanism under oxidative stress condition.

Halotolerance mechanisms in salt­tolerant cyanobacteria.

Cyanobacteria are ubiquitously distributed in nature and are the most abundant photoautotrophs on Earth. Their long evolutionary history reveals that cyanobacteria have a remarkable capacity and strong adaptive tendencies to thrive in a variety of conditions. Thus, they can survive successfully, especially in harsh environmental conditions such as salty environments, high radiation, or extreme temperatures. Among others, salt stress because of excessive salt accumulation in salty environments, is the most common abiotic stress in nature and hampers agricultural growth and productivity worldwide. These detrimental effects point to the importance of understanding the molecular mechanisms underlying the salt stress response. While it is generally accepted that the stress response mechanism is a complex network, fewer efforts have been made to represent it as a network. Substantial evidence revealed that salt-tolerant cyanobacteria have evolved genomic specific mechanisms and high adaptability in response to environmental changes. For example, extended gene families and/or clusters of genes encoding proteins involved in the adaptation to high salinity have been collectively reported. This chapter focuses on recent advances and provides an overview of the molecular basis of halotolerance mechanisms in salt­tolerant cyanobacteria as well as multiple regulatory pathways. We elaborate on the major protective mechanisms, molecular mechanisms associated with halotolerance, and the global transcriptional landscape to provide a gateway to uncover gene regulation principles. Both knowledge and omics approaches are utilized in this chapter to decipher the mechanistic insights into halotolerance. Collectively, this chapter would have a profound impact on providing a comprehensive understanding of halotolerance in salt­tolerant cyanobacteria.

Halotolerance, stress mechanisms, and circadian clock of salt-tolerant cyanobacteria.

Cyanobacteria harbor a high level of physiological flexibility, which enables them to reside in virtually all available environmental niches, including extreme environments. In this review, we summarize the recent advancements in stress mechanisms of salt-tolerant (a.k.a. halotolerant) cyanobacteria. Omics approaches have been extensively employed in recent years to decipher mechanisms of halotolerance and to understand the relevance of halotolerance-associated gene regulatory networks. The vast knowledge from genome mining disclosed that halotolerant cyanobacteria possess extended gene families and/or clusters, encoding enzymes that synthesize unique osmoprotectants, including glycine betaine (GB), betaine derivatives, and mycosporine-like amino acids (MAAs). Comprehensive transcriptomic analyses were conducted using Halothece sp. PCC7418 (hereafter referred to as Halothece), a cyanobacterium that exhibits remarkable halotolerance. These studies revealed a specific transcriptional response when Halothece was subjected to salt stress, whereas salt and osmotic stresses were found to share a common transcriptomic response. Transcriptome and metabolite analyses of Halothece illustrated a complex dynamic relationship between the biosyntheses of osmoprotectants, as well as corresponding and ancillary pathways. Lastly, novel insights highlight the relationship between the molecular regulation of the circadian rhythm and salt stress tolerance. Since the circadian rhythm of gene expression was distorted under salt stress, halotolerant cyanobacteria may prioritize the adaptation to salt stress by attenuation of circadian rhythmicity. KEY POINTS: • Recent advancements in the understanding of stress mechanisms in halotolerant cyanobacteria are described based on omics analyses. • Transcriptome and metabolite analyses of Halothece illustrated a complex dynamic relationship between the biosyntheses of osmoprotectants, as well as corresponding and ancillary pathways. • Since salt stress affects the molecular regulation among clock-related proteins, salt stress may attenuate circadian rhythmicity.

Global transcriptome analyses and regulatory mechanisms in Halothece sp. PCC 7418 exposed to abiotic stresses.

