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

2022 ◽  
Author(s):  
Jilong Qin ◽  
Yaoqin Hong ◽  
Karthik Pullela ◽  
Renato Morona ◽  
Ian R. Henderson ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Protein Complexes ◽  
Fold Increase ◽  
Polymyxin B ◽  
Transformation Frequency ◽  
Clinical Isolates ◽  
Transposon Insertion ◽  
Efficient Delivery ◽  
Natural Isolates ◽  
Transposon Insertion Sequencing

Abstract 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.

scholarly journals Molecular phenotyping of infection-associated small non-coding RNAs

2016 ◽  
Vol 371 (1707) ◽  
pp. 20160081 ◽  
Author(s):  
Lars Barquist ◽  
Alexander J. Westermann ◽  
Jörg Vogel
Keyword(s):  
Virulence Factors ◽  
High Throughput ◽  
Genetic Manipulation ◽  
Genetic Interaction ◽  
Forward Genetics ◽  
Model Systems ◽  
Transposon Insertion ◽  
Culture Models ◽  
Molecular Phenotypes ◽  
Transposon Insertion Sequencing

Infection is a complicated balance, with both pathogen and host struggling to tilt the result in their favour. Bacterial infection biology has relied on forward genetics for many of its advances, defining phenotype in terms of replication in model systems. However, many known virulence factors fail to produce robust phenotypes, particularly in the systems most amenable to genetic manipulation, such as cell-culture models. This has particularly been limiting for the study of the bacterial regulatory small RNAs (sRNAs) in infection. We argue that new sequencing-based technologies can work around this problem by providing a ‘molecular phenotype’, defined in terms of the specific transcriptional dysregulation in the infection system induced by gene deletion. We illustrate this using the example of our recent study of the PinT sRNA using dual RNA-seq, that is, simultaneous RNA sequencing of host and pathogen during infection. We additionally discuss how other high-throughput technologies, in particular genetic interaction mapping using transposon insertion sequencing, may be used to further dissect molecular phenotypes. We propose a strategy for how high-throughput technologies can be integrated in the study of non-coding regulators as well as bacterial virulence factors, enhancing our ability to rapidly generate hypotheses with regards to their function. This article is part of the themed issue ‘The new bacteriology’.

scholarly journals Reproducible and accessible analysis of transposon insertion sequencing in Galaxy for qualitative essentiality analyses

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Delphine Larivière ◽  
Laura Wickham ◽  
Kenneth Keiler ◽  
Anton Nekrutenko ◽  
Keyword(s):  
Data Analysis ◽  
Promoter Sequence ◽  
Entire Genome ◽  
Link Type ◽  
Transposon Insertion ◽  
Control Procedures ◽  
Reproducible Analysis ◽  
Using Data ◽  
Transposon Insertion Sequencing ◽  
The Impact

Abstract Background Significant progress has been made in advancing and standardizing tools for human genomic and biomedical research. Yet, the field of next-generation sequencing (NGS) analysis for microorganisms (including multiple pathogens) remains fragmented, lacks accessible and reusable tools, is hindered by local computational resource limitations, and does not offer widely accepted standards. One such “problem areas” is the analysis of Transposon Insertion Sequencing (TIS) data. TIS allows probing of almost the entire genome of a microorganism by introducing random insertions of transposon-derived constructs. The impact of the insertions on the survival and growth under specific conditions provides precise information about genes affecting specific phenotypic characteristics. A wide array of tools has been developed to analyze TIS data. Among the variety of options available, it is often difficult to identify which one can provide a reliable and reproducible analysis. Results Here we sought to understand the challenges and propose reliable practices for the analysis of TIS experiments. Using data from two recent TIS studies, we have developed a series of workflows that include multiple tools for data de-multiplexing, promoter sequence identification, transposon flank alignment, and read count repartition across the genome. Particular attention was paid to quality control procedures, such as determining the optimal tool parameters for the analysis and removal of contamination. Conclusions Our work provides an assessment of the currently available tools for TIS data analysis. It offers ready to use workflows that can be invoked by anyone in the world using our public Galaxy platform (https://usegalaxy.org). To lower the entry barriers, we have also developed interactive tutorials explaining details of TIS data analysis procedures at https://bit.ly/gxy-tis.

