scholarly journals Identification and Upregulation of Biosynthetic Genes Required for Accumulation of Mycosporine-2-Glycine under Salt Stress Conditions in the Halotolerant Cyanobacterium Aphanothece halophytica

2013 ◽  
Vol 80 (5) ◽  
pp. 1763-1769 ◽  
Author(s):  
Rungaroon Waditee-Sirisattha ◽  
Hakuto Kageyama ◽  
Warangkana Sopun ◽  
Yoshito Tanaka ◽  
Teruhiro Takabe
Keyword(s):  
Salt Stress ◽  
High Salinity ◽  
Dry Weight ◽  
Transcript Level ◽  
Aphanothece Halophytica ◽  
Biosynthetic Genes ◽  
Content Type ◽  
Native Promoter ◽  
Halotolerant Cyanobacterium

ABSTRACTMycosporine-like amino acids (MAAs) are valuable molecules that are the basis for important photoprotective constituents. Here we report molecular analysis of mycosporine-like amino acid biosynthetic genes from the halotolerant cyanobacteriumAphanothece halophytica, which can survive at high salinity and alkaline pH. This extremophile was found to have a unique MAA core (4-deoxygadusol)-synthesizing gene separated from three other genes.In vivoanalysis showed accumulation of the mycosporine-2-glycine but not shinorine or mycosporine-glycine. Mycosporine-2-glycine accumulation was stimulated more under the stress condition of high salinity than UV-B radiation. TheAphanotheceMAA biosynthetic genes also manifested a strong transcript level response to salt stress. Furthermore, the transformedEscherichia coliandSynechococcusstrains expressing four putativeAphanotheceMAA genes under the control of a native promoter were found to be capable of synthesizing mycosporine-2-glycine. The accumulation level of mycosporine-2-glycine was again higher under the high-salinity condition. In the transformedE. colicells, its level was approximately 85.2 ± 0.7 μmol/g (dry weight). Successful production of a large amount of mycosporine in these cells provides a new opportunity in the search for an alternative natural sunscreen compound source.

scholarly journals An Alkaline Phosphatase/Phosphodiesterase, PhoD, Induced by Salt Stress and Secreted Out of the Cells of Aphanothece halophytica, a Halotolerant Cyanobacterium

2011 ◽  
Vol 77 (15) ◽  
pp. 5178-5183 ◽  
Author(s):  
Hakuto Kageyama ◽  
Keshawanand Tripathi ◽  
Ashwani K. Rai ◽  
Suriyan Cha-um ◽  
Rungaroon Waditee-Sirisattha ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Salt Stress ◽  
Functional Characterization ◽  
Gene Families ◽  
Aphanothece Halophytica ◽  
Alkaline Phosphatases ◽  
Content Type ◽  
N Terminus ◽  
Consensus Motifs ◽  
Halotolerant Cyanobacterium

ABSTRACTAlkaline phosphatases (APases) are important enzymes in organophosphate utilization. Three prokaryotic APase gene families, PhoA, PhoX, and PhoD, are known; however, their functional characterization in cyanobacteria largely remains to be clarified. In this study, we cloned thephoDgene from a halotolerant cyanobacterium,Aphanothece halophytica(phoDAp). The deduced protein, PhoDAp, contains Tat consensus motifs and a peptidase cleavage site at the N terminus. The PhoDApenzyme was activated by Ca2+and exhibited APase and phosphodiesterase (APDase) activities. Subcellular localization experiments revealed the secretion and processing of PhoDApin a transformed cyanobacterium. Expression of thephoDApgene inA. halophyticacells was upregulated not only by phosphorus (P) starvation but also under salt stress conditions. Our results suggest thatA. halophyticacells possess a PhoD that participates in the assimilation of P under salinity stress.

scholarly journals High-Level Production of the Industrial Product Lycopene by the Photosynthetic Bacterium Rhodospirillum rubrum