Halotolerant species are of interest since they occur naturally in environments with excess toxic ions. The cyanobacterium Halothece sp. PCC 7418 (hereafter referred to as Halothece) exhibits remarkable halotolerance and was used to examine stress-responsive regulatory mechanisms. The effects of five different stimuli on Halothece transcriptomes were examined using RNA sequencing. In response to diverse stresses, there were both common and stress-specific transcriptional responses. A common upregulated gene set under all stresses consisted of nine differentially expressed genes (DEGs). We also found that osmotic stress elicited the largest set of DEGs. Salt- and osmotic-responsive regulatory mechanisms shared common pathways. DEGs that were upregulated under salt stress encoded proteins involved in photosynthesis and related machineries. Furthermore, DEGs encoding two-component system (TCS) factors, transcriptional factors, scaffolds for protein-protein interactions, transporters, protein turnover factors, and lipid biosynthesis enzymes were also identified under salt stress. Notably, one-carbon (1C) metabolism factors, glycine betaine (GB) synthesis enzymes, and GB transporters were upregulated under salt stress. Metabolic analyses revealed that GB accumulated under salt stress, while mycosporine-2-glycine (M2G) accumulated under salt or osmotic stress. None of the nutrient starvations induced GB nor M2G accumulation. These results suggested that GB and M2G are two osmoprotectants that contribute to halotolerance. Based on our results, we proposed regulatory mechanisms that are crucial for halotolerance, which are coordinated with the GB, M2G, 1C, amino acid, and central carbon interlinking metabolic pathways. 1C metabolism directly fulfills the high metabolite requirements for halotolerance together with the ancillary role of several metabolic pathways.Key Points• Global transcriptome surveys together with molecular and metabolite analyses provide insights into regulatory networks that are crucial for halotolerance• Regulatory networks that are crucial for halotolerance coordinated with the two key osmoprotectants, one carbon, amino acid, and central carbon interlinking metabolic pathways• The findings have translational relevance in genomic and transcriptomic mechanisms of halotolerance.

Molecular and functional insights into glutathione S-transferase genes associated with salt stress in Halothece sp. PCC7418.

Evolution and function of glutathione S-transferase (GST) in primordial oxygenic phototrophs such as cyanobacteria are poorly understood. In this study, we identified and functionally characterized the GST gene family in the halotolerant cyanobacterium Halothece sp. PCC7418. Four putative Halothece-GSTs had very low homology, which implies evolutionary divergence. Of these, H0647, H0729 and H3557 were differentially expressed by oxidative stress whereas H3557 was highly and specifically upregulated under salt stress. In vitro analysis revealed that the recombinant H3557 exhibited GST activity toward 1-chloro-2, 4-dinitrobenzene (CDNB) and glutathione (GSH). H3557 displayed a broad range of activity at pH 6.5-10.5. Kinetic parameters showed the apparent Km for CDNB and GSH was 0.14 and 0.75 mM, respectively. H3557 remained catalytically active in the presence of NaCl. Structural modelling supported that H3557 is salt-adaptive enzyme with highly acidic residues on the protein surface. The vital function of H3557 in heterologous expression system was evaluated. The H3557-expressing cells were more tolerant to H2 O2 -induced oxidative stress compared with other GST-expressing cells and conferred salt tolerance. Taken together, the findings of this study provide insights into the molecular and cellular functions of GST in cyanobacteria, particularly under salt stress, which is less understood compared with other species.

Paper-based sensor from pyrrolidinyl peptide nucleic acid for the efficient detection of Bacillus cereus.

Bacillus cereus is one of the most common foodborne pathogens found in various kinds of staple foods such as rice and wheat. A rapid and accurate detection method for this pathogen is highly desirable for the sustainable production of relevant food products. While several classical and molecular-based detection methods are available for the identification of B. cereus, they suffered one or more limitations such as the requirement for a tedious and time-consuming process, less than ideal specificity, and the lack of portability. Herein, we developed the first paper-based sensing device that exhibits high species specificity with sufficiently low limit of detection for the visual detection of specific DNA sequences of B. cereus. The success is attributed to the strategic planning of fabrication in various dimensions including thorough bioinformatics search for highly specific genes, the use of the pyrrolidinyl peptide nucleic acid (PNA) probe whose selectivity advantage is well documented, and an effective PNA immobilization and DNA-binding visualization method with an internal cross-checking system for validating the results. Testing in rice matrices indicates that the sensor is capable of detecting and distinguishing B. cereus from other bacterial species. Hence, this paper-based sensor has potential to be adopted as a practical means to detect B. cereus in food industries.

