Histone mRNA is subject to 3’ uridylation and re-adenylation in Aspergillus nidulans

The role of post‐transcriptional RNA modification is of growing interest. One example is the addition of non‐templated uridine residues to the 3′ end of transcripts. In mammalian systems, uridylation is integral to cell cycle control of histone mRNA levels. This regulatory mechanism is dependent on the nonsense‐mediated decay (NMD) component, Upf1, which promotes histone mRNA uridylation and degradation in response to the arrest of DNA synthesis. We have identified a similar system in Aspergillus nidulans, where Upf1 is required for the regulation of histone mRNA levels. However, other NMD components are also implicated, distinguishing it from the mammalian system. As in human cells, 3′ uridylation of histone mRNA is induced upon replication arrest. Disruption of this 3′ tagging has a significant but limited effect on histone transcript regulation, consistent with multiple mechanisms acting to regulate mRNA levels. Interestingly, 3′ end degraded transcripts are also subject to re‐adenylation. Both mRNA pyrimidine tagging and re‐adenylation are dependent on the same terminal‐nucleotidyltransferases, CutA, and CutB, and we show this is consistent with the in vitro activities of both enzymes. Based on these data we argue that mRNA 3′ tagging has diverse and distinct roles associated with transcript degradation, functionality and regulation.publishe

Pengenalpastian bakteria tanah yang mempunyai aktiviti antikulat terhadap patogen kelapa sawit, Ganoderma boninense

Penyakit reput pangkal batang (BSR) yang berpunca daripada kulat fitopatogen Ganoderma boninense merupakan ancaman terbesar kepada tanaman kelapa sawit di Malaysia. BSR menyebabkan pengurangan hasil tandan sawit dan kematian terhadap pokok kelapa sawit terjangkit, seterusnya menyebabkan kerugian yang besar kepada ekonomi negara pengeluar. Penggunaan racun kimia juga telah diaplikasikan bagi menangani BSR, namun begitu, kaedah ini tidak mampu mengatasi serangan kulat ini. Beberapa kajian telah dilakukan untuk mengenal pasti potensi penggunaan agen kawalan biologi seperti mikroorganisma antagonis dalam mengawal penyakit BSR. Kajian ini bertujuan untuk mengenal pasti mikroorganisma pencilan tempatan yang mempunyai kesan antagonistik terhadap G. boninense. Sebanyak 49 kultur bakteria tulen telah dipencilkan dari kawasan sekitar Bangi, Selangor, Malaysia. Tiga pencilan iaitu masing-masing dua strain daripada genus Burkholderia dan satu daripada Streptomyces menunjukkan aktiviti antagonistik yang kuat terhadap miselia dwikarion G. boninense strain PER71 dengan nilai Peratusan Perencatan Pertumbuhan Radius (PIRG) melebihi 70% dalam dua asai antikulat yang berbeza. Analisis jujukan DNA bagi semua pencilan telah menunjukkan spesies bakteria tersebut mempunyai kesamaan yang tinggi dengan jujukan nukleotida 16S rRNA daripada Burkholderia stagnalis, B. cepacia dan Streptomyces gelaticus. Bakteria yang digunakan di dalam kajian ini mempunyai potensi yang tinggi untuk digunakan sebagai agen kawalan biologi dalam menghalang penyebaran G. boninense terutamanya di dalam ladang kelapa sawit di Malaysia

Evaluation and improvement of protocols for Ganoderma boninense protoplast isolation and regeneration

Ganoderma boninense is the causal agent of basal stem rot (BSR) disease of oil palm. The BSR disease reduces oil palm yield by up to 80% of the average oil yield. Attempts to control the disease caused by this fungus in the field showed varying levels of success and cases of infection increased from year to year. Hence, the development of new efficient methods to control the spread of this fungus should be commenced promptly. To ensure a better strategy is created, more thorough research on the method deploy by this fungus to infect the host at the molecular level need to be carried out first. However, the major limitation in endeavoring into the functional analysis of virulence genes related to the pathogenicity of this fungus was hampered by the unavailability of established methods for protoplast isolation with a high regeneration rate to be used in the genetic manipulation analysis. Thus, in this paper, we report an efficient protocol for protoplast isolation and regeneration in G. boninense and successfully used the isolated protoplasts in PEG-mediated transformation analysis. A large quantity of protoplast was obtained using the protocol that utilizes the following parameters: 3 to 4-day-old mycelia, treated with 1% lysing enzyme and 0.02% Driselase, incubated at 30 °C in an osmotic medium containing 0.6 M mannitol at pH 5.8 for 2 h. The highest protoplast yield was in the range of 8.95 × 109 to 3.12 × 1010 cells/mL per 5 g of mycelia used. The regeneration rate ranged from 9.03% to 22.55%, depending on the regeneration media used. By using 5 μg of vector to transform into 1.0 × 107 protoplast/mL, around 3 – 10 mitotically stable putative transformants were successfully obtained and verified via PCR. This protocol will find useful applications in genetic studies to enhance insight into this poorly characterized and understood phytopathogen

