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Rhodophyta (red algae) comprises over 6000 species, however, there have only been a few comparative transcriptomic studies due to their under-representation in genomic databases. Kappaphycus alvarezii, a Gigartinales algae, is a valuable source of carrageenan and is extensively cultivated in many countries. The majority of seaweed farming in Southeast Asia is done in intertidal zones under varying light (i.e., spectra and irradiance) and carbon dioxide (CO2) conditions, which affects the rate of photosynthesis. This study conducted transcriptome profiling to investigate the photosynthetic mechanisms in K. alvarezii exposed to different wavelengths of light (i.e., blue, green, and red light, in comparison to white light) and CO2 availability. We analyzed the responses of photosynthetic protein complexes to light and observed that light of different wavelengths regulates a similar set of photosynthetic apparatuses. Under CO2 enrichment, genes encoding C3 and C4 enzymes were found to be actively transcribed, suggesting the likely shift in the carbon metabolism pathway or the involvement of these genes in adaptive physiological processes. This study contributes to the understanding of the regulatory mechanisms of photosynthetic carbon metabolism in red algae and has implications for the culture and commercial production of these economically valuable macroalgae.

SIB1 FKBP22 is a peptidyl prolyl cis-trans isomerase (PPIase) member from a psychrotrophic bacterium, Shewanella sp. SIB1, consisting of N- and C-domains responsible for dimerization and catalytic PPIase activity, respectively. This protein was assumed to be involved in cold adaptation of SIB1 cells through its dual activity of PPIase activity and chaperone like-function. Nevertheless, the catalytic inhibition by FK506 and its substrate specificity remain unknown. Besides, ability of SIB1 FKBP22 to inhibit phosphatase activity of calcinuerin is also interesting to be studied since it may reflect wider cellular functions of SIB1 FKBP22. In this study, we found that wild type (WT) SIB1 FKBP22 bound to FK506 with IC50 of 77.55 nM. This value is comparable to that of monomeric mutants (NNC-FKBP22, C-domain+ and V37R/L41R mutants), yet significantly higher than that of active site mutant (R142A). In addition, WT SIB1 FKBP22 and monomeric variants were found to prefer hydrophobic residues preceding proline. Meanwhile, R142A mutant has wider preferences on bulkier hydrophobic residues due to increasing hydrophobicity and binding pocket space. Surprisingly, in the absence of FK506, SIB1 FKBP22 and its variants inhibited, with the exception of N-domain, calcineurin phosphatase activity, albeit low. The inhibition of SIB1 FKBP22 by FK506 is dramatically increased in the presence of FK506. Altogether, we proposed that local structure at substrate binding pocket of C-domain plays crucial role for the binding of FK506 and peptide substrate preferences. In addition, C-domain is essential for inhibition, while dimerization state is important for optimum inhibition through efficient binding to calcineurin.

The adaptive process in bacteria is driven by specific genetic elements which regulate phenotypic characteristics such as tolerance to high metal ion concentrations and the secretion of protective biofilms. Extreme environments such as those associated with heavy metal pollution and extremes of acidity offer opportunities to study the adaptive mechanisms of microorganisms. This study focused on the genome analysis of Bacillus thuringiensis (Bt MCMY1), a gram positive rod shaped bacterium isolated from an acid mine drainage site in Sabah, Malaysia by using a combination of Single Molecule Real Time DNA Sequencing, Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). The genome size of Bt MCMY1 was determined to be 5,458,152 bases which was encoded on a single chromosome. Analysis of the genome revealed genes associated with resistance to Copper, Mercury, Arsenic, Cobalt, Zinc, Cadmium and Aluminum. Evidence from SEM and FTIR indicated that the bacterial colonies form distinct films which bear the signature of polyhydroxyalkanoates (PHA) and this finding was supported by the genome data indicating the presence of a genetic pathway associated with the biosynthesis of PHAs. This is the first report of a Bacillus sp. isolated from an acid mine drainage site in Sabah, Malaysia and the genome sequence will provide insights into the manner in which B. thuringiensis adapts to acid mine drainage.

The chemical mutagen ethyl methanesulfonate (EMS) has been generally utilised for the development of novel variants of commercially cultivated crop plants. The general approach involves the application of the mutagen at sub-lethal concentrations, followed by the selection of novel phenotypes based on morphometric characterization. This study was directed towards the evaluation of the effects of EMS at a concentration of 0.5 % on Solanum lycopersicum. Mutants were selected based on phenotypic variation, following which nine specific genes such as NRP2 (RNA polymerase II), HKX (hexokinase), P2D1 (photosystem II D1), NEXP (expansin), PHYSN (phytoene synthase), PCANH (carbonic anhydrase), NAGO (argonaute), PRUB (RuBisCO) and NGAG (gag-pol) were isolated via polymerase chain reaction and sequenced to determine the presence of mutations. The results demonstrated that six genes: NRP2, HKX, P2D1, NAGO, NGAG, and NEXP contained mutations that altered the sequence of translated amino acid. In addition to this, a wide range of phenotypic variants was observed. This study concluded that the selected genes were under selection pressure and that undefined physiological processes may be involved in maintaining genetic stability during exposure to chemical mutagens.

