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Background Gene doctoring is an efficient recombination-based genetic engineering approach to mutagenesis of the bacterial chromosome that combines the λ-Red recombination system with a suicide donor plasmid that is cleaved in vivo to generate linear DNA fragments suitable for recombination. The use of a suicide donor plasmid makes Gene Doctoring more efficient than other recombineering technologies. However, generation of donor plasmids typically requires multiple cloning and screening steps. Results We constructed a simplified acceptor plasmid, called pDOC-GG, for the assembly of multiple DNA fragments precisely and simultaneously to form a donor plasmid using Golden Gate assembly. Successful constructs can easily be identified through blue-white screening. We demonstrated proof of principle by inserting a gene for green fluorescent protein into the chromosome of Escherichia coli . We also provided related genetic parts to assist in the construction of mutagenesis cassettes with a tetracycline-selectable marker. Conclusions Our plasmid greatly simplifies the construction of Gene Doctoring donor plasmids and allows for the assembly of complex, multi-part insertion or deletion cassettes with a free choice of target sites and selection markers. The tools we developed are applicable to gene editing for a wide variety of purposes in Enterobacteriaceae and potentially in other diverse bacterial families .

Flagella, the primary means of motility in bacteria, are helical filaments that function as microscopic propellers composed of thousands of copies of the protein flagellin. Here, we show that many bacteria encode \"giant\" flagellins, greater than a thousand amino acids in length, and that two species that encode giant flagellins, the marine γ-proteobacteria Bermanella marisrubri and Oleibacter marinus, produce monopolar flagellar filaments considerably thicker than filaments composed of shorter flagellin monomers. We confirm that the flagellum from B. marisrubri is built from its giant flagellin. Phylogenetic analysis reveals that the mechanism of evolution of giant flagellins has followed a stepwise process involving an internal domain duplication followed by insertion of an additional novel insert. This work illustrates how \"the\" bacterial flagellum should not be seen as a single, idealised structure, but as a continuum of evolved machines adapted to a range of niches.

Purpose It has previously been shown that organic acids produced by Escherichia coli suppress the growth of Pseudomonas aeruginosa in co-cultures under conditions of glucose excess, due to overflow metabolism. Inactivation of genes involved in central carbon metabolism favours fermentation of glucose over respiration and therefore increases production of organic acid by-products such as acetate and lactate. We sought to extend and refine the list of genes known to contribute to the metabolic balance between respiration and fermentation, to better understand the role of overflow metabolism in competitive survival of E. coli . Methods We confirmed the previous finding that E. coli excludes P. aeruginosa from co-cultures by producing organic acids in the presence of glucose. Using a genome-wide transposon screen we identified E. coli genes that are important for survival in co-cultures with P. aeruginosa , both with and without glucose supplementation. Results Central carbon metabolism was the dominant gene function under selection in our experimental conditions, indicating that the observed inhibition is a side-effect of overflow metabolism adopted by E. coli as a response to high glucose concentrations. The presence of a competing species increased the selective pressure for central carbon metabolism genes, with 31 important for growth in the presence of P. aeruginosa and glucose, while only 9 were significant for pure E. coli cultures grown with glucose. In our experiments, each transposon mutant was competed against all others in the pool, suggesting that overflow metabolism provides benefits to individual E. coli cells in addition to competitive inhibition derived from acidification of the growth medium. Conclusion Co-culture assays using transposon mutant libraries can provide insight into the selective pressures present in mixed species competition. This work demonstrates central carbon metabolism is the dominant gene function under selection in E. coli for aerobic growth in glucose and a side-effect of this is overflow metabolism which can inhibit growth of bystander species.

The genus Escherichia has been extensively studied and it is known to encompass a range of commensal and pathogenic bacteria that primarily inhabit the gastrointestinal tracts of warm-blooded vertebrates. However, the presence of E. coli as a model organism and potential pathogen has diverted attention away from commensal strains and other species in the genus. To investigate the diversity of Escherichia in healthy chickens, we collected fecal samples from antibiotic-free Lohmann Brown layer hens and determined the genome sequences of 100 isolates, 81 of which were indistinguishable at the HC0 level of the Hierarchical Clustering of Core Genome Multi-Locus Sequence Typing scheme. Despite initial selection on CHROMagar Orientation medium, which is considered selective for E. coli , in silico phylotyping and core genome single nucleotide polymorphism analysis revealed the presence of at least one representative of all major clades of Escherichia , except for E. albertii, Shigella , and E. coli phylogroup B2 and cryptic clade I. The most frequent phylogenomic groups were E. coli phylogroups A and B1 and E. ruysiae (clades III and IV). We compiled a collection of reference strains isolated from avian sources (predominantly chicken), representing every Escherichia phylogroup and species, and used it to confirm the phylogeny and diversity of our isolates. Overall, the isolates carried low numbers of the virulence and antibiotic resistance genes typically seen in avian pathogenic E. coli . Notably, the clades not recovered are ones that have been most strongly associated with virulence by other studies.

