VIRULENCE GENE PROFILING AND PATHOGENICITY OF Streptococcus agalactiae ISOLATED FROM TILAPIA, Oreochromis niloticus FARMS IN INDONESIA

Streptococcosis caused by Streptococcus agalactiae has become a major disease problem in tilapia culture in Indonesia. This study aimed to detect virulence genes of S. agalactiae during streptococcosis disease outbreaks in several tilapia farms in Indonesia and evaluate the correlation between biotype and virulence genes to bacterial virulence. The presence of virulence genes was determined in 10 strains of S. agalactiae isolated from farm-raised tilapia. Polymerase chain reaction (PCR) protocol was used to determine genes for cylE, hylB, lmb, bib A, PI-2b, fbs A, fbs B, gap, PI-1, and cfb in the template DNA. Pathogenicity test was carried out by intraperitoneal injection of 24 hour-cultured S. agalactiae to tilapia with 108 CFU/fish. Four isolates have seven of virulence genes (cylE, hylB, bibA, PI-2b, fbs A, fbs B, and gap genes), three isolates have six virulence genes (hylB, bib A, fbs A, fbs B, gap, cfb genes), one isolate has four virulence gene (hyl B, bib A, fbs, and cfb genes), and one isolate has one virulence gene (PI-2b gene). None of the isolates has lmb or PI-1 genes. Bacteria with more virulence genes showed higher pathogenicity post injection. Mortality of tilapia injected with b-hemolytic bacteria was 100% within the period of 14-19 hours, while non-hemolytic bacteria was 53.3%-86.6% on 14 days post-injection. Pathological changes associated with the infection by either isolate included melanosis, slow response, anorexia, ocular opacity, gasping, erratic, C-shape, and whirling. It can be concluded that S. agalactiae with more virulence genes show a higher level of pathogenicity. The presence of a virulent gene has the potential to be used as a basis for selecting candidate isolates and designing vaccine compositions as an effort to prevent streptococcosis infection in tilapia in Indonesia.

Comparison of materials for rapid passive collection of environmental DNA

Passive collection is an emerging sampling method for environmental DNA (eDNA) in aquatic systems. Passive eDNA collection is inexpensive, efficient, and requires minimal equipment, making it suited to high density sampling and remote deployment. Here, we compare the effectiveness of nine membrane materials for passively collecting fish eDNA from a 3 million litre marine mesocosm. We submerged materials (cellulose, cellulose with 1% and 3% chitosan, cellulose overlayed with electrospun nanofibers and 1% chitosan, cotton fibres, hemp fibres and sponge with either zeolite or active carbon) for intervals between five and 1080 minutes. We show that for most materials, with as little as five minutes submersion, mitochondrial fish eDNA measured with qPCR, and fish species richness measured with metabarcoding, was comparable to that collected by conventional filtering. Furthermore, PCR template DNA concentrations and species richness were generally not improved significantly by longer submersion. Species richness detected for all materials ranged between 11 to 37 species, with a median of 27, which was comparable to the range for filtered eDNA (19-32). Using scanning electron microscopy, we visualised biological matter adhered to the surface of materials, rather than entrapped, with images also revealing a diversity in size and structure of putative eDNA particles. Environmental DNA can be collected rapidly from seawater with a passive approach and using a variety of materials. This will suit cost and time-sensitive biological surveys, and where access to equipment is limited.

Modeling DNA Opening in the Eukaryotic Transcription Initiation Complexes via Coarse-Grained Models

Recently, the molecular mechanisms of transcription initiation have been intensively studied. Especially, the cryo-electron microscopy revealed atomic structure details in key states in the eukaryotic transcription initiation. Yet, the dynamic processes of the promoter DNA opening in the pre-initiation complex remain obscured. In this study, based on the three cryo-electron microscopic yeast structures for the closed, open, and initially transcribing complexes, we performed multiscale molecular dynamics (MD) simulations to model structures and dynamic processes of DNA opening. Combining coarse-grained and all-atom MD simulations, we first obtained the atomic model for the DNA bubble in the open complexes. Then, in the MD simulation from the open to the initially transcribing complexes, we found a previously unidentified intermediate state which is formed by the bottleneck in the fork loop 1 of Pol II: The loop opening triggered the escape from the intermediate, serving as a gatekeeper of the promoter DNA opening. In the initially transcribing complex, the non-template DNA strand passes a groove made of the protrusion, the lobe, and the fork of Rpb2 subunit of Pol II, in which several positively charged and highly conserved residues exhibit key interactions to the non-template DNA strand. The back-mapped all-atom models provided further insights on atomistic interactions such as hydrogen bonding and can be used for future simulations.