The evolutionarily conserved HtrA is associated with stress tolerance and protein homeostasis in the halotolerant cyanobacterium Halothece sp. PCC7418.

The HtrA protein family represents an important class of serine proteases that are widely distributed across taxa. These evolutionarily conserved proteins are crucial for survival and function as monitors of protein synthesis during various stresses. Here, we performed gene expression analysis of the entire set of putative serine protease genes in Halothece sp. PCC7418 under salt stress conditions. The gene-encoding HtrA2 (H3553) was highly upregulated. This gene was cloned and functionally characterized, and its sub-cellular localization was determined. The recombinant H3553 protein (rH3553) displayed a pH optimum of 8.0, remained stable at 45 °C, and its proteolytic activity was not affected by salts. H3553 completely degraded the unfolded model protein, ß-casein. In contrast, the folded model substrates (lysozyme or BSA) were not degraded by rH3553. Denaturation of BSA at a high temperature significantly increased its degradation by rH3553. H3553 was detected in the soluble protein fraction as well as the plasma membrane and thylakoid membrane fractions. Interestingly, the majority of H3553 was present in the plasma membrane under salt and heat stress conditions. Thus, H3553 resides in multiple sub-cellular locations and its localization drastically changes after exposure to stresses. Taken together, H3553 underpins protein quality-control process and is involved in the response and adaptation to salinity and heat stresses.

Morphological plasticity of hyperelongated cells caused by overexpression of translation elongation factor P in Synechococcus elongatus PCC7942.

Translation elongation factors (EFs) are proteins that play important roles during the elongation stage of protein synthesis. In prokaryotes, at least four EFs function in repetitive reactions (EF-Tu, EF-Ts, EF-G, and EF-P). EF-P plays a vital role in the specialized translation of consecutive proline amino acid motifs. It was also recently recognized that EF-P acts throughout translation elongation. Here, we demonstrated for the first time that cell division and morphology are intimately linked to the control of EF-P in the model cyanobacterium Synechococcus elongatus PCC7942. We constructed the overexpression of a wild-type gene product for EF-P (Synpcc7942_2565) as a tool to identify EF-P functionality. The overexpression of EF-P resulted in the morphological plasticity of hyperelongated cells. During the stationary phase, EF-P overexpressors displayed cell lengths of 150 µm or longer, approximately 35 times longer than the control. Total cellular protein and amino acid content were also increased in overexpressors. To explore the molecular mechanisms underlying hyperelongation, gene expression analysis was performed. The results revealed that cell division genes, including ftn6, minD, mreB, mreC, and ftsZ, were modulated in overexpressors. Strikingly, ftn6 was severely down-regulated. Little is known regarding EF-P in prokaryotic photosynthetic organisms. Our results suggest that cyanobacterial EF-P participates in the acceleration of protein synthesis and also regulates cell division processes. These findings suggest new ways to modify translation and metabolism in cyanobacteria. Phenotypic and metabolic alterations caused by overexpressing EF-P may also be beneficial for applications such as low-cost, green molecular factories. KEY POINTS: • Cell division and cell morphology in the cyanobacterium Synechococcus elongatus PCC7942 are closely linked with the control of translation elongation factor P (EF-P). • Overexpression of EF-P leads to morphological plasticity in hyperelongated cells. • Cyanobacterial EF-P is involved in the acceleration of protein synthesis and the regulation of cell division processes.

Mycosporine-2-glycine exerts anti-inflammatory and antioxidant effects in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.

