Characterization of macrophages associated with human skin models exposed to UV radiation.

Skin macrophages play important roles in the response to external stimuli. Human skin equivalents (HSEs) incorporating the human monocytic cell line THP-1 were fabricated to generate immunocompetent human skin models. These HSEs were used to investigate the influence of the skin microenvironment and ultraviolet A (UVA) on macrophages. Transcriptomic analysis revealed that THP-1 cells in HSEs were enriched in extracellular matrix interaction hallmark but downregulated in DNA replication hallmark. Upon UVA exposure, immunocompetent HSEs presented epidermal distortion and increased DNA double-strand breaks (DSBs). The genes associated with oxidative stress and the inflammatory response were significantly upregulated in THP-1 cells. When the photoprotective agent mycosporine-2-glycine from cyanobacteria was applied to HSEs, the incidence of UVA-induced DSBs was significantly lower, and inflammatory and UV response hallmarks were downregulated in THP-1 cells. Taken together, these results suggest that immunocompetent HSEs can be used to investigate the responses of skin-resident macrophages to external stimuli.

Novel NhaC Na+/H+ antiporter in cyanobacteria contributes to key molecular processes for salt tolerance.

Genome mining has revealed the halotolerant cyanobacterium Halothece sp. PCC7418 harbors considerable enrichment in the ion transport gene family for putative Na+/H+ antiporters. Here, we compared transcriptomic profiles of these encoding genes under various abiotic stresses and discovered that Halothece NhaC (hnhaC) was one of 24 genes drastically upregulated under salt stress. Critical roles of HnhaC in salt-stress protection and response were identified by a complementation assay using the salt-sensitive mutant Escherichia coli strain TO114. Expression of HnhaC rendered this mutant more tolerant to high concentrations of NaCl and LiCl. Antiporter activity assays showed that HnhaC protein predominantly exhibited Na+/H+ and Li+/H+ antiporter activities under neutral or alkaline pH conditions. Furthermore, expression of HnhaC conferred adaptive benefits onto E. coli by enabling a conditional filamentation phenotype. Dissecting the molecular mechanism of this phenotype revealed that differentially expressed genes were associated with clusters of SOS-cell division inhibitor, SOS response repair, and Z-associated proteins. Together, these results strongly indicate that HnhaC is an Na+/H+ antiporter that contributes to salt tolerance. The ubiquitous existence of several Na+/H+ antiporters represents a complex molecular system in halotolerant cyanobacteria, which can be deployed differently in response to growth and to environmental stresses.

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.

Identification of Desiccation Stress-Inducible Antioxidative and Antiglycative Ultraviolet-Absorbing Oxylipins, Saclipin A and Saclipin B, in an Edible Cyanobacterium Aphanothece sacrum.

Aphanothece sacrum, a freshwater cyanobacterium, is an edible cyanobacterial strain. We identified two compounds belonging to the oxylipin family that possess UV-absorbing abilities and accumulate in the dried sample of A. sacrum. The compounds, named saclipin A and saclipin B, exhibited strong UV-absorption properties with the absorption maxima at 316 and 319 nm, respectively, and the molar extinction coefficients of 26,454 and 30,555 M-1 cm-1, respectively. The chemical structures of saclipins A and B have been elucidated, revealing that they have an all-E and a 12Z isomeric relationship within the triene structure. The saclipins could be isomerized by photoirradiation, with the cis-form saclipin B proving to be more stable in methanol, ethanol, or acetonitrile. Under drought stress conditions, the accumulation of saclipins A and B in A. sacrum was found to be increased 20- and 10-fold, respectively. Purified saclipins from A. sacrum showed biocompatibility and valuable bioactivities. Specifically, saclipins exhibited radical scavenging activity, maintaining their activity even 40 min after the reaction began. Additionally, they demonstrated inhibitory activity against glycation of elastin and collagen, which are constituents of dermal tissue. Notably, saclipins showed higher activity than the well-known glycation inhibitor aminoguanidine against collagen glycation.