2012 ◽  
Vol 78 (20) ◽  
pp. 7205-7215 ◽  
Author(s):  
Guo-Shu Wang ◽  
Hartmut Grammel ◽  
Khaled Abou-Aisha ◽  
Rudolf Sägesser ◽  
Robin Ghosh
Keyword(s):  
Rhodospirillum Rubrum ◽  
Purple Bacteria ◽  
Dry Weight ◽  
Photosynthetic Bacterium ◽  
Downstream Processing ◽  
Kanamycin Resistance ◽  
Content Type ◽  
Light Harvesting Complex ◽  
Kanamycin Resistance Cassette

ABSTRACTThe biosynthesis of the major carotenoid spirilloxanthin by the purple nonsulfur bacteriumRhodospirillum rubrumis thought to occur via a linear pathway proceeding through phytoene and, later, lycopene as intermediates. This assumption is based solely on early chemical evidence (B. H. Davies, Biochem. J. 116:93–99, 1970). In most purple bacteria, the desaturation of phytoene, catalyzed by the enzyme phytoene desaturase (CrtI), leads to neurosporene, involving only three dehydrogenation steps and not four as in the case of lycopene. We show here that the chromosomal insertion of a kanamycin resistance cassette into thecrtC-crtDregion of the partial carotenoid gene cluster, whose gene products are responsible for the downstream processing of lycopene, leads to the accumulation of the latter as the major carotenoid. We provide spectroscopic and biochemical evidence thatin vivo, lycopene is incorporated into the light-harvesting complex 1 as efficiently as the methoxylated carotenoids spirilloxanthin (in the wild type) and 3,4,3′,4′-tetrahydrospirilloxanthin (in acrtDmutant), both under semiaerobic, chemoheterotrophic, and photosynthetic, anaerobic conditions. Quantitative growth experiments conducted in dark, semiaerobic conditions, using a growth medium for high cell density and high intracellular membrane levels, which are suitable for the conventional industrial production in the absence of light, yielded lycopene at up to 2 mg/g (dry weight) of cells or up to 15 mg/liter of culture. These values are comparable to those of many previously describedEscherichia colistrains engineered for lycopene production. This study provides the first genetic proof that theR. rubrumCrtI produces lycopene exclusively as an end product.

scholarly journals Functional characterization of multiple PAS domain-containing diguanylate cyclases in Synechocystis sp. PCC 6803

Microbiology ◽  
2020 ◽  
Vol 166 (7) ◽  
pp. 659-668
Author(s):  
Ko Ishikawa ◽  
Chihiro Chubachi ◽  
Saeko Tochigi ◽  
Naomi Hoshi ◽  
Seiji Kojima ◽  
...  
Keyword(s):  
Salt Stress ◽  
Catalytic Domain ◽  
Functional Characterization ◽  
Active Form ◽  
Guanosine Monophosphate ◽  
Content Type ◽  
Link Type ◽  
Pcc 6803

Bis-(3′–5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) is a second messenger known to control a variety of bacterial processes. The model cyanobacterium, Synechocystis sp. PCC 6803, has a score of genes encoding putative enzymes for c-di-GMP synthesis and degradation. However, most of them have not been functionally characterized. Here, we chose four genes in Synechocystis (dgcA–dgcD), which encode proteins with a GGDEF, diguanylate cyclase (DGC) catalytic domain and multiple Per-ARNT-Sim (PAS) conserved regulatory motifs, for detailed analysis. Purified DgcA, DgcB and DgcC were able to catalyze synthesis of c-di-GMP from two GTPs in vitro. DgcA had the highest activity, compared with DgcB and DgcC. DgcD did not show detectable activity. DgcA activity was specific for GTP and stimulated by the divalent cations, magnesium or manganese. Full activity of DgcA required the presence of the multiple PAS domains, probably because of their role in protein dimerization or stability. Synechocystis mutants carrying single deletions of dgcA–dgcD were not affected in their growth rate or biofilm production during salt stress, suggesting that there was functional redundancy in vivo. In contrast, overexpression of dgcA resulted in increased biofilm formation in the absence of salt stress. In this study, we characterize the enzymatic and physiological function of DgcA–DgcD, and propose that the PAS domains in DgcA function in maintaining the enzyme in its active form.