Identification and expression profiles of amino acid biosynthesis genes from psychrophilic yeast, glaciozyma antarctica

The mechanism of amino acid uptake and synthesis in the psychrophilic microorganism lives and proliferate in the extreme low-temperature environment is still not well understood. The aim of this study was to identify genes involved in amino acid generation for psychrophilic yeast, Glaciozyma antarctica and to determine their expression profiles when cells grow in media rich in amino acids or with limited amount of amino acids. The identification of genes was carried out by generating expressed sequence tags (EST) from two cDNA libraries generated from cells grown in complex growth medium and minimal growth medium without amino acids. A total of 3552 cDNA clones from each library was randomly picked and sequenced, generating 1492 unique transcripts (complex medium) and 1928 unique transcripts (minimal medium). Homology analyses have identified genes encoding proteins required for free amino acid uptake, biosynthesis of amino acids and recycling of amino acids based on the pathway used in the model yeast, Saccharomyces cerevisiae. Gene expression analysis by RT-qPCR showed that genes required for free amino acid uptake showed a higher expression profile in the complex medium, whereas the expression of most genes encode for proteins essential for biosynthesis and recycling of amino acids are higher in the minimal medium. In summary, genes that are involved in the generation and the uptake of amino acids for psychrophilic microorganism are conserved as in their mesophilic counterparts and the expression of these genes are regulated in the presence or absent of free amino acids in the surrounding

Pengenalpastian dan profil pengekspresan gen biosintesis asid amino yis psikrofil, Glaciozyma antarctica

Mekanisme pengambilan dan penghasilan asid amino bagi mikroorganisma psikrofil yang bermandiri dan berpoliferasi pada persekitaran sejuk melampau masih belum difahami sepenuhnya. Objektif kajian ini ialah untuk mengenal pasti gen yang terlibat dalam penjanaan asid amino bagi yis psikrofil, Glaciozyma antarctica serta menentukan pengekspresan gen tersebut semasa kehadiran dan kekurangan asid amino dalam medium pertumbuhan. Pengenalpastian gen telah dilakukan melalui penjanaan penanda jujukan terekspres (ESTs) daripada dua perpustakaan cDNA yang dibina daripada sel yang dikultur dalam medium pertumbuhan kompleks dan medium pertumbuhan minimum tanpa asid amino. Sebanyak 3552 klon cDNA daripada setiap perpustakaan dipilih secara rawak untuk dijujuk menghasilkan 1492 transkrip unik (medium kompleks) dan 1928 transkrip unik (medium minimum). Analisis pemadanan telah mengenl pasti gen mengekod protein yang terlibat di dalam pengambilan asid amino bebas, biosintesis asid amino serta gen yang terlibat dengan kitar semula asid amino berdasarkan tapak jalan yang digunakan oleh yis model, Saccharomyces cerevisiae. Analisis pengekspresan gen menggunakan kaedah RT-qPCR menunjukkan pengekspresan gen mengekod protein yang terlibat di dalam pengambilan asid amino bebas iaitu permease adalah tinggi pada medium kompleks manakala pengekspresan kebanyakan gen mengekod protein yang terlibat dalam kitar semula dan biosintesis asid amino adalah tinggi di dalam medium minimum. Kesimpulannya, gen yang terlibat dalam penjanaan dan pengambilan asid amino bagi mikroorganisma psikrofil adalah terpulihara seperti mikroorganisma mesofil dan pengekspresan gen-gen ini adalah diaruh oleh kehadiran atau ketiadaan asid amino bebas pada persekitaran

Biochemical characterisation and structure determination of a novel cold-active Proline iminopeptidase from the Psychrophilic yeast, Glaciozyma antarctica PI12