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.

Development of peptide nucleic acid-based bead array technology for Bacillus cereus detection.

Numerous novel methods to detect foodborne pathogens have been extensively developed to ensure food safety. Among the important foodborne bacteria, Bacillus cereus was identified as a pathogen of concern that causes various food illnesses, leading to interest in developing effective detection methods for this pathogen. Although a standard method based on culturing and biochemical confirmative test is available, it is time- and labor-intensive. Alternative PCR-based methods have been developed but lack high-throughput capacity and ease of use. This study, therefore, attempts to develop a robust method for B. cereus detection by leveraging the highly specific pyrrolidinyl peptide nucleic acids (PNAs) as probes for a bead array method with multiplex and high-throughput capacity. In this study, PNAs bearing prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbone with groEL, motB, and 16S rRNA sequences were covalently coupled with three sets of fluorescently barcoded beads to detect the three B. cereus genes. The developed acpcPNA-based bead array exhibited good selectivity where only signals were detectable in the presence of B. cereus, but not for other species. The sensitivity of this acpcPNA-based bead assay in detecting genomic DNA was found to be 0.038, 0.183 and 0.179 ng for groEL, motB and 16S rRNA, respectively. This performance was clearly superior to its DNA counterpart, hence confirming much stronger binding strength of acpcPNA over DNA. The robustness of the developed method was further demonstrated by testing artificially spiked milk and pickled mustard greens with minimal interference from food metrices. Hence, this proof-of-concept acpcPNA-based bead array method has been proven to serve as an effective alternative nucleic acid-based method for foodborne pathogens.

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, 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.

Global transcriptional and circadian regulation in a halotolerant cyanobacterium Halothece sp. PCC7418.

Substantial evidence has been accumulated about the molecular basis underlying halotolerance; however, insights into the regulatory networks for relevant genes and mechanisms of their interplay remain elusive. Here, we present a comprehensive transcriptome investigation, using RNA sequencing, of specific metabolic pathways and networks in a halotolerant cyanobacterium, Halothece sp. PCC7418, including the circadian rhythm profile. Dissecting the transcriptome presented the intracellular regulation of gene expressions, which was linked with ion homeostasis, protein homeostasis, biosynthesis of compatible solutes, and signal transduction, for adaptations to high-salinity environments. The efficient production and distribution of energy were also implicated in this acclimation process. Furthermore, we found that high-salinity environments had a dramatic effect on the global transcriptional expression regulated by the circadian clock. Our findings can provide a comprehensive transcriptome for elucidating the molecular mechanisms underlying halotolerance in cyanobacteria.

Potential Probiotic Enterococcus faecium OV3-6 and Its Bioactive Peptide as Alternative Bio-Preservation.

Probiotic Enterococcus faecium OV3-6 and its secreted active peptide were characterized and investigated. The strain survived in simulated gastric and small intestinal conditions at 88.16% and 94.33%, respectively. The safety assessment revealed that the strain was shown α-hemolysis and susceptible to most clinically relevant antibiotics, but intermediate sensitivity to erythromycin and kanamycin was found. It does not harbor any virulence genes except for the efaAfm gene. Both of its living cells and the cell-free supernatants (CFS) of the strain significantly reduced the adhesion of E. coli and S. Typhi on Caco-2 cells. The strain can regulate the secretion of pro and inflammatory cytokines, IL-6 and IL-12 and induce the secretion of anti-inflammatory IL-10 of the Caco-2 cell. The strain can prevent the growth of Gram-positive strains belonging to the genera Bacillus, Carnobacterium, Listeria, and Staphylococcus. It also presented the entP gene that involves the production of bacteriocin named enterocin P. The antimicrobial peptide was matched 40% with 50S ribosomal proteins L29 (7.325 kDa), as revealed by LC-MS/MS. This active peptide exhibits heat stability, is stable over a wide pH range of 2-10, and maintains its activity at -20 and 4 °C for 12 weeks of storage. Altogether, E. faecium OV3-6 thus has potential for consideration as a probiotic and bio-preservative for applied use as a fermented food starter culture and in functional food or feed industries.

