Degradation of Plant Lignin by Pseudomonas sp. Strain YS-1p And Phanerochaete chrysosposium RP-78 Co-Cultures

Delignification is a critical step in the bioconversion of lignocellulosic biomass into useful monomers. Fungi and bacteria both have evolved pathways to degrade lignin and are known to play active roles in decomposition of plant biomass. However, little efforts have been made to study them together as an integrated microbial system. There is limited knowledge about what specific roles they play and what kind of interactions they have during the decomposition process. The major focus of this research is to study lignin degradation using Pseudomonas sp. strain YS-1p (bacteria) and Phanerochaete chrysosporium RP-78 (fungi) when inoculated as individual cultures as well as in co-cultures in mineral salts medium supplemented with plant biomass: sugarcane bagasse and sorghum bagasse. Flasks were inoculated with strain RP-78 (F) and strain YS-1p (B) at various fungal-to-bacterial ratios including 0:1, 1:0, 1:1, 1:10 and 1:50, respectively. Flasks were sacrificed periodically and monitored for population dynamics. Culture supernatant was assayed for major lignin degrading enzymes including lignin peroxidase, dyp peroxidase and laccase. Additionally, extracellular enzymes were concentrated and subjected to 1-D PAGE-LC-MS/MS for proteome analysis, while recovered plant biomass was used for Py-GC/MS analysis. Our results show that Pseudomonas grew best in co-cultures with P. chrysosporium whereas the growth of latter was suppressed in the presence of bacteria. This also affected their abilities to produce extracellular enzymes involved in lignocellulose degradation as shown by our results of proteome analysis. Most of the enzymes involved in degradation of lignin, cellulose and hemicellulose were produced by the fungi, while the bacteria produced few enzymes at low levels. This suggests that bacteria were able to derive benefit from fungal enzymes for accessing degradation products and promoting their growth without contributing as much to the degradation process. Enzyme activities of lignin degrading enzymes showed that the two peroxidases: Lip and Dyp were expressed maximum in co-cultures while laccase was expressed in highest amounts in bacterial monocultures. Furthermore, analysis of plant biomass by Py-GC/MS revealed that microbial pretreatment altered the composition of lignocellulose in plant biomass with plant biomass showing significant decrease in lignin derivatives than in untreated controls.Microbiology, Cell and Molecular Biolog

An enhanced CRISPR repressor for targeted mammalian gene regulation.

The RNA-guided endonuclease Cas9 can be converted into a programmable transcriptional repressor, but inefficiencies in target-gene silencing have limited its utility. Here we describe an improved Cas9 repressor based on the C-terminal fusion of a rationally designed bipartite repressor domain, KRAB-MeCP2, to nuclease-dead Cas9. We demonstrate the system's superiority in silencing coding and noncoding genes, simultaneously repressing a series of target genes, improving the results of single and dual guide RNA library screens, and enabling new architectures of synthetic genetic circuits

Viral Aggregation: The Knowns and Unknowns

Viral aggregation is a complex and pervasive phenomenon affecting many viral families. An increasing number of studies have indicated that it can modulate critical parameters surrounding viral infections, and yet its role in viral infectivity, pathogenesis, and evolution is just beginning to be appreciated. Aggregation likely promotes viral infection by increasing the cellular multiplicity of infection (MOI), which can help overcome stochastic failures of viral infection and genetic defects and subsequently modulate their fitness, virulence, and host responses. Conversely, aggregation can limit the dispersal of viral particles and hinder the early stages of establishing a successful infection. The cost–benefit of viral aggregation seems to vary not only depending on the viral species and aggregating factors but also on the spatiotemporal context of the viral life cycle. Here, we review the knowns of viral aggregation by focusing on studies with direct observations of viral aggregation and mechanistic studies of the aggregation process. Next, we chart the unknowns and discuss the biological implications of viral aggregation in their infection cycle. We conclude with a perspective on harnessing the therapeutic potential of this phenomenon and highlight several challenging questions that warrant further research for this field to advance

Fluorescent Guide RNAs Facilitate Development of Layered Pol II‑Driven CRISPR Circuits

Efficient clustered regularly interspaced short palindromic repeat (CRISPR) guide RNA (gRNA) expression from RNA Polymerase II (Pol II) promoters will aid in construction of complex CRISPR-based synthetic gene networks. Yet, we require tools to properly visualize gRNA directly to quantitatively study the corresponding network behavior. To address this need, we employed a fluorescent gRNA (fgRNA) to visualize synthetic CRISPR network dynamics without affecting gRNA functionality. We show that studying gRNA dynamics directly enables circuit modification and improvement of network function in Pol II-driven CRISPR circuits. This approach generates information necessary for optimizing the overall function of these networks and provides insight into the hurdles remaining in Pol II-regulated gRNA expression

Glycerol gradient preparation and RZC purification (S1 Movie in S1 File).

