A frameshift in Yersinia pestis rcsD alters canonical Rcs signalling to preserve flea-mammal plague transmission cycles.

Multiple genetic changes in the enteric pathogen Yersinia pseudotuberculosis have driven the emergence of Yesinia pestis, the arthropod-borne, etiological agent of plague. These include developing the capacity for biofilm-dependent blockage of the flea foregut to enable transmission by flea bite. Previously, we showed that pseudogenization of rcsA, encoding a component of the Rcs signalling pathway, is an important evolutionary step facilitating Y. pestis flea-borne transmission. Additionally, rcsD, another important gene in the Rcs system, harbours a frameshift mutation. Here, we demonstrated that this rcsD mutation resulted in production of a small protein composing the C-terminal RcsD histidine-phosphotransferase domain (designated RcsD-Hpt) and full-length RcsD. Genetic analysis revealed that the rcsD frameshift mutation followed the emergence of rcsA pseudogenization. It further altered the canonical Rcs phosphorylation signal cascade, fine-tuning biofilm production to be conducive with retention of the pgm locus in modern lineages of Y. pestis. Taken together, our findings suggest that a frameshift mutation in rcsD is an important evolutionary step that fine-tuned biofilm production to ensure perpetuation of flea-mammal plague transmission cycles.

Yersinia pestis, the agent responsible for the plague, emerged 6,000 to 7,000 years ago from Yersinia pseudotuberculosis, another type of bacteria which still exists today. Although they are highly similar genetically, these two species are strikingly different. While Y. pseudotuberculosis spreads via food and water and causes mild stomach distress, Y. pestis uses fleas to infect new hosts and has killed millions. A small set of genetic changes has contributed to the emergence of Y. pestis by allowing it to thrive inside a flea and maximise its transmission. In particular, some of these mutations have led to the bacteria being able to come together to form a sticky layer that adheres to the gut of the insect, with this 'biofilm' stopping the flea from feeding on blood. The starving flea keeps trying to feed, and with each bite comes another opportunity for Y. pestis to jump host. However, it remains unclear exactly how the mutations have influenced biofilm formation to allow for this new transmission mechanism to take place. To examine this phenomenon, Guo et al. focused on rcsD, a gene that codes for a component of the signalling system that controls biofilm creation. In Y. pestis this sequence has been mutated to become a 'pseudogene', a type of sequence which is often thought to be non-functional. However, the experiments showed that, in Y. pestis, rcsD could produce small amounts of a full-length RcsD protein similar to the one found in Y. pseudotuberculosis. However, the gene mostly produces a short 'RcsD-Hpt' protein that can, in turn, alter the expression of many genes, including those that decrease biofilm formation. This may prove to be beneficial for Y. pestis, for example when the bacteria switches from living in fleas to living in humans, where it does not require a biofilm. Guo et al. further investigated the impact of rcsD becoming a pseudogene inY. pestis, showing that if normal amounts of the full-length RcsD protein are produced, the bacteria quickly lose the gene that allows them to form biofilm in fleas, and cause disease in humans. In fact, additional analyses revealed that all sequenced strains of ancient and modern Y. pestis bacteria can produce RcsD-Hpt, even if they do not carry the same exact rcsD mutation. Overall, these results indicate that rcsD turning into a pseudogene marked an important step in the emergence of Y. pestis strains that can cause lasting plague outbreaks. They also point towards pseudogenes having more important roles in evolution than previously thought.

CRISPR-Cas12a-Assisted Recombineering in Bacteria.

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a (Cpf1) has emerged as an effective genome editing tool in many organisms. Here, we developed and optimized a CRISPR-Cas12a-assisted recombineering system to facilitate genetic manipulation in bacteria. Using this system, point mutations, deletions, insertions, and gene replacements can be easily generated on the chromosome or native plasmids in Escherichia coli, Yersinia pestis, and Mycobacterium smegmatis Because CRISPR-Cas12a-assisted recombineering does not require introduction of an antibiotic resistance gene into the chromosome to select for recombinants, it is an efficient approach for generating markerless and scarless mutations in bacteria.IMPORTANCE The CRISPR-Cas9 system has been widely used to facilitate genome editing in many bacteria. CRISPR-Cas12a (Cpf1), a new type of CRISPR-Cas system, allows efficient genome editing in bacteria when combined with recombineering. Cas12a and Cas9 recognize different target sites, which allows for more precise selection of the cleavage target and introduction of the desired mutation. In addition, CRISPR-Cas12a-assisted recombineering can be used for genetic manipulation of plasmids and plasmid curing. Finally, Cas12a-assisted recombineering in the generation of point mutations, deletions, insertions, and replacements in bacteria has been systematically analyzed. Taken together, our findings will guide efficient Cas12a-mediated genome editing in bacteria.