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.

Protective effects of mycosporine-like amino acid-containing emulsions on UV-treated mouse ear tissue from the viewpoints of antioxidation and antiglycation.

Mycosporine-like amino acids (MAAs) are promising natural antioxidative compounds with cosmetic applications for the prevention of skin aging. In this study, we evaluated the protective effects of natural resources-derived MAA-containing emulsions on mouse ear tissue exposed to UV irradiation. DBA/2CrSlc male mice were irradiated by UV light at 120 mJ/cm2/day for 9 days. MAA-containing emulsions were prepared using mycosporine-2-glycine (M2G), shinorine (SHI), or porphyra-334 (P334) and applied to mice ears at a dose of 50 mg/ear/day. After that, collected ear skin tissues were subjected to the observation of melanocytes, investigation for antioxidative stress markers, and measurement of advanced glycation-end products (AGEs). In addition, the antiglycative effects of MAAs were investigated in vitro. MAA-containing emulsions prepared in this study upregulated the activities of total superoxide dismutase (SOD) and catalase (CAT) in mouse ear tissue exposed to UV irradiation. Increased accumulation of copper/zinc (Cu/Zn) -SOD and/or CAT was also found in mouse ear tissue on which M2G- or P334-containing emulsion had been applied. Furthermore, P334 exhibited an antiglycative effect on elastin in vitro. Although MAA-containing emulsions have antioxidative effects as well as in vitro antiglycation, a protective effect by the accumulation of AGEs in mice ears exposed to UV was not observed. Thus, application of MAA-containing emulsions stimulated or protected the expression of antioxidant-associated proteins, thereby leading to upregulation of antioxidative activities in mouse ear skin samples tissues under UV irradiation. Additional optimization of MAA-containing emulsions, including composition, process, and dosage should be considered for further improvement of efficacy.

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.

Strategies for Poly(3-hydroxybutyrate) Production Using a Cold-Shock Promoter in Escherichia coli.

The present study attempted to increase poly(3-hydroxybutyrate) (PHB) production by improving expression of PHB biosynthesis operon derived from Cupriavidus necator strain A-04 using various types of promoters. The intact PHB biosynthesis operon of C. necator A-04, an alkaline tolerant strain isolated in Thailand with a high degree of 16S rRNA sequence similarity with C. necator H16, was subcloned into pGEX-6P-1, pColdI, pColdTF, pBAD/Thio-TOPO, and pUC19 (native promoter) and transformed into Escherichia coli JM109. While the phaC A-04 gene was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that the cold-shock cspA promoter enhanced phaCA-04 protein expression and the chaperone function of TF play critical roles in increasing soluble phaCA-04 protein. Induction strategies and parameters in flask experiments were optimized to obtain high expression of soluble PhaCA-04 protein with high YP/S and PHB productivity. Soluble phaCA-04 was purified through immobilized metal affinity chromatography (IMAC). The results demonstrated that the soluble phaCA-04 from pColdTF-phaCAB A-04 was expressed at a level of as high as 47.4 ± 2.4% of total protein and pColdTF-phaCAB A-04 enhanced soluble protein formation to approximately 3.09-4.1 times higher than that from pColdI-phaCAB A-04 by both conventional method and short induction method developed in this study. Cultivation in a 5-L fermenter led to PHB production of 89.8 ± 2.3% PHB content, a YP/S value of 0.38 g PHB/g glucose and a productivity of 0.43 g PHB/(L.h) using pColdTF-phaCAB A-04. The PHB film exhibited high optical transparency and possessed Mw 5.79 × 105 Da, Mn 1.86 × 105 Da, and PDI 3.11 with normal melting temperature and mechanical properties.

Insights into the phylogeny and transcriptional response of serine proteases in a halotolerant cyanobacterium Halothece sp. PCC7418.

