scholarly journals Electroporation-Based Genetic Manipulation in Type I Methanotrophs

2016 ◽  
Vol 82 (7) ◽  
pp. 2062-2069 ◽  
Author(s):  
Xin Yan ◽  
Frances Chu ◽  
Aaron W. Puri ◽  
Yanfen Fu ◽  
Mary E. Lidstrom
Keyword(s):  
Process Development ◽  
Transformation Efficiency ◽  
Genetic Manipulation ◽  
Industrial Process ◽  
Type I ◽  
Content Type ◽  
High Transformation Efficiency ◽  
New Antibiotics ◽  
Industrial Strains ◽  
High Transformation

ABSTRACTMethane is becoming a major candidate for a prominent carbon feedstock in the future, and the bioconversion of methane into valuable products has drawn increasing attention. To facilitate the use of methanotrophic organisms as industrial strains and accelerate our ability to metabolically engineer methanotrophs, simple and rapid genetic tools are needed. Electroporation is one such enabling tool, but to date it has not been successful in a group of methanotrophs of interest for the production of chemicals and fuels, the gammaproteobacterial (type I) methanotrophs. In this study, we developed electroporation techniques with a high transformation efficiency for three different type I methanotrophs:Methylomicrobium buryatense5GB1C,Methylomonassp. strain LW13, andMethylobactertundripaludum21/22. We further developed this technique inM. buryatense, a haloalkaliphilic aerobic methanotroph that demonstrates robust growth with a high carbon conversion efficiency and is well suited for industrial use for the bioconversion of methane. On the basis of the high transformation efficiency ofM. buryatense, gene knockouts or integration of a foreign fragment into the chromosome can be easily achieved by direct electroporation of PCR-generated deletion or integration constructs. Moreover, site-specific recombination (FLP-FRT [FLP recombination target] recombination) andsacBcounterselection systems were employed to perform marker-free manipulation, and two new antibiotics, zeocin and hygromycin, were validated to be antibiotic markers in this strain. Together, these tools facilitate the rapid genetic manipulation ofM. buryatenseand other type I methanotrophs, promoting the ability to perform fundamental research and industrial process development with these strains.

Preparation of electrocompetent cells v2

2021 ◽  
Author(s):  
Shuning Guo
Keyword(s):  
Transformation Efficiency ◽  
High Transformation Efficiency ◽  
High Transformation

This protocol is used to prepare electrocompetent cells with high transformation efficiency.

Preparation of electrocompetent cells v2

2021 ◽  
Author(s):  
Shuning Guo
Keyword(s):  
Transformation Efficiency ◽  
High Transformation Efficiency ◽  
High Transformation

This protocol is used to prepare electrocompetent cells with high transformation efficiency.

scholarly journals Natural Transformation of Gallibacterium anatis

2012 ◽  
Vol 78 (14) ◽  
pp. 4914-4922 ◽  
Author(s):  
Bodil M. Kristensen ◽  
Sunita Sinha ◽  
John D. Boyce ◽  
Anders M. Bojesen ◽  
Joshua C. Mell ◽  
...  
Keyword(s):  
Bioinformatics Analysis ◽  
Natural Transformation ◽  
Genetic Manipulation ◽  
Targeted Mutagenesis ◽  
Chromosomal Dna ◽  
Natural Competence ◽  
Content Type ◽  
Transformation Procedure ◽  
High Transformation ◽  
Dna Types

ABSTRACTGallibacterium anatisis a pathogen of poultry. Very little is known about its genetics and pathogenesis. To enable the study of gene function inG. anatis, we have established methods for transformation and targeted mutagenesis. The genusGallibacteriumbelongs to thePasteurellaceae, a group with several naturally transformable members, includingHaemophilus influenzae. Bioinformatics analysis identifiedG. anatishomologs of theH. influenzaecompetence genes, and natural competence was induced inG. anatisby the procedure established forH. influenzae: transfer from rich medium to the starvation medium M-IV. This procedure gave reproducibly high transformation frequencies withG. anatischromosomal DNA and with linearized plasmid DNA carryingG. anatissequences. Both DNA types integrated into theG. anatischromosome by homologous recombination. Targeted mutagenesis gave transformation frequencies of >2 × 10−4transformants CFU−1. Transformation was also efficient with circular plasmid containing noG. anatisDNA; this resulted in the establishment of a self-replicating plasmid. Nine diverseG. anatisstrains were found to be naturally transformable by this procedure, suggesting that natural competence is common and the M-IV transformation procedure widely applicable for this species. TheG. anatisgenome is only slightly enriched for the uptake signal sequences identified in other pasteurellaceaen genomes, butG. anatisdid preferentially take up its own DNA over that ofEscherichia coli. Transformation by electroporation was not effective for chromosomal integration but could be used to introduce self-replicating plasmids. The findings described here provide important tools for the genetic manipulation ofG. anatis.