scholarly journals OmpR and LeuO Positively Regulate the Salmonella enterica Serovar Typhi ompS2 Porin Gene

2004 ◽  
Vol 186 (10) ◽  
pp. 2909-2920 ◽  
Author(s):  
Marcos Fernández-Mora ◽  
José Luis Puente ◽  
Edmundo Calva
Keyword(s):  
Salmonella Enterica ◽  
Type Strain ◽  
Wild Type Strain ◽  
Phosphorylation Site ◽  
Regulatory Region ◽  
Random Mutagenesis ◽  
Fold Increase ◽  
Upstream Regulatory Region ◽  
Wild Type ◽  
Transposon Insertion

ABSTRACT The Salmonella enterica serovar Typhi ompS2 gene codes for a 362-amino-acid outer membrane protein that contains motifs common to the porin superfamily. It is expressed at very low levels compared to the major OmpC and OmpF porins, as observed for S. enterica serovar Typhi OmpS1, Escherichia coli OmpN, and Klebsiella pneumoniae OmpK37 quiescent porins. A region of 316 bp, between nucleotides −413 and −97 upstream of the transcriptional start point, is involved in negative regulation, as its removal resulted in a 10-fold increase in ompS2 expression in an S. enterica serovar Typhi wild-type strain. This enhancement in expression was not observed in isogenic mutant strains, which had specific deletions of the regulatory ompB (ompR envZ) operon. Furthermore, ompS2 expression was substantially reduced in the presence of the OmpR D55A mutant, altered in the major phosphorylation site. Upon random mutagenesis, a mutant where the transposon had inserted into the upstream regulatory region of the gene coding for the LeuO regulator, showed an increased level of ompS2 expression. Augmented expression of ompS2 was also obtained upon addition of cloned leuO to the wild-type strain, but not in an ompR isogenic derivative, consistent with the notion that the transposon insertion had increased the cellular levels of LeuO and with the observed dependence on OmpR. Moreover, LeuO and OmpR bound in close proximity, but independently, to the 5′ upstream regulatory region. Thus, the OmpR and LeuO regulators positively regulate ompS2.

scholarly journals Transposon-insertion sequencing screens unveil requirements for EHEC growth and intestinal colonization

2019 ◽  
Vol 15 (8) ◽  
pp. e1007652 ◽  
Author(s):  
Alyson R. Warr ◽  
Troy P. Hubbard ◽  
Diana Munera ◽  
Carlos J. Blondel ◽  
Pia Abel zur Wiesch ◽  
...  
Keyword(s):  
Intestinal Colonization ◽  
Transposon Insertion ◽  
Transposon Insertion Sequencing

scholarly journals ARTIST: High-Resolution Genome-Wide Assessment of Fitness Using Transposon-Insertion Sequencing

PLoS Genetics ◽  
2014 ◽  
Vol 10 (11) ◽  
pp. e1004782 ◽  
Author(s):  
Justin R. Pritchard ◽  
Michael C. Chao ◽  
Sören Abel ◽  
Brigid M. Davis ◽  
Catherine Baranowski ◽  
...  
Keyword(s):  
High Resolution ◽  
Transposon Insertion ◽  
Genome Wide ◽  
Transposon Insertion Sequencing

scholarly journals Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

2020 ◽  
Vol 48 (8) ◽  
pp. 4585-4600
Author(s):  
Gabriel A Suárez ◽  
Kyle R Dugan ◽  
Brian A Renda ◽  
Sean P Leonard ◽  
Lakshmi Suryateja Gangavarapu ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Single Gene ◽  
Acinetobacter Baylyi ◽  
Gene Deletions ◽  
Multiple Gene ◽  
Transposon Insertion ◽  
A Genome ◽  
Acinetobacter Baylyi Adp1 ◽  
Transposon Insertion Sequencing ◽  
Improved Methods

Abstract One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 18 successful multiple-gene deletions ranged in size from 21 to 183 kb and collectively accounted for 23.4% of its genome. The success of each multiple-gene deletion attempt could only be partially predicted on the basis of an existing collection of viable ADP1 single-gene deletion strains and a new transposon insertion sequencing (Tn-Seq) dataset that we generated. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

scholarly journals Transposon Insertion Sequencing in a Clinical Isolate of Legionella pneumophila Identifies Essential Genes and Determinants of Natural Transformation

