scholarly journals Genetic Tools for the Industrially Promising Methanotroph Methylomicrobium buryatense

2014 ◽  
Vol 81 (5) ◽  
pp. 1775-1781 ◽  
Author(s):  
Aaron W. Puri ◽  
Sarah Owen ◽  
Frances Chu ◽  
Ted Chavkin ◽  
David A. C. Beck ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Glycogen Synthase ◽  
Broad Host Range ◽  
Type I ◽  
Incompatibility Group ◽  
Content Type ◽  
Genetic Tools ◽  
Ambient Temperatures ◽  
Aerobic Methanotrophs ◽  
Native Plasmid

ABSTRACTAerobic methanotrophs oxidize methane at ambient temperatures and pressures and are therefore attractive systems for methane-based bioconversions. In this work, we developed and validated genetic tools forMethylomicrobium buryatense, a haloalkaliphilic gammaproteobacterial (type I) methanotroph.M. buryatensewas isolated directly on natural gas and grows robustly in pure culture with a 3-h doubling time, enabling rapid genetic manipulation compared to many other methanotrophic species. As a proof of concept, we used a sucrose counterselection system to eliminate glycogen production inM. buryatenseby constructing unmarked deletions in two redundant glycogen synthase genes. We also selected for a more genetically tractable variant strain that can be conjugated with small incompatibility group P (IncP)-based broad-host-range vectors and determined that this capability is due to loss of the native plasmid. These tools makeM. buryatensea promising model system for studying aerobic methanotroph physiology and enable metabolic engineering in this bacterium for industrial biocatalysis of methane.

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 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.

scholarly journals Genetic Modification of Sodalis Species by DNA Transduction

mSphere ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Chelsea M. Keller ◽  
Christopher G. Kendra ◽  
Roberto E. Bruna ◽  
David Craft ◽  
Mauricio H. Pontes
Keyword(s):  
Host Range ◽  
Genetic Study ◽  
Genetic Manipulation ◽  
Bacterial Species ◽  
Broad Host Range ◽  
Insect Host ◽  
Exogenous Dna ◽  
Content Type ◽  
Sodalis Glossinidius ◽  
Genetically Manipulated

ABSTRACT Bacteriophages (phages) are ubiquitous in nature. These viruses play a number of central roles in microbial ecology and evolution by, for instance, promoting horizontal gene transfer (HGT) among bacterial species. The ability of phages to mediate HGT through transduction has been widely exploited as an experimental tool for the genetic study of bacteria. As such, bacteriophage P1 represents a prototypical generalized transducing phage with a broad host range that has been extensively employed in the genetic manipulation of Escherichia coli and a number of other model bacterial species. Here we demonstrate that P1 is capable of infecting, lysogenizing, and promoting transduction in members of the bacterial genus Sodalis, including the maternally inherited insect endosymbiont Sodalis glossinidius. While establishing new tools for the genetic study of these bacterial species, our results suggest that P1 may be used to deliver DNA to many Gram-negative endosymbionts in their insect host, thereby circumventing a culturing requirement to genetically manipulate these organisms. IMPORTANCE A large number of economically important insects maintain intimate associations with maternally inherited endosymbiotic bacteria. Due to the inherent nature of these associations, insect endosymbionts cannot be usually isolated in pure culture or genetically manipulated. Here we use a broad-host-range bacteriophage to deliver exogenous DNA to an insect endosymbiont and a closely related free-living species. Our results suggest that broad-host-range bacteriophages can be used to genetically alter insect endosymbionts in their insect host and, as a result, bypass a culturing requirement to genetically alter these bacteria.

