A method for increasing electroporation competency of Gram-negative clinical isolates by Polymyxin B nonapeptide

The study of clinically relevant bacterial pathogens relies on molecular and genetic approaches. However, the generally low transformation frequency among natural isolates poses technical hurdles to widely applying common methods in molecular biology, including transformation of large constructs, chromosomal genetic manipulation, and dense mutant library construction. Here we demonstrate that culturing clinical isolates in the presence of polymyxin B nonapeptide (PMBN) improves their transformation frequency via electroporation by up to 100-fold in a dose-dependent and reversible manner. The effect was observed for PMBN-binding uropathogenic Escherichia coli (UPEC) and Salmonella enterica strains but not naturally polymyxin resistant Proteus mirabilis. Using our PMBN electroporation method we show efficient delivery of large plasmid constructs into UPEC, which otherwise failed using a conventional electroporation protocol. Moreover, we show a 5-fold increase in the yield of engineered mutant colonies obtained in S. enterica with the widely used lambda-Red recombineering method, when cells are cultured in the presence of PMBN. Lastly, we demonstrate that PMBN treatment can enhance the delivery of DNA-transposase complexes into UPEC and increase transposon mutant yield by 8-fold when constructing Transposon Insertion Sequencing (TIS) libraries. Therefore, PMBN can be used as a powerful electropermeabilisation adjuvant to aid the delivery of DNA and DNA-protein complexes into clinically important bacteria.

Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance

Alteration of antibiotic binding sites through modification of ribosomal RNA (rRNA) is a common form of resistance to ribosome-targeting antibiotics. The rRNA-modifying enzyme Cfr methylates an adenosine nucleotide within the peptidyl transferase center, resulting in the C-8 methylation of A2503 (m8A2503). Acquisition of cfr results in resistance to eight classes of ribosome-targeting antibiotics. Despite the prevalence of this resistance mechanism, it is poorly understood whether and how bacteria modulate Cfr methylation to adapt to antibiotic pressure. Moreover, direct evidence for how m8A2503 alters antibiotic binding sites within the ribosome is lacking. In this study, we performed directed evolution of Cfr under antibiotic selection to generate Cfr variants that confer increased resistance by enhancing methylation of A2503 in cells. Increased rRNA methylation is achieved by improved expression and stability of Cfr through transcriptional and post-transcriptional mechanisms, which may be exploited by pathogens under antibiotic stress as suggested by natural isolates. Using a variant that achieves near-stoichiometric methylation of rRNA, we determined a 2.2 Å cryo-electron microscopy structure of the Cfr-modified ribosome. Our structure reveals the molecular basis for broad resistance to antibiotics and will inform the design of new antibiotics that overcome resistance mediated by Cfr.

Biosynthesis of Polyhydroxyalkanoate by Recombinant Bacteria: A Review

Polyhydroxyalkanoates (PHAs), a biodegradable polymer, has many industrial and medical applications such as heart valves, scaffold, suture and drug delivery. Because of their structural diversity and close analogy to plastics, PHAs have gained major interest over the world. Natural isolates, recombinant bacteria, and plants have all been found to improve the quality, quantity, and economics of PHA production. Their biodegradability makes them an especially appealing synthetic plastic substitute. PHB biosynthetic genes phbA, phbB, and phbC are grouped and systematised into a single phbCAB operon. In terms of alignment and clustering of the relevant genes, the PHB pathway varies greatly across bacterial genera. In this regard, the enzymes appear to have a high degree of sequence preservation. The structural studies further improve the mechanism of action of these enzymes and helped in improving and choosing the good candidates for increased production of PHB.

Diverse mating phenotypes impact the spread of wtf meiotic drivers in Schizosaccharomyces pombe

Meiotic drivers are genetic elements that break Mendel's law of segregation to be transmitted into more than half of the offspring produced by a heterozygote. The success of a driver relies on outcrossing (mating between individuals from distinct lineages) because drivers gain their advantage in heterozygotes. It is, therefore, curious that Schizosaccharomyces pombe, a species reported to rarely outcross, harbors many meiotic drivers. To address this paradox, we measured mating phenotypes in S. pombe natural isolates. We found that the propensity for cells from distinct clonal lineages to mate varies between natural isolates and can be affected both by cell density and by the available sexual partners. Additionally, we found that the observed levels of preferential mating between cells from the same clonal lineage can slow, but not prevent, the spread of a wtf meiotic driver in the absence of additional fitness costs linked to the driver. These analyses reveal parameters critical to understanding the evolution of S. pombe and help explain the success of meiotic drivers in this species.

KTD1 is a yeast defense factor against K28 killer toxin

Secreted protein toxins are widely used weapons in conflicts between organisms. Killer yeast produce killer toxins that inhibit the growth of nearby sensitive yeast. We investigated variation in resistance to the killer toxin K28 across diverse natural isolates of the Saccharomyces cerevisiae population and discovered a novel defense factor, which we named KTD1, that is an important determinant of K28 toxin resistance. KTD1 is a member of the DUP240 gene family of unknown function. We uncovered a putative role of DUP240 proteins in killer toxin defense and identified a region that is undergoing rapid evolution and is critical to KTD1's protective ability. Our findings implicate KTD1 as a key factor in the defense against killer toxin K28.