Comparison of materials for rapid passive collection of environmental DNA

Passive collection is an emerging sampling method for environmental DNA (eDNA) in aquatic systems. Passive eDNA collection is inexpensive, efficient, and requires minimal equipment, making it suited to high density sampling and remote deployment. Here, we compare the effectiveness of nine membrane materials for passively collecting fish eDNA from a 3 million litre marine mesocosm. We submerged materials (cellulose, cellulose with 1% and 3% chitosan, cellulose overlayed with electrospun nanofibers and 1% chitosan, cotton fibres, hemp fibres and sponge with either zeolite or active carbon) for intervals between five and 1080 minutes. We show that for most materials, with as little as five minutes submersion, mitochondrial fish eDNA measured with qPCR, and fish species richness measured with metabarcoding, was comparable to that collected by conventional filtering. Furthermore, PCR template DNA concentrations and species richness were generally not improved significantly by longer submersion. Species richness detected for all materials ranged between 11 to 37 species, with a median of 27, which was comparable to the range for filtered eDNA (19-32). Using scanning electron microscopy, we visualised biological matter adhered to the surface of materials, rather than entrapped, with images also revealing a diversity in size and structure of putative eDNA particles. Environmental DNA can be collected rapidly from seawater with a passive approach and using a variety of materials. This will suit cost and time-sensitive biological surveys, and where access to equipment is limited.

A thermodynamic model of bacterial transcription

Transcriptional pausing is highly regulated by the template DNA and nascent transcript sequences. Here, we propose a thermodynamic model of transcriptional pausing, based on the thermal energy of transcription bubbles and nascent RNA structures, to describe the kinetics of the reaction pathways between active translocation, intermediate, backtracked, and hairpin-stabilized pauses. The model readily predicts experimentally detected pauses in high-resolution optical tweezers measurements of transcription. Unlike other models, it also predicts the effect of tension and the GreA transcription factor on pausing.

Production of Recombinant Horseradish Peroxidase in an Engineered Cell-free Protein Synthesis System

One of the main advantages of a cell-free synthesis system is that the synthetic machinery of cells can be modularized and re-assembled for desired purposes. In this study, we attempted to combine the translational activity of Escherichia coli extract with a heme synthesis pathway for the functional production of horseradish peroxidase (HRP). We first optimized the reaction conditions and the sequence of template DNA to enhance protein expression and folding. The reaction mixture was then supplemented with 5-aminolevulinic acid synthase to facilitate co-synthesis of the heme prosthetic group from glucose. Combining the different synthetic modules required for protein synthesis and cofactor generation led to successful production of functional HRP in a cell-free synthesis system.

5' modifications improve potency and efficacy of DNA donors for precision genome editing

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5′-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.

Metabolic Engineering of Escherichia Coli BL21 Strain Using Simplified CRISPR-Cas9 and Asymmetric Homolog Arms Recombineering

BackgroundThe recent CRISPR-Cas coupled with λ recombinase mediated genome recombineering has become a common laboratory practice to modify bacterial genomes. It requires supplying a template DNA or homolog arms for precise genome editing. However, it is often overlooked the process to generate the homolog arms which is a time-consuming, costly and inefficient step.ResultsIn this study, we first optimized CRISPR-Cas protocol in BL21 strain and successfully deleted 10 kb gene from the genome in one round of editing. To further simplify the protocol, asymmetric homolog arms as PCR fragments was used. It can be obtained by one-step PCR reaction with two primers and purified with desalting columns. Unlike conventional homolog arms that are prepared through overlapping PCR, cloning to plasmid or annealing synthetic DNA fragments, our method significantly shortened the time taken and reduced the cost to prepare the homolog arms. To test the robustness of the optimized workflow, we successfully deleted 26 / 27 genes across BL21 genome. Noteworthy, gRNA design is important for CRISPR-Cas system and a general heuristic gRNA design was proposed in the study. To apply our established protocol, we targeted 16 genes and iteratively deleted 7 genes from BL21 genome. The resulting strain increased lycopene production from ~15,000 ppm to > 40,000 ppm.ConclusionsOur work has optimized the homolog arms design for gene deletion in BL21 strains. The protocol efficiently edited BL21 strain to improve lycopene production. The same workflow is applicable to all E. coli strain which would be useful for genome rewiring to further increase metabolite production in microbial cell factories.