We present CoronaHiT, a platform and throughput flexible method for sequencing SARS-CoV-2 genomes (≤ 96 on MinION or > 96 on Illumina NextSeq) depending on changing requirements experienced during the pandemic. CoronaHiT uses transposase-based library preparation of ARTIC PCR products. Method performance was demonstrated by sequencing 2 plates containing 95 and 59 SARS-CoV-2 genomes on nanopore and Illumina platforms and comparing to the ARTIC LoCost nanopore method. Of the 154 samples sequenced using all 3 methods, ≥ 90% genome coverage was obtained for 64.3% using ARTIC LoCost, 71.4% using CoronaHiT-ONT and 76.6% using CoronaHiT-Illumina, with almost identical clustering on a maximum likelihood tree. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally.

Wet scanning‐transmission electron microscopy (STEM) is a technique that allows high‐resolution transmission imaging of biological samples in a hydrated state, with minimal sample preparation. However, it has barely been used for the study of bacterial cells. In this study, we present an analysis of the advantages and disadvantages of wet STEM compared with standard transmission electron microscopy (TEM). To investigate the potential applications of wet STEM, we studied the growth of polyhydroxyalkanoate and triacylglycerol carbon storage inclusions. These were easily visible inside cells, even in the early stages of accumulation. Although TEM produces higher resolution images, wet STEM is useful when preservation of the sample is important or when studying the relative sizes of different features, since samples do not need to be sectioned. Furthermore, under carefully selected conditions, it may be possible to maintain cell viability, enabling new types of experiments to be carried out. To our knowledge, internal features of bacterial cells have not been imaged previously by this technique. SCANNING 33: 59–68, 2011. © 2011 Wiley Periodicals, Inc.

In this approach, Z score values of two and six are used as the cut-off values for assessing practical significance. In cases where a Z score is larger than six, as illustrated conceptually in Figure 3(a), there are no practically significant parameters. In cases where a Z score is less than two as illustrated conceptually in Figure 3(c), it is generally appropriate to conclude that every statistically significant parameter is practically significant.

Abstract The COVID-19 pandemic has spread to almost every country in the world since it started in China in late 2019. Controlling the pandemic requires a multifaceted approach including whole genome sequencing to support public health interventions at local and national levels. One of the most widely used methods for sequencing is the ARTIC protocol, a tiling PCR approach followed by Oxford Nanopore sequencing (ONT) of up to 96 samples at a time. There is a need, however, for a flexible, platform agnostic, method that can provide multiple throughput options depending on changing requirements as the pandemic peaks and troughs. Here we present CoronaHiT, a method capable of multiplexing up to 96 small genomes on a single MinION flowcell or >384 genomes on Illumina NextSeq, using transposase mediated addition of adapters and PCR based addition of barcodes to ARTIC PCR products. We demonstrate the method by sequencing 95 and 59 SARS-CoV-2 genomes for routine and rapid outbreak response runs, respectively, on Nanopore and Illumina platforms and compare to the standard ARTIC LoCost nanopore method. Of the 154 samples sequenced using the three approaches, genomes with ≥ 90% coverage (GISAID criteria) were generated for 64.3% of samples for ARTIC LoCost, 71.4% for CoronaHiT-ONT, and 76.6% for CoronaHiT-Illumina and have almost identical clustering on a maximum likelihood tree. In conclusion, we demonstrate that CoronaHiT can multiplex up to 96 SARS-CoV-2 genomes per MinION flowcell and that Illumina sequencing can be performed on the same libraries, which will allow significantly higher throughput. CoronaHiT provides increased coverage for higher Ct samples, thereby increasing the number of high quality genomes that pass the GISAID QC threshold. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally, to help control the pandemic. Competing Interest Statement LG received a partial support for his PhD from Roche. The use of Roche technology for diagnostics in NNUH is coincidental. Footnotes * Improved protocol and updated comparison to LoCost method