Mycosporine-like amino acids (MAAs) are a group of water-soluble low-molecular-weight secondary metabolites, which are well-documented UV-screening molecules and antioxidants. We have recently demonstrated that a rare MAA, mycosporine-2-glycine (M2G), efficiently inhibited the formation of advanced glycation end-products (AGEs). Because AGEs contribute significantly to the aging process, including the pathogenesis and progression of age-related diseases, the present study further evaluated anti-inflammatory effects of M2G using an in vitro model of RAW 264.7 macrophages. We measured the inflammatory signaling molecule nitric oxide (NO) under inflammatory stimulation by lipopolysaccharide (LPS), revealing that M2G diminished LPS-induced NO production. M2G inhibited NO production approximately 2-3-fold more potently than other MAAs, including shinorine, porphyra-334, and palythine. Transcriptional analyses revealed that M2G significantly suppressed iNOS and COX-2 expression. Therefore, M2G inhibits the production of inflammatory mediators by suppressing the NF-κB pathway. Furthermore, under H2O2-induced oxidative stress, M2G down-regulated Sod1, Cat, and Nrf2 expression. Our findings clearly demonstrate anti-inflammatory and antioxidant effects of M2G in LPS-stimulated RAW 264.7 macrophages. Structure-activity relationships of biologically active MAAs are also discussed.

A class I fructose-1,6-bisphosphate aldolase is associated with salt stress tolerance in a halotolerant cyanobacterium Halothece sp. PCC 7418.

Fructose-1,6-bisphosphate aldolase (FBA) is a key metabolic enzyme, which is involved in glycolysis, gluconeogenesis and the Calvin cycle. The distinct physiological roles of FBAs in various organisms have been reported; however, in cyanobacteria, the functional characterization of FBAs and investigation of the intracellular dynamics of FBAs largely remains unknown. Here, we utilized a two-step chromatographic technique to identify a class I FBA (CI-FBA), which we named H2846. H2846 was induced by salt stress in the halotolerant cyanobacterium Halothece sp. PCC 7418 (hereafter referred to as Halothece 7418). Phylogenetic analysis showed that H2846-like CI-FBAs existed mainly in cyanobacterial species that inhabit hypersaline environments. Subcellular fractionation revealed that H2846 localized in the cytosolic and periplasmic spaces and size-exclusion chromatography suggested that H2846 formed a homohexamer. The CI-FBA activity of recombinant H2846-mediated cleavage of fructose bisphosphate (FBP) was characterized using a coupled enzymatic assay. This analysis allowed us to determine the Km and Vmax values of recombinant H2846, which were then compared to previously reported Km and Vmax values of several FBAs. Our data suggested that H2846 was likely responsible for the salt stress-induced CI-FBA activity from the total soluble protein extracts derived from Halothece 7418 cells. Moreover, heterologous expression of H2846 but not H2847, a class II FBA (CII-FBA), conferred salt stress tolerance to the salt-sensitive freshwater cyanobacterium, Synechococcus elongatus PCC 7942, which only contains the CII-FBA, S1443. S. elongatus PCC 7942 with a S1443 gene deletion was complemented by H2847 expression, but was not complemented by expression of H2846. Taken together, these results indicate the functional differences between two distinct sets of FBAs in cyanobacteria. H2846 is an active CI-FBA that contributes to the mechanism of salt stress tolerance in Halothece 7418.

Effects of Potassium Chloride-Induced Stress on the Carotenoids Canthaxanthin, Astaxanthin, and Lipid Accumulations in the Green Chlorococcal Microalga Strain TISTR 9500.