scholarly journals Bypassing the Restriction System To Improve Transformation of Staphylococcus epidermidis

2017 ◽  
Vol 199 (16) ◽  
Author(s):  
Stephen K. Costa ◽  
Niles P. Donegan ◽  
Anna-Rita Corvaglia ◽  
Patrice François ◽  
Ambrose L. Cheung
Keyword(s):  
Staphylococcus Epidermidis ◽  
Genetic Manipulation ◽  
Genetically Modify ◽  
Clinical Isolates ◽  
Type I ◽  
Genetic Barrier ◽  
Content Type ◽  
Genetic Barriers ◽  
Underlying Mechanisms ◽  
Considerable Morbidity

ABSTRACT Staphylococcus epidermidis is the leading cause of infections on indwelling medical devices worldwide. Intrinsic antibiotic resistance and vigorous biofilm production have rendered these infections difficult to treat and, in some cases, require the removal of the offending medical prosthesis. With the exception of two widely passaged isolates, RP62A and 1457, the pathogenesis of infections caused by clinical S. epidermidis strains is poorly understood due to the strong genetic barrier that precludes the efficient transformation of foreign DNA into clinical isolates. The difficulty in transforming clinical S. epidermidis isolates is primarily due to the type I and IV restriction-modification systems, which act as genetic barriers. Here, we show that efficient plasmid transformation of clinical S. epidermidis isolates from clonal complexes 2, 10, and 89 can be realized by employing a plasmid artificial modification (PAM) in Escherichia coli DC10B containing a Δdcm mutation. This transformative technique should facilitate our ability to genetically modify clinical isolates of S. epidermidis and hence improve our understanding of their pathogenesis in human infections. IMPORTANCE Staphylococcus epidermidis is a source of considerable morbidity worldwide. The underlying mechanisms contributing to the commensal and pathogenic lifestyles of S. epidermidis are poorly understood. Genetic manipulations of clinically relevant strains of S. epidermidis are largely prohibited due to the presence of a strong restriction barrier. With the introductions of the tools presented here, genetic manipulation of clinically relevant S. epidermidis isolates has now become possible, thus improving our understanding of S. epidermidis as a pathogen.

scholarly journals Improved transformation efficiency of group A Streptococcus by inactivation of a type I restriction modification system

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0248201
Author(s):  
Meredith B. Finn ◽  
Kathryn M. Ramsey ◽  
Hunter J. Tolliver ◽  
Simon L. Dove ◽  
Michael R. Wessels
Keyword(s):  
Genetic Transformation ◽  
Parent Strain ◽  
Transformation Efficiency ◽  
Genetic Manipulation ◽  
Spontaneous Mutation ◽  
Type I ◽  
Modification System ◽  
Restriction Modification System ◽  
Group A ◽  
Restriction Modification

Streptococcus pyogenes or group A Streptococcus (GAS) is a leading cause of bacterial pharyngitis, skin and soft tissue infections, life-threatening invasive infections, and the post-infectious autoimmune syndromes of acute rheumatic fever and post-streptococcal glomerulonephritis. Genetic manipulation of this important pathogen is complicated by resistance of the organism to genetic transformation. Very low transformation efficiency is attributed to recognition and degradation of introduced foreign DNA by a type I restriction-modification system encoded by the hsdRSM locus. DNA sequence analysis of this locus in ten GAS strains that had been previously transformed with an unrelated plasmid revealed that six of the ten harbored a spontaneous mutation in hsdR, S, or M. The mutations were all different, and at least five of the six were predicted to result in loss of function of the respective hsd gene product. The unexpected occurrence of such mutations in previously transformed isolates suggested that the process of transformation selects for spontaneous inactivating mutations in the Hsd system. We investigated the possibility of exploiting the increased transformability of hsd mutants by constructing a deletion mutation in hsdM in GAS strain 854, a clinical isolate representative of the globally dominant M1T1 clonal group. Mutant strain 854ΔhsdM exhibited a 5-fold increase in electrotransformation efficiency compared to the wild type parent strain and no obvious change in growth or off-target gene expression. We conclude that genetic transformation of GAS selects for spontaneous mutants in the hsdRSM restriction modification system. We propose that use of a defined hsdM mutant as a parent strain for genetic manipulation of GAS will enhance transformation efficiency and reduce the likelihood of selecting spontaneous hsd mutants with uncharacterized genotypes.