Serine proteases are a class of versatile proteolytic enzymes. They are necessary for protein catabolism, intracellular amino acid turnover, and regulation of proteins involved in diverse molecular and cellular processes across taxa. In this study, bioinformatic analyses revealed a significantly large number of serine proteases in the halotolerant cyanobacterium Halothece sp. PCC7418 (hereafter referred to as Halothece 7418) compared to the model freshwater cyanobacterium Synechococcus elongatus PCC7942 (hereafter referred to as S. elongatus 7942). The cyanobacterial serine proteases are likely derived from different linages since no conserved motifs were detected. The presence of highly diverse serine proteases in Halothece 7418 implicated an evolutionary-mediated modification of several proteases, which may play numerous physiological roles. We also examined the gene expression patterns of 34 serine protease encoding genes in Halothece 7418 exposed to salt stress. Our results revealed that several serine protease genes were drastically up-regulated under salt with high concentration but remained unchanged under salt with low concentration. All four clp genes (H1996, H1997, H0950, and H3375) and H3553 gene (which encodes a putative HtrA protease) were significantly induced upon salt stress. These responses support the roles of the housekeeping pathways in both the degradation of damaged proteins induced by salt stress and regulation of proteins involved in the molecular recovery from salt stress. Since serine proteases share several biochemical features and physiological functions, the results from this study provide an insight into diversification of serine proteases in cyanobacteria. Further, these results will increase our understanding of several mechanisms at the subcellular level.

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.

Antioxidative and Antiglycative Properties of Mycosporine-Like Amino Acids-Containing Aqueous Extracts Derived from Edible Terrestrial Cyanobacteria.

The terrestrial filamentous cyanobacterium, Nostoc commune, has been used as a food source in many countries, especially countries in Asia. In this study, N. commune-derived aqueous extracts were evaluated with regard to their antioxidative and antiglycative properties. The antioxidative activity was significantly higher in N. commune colonies isolated from the field than in extracts from colonies cultured in the laboratory. The antioxidative compound content of extracts, including phenolic compounds and phycobiliproteins, was correlated with their antioxidative power. In addition, two mycosporine-like amino acids (MAAs), specifically detected in colonies isolated from the field, were purified. In addition to assessing their antioxidative properties, the antiglycative activity of these MAAs was also assessed. Their inhibitory effects on glycation-dependent protein cross-linking might contribute to the antiglycative power of the extract prepared from field colonies. Taken together, the results from this study revealed that N. commune may have beneficial properties for functional food applications, both by preventing oxidative stress and suppressing the formation of advanced glycation end-products.

The Effects of Salts and Osmoprotectants on Enzyme Activities of Fructose-1,6-biphosphate Aldolases in a Halotolerant Cyanobacterium, Halothece sp. PCC 7418.

The halotolerant cyanobacterium, Halothece sp. PCC 7418, possesses two classes of fructose-1,6-bisphosphate aldolase (FBA): H2846 and H2847. Though class I (CI)-FBA H2846 is thought to be associated with salt tolerance, the regulatory mechanisms, molecular characteristics, and expression profiles between H2846 and class II (CII)-FBA H2847 have scarcely been investigated. Here, we show that the accumulation of the H2846 protein is highly responsive to both up- and down-shock with NaCl, whereas H2847 is constitutively expressed. The activity of CI- and CII-FBA in cyanobacterial extracts is correlated with the accumulation patterns of H2846 and H2847, respectively. In addition, it was found that these activities were inhibited by NaCl and KCl, with CII-FBA activity strikingly inhibited. It was also found that the CI-FBA activity of recombinant H2846 was hindered by salts and that this hindrance could be moderated by the addition of glycine betaine (GB), whereas no moderation occurred with other potential osmoprotectant molecules (proline, sucrose, and glycerol). In addition, a phylogenetic analysis showed that CI-FBAs with higher similarities to H2846 tended to be distributed among potential GB-synthesizing cyanobacteria. Taken together, our results provide insights into the independent evolution of the CI- and CII-FBA gene families, which show distinct expression profiles and functions following salt stress.

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.