Microalgae are a diverse group of photosynthetic eukaryotic organisms that are widely distributed globally. They are prolific sources of highly valuable compounds with fascinating chemical structures. Due to their balanced nutritional compositions and health benefits, they are increasingly being used as functional food ingredients. Carotenoid-based pigments and polyunsaturated fatty acids (PUFAs) are examples of high-value nutrients that can be accumulated abundantly in microalgae. Here, the effects of potassium chloride-induced stress on the productions of lipids and carotenoids in the green microalga of the Chlorococcaceae family were investigated. Under normal BG11 medium, this green microalga strain TISTR 9500 accumulated high levels of PUFA and primary carotenoid lutein. Stress tests revealed that KCl enhanced and modulated lipid and carotenoid accumulation levels. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that secondary carotenoids astaxanthin and canthaxanthin were robustly produced under KCl stress with the similar content of lutein. Further, this stress led to a significant increase in the total FA amount with the higher proportion of unsaturated FA than saturated FA. Thus, this green microalga could be an attractive and alternative natural biosource for canthaxanthin and astaxanthin, as well as for functional lipids.

Antioxidative, Anti-Inflammatory, and Anti-Aging Properties of Mycosporine-Like Amino Acids: Molecular and Cellular Mechanisms in the Protection of Skin-Aging.

Prolonged exposure to ultraviolet (UV) radiation causes photoaging of the skin and induces a number of disorders, including sunburn, fine and coarse wrinkles, and skin cancer risk. Therefore, the application of sunscreen has gained much attention to reduce the harmful effects of UV irradiation on our skin. Recently, there has been a growing demand for the replacement of chemical sunscreens with natural UV-absorbing compounds. Mycosporine-like amino acids (MAAs), promising alternative natural UV-absorbing compounds, are a group of widely distributed, low molecular-weight, water-soluble molecules that can absorb UV radiation and disperse the absorbed energy as heat, without generating reactive oxygen species (ROS). More than 30 MAAs have been characterized, from a variety of organisms. In addition to their UV-absorbing properties, there is substantial evidence that MAAs have the potential to protect against skin aging, including antioxidative activity, anti-inflammatory activity, inhibition of protein-glycation, and inhibition of collagenase activity. This review will provide an overview of MAAs, as potential anti-aging ingredients, beginning with their structure, before moving on to discuss the most recent experimental observations, including the molecular and cellular mechanisms through which MAAs might protect the skin. In particular, we focus on the potential anti-aging activity of mycosporine-2-glycine (M2G).

Enhanced Lipid Production and Molecular Dynamics under Salinity Stress in Green Microalga Chlamydomonas reinhardtii (137C).

Microalgal lipids are a source of valuable nutritional ingredients in biotechnological industries, and are precursors to biodiesel production. Here, the effects of salt-induced stresses, including NaCl, KCl, and LiCl stresses, on the production of lipid in green microalga Chlamydomonas reinhardtii (137c) were investigated. NaCl stress dramatically increased saturated fatty acids (SFAs), which accounted for 70.2% of the fatty acid methyl ester (FAMEs) under stress. In contrary, KCl stress led to a slight increase in SFAs (47.05%) with the remaining being polyunsaturated fatty acids (PUFAs) (45.77%). RT-PCR analysis revealed that the genes involved in FA biosynthesis, such as PDH2, ACCase, MAT and KAS2, were up-regulated by NaCl-induced stress. Conversely, the genes responsible for the Kennedy pathway were suppressed. The alteration of FA homeostasis was further assessed by overexpressing MAT, the enzyme responsible for the production of malonyl-ACP, a key building block for FA biosynthesis, in the cyanobacterium Synechococcus elongatus PCC 7942. Intracellular FA composition was affected, with a predominant synthesis of SFAs in transformed cells. Owing to the diversity and relative abundance of SFAs, monounsaturated fatty acid (MUFAs) and PUFAs enable the feasibility of using microorganisms as a source of microalgal lipids or valuable nutritional ingredients; salt-induced stress and expression of MAT are useful in providing precursors for enhanced lipid production.

Nutrient Deprivation-Associated Changes in Green Microalga Coelastrum sp. TISTR 9501RE Enhanced Potent Antioxidant Carotenoids.