scholarly journals Complete Bypass of Restriction Systems for Major Staphylococcus aureus Lineages

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Ian R. Monk ◽  
Jai J. Tree ◽  
Benjamin P. Howden ◽  
Timothy P. Stinear ◽  
Timothy J. Foster
Keyword(s):  
Staphylococcus Aureus ◽  
Plasmid Dna ◽  
High Efficiency ◽  
Recombinant Dna ◽  
Genetic Manipulation ◽  
Bacterial Pathogen ◽  
Type I ◽  
Content Type ◽  
E Coli ◽  
Adenine Methylation

ABSTRACTStaphylococcus aureusis a prominent global nosocomial and community-acquired bacterial pathogen. A strong restriction barrier presents a major hurdle for the introduction of recombinant DNA into clinical isolates ofS. aureus. Here, we describe the construction and characterization of the IMXXB series ofEscherichia colistrains that mimic the type I adenine methylation profiles ofS. aureusclonal complexes 1, 8, 30, and ST93. The IMXXB strains enable direct, high-efficiency transformation and streamlined genetic manipulation of majorS. aureuslineages.IMPORTANCEThe genetic manipulation of clinicalS. aureusisolates has been hampered due to the presence of restriction modification barriers that detect and subsequently degrade inappropriately methylated DNA. Current methods allow the introduction of plasmid DNA into a limited subset ofS. aureusstrains at high efficiency after passage of plasmid DNA through the restriction-negative, modification-proficient strain RN4220. Here, we have constructed and validated a suite ofE. colistrains that mimic the adenine methylation profiles of different clonal complexes and show high-efficiency plasmid DNA transfer. The ability to bypass RN4220 will reduce the cost and time involved for plasmid transfer intoS. aureus. The IMXXB series ofE. colistrains should expedite the process of mutant construction in diverse genetic backgrounds and allow the application of new techniques to the genetic manipulation ofS. aureus.

Preparation of electrocompetent cells v2

2021 ◽  
Author(s):  
Shuning Guo
Keyword(s):  
Transformation Efficiency ◽  
High Transformation Efficiency ◽  
High Transformation

This protocol is used to prepare electrocompetent cells with high transformation efficiency.

scholarly journals Transforming the Untransformable: Application of Direct Transformation To Manipulate Genetically Staphylococcus aureus and Staphylococcus epidermidis

mBio ◽  
2012 ◽  
Vol 3 (2) ◽  
Author(s):  
Ian R. Monk ◽  
Ishita M. Shah ◽  
Min Xu ◽  
Man-Wah Tan ◽  
Timothy J. Foster
Keyword(s):  
Staphylococcus Aureus ◽  
Staphylococcus Epidermidis ◽  
High Efficiency ◽  
Genetic Manipulation ◽  
Laboratory Strain ◽  
Small Subset ◽  
Type I ◽  
Major Barrier ◽  
Content Type ◽  
Functional Genomic Analysis

ABSTRACTThe strong restriction barrier present inStaphylococcus aureusandStaphylococcus epidermidishas limited functional genomic analysis to a small subset of strains that are amenable to genetic manipulation. Recently, a conserved type IV restriction system termed SauUSI (which specifically recognizes cytosine methylated DNA) was identified as the major barrier to transformation with foreign DNA. Here we have independently corroborated these findings in a widely used laboratory strain ofS. aureus. Additionally, we have constructed a DNA cytosine methyltransferase mutant in the high-efficiencyEscherichia colicloning strain DH10B (called DC10B). Plasmids isolated from DC10B can be directly transformed into clinical isolates ofS. aureusandS. epidermidis. We also show that the loss of restriction (both type I and IV) in anS. aureusUSA300 strain does not have an impact on virulence. Circumventing the SauUSI restriction barrier, combined with an improved deletion and transformation protocol, has allowed the genetic manipulation of previously untransformable strains of these important opportunistic pathogens.IMPORTANCEStaphylococcal infections place a huge burden on the health care sector due both to their severity and also to the economic impact of treating the infections because of prolonged hospitalization. To improve the understanding ofStaphylococcus aureusandStaphylococcus epidermidisinfections, we have developed a series of improved techniques that allow the genetic manipulation of strains that were previously refractory to transformation. These developments will speed up the process of mutant construction and increase our understanding of these species as a whole, rather than just a small subset of strains that could previously be manipulated.

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