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.

scholarly journals Development of a CRISPR/Cas9 System forMethylococcus capsulatusIn VivoGene Editing

2019 ◽  
Vol 85 (11) ◽  
Author(s):  
Timothy Tapscott ◽  
Michael T. Guarnieri ◽  
Calvin A. Henard
Keyword(s):  
Natural Gas ◽  
Host Range ◽  
Gene Editing ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Broad Host Range ◽  
Methanotrophic Bacteria ◽  
Methylococcus Capsulatus ◽  
Content Type ◽  
Genetic Tools

ABSTRACTMethanotrophic bacteria play a crucial role in the Earth’s biogeochemical cycle and have the potential to be employed in industrial biomanufacturing processes due to their capacity to use natural gas- and biogas-derived methane as a sole carbon and energy source. Advanced gene-editing systems have the potential to enable rapid, high-throughput methanotrophic genetics and biocatalyst development. To this end, we employed a series of broad-host-range expression plasmids to construct a conjugatableclusteredregularlyinterspacedshortpalindromicrepeats (CRISPR)/Cas9 gene-editing system inMethylococcus capsulatus(Bath). Heterologous coexpression of theStreptococcus pyogenesCas9 endonuclease and a synthetic single guide RNA (gRNA) showed efficient Cas9 DNA targeting and double-stranded DNA (dsDNA) cleavage that resulted in cell death. We demonstrated effectivein vivoediting of plasmid DNA using both Cas9 and Cas9D10Anickase to convert green fluorescent protein (GFP)- to blue fluorescent protein (BFP)-expressing cells with 71% efficiency. Further, we successfully introduced a premature stop codon into the soluble methane monooxygenase (sMMO) hydroxylase component-encodingmmoXgene with the Cas9D10Anickase, disrupting sMMO function. These data provide proof of concept for CRISPR/Cas9-mediated gene editing inM. capsulatus. Given the broad-host-range replicons and conjugation capability of these CRISPR/Cas9 tools, they have potential utility in other methanotrophs and a wide array of Gram-negative microorganisms.IMPORTANCEIn this study, we targeted the development and evaluation of broad-host-range CRISPR/Cas9 gene-editing tools in order to enhance the genetic-engineering capabilities of an industrially relevant methanotrophic biocatalyst. The CRISPR/Cas9 system developed in this study expands the genetic tools available to define molecular mechanisms in methanotrophic bacteria and has the potential to foster advances in the generation of novel biocatalysts to produce biofuels, platform chemicals, and high-value products from natural gas- and biogas-derived methane. Further, due to the broad-host-range applicability, these genetic tools may also enable innovative approaches to overcome the barriers associated with genetically engineering diverse, industrially promising nonmodel microorganisms.

scholarly journals Mining the Methylome Reveals Extensive Diversity in Staphylococcus epidermidis Restriction Modification

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Jean Y. H. Lee ◽  
Glen P. Carter ◽  
Sacha J. Pidot ◽  
Romain Guérillot ◽  
Torsten Seemann ◽  
...  
Keyword(s):  
Staphylococcus Epidermidis ◽  
Genetic Manipulation ◽  
Gene Arrangement ◽  
Type I ◽  
Content Type ◽  
Efficient Approach ◽  
Molecular Studies ◽  
Foreign Dna ◽  
Clinically Significant ◽  
Restriction Modification

ABSTRACT Staphylococcus epidermidis is a significant opportunistic pathogen of humans. Molecular studies in this species have been hampered by the presence of restriction-modification (RM) systems that limit introduction of foreign DNA. Here, we establish the complete genomes and methylomes for seven clinically significant, genetically diverse S. epidermidis isolates and perform the first systematic genomic analyses of the type I RM systems within both S. epidermidis and Staphylococcus aureus. Our analyses revealed marked differences in the gene arrangement, chromosomal location, and movement of type I RM systems between the two species. Unlike S. aureus, S. epidermidis type I RM systems demonstrate extensive diversity even within a single genetic lineage. This is contrary to current assumptions and has important implications for approaching the genetic manipulation of S. epidermidis. Using Escherichia coli plasmid artificial modification (PAM) to express S. epidermidis hsdMS, we readily overcame restriction barriers in S. epidermidis and achieved electroporation efficiencies equivalent to those of modification-deficient mutants. With these functional experiments, we demonstrated how genomic data can be used to predict both the functionality of type I RM systems and the potential for a strain to be electroporation proficient. We outline an efficient approach for the genetic manipulation of S. epidermidis strains from diverse genetic backgrounds, including those that have hitherto been intractable. Additionally, we identified S. epidermidis BPH0736, a naturally restriction-defective, clinically significant, multidrug-resistant ST2 isolate, as an ideal candidate for molecular studies. IMPORTANCE Staphylococcus epidermidis is a major cause of hospital-acquired infections, especially those related to implanted medical devices. Understanding how S. epidermidis causes disease and devising ways to combat these infections have been hindered by an inability to genetically manipulate clinically significant hospital-adapted strains. Here, we provide the first comprehensive analyses of the barriers to the uptake of foreign DNA in S. epidermidis and demonstrate that these are distinct from those described for S. aureus. Using these insights, we demonstrate an efficient approach for the genetic manipulation of S. epidermidis to enable the study of clinical isolates for the first time.