The utilization of microalgae as a source of carotenoid productions has gained increasing popularity due to its advantages, such as a relatively fast turnaround time. In this study, a newly discovered Coelastrum sp. TISTR 9501RE was characterized and investigated for its taxonomical identity and carotenoid profile. To the best of our knowledge, this report was the first to fully investigate the carotenoid profiles in a microalga of the genus Coelastrum. Upon use of limited nutrients as a stress condition, the strain was able to produce astaxanthin, canthaxanthin, and lutein, as the major carotenoid components. Additionally, the carotenoid esters were found to be all astaxanthin derivatives, and ß-carotene was not significantly present under this stress condition. Importantly, we also demonstrated that this practical stress condition could be combined with simple growing factors, such as ambient sunlight and temperature, to achieve even more focused carotenoid profiles, i.e., increased overall amounts of the aforementioned carotenoids with fewer minor components and chlorophylls. In addition, this green microalga was capable of tolerating a wide range of salinity. Therefore, this study paved the way for more investigations and developments on this fascinating strain, which will be reported in due course.

Dimethylsulfoniopropionate biosynthesis in a diatom Thalassiosira pseudonana: Identification of a gene encoding MTHB-methyltransferase.

Dimethylsulfoniopropionate (DMSP) is one of the most abundant molecules on earth and plays a pivotal role in the marine sulfur cycle. DMSP is believed to be synthesized from methionine by a four-step reaction pathway in marine algae. The genes responsible for biosynthesis of DMSP remain unidentified. A diatom Thalassiosira pseudonana CCMP1335 is an important component of marine ecosystems and contributes greatly to the world's primary production. In this study, through genome search, in vivo activity and functional studies of cDNA products, a gene encoding Thalassiosira methyltransferase (TpMMT) which catalyzes the key step of DMSP synthesis formation of 4-methylthio-2-hydroxybutyrate (DMSHB) from 4-methylthio-2-oxobutyrate (MTHB), was identified. The amino acid sequence of TpMMT was homologous to the methyltransferase from Phaeodactylum tricornutum CCAP 1055/1, but not the recently identified bacterium gene. High salinity and nitrogen limitation stresses caused the increase of DMSP content and TpMMT protein in Thalassiosira. In addition to TpMMT, the enzyme activities for the first three steps could be detected and enhanced under high salinity, suggesting the importance of four-step DMSP synthetic pathway in Thalassiosira.

Functional characterization of the NhaA Na+/H+ antiporter from the green picoalga Ostreococcus tauri.

Transmembrane ion transport is a critical process in the cellular response to salt stress. Among the known functional membrane transporters that are involved in the salt stress response, Na+/H+ antiporters have been extensively studied. These ubiquitous membrane proteins are crucial for salt tolerance and are associated with the regulation of internal pH, cell volume, morphogenesis, and vesicular trafficking. Molecular and functional analyses of Na+/H+ antiporters have been characterized among taxa but little is known about algal Na+/H+ antiporters. Here, we analyzed putative Na+/H+ antiporters from the complete genome sequence of the marine picoalga Ostreococcus tauri. At least 10 putative Na+/H+ antiporters belonging to the SOS1, NHX, and KEA/Kef families were found. Surprisingly, a bacterial type NhaA sequence (OtNhaA) was also found. Topological modeling of OtNhaA predicted 12 possible transmembrane segments with a long N-terminus. The full-length (FL_OtNhaA) and N-terminal truncated (ΔN112_OtNhaA) versions of OtNhaA were constructed, expressed in the salt-sensitive mutant Escherichia coli TO114, and functionally characterized. Complementation analysis revealed that FL_OtNhaA- and ΔN112_OtNhaA-expressing cells exhibited increased tolerance to high NaCl concentrations up to 700 mM. Antiporter activity assays showed that both FL_OtNhaA and ΔN112_OtNhaA proteins predominantly exhibited Na+/H+ and Ca2+/H+ antiporter activities at alkaline pH conditions. Intriguingly, the ΔN112_OtNhaA exhibited higher Na+/H+ and Ca2+/H+ antiporter activities compared to FL_OtNhaA. Kinetic analysis revealed that FL_OtNhaA has a high affinity for Na+ and Ca2+ ions with a Km of 1.1 ±â€¯0.23 mM for Na+ (at pH 8.5) and a Km of 0.3 ±â€¯0.07 mM for Ca2+ (at pH 8.5). Since NhaA has shown striking diversity among taxa, our results provide insight into the functional properties of the algal NhaA Na+/H+ antiporter. These results will contribute to the understanding of Na+/H+ antiporters that have various implications in all kingdoms of life.

