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Pili and Hosts
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
Recently, Ellison et al. (2019) performed transposon sequencing of mutant C. crescentus libraries infected with the phage φCB5 to identify genes required for the phage infection. They found that the φCB5 infection, in contrast to the φCbK ones, was 75% prevented when pilus retraction was obstructed.
The application of machine learning techniques to innovative antibacterial discovery and development
Published in Expert Opinion on Drug Discovery, 2020
Mateus Sá Magalhães Serafim, Thales Kronenberger, Patrícia Rufino Oliveira, Antti Poso, Káthia Maria Honório, Bruno Eduardo Fernandes Mota, Vinícius Gonçalves Maltarollo
Moradigaravand et al., 2018 aimed to overcome the limitation of using known resistance determinants to identify resistant-strains, by using whole-genome sequences [105]. This work studied population structure, isolation year, gene content, and polymorphism information, and treated them as unbiased predictors characters from 1,936 E. coli genomic samples exposed to eleven different antibiotics. They employed gradient boosted decision trees, which out-performed others such as deep neural networks and displayed maximum sensitivity by including information about genetic content in their models. The gene content encompassed not only the SNP information but also chromosomic variations, such as small insertions and deletions (indels). As another example, Santiago et al. [106], focused on transposon sequencing (Tn-Seq) of S. aureus strains resistant to different antibiotics to unveil their mechanism of resistance, beyond the gene-wise approach. Transposon insertion can lead to knock-out phenotypes and, occasionally, to up-regulation of upstream targets. Tn-Seq data was further explored using a supervised machine learning model based on K-nearest neighbors algorithm, using the transposon-induced gene upregulation and inactivation mutant fitness data. As a case of study, a group of strains treated with lysocin derivatives was selected and their model predicted, which later confirmed the cell wall precursor Lipid II to be their molecular target. This work highlights the importance of integrating high-quality phenotypical data with target-engagement approaches to confirm proposed molecular targets for antibiotics.
Transposon mutagenesis in oral streptococcus
Published in Journal of Oral Microbiology, 2022
Yixin Zhang, Zhengyi Li, Xin Xu, Xian Peng
Although transposon sequencing has some applicability, it still has some limitations. Traditional TIS is mainly used to study the functions of nonessential genes and identify essential genes. However, libraries with large numbers of mutants have a bottleneck effect; insertion mutants may be randomly lost during selective growth for reasons unrelated to fitness, especially in animal models. Liu et al. designed single-guide RNA (sgRNA) sequences targeting core genes identified by Tn-Seq and developed an IPTG-induced CRISPR interference (CRISPRi) system for functional studies of essential S. pneumoniae D39V genes in vitro [90]. Bosch et al. developed a CRISPRi platform for S. thermophilus to provide a genome-level assessment of gene vulnerability, which links the degree of gene inhibition to its effect on fitness [91]. Liu et al. recently developed a doxycycline-induced CRISPRi system and constructed a pooled CRISPRi library that targets almost all operons of S. pneumoniae D39V and can be easily combined with Illumina sequencing (CRISPRi-Seq) [92]. By selecting a sgRNA for each operon, CRISPRi-Seq was used to assess bottlenecks and identify pneumococcal genes that are important in a murine pneumonia model. Genome-wide CRISPR screening can be used to systematically investigate gene functions. However, an sgRNA library is large, and its synthesis is expensive. Jiang et al. used the CRISPR-CAS adaptation mechanism of S. pyogenes to develop CRISPR adaptation-mediated library manufacturing, which transforms bacterial cells into ‘factories’ that generate hundreds of thousands of CRISPR RNAs, covering 95% of all targeted genomic sites [93]. However, this method also produces numerous mutants resulting in bottlenecks. Moreover, when operons contain multiple essential genes, the CRISPRi system results in polarity effects that inhibit the expression of downstream genes. The insertion of transposons within operons in Tn-Seq, owing to the lack of transcriptional terminators, allows for read-through transcription and, thus, minimises polarity effects. With the development of TIS, the functions of essential genes can be studied using transposon libraries with outwards promoters that promote gene overexpression [94]. Along with technical and analytical development, the bottleneck of saturated libraries between different conditions gradually decreases. In general, TIS is simple to operate and inexpensive and is still a powerful tool for high-throughput quantitative studies of microbial genotypes influencing their phenotypes. The CRISPRi system is indispensable for the functional study of essential genes in microorganisms.