Microbial proteases constitute one of the most important groups of industrially relevant enzymes. Proline iminopeptidases (PIPs) that specifically release amino-terminal proline from peptides are of major interest for applications in food biotechnology. Proline iminopeptidase has been extensively characterised in bacteria and filamentous fungi. However, no similar reports exist for yeasts. In this study, a protease gene from Glaciozyma antarctica designated as GaPIP was cloned and overexpressed in Escherichia coli. Sequence analyses of the gene revealed a 960 bp open reading frame encoding a 319 amino acid protein (35,406 Da). The purified recombinant GaPIP showed a specific activity of 3561 Umg−1 towards L-proline-p-nitroanilide, confirming its identity as a proline iminopeptidase. GaPIP is a cold-active enzyme with an optimum activity of 30◦ C at pH 7.0. The enzyme is stable between pH 7.0 and 8.0 and able to retain its activity at 10–30◦ C. Although GaPIP is a serine protease, only 25% inhibition by the serine protease inhibitor, phenylmethanesulfonylfluoride (PMSF) was recorded. This enzyme is strongly inhibited by the presence of EDTA, suggesting that it is a metalloenzyme. The dimeric structure of GaPIP was determined at a resolution of 2.4 Å. To date, GaPIP is the first characterised PIP from yeasts and the structure of GaPIP is the first structure for PIP from eukaryotes

Cold adaptation strategies and the potential of psychrophilic enzymes from the antarctic yeast, glaciozyma antarctica PI12

Psychrophilic organisms possess several adaptive strategies which allow them to sustain life at low temperatures between −20 to 20 °C. Studies on Antarctic psychrophiles are interesting due to the multiple stressors that exist on the permanently cold continent. These organisms produce, among other peculiarities, cold-active enzymes which not only have tremendous biotechnological potential but are valuable models for fundamental research into protein structure and function. Recent innovations in omics technologies such as genomics, transcriptomics, proteomics and metabolomics have contributed a remarkable perspective of the molecular basis underpinning the mechanisms of cold adaptation. This review critically discusses similar and different strategies of cold adaptation in the obligate psychrophilic yeast, Glaciozyma antarctica PI12 at the molecular (genome structure, proteins and enzymes, gene expression) and physiological (antifreeze proteins, membrane fluidity, stress-related proteins) levels. Our extensive studies on G. antarctica have revealed significant insights towards the innate capacity of- and the adaptation strategies employed by this psychrophilic yeast for life in the persistent cold. Furthermore, several cold-active enzymes and proteins with biotechnological potential are also discussed

Whole genome sequencing analysis of salmonella enterica serovar typhi: history and current approaches

In recent years, the advance in whole-genome sequencing technology has changed the study of infectious diseases. The emergence of genome sequencing has improved the understanding of infectious diseases, which has revamped many fields, such as molecular microbiology, epidemiology, infection control, and vaccine production. In this review we discuss the findings of Salmonella enterica serovar Typhi genomes, publicly accessible from the initial complete genome to the recent update of Salmonella enterica serovar Typhi genomes, which has greatly improved Salmonella enterica serovar Typhi and other pathogen genomic research. Significant information on genetic changes, evolution, antimicrobial resistance, virulence, pathogenesis, and investigation from the genome sequencing of S. Typhi is also addressed. This review will gather information on the variation of the Salmonella enterica serovar Typhi genomes and hopefully facilitate our understanding of their genome evolution, dynamics of adaptation, and pathogenesis for the development of the typhoid point-of-care diagnostics, medications, and vaccines

A Review of Entomopathogenic Nematodes as a Biological Control Agent for Red Palm Weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae)

Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) is a severe pest of palm trees worldwide. The development and feeding activities of R. ferrugineus larvae inside the trunk damage palm trees. However, the absence of noticeable infestation signs at an early stage contributes to the spread of the attack. Integrated pest management (IPM) has been introduced to control R. ferrugineus infestation by implementing various approaches and techniques. The application of chemical pesticides has shown impressive results. However, biological control should be applied as an alternative solution due to adverse environmental impacts and pest resistance issues. One example is the use of entomopathogenic nematodes (EPNs) as biological control agents, which can forage and attack targeted pests without compromising the environment and other nontarget organisms. EPNs and their symbiotic bacteria have a mutualistic interaction that can kill the host within a short period of time. Therefore, this review emphasizes the effectiveness of entomopathogenic nematodes and their symbiotic bacteria against R. ferrugineus