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.

scholarly journals Klebsiella pneumoniae Reduces SUMOylation To Limit Host Defense Responses

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Joana Sá-Pessoa ◽  
Kornelia Przybyszewska ◽  
Filipe Nuno Vasconcelos ◽  
Amy Dumigan ◽  
Christian G. Frank ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Klebsiella Pneumoniae ◽  
Glycogen Synthase ◽  
Defense Responses ◽  
Multidrug Resistant ◽  
Lung Epithelial Cells ◽  
Type I ◽  
Dependent Manner ◽  
Content Type ◽  
Medical Needs

ABSTRACT Klebsiella pneumoniae is an important cause of multidrug-resistant infections worldwide. Understanding the virulence mechanisms of K. pneumoniae is a priority and timely to design new therapeutics. Here, we demonstrate that K. pneumoniae limits the SUMOylation of host proteins in epithelial cells and macrophages (mouse and human) to subvert cell innate immunity. Mechanistically, in lung epithelial cells, Klebsiella increases the levels of the deSUMOylase SENP2 in the cytosol by affecting its K48 ubiquitylation and its subsequent degradation by the ubiquitin proteasome. This is dependent on Klebsiella preventing the NEDDylation of the Cullin-1 subunit of the ubiquitin ligase complex E3-SCF-βTrCP by exploiting the CSN5 deNEDDylase. Klebsiella induces the expression of CSN5 in an epidermal growth factor receptor (EGFR)-phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-extracellular signal-regulated kinase (ERK)-glycogen synthase kinase 3 beta (GSK3β) signaling pathway-dependent manner. In macrophages, Toll-like receptor 4 (TLR4)-TRAM-TRIF-induced type I interferon (IFN) via IFN receptor 1 (IFNAR1)-controlled signaling mediates Klebsiella-triggered decrease in the levels of SUMOylation via let-7 microRNAs (miRNAs). Our results revealed the crucial role played by Klebsiella polysaccharides, the capsule, and the lipopolysaccharide (LPS) O-polysaccharide, to decrease the levels of SUMO-conjugated proteins in epithelial cells and macrophages. A Klebsiella-induced decrease in SUMOylation promotes infection by limiting the activation of inflammatory responses and increasing intracellular survival in macrophages. IMPORTANCE Klebsiella pneumoniae has been singled out as an urgent threat to human health due to the increasing isolation of strains resistant to “last-line” antimicrobials, narrowing the treatment options against Klebsiella infections. Unfortunately, at present, we cannot identify candidate compounds in late-stage development for treatment of multidrug-resistant Klebsiella infections; this pathogen is exemplary of the mismatch between unmet medical needs and the current antimicrobial research and development pipeline. Furthermore, there is still limited evidence on K. pneumoniae pathogenesis at the molecular and cellular levels in the context of the interactions between bacterial pathogens and their hosts. In this research, we have uncovered a sophisticated strategy employed by Klebsiella to subvert the activation of immune defenses by controlling the modification of proteins. Our research may open opportunities to develop new therapeutics based on counteracting this Klebsiella-controlled immune evasion strategy.