Bifunctional alanine dehydrogenase from the halotolerant cyanobacterium Aphanothece halophytica: characterization and molecular properties.

A link between carbon and nitrogen metabolism is important for serving as metabolic ancillary reactions. Here, we identified and characterized the alanine dehydrogenase gene in Aphanothece halophytica (ApalaDH) that is involved in alanine assimilation/dissimilation. Functional analysis revealed that ApalaDH encodes a bifunctional protein catalyzing the reversible reaction of pyruvate to L-alanine via its pyruvate reductive aminase (PvRA) activity, the reaction of L-alanine to pyruvate via its alanine oxidative dehydrogenase activity, and the non-reversible reaction of glyoxylate to glycine via its glyoxylate reductive aminase (GxRA) activity. Kinetic analysis showed the lowest affinity for pyruvate followed by L-alanine and glyoxylate with a Km of 0.22 ± 0.02, 0.72 ± 0.04, and 1.91 ± 0.43 mM, respectively. ApalaDH expression was upregulated by salt. Only PvRA and GxRA activities were detected in vivo and both activities increased about 1.2- and 2.7-fold upon salt stress. These features implicate that the assimilatory/dissimilatory roles of ApAlaDH are not only selective for L-alanine and pyruvate, but also, upon salt stress, can catabolize glyoxylate to generate glycine.

Overexpression of halophilic serine hydroxymethyltransferase in fresh water cyanobacterium Synechococcus elongatus PCC7942 results in increased enzyme activities of serine biosynthetic pathways and enhanced salinity tolerance.

Serine hydroxymethyltransferase (SHMT) catalyzes the conversion of serine to glycine and provides activated one-carbon units required for synthesis of nucleic acids, proteins and numerous biological compounds. SHMT is involved in photorespiratory pathway of oxygenic photosynthetic organisms. Accumulating evidence revealed that SHMT plays vital role for abiotic stresses such as low CO2 and high salinity in plants, but its role in cyanobacteria remains to be clarified. In this study, we examined to overexpress the SHMT from halotolerant cyanobacterium Aphanothece halophytica in freshwater cyanobacterium, Synechococcus elongatus PCC7942. The transformed cells did not show an obvious phenotype under non-stress condition, but exhibited more tolerance to salinity than the control cells harboring vector only under high salinity. Elevated levels of enzymes in phosphorylated serine biosynthetic pathway and photorespiration pathway were observed in the transformed cells. Glycine level was also increased in the transformed cells. Physiological roles of SHMT for salt tolerance were discussed.

Caleosin from Chlorella vulgaris TISTR 8580 is salt-induced and heme-containing protein.

Physiological and functional properties of lipid droplet-associated proteins in algae remain scarce. We report here the caleosin gene from Chlorella vulgaris encodes a protein of 279 amino acid residues. Amino acid sequence alignment showed high similarity to the putative caleosins from fungi, but less to plant caleosins. When the C. vulgaris TISTR 8580 cells were treated with salt stress (0.3 M NaCl), the level of triacylglycerol increased significantly. The mRNA contents for caleosin in Chlorella cells significantly increased under salt stress condition. Caleosin gene was expressed in E. coli. Crude extract of E. coli cells exhibited the cumene hydroperoxide-dependent oxidation of aniline. Absorption spectroscopy showed a peak around 415 nm which was decreased upon addition of cumene hydroperoxide. Native polyacrylamide gel electrophoresis suggests caleosin existed as the oligomer. These data indicate that a fresh water C. vulgaris TISTR 8580 contains a salt-induced heme-protein caleosin.