Biotechnological Approaches to Lowering the Ethanol Yield during Wine Fermentation

One of the most prominent consequences of global climate warming for the wine industry is a clear increase of the sugar content in grapes, and thus the alcohol level in wines. Among the several approaches to address this important issue, this review focuses on biotechnological solutions, mostly relying on the selection and improvement of wine yeast strains for reduced ethanol yields. Other possibilities are also presented. Researchers are resorting to both S. cerevisiae and alternative wine yeast species for the lowering of alcohol yields. In addition to the use of selected strains under more or less standard fermentation conditions, aerobic fermentation is increasingly being explored for this purpose. Genetic improvement is also playing a role in the development of biotechnological tools to counter the increase in the wine alcohol levels. The use of recombinant wine yeasts is restricted to research, but its contribution to the advancement of the field is still relevant. Furthermore, genetic improvement by non-GMO approaches is providing some interesting results, and will probably result in the development of commercial yeast strains with a lower alcohol yield in the near future. The optimization of fermentation processes using natural isolates is, anyway, the most probable source of advancement in the short term for the production of wines with lower alcohol contents.

High-throughput imaging of Caenorhabditis elegans aging using collective activity monitoring

The genetic manipulability and short lifespan of C. elegans make it an important model for aging research. Widely applied methods for measurements of worm aging based on manual observation are labor intensive and low-throughput. Here, we describe the Worm Collective Activity Monitoring Platform (WormCamp), a system for assaying aging in C. elegans by monitoring activity of populations of worms in standard 24-well plates. We show that metrics based on the rate of decline in collective activity can be used to estimate the average lifespan and locomotor healthspan in the population. Using the WormCamp, we assay a panel of highly divergent natural isolates of C. elegans and show that both lifespan and locomotor healthspan display substantial heritability. To facilitate analysis of large numbers of worms, we developed a robotic imaging system capable of simultaneous automated monitoring of activity, lifespan, and locomotor healthspan in up to 2,304 populations containing a total of ~90,000 animals. We applied the automated system to conduct a large-scale RNA interference screen for genes that affect lifespan and locomotor healthspan. The WormCamp system is complementary to other current automated methods for assessing C. elegans aging and is well suited for efficiently screening large numbers of conditions.

Genomic population structure associated with repeated escape of Salmonella enterica ATCC14028s from the laboratory into nature

Salmonella enterica serovar Typhimurium strain ATCC14028s is commercially available from multiple national type culture collections, and has been widely used since 1960 for quality control of growth media and experiments on fitness (“laboratory evolution”). ATCC14028s has been implicated in multiple cross-contaminations in the laboratory, and has also caused multiple laboratory infections and one known attempt at bioterrorism. According to hierarchical clustering of 3002 core gene sequences, ATCC14028s belongs to HierCC cluster HC20_373 in which most internal branch lengths are only one to three SNPs long. Many natural Typhimurium isolates from humans, domesticated animals and the environment also belong to HC20_373, and their core genomes are almost indistinguishable from those of laboratory strains. These natural isolates have infected humans in Ireland and Taiwan for decades, and are common in the British Isles as well as the Americas. The isolation history of some of the natural isolates confirms the conclusion that they do not represent recent contamination by the laboratory strain, and 10% carry plasmids or bacteriophages which have been acquired in nature by HGT from unrelated bacteria. We propose that ATCC14028s has repeatedly escaped from the laboratory environment into nature via laboratory accidents or infections, but the escaped micro-lineages have only a limited life span. As a result, there is a genetic gap separating HC20_373 from its closest natural relatives due to a divergence between them in the late 19th century followed by repeated extinction events of escaped HC20_373.

Genetic diversity in Chrysodeixis includens nucleopolyhedrovirus and factors involved in its generation

In the present thesis, the genetic variability of field-collected isolates of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) was examined. These results have revealed the great variability present within natural ChinNPV isolates, confirm the differences between two different genetic variants from these isolates and, more important, appear to identify the ChinNPV-K variant as a generator of variability in these isolates. In conclusion, a great variability has been revealed within ChinNPV natural isolates, with a single variant showing a key role in the generation of this diversity in a concentration-dependent manner.

Generation of Variability in Chrysodeixis includens Nucleopolyhedrovirus (ChinNPV): The Role of a Single Variant

The mechanisms generating variability in viruses are diverse. Variability allows baculoviruses to evolve with their host and with changes in their environment. We examined the role of one genetic variant of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) and its contribution to the variability of the virus under laboratory conditions. A mixture of natural isolates (ChinNPV-Mex1) contained two genetic variants that dominated over other variants in individual larvae that consumed high (ChinNPV-K) and low (ChinNPV-E) concentrations of inoculum. Studies on the ChinNPV-K variant indicated that it was capable of generating novel variation in a concentration-dependent manner. In cell culture, cells inoculated with high concentrations of ChinNPV-K produced OBs with the ChinNPV-K REN profile, whereas a high diversity of ChinNPV variants was recovered following plaque purification of low concentrations of ChinNPV-K virion inoculum. Interestingly, the ChinNPV-K variant could not be recovered from plaques derived from low concentration inocula originating from budded virions or occlusion-derived virions of ChinNPV-K. Genome sequencing revealed marked differences between ChinNPV-K and ChinNPV-E, with high variation in the ChinNPV-K genome, mostly due to single nucleotide polymorphisms. We conclude that ChinNPV-K is an unstable genetic variant that is responsible for generating much of the detected variability in the natural ChinNPV isolates used in this study.