The division Rhodophyta includes members from the commercially important genera Kappaphycus and Eucheuma. The efficient cultivation of seaweeds depends upon the development of genomic molecular markers which can be applied to detect varietal differences and select strains which exhibit ideal growth characteristics. We developed 117 single loci DNA markers from shotgun genomic libraries derived from Kappaphycus alvarezii, Kappaphycus striatus and Eucheuma denticulatum and tested them across twelve phenotypes which are commercially cultured in Sabah, Malaysia. Sixteen of the markers amplified across all varieties of Kappaphycus implying a shared genetic identity. Eight of the markers could successfully differentiate between varieties of Eucheuma and Kappaphycus on the basis of DNA fingerprinting profiles. These markers will serve as resource for the identification and selection of red algae for commercial cultivation and their application can be extended to marker assisted selection (MAS) and hybrid development.

Environmental pH is one of the factors contributing to abiotic stress which in turn influences the growth and development of macroalgae. This study was conducted in order to assess the growth and physiological changes in Kappaphycus alvarezii under different pH conditions: pHs 6, ∼8.4 (control) and 9. K. alvarezii explants exhibited a difference in the daily growth rate (DGR) among the different pH treatments (p≤0.05). The highest DGR was observed in control culture with pH ∼8.4 followed by alkaline (pH 9) and acidic (pH 6) induced stress cultures. Protein expression profile was generated from different pH induced K. alvarezii cultures using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by protein identification and analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) and Mascot software. Ribulose bisphosphate carboxylase (Rubisco) large chain was identified to be up-regulated under acidic (pH 6) condition during the second and fourth week of culture. The findings indicated that Rubisco can be employed as a biomarker for pH induced abiotic stress. Further study on the association between the expression levels of Rubisco large chain and their underlying mechanisms under pH stress conditions is recommended.

A novel electrochemical glucose biosensor was developed by depositing an ionic liquid (IL) (e.g., 1-ethyl-3-methylimidazolium trifluoromethanesulfonate; [EMIM][Otf]), ZnO nanoparticles (ZnONPs) and eggshell membrane (ESM) on a modified glassy carbon electrode (GCE) for determination of glucose. Glucose oxidase (GOx) was covalently immobilized on eggshell membrane with glutaraldehyde as a cross-linker. Methylene blue was used as a redox indicator to enhance the electron transfer capacity and to ensure stability of both the oxidized and reduced forms in the reaction of enzyme and substrate. The morphological characteristics of microstructures eggshell membranes, chitosan, GOx/ESM, GOx/ZnONPs/IL/ESM and GOx/ZnONPs-IL/CHIT were observed using scanning electron microscopy (SEM). The effects of scan rate, time and pH on the response of glucose biosensors were studied in detail. Under optimal conditions (pH 6.5, 50 s), cyclic voltammetry showed different glucose concentrations on the range of 1 × 10−12 to 0.6 M, with a detection limit of 1 × 10−13 M. The GOx/ZnONPs/IL/ESM was found to be more sensitive as compared to GOx/ZnONPs-IL/CHIT. This developed glucose biosensor detection approach has several advantages such as fast, simple and convenient method, sensitivity, low cost, eco-friendly, low concentrations and remarkable catalytic activities of current signals during glucose reaction.

Jatropha curcas is an important plant belonging to the family Euphorbiaceae which is a potential candidate for biofuel production. Genetic transformation protocol for J. curcas callus mediated by Agrobacterium tumefaciens were optimized using a pCAMBIA1303 plasmid which carries green fluorescent protein (GFP) gene as a reporter. Results obtained were based on the highest percentage of GFP expression which was observed three days post-transformation. Immersion of callus into 1×105 cfu ml-1 (OD600nm 0.6) of A. tumefaciens LBA4404 with addition of 300 µM of acetosyringone for 45 min, two days of pre-culture and three days of co-cultivation periods were determined to be ideal for J. curcas callus transformation. Putative transformants were selected in the presence of 25 mg/l hygromycin. Surviving calli were transferred into proliferation media (MS with 1 mg/l NAA and 1 mg/l BAP) to proliferate the callus for further molecular analyses and to confirm the presence of the target GFP transgene in the putative transformants. Polymerase chain reaction (PCR) was carried out using a 35S specific primer pair confirmed the presence of the 454 bp of 35S promoter region from the transformed callus. Quantitative real-time PCR (qRT-PCR) was carried out to demonstrate the integration and copy number of the 35S promoter in the putative tranformants. The 35S promoter gene (178 bp) as a target gene and J. curcas actin gene (179 bp) which functions as reference gene was designed to detect the positive transformants and control sample in real-time PCR reaction analysis. The results indicated that the actin specific PCR product was present in both the control and transformed calli, however the 35S PCR product was found only in the positive transformants. The similarity in CT values confirmed that both the genes were present as single copy thus confirming a single integration event.