Regulation of Glycinebetaine Accumulation in the Halotolerant Cyanobacterium Aphanothece halophytica

1993 ◽  
Vol 20 (6) ◽  
pp. 693 ◽  
Author(s):  
M Ishitani ◽  
T Takabe ◽  
K Kojima ◽  
T Takabe
Keyword(s):  
Salt Stress ◽  
Co2 Fixation ◽  
Photosynthetic Activity ◽  
Higher Plants ◽  
Control Culture ◽  
Ultrastructural Changes ◽  
Intracellular Level ◽  
Aphanothece Halophytica ◽  
Order Of Magnitude ◽  
Halotolerant Cyanobacterium

When cells of the halotolerant cyanobacterium, Aphanothece halophytica were transferred from control culture medium that contained 0.5 M NaCl to a hypersaline medium with either 1.5 M or 2.0 M NaCl, the rate of photosynthetic CO2 fixation fell instantaneously. Subsequently, the photosynthetic activity returned to almost the original level within 1 day. Under salt stress, ultrastructural changes in the cells were observed by electron microscopy, these cells appeared to recover in parallel with the recovery of their photosynthetic activity. However, the intracellular level of glycinebetaine increased more slowly than the recovery of the photosynthetic CO2 fixation. The maximum rate of accumulation of betaine was estimated to be approximately 60 nmol (mg protein)-1 h-1. This rate is at least one order of magnitude greater than rates reported previously in leaves of salt-stressed higher plants. The accumulation of betaine did not take place in the dark. The intracellular level of betaine decreased as a result of the transfer of the cells to a hypo-osmotic NaCl-containing medium. The accumulation of betaine was also induced by an organic osmoticum, sorbitol. Nitrate enhances the accumulation of betaine under salt stress.

scholarly journals Salt Stress Response of Sulfolobus acidocaldarius Involves Complex Trehalose Metabolism Utilizing a Novel Trehalose-6-Phosphate Synthase (TPS)/Trehalose-6-Phosphate Phosphatase (TPP) Pathway

2020 ◽  
Vol 86 (24) ◽  
Author(s):  
Christina Stracke ◽  
Benjamin H. Meyer ◽  
Anna Hagemann ◽  
Eunhye Jo ◽  
Areum Lee ◽  
...  
Keyword(s):  
Salt Stress ◽  
Stress Response ◽  
Extreme Environments ◽  
Compatible Solutes ◽  
Sulfolobus Acidocaldarius ◽  
Triple Mutant ◽  
Salt Stress Response ◽  
Content Type ◽  
Trehalose Metabolism

ABSTRACT The crenarchaeon Sulfolobus acidocaldarius has been described to synthesize trehalose via the maltooligosyltrehalose synthase (TreY) and maltooligosyltrehalose trehalohydrolase (TreZ) pathway, and the trehalose glycosyltransferring synthase (TreT) pathway has been predicted. Deletion mutant analysis of strains with single and double deletions of ΔtreY and ΔtreT in S. acidocaldarius revealed that in addition to these two pathways, a third, novel trehalose biosynthesis pathway is operative in vivo: the trehalose-6-phosphate (T6P) synthase/T6P phosphatase (TPS/TPP) pathway. In contrast to known TPS proteins, which belong to the GT20 family, the S. acidocaldarius TPS belongs to the GT4 family, establishing a new function within this group of enzymes. This novel GT4-like TPS was found to be present mainly in the Sulfolobales. The ΔtreY ΔtreT Δtps triple mutant of S. acidocaldarius, which lacks the ability to synthesize trehalose, showed no altered phenotype under standard conditions or heat stress but was unable to grow under salt stress. Accordingly, in the wild-type strain, a significant increase of intracellular trehalose formation was observed under salt stress. Quantitative real-time PCR showed a salt stress-mediated induction of all three trehalose-synthesizing pathways. This demonstrates that in Archaea, trehalose plays an essential role for growth under high-salt conditions. IMPORTANCE The metabolism and function of trehalose as a compatible solute in Archaea was not well understood. This combined genetic and enzymatic approach at the interface of microbiology, physiology, and microbial ecology gives important insights into survival under stress, adaptation to extreme environments, and the role of compatible solutes in Archaea. Here, we unraveled the complexity of trehalose metabolism, and we present a comprehensive study on trehalose function in stress response in S. acidocaldarius. This sheds light on the general microbiology and the fascinating metabolic repertoire of Archaea, involving many novel biocatalysts, such as glycosyltransferases, with great potential in biotechnology.