scholarly journals A New Group of Phage Anti-CRISPR Genes Inhibits the Type I-E CRISPR-Cas System of Pseudomonas aeruginosa

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
April Pawluk ◽  
Joseph Bondy-Denomy ◽  
Vivian H. W. Cheung ◽  
Karen L. Maxwell ◽  
Alan R. Davidson
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Genetic Manipulation ◽  
Sequence Similarity ◽  
Adaptive Immune System ◽  
Resistance Mechanisms ◽  
Type I ◽  
Content Type ◽  
Genetic Elements ◽  
Small Proteins ◽  
Genomic Regions

ABSTRACT CRISPR-Cas systems are one of the most widespread phage resistance mechanisms in prokaryotes. Our lab recently identified the first examples of phage-borne anti-CRISPR genes that encode protein inhibitors of the type I-F CRISPR-Cas system of Pseudomonas aeruginosa. A key question arising from this work was whether there are other types of anti-CRISPR genes. In the current work, we address this question by demonstrating that some of the same phages carrying type I-F anti-CRISPR genes also possess genes that mediate inhibition of the type I-E CRISPR-Cas system of P. aeruginosa. We have discovered four distinct families of these type I-E anti-CRISPR genes. These genes do not inhibit the type I-F CRISPR-Cas system of P. aeruginosa or the type I-E system of Escherichia coli. Type I-E and I-F anti-CRISPR genes are located at the same position in the genomes of a large group of related P. aeruginosa phages, yet they are found in a variety of combinations and arrangements. We have also identified functional anti-CRISPR genes within nonprophage Pseudomonas genomic regions that are likely mobile genetic elements. This work emphasizes the potential importance of anti-CRISPR genes in phage evolution and lateral gene transfer and supports the hypothesis that more undiscovered families of anti-CRISPR genes exist. Finally, we provide the first demonstration that the type I-E CRISPR-Cas system of P. aeruginosa is naturally active without genetic manipulation, which contrasts with E. coli and other previously characterized I-E systems. IMPORTANCE The CRISPR-Cas system is an adaptive immune system possessed by the majority of prokaryotic organisms to combat potentially harmful foreign genetic elements. This study reports the discovery of bacteriophage-encoded anti-CRISPR genes that mediate inhibition of a well-studied subtype of CRISPR-Cas system. The four families of anti-CRISPR genes described here, which comprise only the second group of anti-CRISPR genes to be identified, encode small proteins that bear no sequence similarity to previously studied phage or bacterial proteins. Anti-CRISPR genes represent a newly discovered and intriguing facet of the ongoing evolutionary competition between phages and their bacterial hosts.

Development of techniques for the genetic manipulation of the gliding bacteria Lysobacter enzymogenes and Lysobacter brunescens

1996 ◽  
Vol 42 (9) ◽  
pp. 896-902 ◽  
Author(s):  
Danli Lin ◽  
Mark J. McBride
Keyword(s):  
Antibiotic Resistance ◽  
Gene Transfer ◽  
Genetic Analysis ◽  
Genetic Manipulation ◽  
Gliding Motility ◽  
Broad Host Range ◽  
Incompatibility Group ◽  
Gram Negative ◽  
Lysobacter Enzymogenes ◽  
Gliding Bacteria

Lysobacter enzymogenes and Lysobacter brunescens are Gram-negative gliding bacteria that belong to the γ subgroup of the proteobacteria. As a first step toward a molecular analysis of Lysobacter gliding motility, we developed techniques to genetically manipulate these bacteria. Cosmid pSUP106 of the broad host range incompatibility group Q (Inc Q) was introduced into L. enzymogenes and L. brunescens by conjugation and electroporation. pSUP106 replicated stably in both organisms and conferred antibiotic resistance. We also identified several other plasmids (pKT210, pH1JI) that functioned in L. enzymogenes and a transposon (mini-Tn5Sp) that functioned in L. brunescens. The identification of these tools allows genetic analysis of Lysobacter gliding motility, exoenzyme production, and production of antibiotics and other secondary metabolites.Key words: Lysobacter, gliding motility, gene transfer.

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