Living organisms have some common and unique strategies to response to thermal stress. However, the amount of data on thermal stress response of certain organism is still lacking, especially psychrophilic yeast from the extreme habitat. Therefore, it is not known whether psychrophilic yeast shares the common responses of other organisms when exposed to thermal stresses. In this work, the cold shock and heat shock responses in Antarctic psychrophilic yeast Glaciozyma antarctica PI12 which had an optimal growth temperature of 12 °C were determined. The expression levels of 14 thermal stress-related genes were measured using real-time quantitative PCR (qPCR) when the yeast cells were exposed to cold shock (0 °C), mild cold shock (5 °C), and heat shock (22 °C) conditions. The expression profiles of the 14 genes at these three temperatures varied indicating that these genes had their specific roles to ensure the survival of the yeast. Under cold shock condition, the afp4 and fad genes were over-expressed possibly as a way for the G. antarctica PI12 to avoid ice crystallization in the cell and to maintain the membrane fluidity. Under the heat shock condition, hsp70 was significantly up-regulated possibly to ensure the proteins fold properly. Among the six oxidative stress-related genes, MnSOD and prx were up-regulated under cold shock and heat shock, respectively, possibly to reduce the negative effects caused by oxidative stress. Interestingly, it was found that the trehalase gene, nth1 that plays a role in degrading excess trehalose, was down-regulated under the heat shock condition possibly as an alternative way to accumulate trehalose in the cells to protecting them from being damaged.

Kappa-carrageenan (K-carrageenan) is an important phycocolloid which is a major constituent of the cell wall of Kappaphycus alvarezii. The chemical structure of K-carrageenan comprises a linear backbone of D-galactose residues linked with alternating α-(1,3) and β-(1,4) linkages which are substituted by one ester-sulphonic group per di-galactose repeating unit. The spectral qualities of light as well as the ambient carbon dioxide concentration, both play an important role in the photosynthetic pathway in plants and this investigation set forth to establish the effect of different wavelengths of light and carbon dioxide supplementation on the chemical structure of K-carrageenan obtained from K. alvarezii. Specimens were cultivated under a range of monochromatic light spectra and assessed for chemical composition using Fourier Transform Infrared (FTIR) spectroscopy.The K. alvarezii control was irradiated with full light spectrum, treatments were carried out using blue (492-455 nm), green (577-492 nm) and red (780-622 nm)light. One experiment was carried out by supplementation with carbon dioxide. Samples were collected after 14 days. The effect of different wavelengths of light on the growth rates of experimental samples was determined. Red light had the most significant impact on the growth rate of K. alvarezii as compared to those treated with blue light. The FTIR fingerprint of the ground seaweed was found to be identical to that of commercial K-carrageenan (Sigma). Special emphasis was given to the 800-1300 cm-1 region, which presents several vibrational modes. All the samples produced similar FTIR spectral profiles, suggesting that genes related to the carrageenan biosynthesis are not affected by different wavelengths of light or CO2. The results obtained from FTIR spectroscopy demonstrated that different wavelengths of light and supplementation with CO2 have no influence to the chemical structure of K-carrageenan in K. alvarezii.

The genus Trichoderma is a soil-borne fungi which in numerous reports has been successfully used as a biological control agent against various plant pathogens. The identification of Trichoderma species worldwide is currently deduced from micro-morphological descriptions which are tedious and prone to error. Electrochemical approaches are currently being developed for the detection and analysis of DNA. In the present study, an electrochemical DNA biosensor was successfully developed based on ionic liquid (e.g., 1-ethyl-3-methylimidazolium trifluoromethanesulfonate ([EMIM][Otf])), ZnO nanoparticles and a chitosan (CHIT) nanocomposite membrane on a modified gold electrode (AuE). A single-stranded DNA probe was immobilized on this electrode. Methylene blue (MB) was used as the hybridization indicator to monitor the hybridization reaction of the target DNA. Under optimal conditions using differential pulse voltammetry (DPV), the target DNA sequences were detectable at concentration ranges of 1.0 A— 10−18–1.82 A— 10−4 mol L−1, and the detectable limit was 1.0 A— 10−19 mol L−1. The developed DNA biosensor enables the study of hybridization with crude DNA fragments and the results of this study confirm that this DNA biosensor provides a fast, sensitive and convenient way for the species level identification of Trichoderma harzianum.