(A) Preparation of glycerol gradient with LEGO gradient mixer and separation of sample by RZC. (B) Layers of glycerol before mixing; the blue glycerol at the bottom is 45% (v/v) and the clear glycerol at the top is 15% (v/v). (C) Linear glycerol gradient after mixing with LEGO gradient mixer. (D) 140 nm green fluorescent beads on top of the glycerol gradient before RZC. (E) Fluorescent beads after RZC concentrated into a thin layer due to separation from the glycerol gradient. All glycerol solutions were diluted in 1× TAE 12.5 mM MgCl2.</p

RZC purification of high and low aspect ratio DNA origami nanostructures.

(A) 3D rendered model of high aspect ratio 6-helix bundle DNA origami nanotube (6-hb) monomers. (B) Gel result of liquid fractions from top to bottom (fractions 1–17) of the 6-hb sample. R is unpurified samples as a negative control lane. Fractions 14 and 15 correspond to the fractions containing the purified monomers. (C− F) AFM images and their corresponding heigh distribution of 6-hb monomer before (C and E) and after (D and F) RZC purification. (G) RZC purified 6-hb monomer SYBR gold stained 6-hb monomer sample after RZC purification. (H) 3D rendered model of a 40-nm DNA origami snub cube (SC). (I) Gel shift assays for indicated RZC-purified SC samples using glycerol gradients prepared by overnight passive diffusion or LEGO gradient mixer, followed by ultrafiltration.</p

This document provides comprehensive supporting information, including detailed protocols, materials, equipment, 5 supplementary figures, 2 supplementary tables, and 1 supplementary movie.

This document provides comprehensive supporting information, including detailed protocols, materials, equipment, 5 supplementary figures, 2 supplementary tables, and 1 supplementary movie.</p

RZC purification of 6-hb dimer.

(A) 3D-rendered model of a 6-hb dimer. (B) Comparison between SYBR gold stained 6-hb monomer (left) and (C) 6-hb dimer (right) purified using a 30%—60% gradient column centrifuged for 3 hours at 50k rpm in 4°C. (D) SYBR gold stained dimer with 6 hours of centrifugation. (E) AFM image of unpurified dimer. (F) Precursor monomer (highlighted blue) and dimer (highlighted light brown) from unpurified dimer. (G) AFM image of RZC purified dimer. (H) Significantly smaller number of monomer (highlighted blue) with similar number of dimer (highlighted light brown) compared to those in unpurified dimer. (I and J) Comparison of precursor monomers (highlighted blue) and dimer (highlighted light brown) between unpurified and purified dimer. Dimer content of unpurified dimer and purified dimer at 21±5% and 63±6% respectively. Standard deviation was calculated with the bootstrapping method (N=273 for unpurified dimer; N=114 for purified dimer).</p

Comparison of different spin time for different gradients.

(A–D) Four different combinations of glycerol concentrations before and after loading into the LEGO gradient mixer. The bottom layer of glycerol is dyed blue for visual inspection, while the top layer is left clear. Four spin times were selected to compare the glycerol gradient formed at the indicated spin times. As can be seen by the color gradient created as the blue-dyed glycerol mixes with the clear glycerol, each gradient, and spin time combination lead to comparable results.</p

Fig 1 -

Side view of the LEGO gradient mixer during (A) its initial position and (B) its horizontal tilting phase. (1) 3D printed centrifuge-tube holder. (2) Spinning motor to rotate the tubes while in horizontal position. (3) Turning servo motor responsible for tilting the tubes horizontally. (4) Large grey gear connecting the two motors with its small gear complement. (5) The scaffold holding the structure together. (6) The LEGO controller for orchestrating the motions of the two motors. The black cables are traced in white for clarity.</p