2020 ◽  
Vol 203 (3) ◽  
Author(s):  
Léo Hardy ◽  
Pierre-Alexandre Juan ◽  
Bénédicte Coupat-Goutaland ◽  
Xavier Charpentier
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Legionella Pneumophila ◽  
Genetic Basis ◽  
Natural Transformation ◽  
Essential Genes ◽  
Content Type ◽  
Transposon Insertion ◽  
Life Traits ◽  
Transposon Insertion Sequencing

ABSTRACT Legionella pneumophila is a Gram-negative bacterium ubiquitous in freshwater environments which, if inhaled, can cause a severe pneumonia in humans. The emergence of L. pneumophila is linked to several traits selected in the environment, the acquisition of some of which involved intra- and interkingdom horizontal gene transfer events. Transposon insertion sequencing (TIS) is a powerful method to identify the genetic basis of selectable traits as well as to identify fitness determinants and essential genes, which are possible antibiotic targets. TIS has not yet been used to its full power in L. pneumophila, possibly because of the difficulty of obtaining a high-saturation transposon insertion library. Indeed, we found that isolates of sequence type 1 (ST1), which includes the commonly used laboratory strains, are poorly permissive to saturating mutagenesis by conjugation-mediated transposon delivery. In contrast, we obtained high-saturation libraries in non-ST1 clinical isolates, offering the prospect of using TIS on unaltered L. pneumophila strains. Focusing on one of them, we then used TIS to identify essential genes in L. pneumophila. We also revealed that TIS could be used to identify genes controlling vertical transmission of mobile genetic elements. We then applied TIS to identify all the genes required for L. pneumophila to develop competence and undergo natural transformation, defining the set of major and minor type IV pilins that are engaged in DNA uptake. This work paves the way for the functional exploration of the L. pneumophila genome by TIS and the identification of the genetic basis of other life traits of this species. IMPORTANCE Legionella pneumophila is the etiologic agent of a severe form of nosocomial and community-acquired pneumonia in humans. The environmental life traits of L. pneumophila are essential to its ability to accidentally infect humans. A comprehensive identification of their genetic basis could be obtained through the use of transposon insertion sequencing. However, this powerful approach had not been fully implemented in L. pneumophila. Here, we describe the successful implementation of the transposon-sequencing approach in a clinical isolate of L. pneumophila. We identify essential genes, potential drug targets, and genes required for horizontal gene transfer by natural transformation. This work represents an important step toward identifying the genetic basis of the many life traits of this environmental and pathogenic species.

scholarly journals The RNA degradosome promotes tRNA quality control through clearance of hypomodified tRNA

2019 ◽  
Vol 116 (4) ◽  
pp. 1394-1403 ◽  
Author(s):  
Satoshi Kimura ◽  
Matthew K. Waldor
Keyword(s):  
Quality Control ◽  
Elongation Factor ◽  
Trna Modification ◽  
Rapid Degradation ◽  
Transposon Insertion ◽  
Specific Manner ◽  
Suppressor Mutants ◽  
Rna Degradosome ◽  
Biochemical Analyses ◽  
Transposon Insertion Sequencing

The factors and mechanisms that govern tRNA stability in bacteria are not well understood. Here, we investigated the influence of posttranscriptional modification of bacterial tRNAs (tRNA modification) on tRNA stability. We focused on ThiI-generated 4-thiouridine (s4U), a modification found in bacterial and archaeal tRNAs. Comprehensive quantification ofVibrio choleraetRNAs revealed that the abundance of some tRNAs is decreased in a ΔthiIstrain in a stationary phase-specific manner. Multiple mechanisms, including rapid degradation of a subset of hypomodified tRNAs, account for the reduced abundance of tRNAs in the absence ofthiI. Through transposon insertion sequencing, we identified additional tRNA modifications that promote tRNA stability and bacterial viability. Genetic analysis of suppressor mutants as well as biochemical analyses revealed that rapid degradation of hypomodified tRNA is mediated by the RNA degradosome. Elongation factor Tu seems to compete with the RNA degradosome, protecting aminoacyl tRNAs from decay. Together, our observations describe a previously unrecognized bacterial tRNA quality control system in which hypomodification sensitizes tRNAs to decay mediated by the RNA degradosome.

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