Growth and flowering in salt-stressed avocado trees

1978 ◽  
Vol 29 (3) ◽  
pp. 523 ◽  
Author(s):  
WJS Downton
Keyword(s):  
Salt Stress ◽  
Sodium Chloride ◽  
Calcium Ion ◽  
High Salinity ◽  
Dry Weight ◽  
Chloride Salt ◽  
Ion Composition ◽  
Low Salinity ◽  
Nitrogen Concentrations ◽  
Stems And Leaves

The effect of salinity (0, 5, 10 and 2 0 mM sodium chloride) on the growth, ion composition and flowering response of Fuerte scion grafted to rootstocks of Mexican, Guatemalan and Mexican × Guatemalan (Zutano) race is described. Scions on the Mexican and Zutano stocks were less tolerant of salinity than scions on the Guatemalan stock based on measurements of stem diameter and biomass. The less tolerant trees also contained higher concentrations of sodium and chloride in leaves. The entry of sodium into scions on the Mexican and Zutano stocks was associated with increased succulence of stems and leaves. This response altered the mineral composition of the leaves. Leaf potassium and nitrogen per unit of dry weight increased on the Mexican stock given 20 mM sodium chloride. By taking account of the changes in succulence, however, it could be demonstrated that potassium and nitrogen concentrations on a leaf water basis remained close to control values. Calcium ion failed to make the adjustment, and the already low calcium levels were further reduced by succulence. Succulence was responsible for a 25–37% dilution of sodium and chloride in the leaves of scions on Zutano and Mexican stocks, given 2 0 mM sodium chloride. Salt stress promoted the flowering of Fuerte scion. Low salinity (5mM sodium chloride) failed to stimulate flowering in trees on the Guatemalan stock. High salinity (20mM sodium chloride) was detrimental to flowering in scions on the Mexican stock and resulted in reduced numbers of floral buds, flowers per bud, inflorescences per bud and flowers per inflorescence. The experimental results are discussed in relation to the salinity situation in the irrigated horticultural areas along the River Murray.

Comparative Study on Photosynthetic Characteristics of Two Ecotypes of Euhalophyte Suaeda salsa L. Grown Under Natural Saline Conditions

2013 ◽  
Vol 726-731 ◽  
pp. 4488-4493
Author(s):  
Na Sui ◽  
Yu Liu ◽  
Bao Shan Wang
Keyword(s):  
Salt Stress ◽  
Intertidal Zone ◽  
High Salinity ◽  
Yellow River ◽  
Yellow River Delta ◽  
Dry Weight ◽  
Suaeda Salsa ◽  
The Yellow River ◽  
Fresh Weight ◽  
The Yellow River Delta