Short-term effect of titanium abutmentversus zirconia abutment on peri-implant tissues in anterior teeth / 中国组织工程研究

BACKGROUND: With the increasing aesthetic pursuit of patients, an aesthetic abutment with certain strength has aroused people's attentions either in the anterior or posterior tooth repair. OBJECTIVE: To evaluate the effects of titanium abutment and zirconia abutment on peri-implant tissue in anterior teeth.METHODS: A total of 40 anterior teeth from 34 patients were selected and received single crown restoration with Ankylos implants. Among them, titanium abutments were used in 20 teeth, and zirconia abutments in the other 20 teeth. All the patients were re-visited at 12 months after crown restoration. Peri-implant marginal bone resorption length (MBL), periodontal probing depth (PD), modified plaque index (mPLI), modified sulcus bleeding index (mSBI), and papilla index (PP) were detected; patient satisfaction investigation was carried out; and aesthetic assessment was done based on the pink esthetic score (PES).RESULTS AND CONCLUSION: There were no differences between two groups in MBL, PD, mPLI, mSBI, PP. Patients in both groups were satisfied with the treatment outcomes with insignificant difference (P > 0.05). The PES value in the zirconia abutment group was significantly higher than that in the titanium abutment group (P < 0.05). Overall, titanium and zirconia abutments show the similar effects on the peri-implant tissues and both can achieve good clinical outcomes. However, zirconia abutments have certain advantages in the aesthetic restoration for anterior teeth, and further investigation on long-term effects is warranted.

Genetic Polymorphisms of 17 Y-STR Loci in Nantong Han Population / 法医学杂志

Objective To analyze the genetic phenotypes of Nantong Han population and evaluate the application value of 17 Y-STR loci in Nantong population.Methods The peripheral blood samples were collected from 343 unrelated Nantong Han males and the genomic DNA were extracted by Chelex-100 method.Genotyping was performed using the AmpF(l)STR YfilerTM Kit.The results were compared with other 12 Han populations,including Anhui,Jiangsu,Jiangxi,Shandong,Shanghai,Zhejiang (1),Lanzhou,Nanyang,Luzhou,Mudanjiang,Shanxi and Zhejiang (2),and 9 minority populations (Mongol,Xibe,Tibetan in Lhasa,Tibetan in Qinghai,Kazak,Uighur,Manchu,Paiwan in Taiwan and Tujia).Results A total of 327 different haplotypes were found in 17 Y-STR loci in Nantong Han population.The haplotype diversity (HD) was 0.999 7.The R,value between Nantong Han and other Chinese populations ranged from-0.000 6 to 0.263 5.The multidimensional scaling results showed that Nantong Han population had no significant differences between most of the Han populations,but had significant differences between most of Chinese minority populations.Conclusion Seventeen Y-STR loci can be a powerful tool for forensic application because of its high polymorphism in Nantong Han population.

Short-term effect of titanium abutmentversus zirconia abutment on peri-implant tissues in anterior teeth / 中国组织工程研究

