Recombination-independent recognition of DNA homology for meiotic silencing in Neurospora crassa

The pairing of homologous chromosomes represents a critical step of meiosis in nearly all sexually reproducing species. In many organisms, pairing involves chromosomes that remain apparently intact. The mechanistic nature of homology recognition at the basis of such pairing is unknown. Using “meiotic silencing by unpaired DNA” (MSUD) as a model process, we demonstrate the existence of a cardinally different approach to DNA homology recognition in meiosis. The main advantage of MSUD over other experimental systems lies in its ability to identify any relatively short DNA fragment lacking a homologous allelic partner. Here, we show that MSUD does not rely on the canonical mechanism of meiotic recombination, yet it is promoted by REC8, a conserved component of the meiotic cohesion complex. We also show that certain patterns of interspersed homology are recognized as pairable during MSUD. Such patterns need to be colinear and must contain short tracts of sequence identity spaced apart at 21 or 22 base pairs. By using these periodicity values as a guiding parameter in all-atom molecular modeling, we discover that homologous DNA molecules can pair by forming quadruplex-based contacts with an interval of 2.5 helical turns. This process requires right-handed plectonemic coiling and additional conformational changes in the intervening double-helical segments. Our results 1) reconcile genetic and biophysical evidence for the existence of direct homologous double-stranded DNA (dsDNA)–dsDNA pairing, 2) identify a role for this process in initiating RNA interference, and 3) suggest that chromosomes can be cross-matched by a precise mechanism that operates on intact dsDNA molecules.

Construction of CRISPR/CAS9 system for industrial Saccharomyces cerevisiae strain and genetic manipulation effect on 2-phenylethanol pathway

Background The market demand for natural 2-phenylethanol (2-PE) continues to increase. Saccharomyces cerevisiae can synthesize 2-PE through the Ehrlich pathway. There are few studies on the improvement of the diploid industrial strains of S. cerevisiae by gene editing technology. There is no report on the comparison of genetic manipulation effect among S.cerevisiae strains with different 2-PE yield background, and the study on knockout of 2-PE downstream product synthesis gene and its effect on the yield of 2-PE have not been found. Results The CRISPR/CAS9 system with high efficiency for diploid S.cerevisiae CWY132 strain for industrial production of 2-PE was constructed. When the length of the homology arm of donor DNA is increased from 60bp to 500bp, the efficiency of gene editing increased from 0–100%. Using CRISPR/CAS9 technology, the branched acetaldehyde dehydrogenase genes ALD2 and ALD3 and the terminal acetyltransferase gene ATF1 in the Ehrlich pathway of S.cerevisiae strains with different 2-PE yields were knocked out. The results showed that in the high-yielding CWY-132 strain, the 2-PE yield decreased from 3.50 g/L to 1.65 g/L when double ALD2 and ALD3 were knocked out, a decrease of 52.8%. When ATF1 was knocked out, the yield of 2-PE decreased to 0.83 g/L, a decrease of 76.2%; In the low-yielding strain PK-2C, the yield of 2-PE increased from 0.21 g/L to 1.20 g/L when ALD2 was knocked out, an increase of 471%. When ATF1 was knocked out, the yield of 2-PE increased to 0.45g/L, an increase of 114%. The results show that the same genetic manipulation strategy for strains with different 2-PE yeilds backgrounds produces significantly different or even opposite effects. In addition, we found that the insufficient supply of NADH in cells can significantly affect the production of 2-PE, and the tolerance of cells to 2-PE is also a key factor that limits the further increase of 2-PE production in high-yielding strain. Conclusions This study shows that the length of the Donor DNA homology arm is a key factor affecting the efficiency of CRISPR/CAS9 gene editing in industrial diploid S. cerevisiae strains. Our result also shows that it is not feasible to increase the 2-PE production in high-yielding strains by blocking the branch pathway in the Ehrlich pathway. Breakthrough in the 2-PE yield of the high-yielding strains requires improved strains’ tolerance to 2-PE and increase the cellular NADH level.

The cloned SPI-1 type 3 secretion system can be functionally expressed outside Salmonella backgrounds

ABSTRACT Due to its potential for use in bacterial engineering applications, we previously cloned the SPI-1 type 3 secretion system (T3SS) genes from the genome of Salmonella enterica serovar Typhimurium strain LT2. We have documented that this clone, while functionally expressed in S. Typhimurium strains, displays a severe expression defect in other Gram negative backgrounds including Escherichia coli. To address this issue, we compared SPI-1 DNA sequence across different backgrounds, fully sequenced the original SPI-1 clone, and cloned SPI-1 from other S. Typhimurium strains. In this process, we were able to successfully obtain SPI-1 clones that are functionally expressed in E. coli indicating the first such result for a full-length SP-1 T3SS clone. We discovered that the original cloning technique using a DNA homology-based capture method was the root of the expression defect and that the FRT-Capture technique is preferable over the homology-based method. This result paves the way for future studies and applications using cloned SPI-1 and other T3SS in non-Salmonella bacterial backgrounds.

Pseudomonas spp.: contamination sources in bulk tanks of dairy farms

ABSTRACT: This study focused on isolating Pseudomonas spp. during milking process in ten dairy farms with manual and mechanical milking systems during dry and rainy seasons, and evaluating DNA homology and patterns of distribution between isolates, in order to identify main sources of milk contamination by Pseudomonas spp. A total of 167 isolates of Pseudomonas spp. were obtained from water, milkers’ hands, cows’ teats, teat cups, cooling tanks and raw milk. Bacteria of Pseudomonas spp. genus were isolated from 85 and 82 sampling points in dairy farms with manual and mechanical milking system, respectively. A significant difference (p=0.02) on Pseudomonas spp. isolation was observed among samples of surface of cows’ teats before and after pre-dipping, but no significant difference (p>0.05) was observed among milking systems or seasons. The possibility of the same Pseudomonas spp. patterns are distributed in different farms and seasons using Amplified Fragment Length Polymorphism (AFLP) technique was demonstrated. Milkers’ hands, surface of cows’ teats, teat cups and cooling tanks were associated with raw milk contamination with Pseudomonas spp. on farms with manual and mechanical milking system, showing that regardless of the type of milking system and season, proper hygiene procedures of equipment, utensils and workers’ hands are essential to avoid contamination of the milk and, therefore, improve milk quality.