Suaeda salsa L. grown in the intertidal zone and those in high salinity soils of the Yellow River Delta were used to investigate the category and characteristic of photosynthesis and fluorescence. Results showed that the water content, Na+ and Cl- contents of the high salinity soils were lower. The temperature on the surface of soil and in the depth of 10 cm from the surface, the content of K+ and Ca2+ of the high salinity soils were significantly higher than those in the intertidal zone soils. Pn, Gs, Fv/Fm, ФPSII, the fresh weight and dry weight per plant of S. salsa grown in the high salinity soils were higher. However, Ci of S. salsa grown in the high salinity soils were lower. These suggested that S. salsa grown in the high salinity soils was mainly suffering from salt stress, while S. salsa in the intertidal zone soils was suffering from waterlogging, low temperature and salt stress together. S. salsa in the intertidal zone soils decreased light absorption and alleviated photoinhibition, but as a result the biomass was reduced.

scholarly journals Two CRISPR/Cas9 Systems Developed in Thermomyces dupontii and Characterization of Key Gene Functions in Thermolide Biosynthesis and Fungal Adaptation

2020 ◽  
Vol 86 (20) ◽  
Author(s):  
Wei-Ping Huang ◽  
Yuan-Jiang Du ◽  
Yun Yang ◽  
Jia-Ning He ◽  
Qian Lei ◽  
...  
Keyword(s):  
Gene Editing ◽  
Thermophilic Bacteria ◽  
Thermophilic Fungi ◽  
Chemical Stress ◽  
Biosynthetic Gene ◽  
Biosynthetic Genes ◽  
Content Type ◽  
Fungal Adaptation ◽  
Thermomyces Dupontii

ABSTRACT Thermomyces dupontii, a widely distributed thermophilic fungus, is an ideal organism for investigating the mechanism of thermophilic fungal adaptation to diverse environments. However, genetic analysis of this fungus is hindered by a lack of available and efficient gene-manipulating tools. In this study, two different Cas9 proteins from mesophilic and thermophilic bacteria, with in vivo expression of a single guide RNA (sgRNA) under the control of tRNAGly, were successfully adapted for genome editing in T. dupontii. We demonstrated the feasibility of applying these two gene editing systems to edit one or two genes in T. dupontii. The mesophilic CRISPR/Cas9 system displayed higher editing efficiency (50 to 86%) than the thermophilic CRISPR/Cas9 system (40 to 67%). However, the thermophilic CRISPR/Cas9 system was much less time-consuming than the mesophilic CRISPR/Cas9 system. Combining the CRISPR/Cas9 systems with homologous recombination, a constitutive promoter was precisely knocked in to activate a silent polyketide synthase-nonribosomal peptide synthase (PKS-NRPS) biosynthetic gene, leading to the production of extra metabolites that did not exist in the parental strains. Metabolic analysis of the generated biosynthetic gene mutants suggested that a key biosynthetic pathway existed for the biosynthesis of thermolides in T. dupontii, with the last two steps being different from those in the heterologous host Aspergillus. Further analysis suggested that these biosynthetic genes might be involved in fungal mycelial growth, conidiation, and spore germination, as well as in fungal adaptation to osmotic, oxidative, and cell wall-perturbing agents. IMPORTANCE Thermomyces represents a unique ecological taxon in fungi, but a lack of flexible genetic tools has greatly hampered the study of gene function in this taxon. The biosynthesis of potent nematicidal thermolides in T. dupontii remains largely unknown. In this study, mesophilic and thermophilic CRISPR/Cas9 gene editing systems were successfully established for both disrupting and activating genes in T. dupontii. In this study, a usable thermophilic CRISPR/Cas9 gene editing system derived from bacteria was constructed in thermophilic fungi. Chemical analysis of the mutants generated by these two gene editing systems identified the key biosynthetic genes and pathway for the biosynthesis of nematocidal thermolides in T. dupontii. Phenotype analysis and chemical stress experiments revealed potential roles of secondary metabolites or their biosynthetic genes in fungal development and adaption to chemical stress conditions. These two genomic editing systems will not only accelerate investigations into the biosynthetic mechanisms of unique natural products and functions of cryptic genes in T. dupontii but also offer an example for setting up CRISPR/Cas9 systems in other thermophilic fungi.

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