BACKGROUND: With the increasing aesthetic pursuit of patients, an aesthetic abutment with certain strength has aroused people's attentions either in the anterior or posterior tooth repair. OBJECTIVE: To evaluate the effects of titanium abutment and zirconia abutment on peri-implant tissue in anterior teeth.METHODS: A total of 40 anterior teeth from 34 patients were selected and received single crown restoration with Ankylos implants. Among them, titanium abutments were used in 20 teeth, and zirconia abutments in the other 20 teeth. All the patients were re-visited at 12 months after crown restoration. Peri-implant marginal bone resorption length (MBL), periodontal probing depth (PD), modified plaque index (mPLI), modified sulcus bleeding index (mSBI), and papilla index (PP) were detected; patient satisfaction investigation was carried out; and aesthetic assessment was done based on the pink esthetic score (PES).RESULTS AND CONCLUSION: There were no differences between two groups in MBL, PD, mPLI, mSBI, PP. Patients in both groups were satisfied with the treatment outcomes with insignificant difference (P > 0.05). The PES value in the zirconia abutment group was significantly higher than that in the titanium abutment group (P < 0.05). Overall, titanium and zirconia abutments show the similar effects on the peri-implant tissues and both can achieve good clinical outcomes. However, zirconia abutments have certain advantages in the aesthetic restoration for anterior teeth, and further investigation on long-term effects is warranted.

HmsC, a periplasmic protein, controls biofilm formation via repression of HmsD, a diguanylate cyclase in Yersinia pestis.

Yersinia pestis, the cause of plague, forms a biofilm in the foregut of its flea vector to enhance transmission. Biofilm formation in Y. pestis is controlled by the intracellular levels of the second messenger molecule cyclic diguanylate (c-di-GMP). HmsT and Y3730, the two diguanylate cyclases (DGC) in Y. pestis, are responsible for the synthesis of c-di-GMP. Y3730, which we name here as HmsD, has little effect on in vitro biofilms, but has a major effect on biofilm formation in the flea. The mechanism by which HmsD plays differential roles in vivo and in vitro is not understood. In this study, we show that hmsD is part of a three-gene operon (y3729-31), which we designate as hmsCDE. Deletion of hmsC resulted in increased, hmsD-dependent biofilm formation, while deletion or overexpression of hmsE did not affect biofilm formation. Localization experiments suggest that HmsC resides in the periplasmic space. In addition, we provide evidence that HmsC might interact directly with the periplasmic domain of HmsD and cause the proteolysis of HmsD. We propose that HmsC senses the environmental signals, which in turn regulates HmsD, and controls the c-di-GMP synthesis and biofilm formation in Y. pestis.

Novel AroA from Pseudomonas putida confers tobacco plant with high tolerance to glyphosate.

Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, also designated as AroA), a key enzyme in the aromatic amino acid biosynthesis pathway in microorganisms and plants. Previously, we reported that a novel AroA (PpAroA1) from Pseudomonas putida had high tolerance to glyphosate, with little homology to class I or class II glyphosate-tolerant AroA. In this study, the coding sequence of PpAroA1 was optimized for tobacco. For maturation of the enzyme in chloroplast, a chloroplast transit peptide coding sequence was fused in frame with the optimized aroA gene (PparoA1(optimized)) at the 5' end. The PparoA1(optimized) gene was introduced into the tobacco (Nicotiana tabacum L. cv. W38) genome via Agrobacterium-mediated transformation. The transformed explants were first screened in shoot induction medium containing kanamycin. Then glyphosate tolerance was assayed in putative transgenic plants and its T(1) progeny. Our results show that the PpAroA1 from Pseudomonas putida can efficiently confer tobacco plants with high glyphosate tolerance. Transgenic tobacco overexpressing the PparoA1(optimized) gene exhibit high tolerance to glyphosate, which suggest that the novel PpAroA1 is a new and good candidate applied in transgenic crops with glyphosate tolerance in future.

Reconstitution of the enzyme AroA and its glyphosate tolerance by fragment complementation.

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase (AroA) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, and is the target of the herbicide glyphosate. Glyphosate tolerance activity of the enzyme could be obtained by natural occurrence or by site-directed mutagenesis. A functional Pseudomonas putida AroA was obtained by co-expression of two protein fragments AroA(P. putida)-N210 and AroA(P. putida)-C212 in Escherichia coli aroA mutant strain AB2829. From sequence analysis, the equivalent split site on E. coli AroA was chosen for further study. The result indicated that functional E. coli AroA could also be reconstituted from two protein fragments AroA(E. coli)-N218 and AroA(E. coli)-C219, under both in vivo and in vitro conditions. This result suggested that the fragment complementation property of this family of enzyme may be general. Additional experiments indicated that the glyphosate tolerance property of AroA could also be reconstituted in parallel with its enzyme activity. The implication of this finding is discussed.