scholarly journals Engineering the Genome of Thermus thermophilus Using a Counterselectable Marker

2015 ◽  
Vol 197 (6) ◽  
pp. 1135-1144 ◽  
Author(s):  
Jennifer F. Carr ◽  
Michael E. Danziger ◽  
Athena L. Huang ◽  
Albert E. Dahlberg ◽  
Steven T. Gregory
Keyword(s):  
Resistance Gene ◽  
Large Scale ◽  
Genetic Manipulation ◽  
Thermus Thermophilus ◽  
Point Mutations ◽  
Trna Synthetase ◽  
Biological Research ◽  
Lethal Mutant ◽  
Α Subunit ◽  
Content Type

ABSTRACTThermus thermophilusis an extremely thermophilic bacterium that is widely used as a model thermophile, in large part due to its amenability to genetic manipulation. Here we describe a system for the introduction of genomic point mutations or deletions using a counterselectable marker consisting of a conditionally lethal mutant allele ofpheSencoding the phenylalanyl-tRNA synthetase α-subunit. Mutant PheS with an A294G amino acid substitution renders cells sensitive to the phenylalanine analogp-chlorophenylalanine. Insertion of the mutantpheSallele via a linked kanamycin resistance gene into a chromosomal locus provides a gene replacement intermediate that can be removed by homologous recombination usingp-chlorophenylalanine as a counterselective agent. This selection is suitable for the sequential introduction of multiple mutations to produce a final strain unmarked by an antibiotic resistance gene. We demonstrated the utility of this method by constructing strains bearing either a point mutation in or a precise deletion of therrsBgene encoding 16S rRNA. We also used this selection to identify spontaneous, large-scale deletions in the pTT27 megaplasmid, apparently mediated by either of theT. thermophilusinsertion elements ISTth7and ISTth8. One such deletion removed 121 kb, including 118 genes, or over half of pTT27, including multiple sugar hydrolase genes, and facilitated the development of a plasmid-encoded reporter system based on β-galactosidase. The ability to introduce mutations ranging from single base substitutions to large-scale deletions provides a potentially powerful tool for engineering the genome ofT. thermophilusand possibly other thermophiles as well.IMPORTANCEThermus thermophilusis an extreme thermophile that has played an important part in the development of both biotechnology and basic biological research. Its suitability as a genetic model system is established by its natural competence for transformation, but the scarcity of genetic tools limits the kinds of manipulations that can currently be performed. We have developed a counterselectable marker that allows the introduction of unmarked deletions and point mutations into theT. thermophilusgenome. We find that this marker can also be used to select large chromosomal deletions apparently resulting from aberrant transposition of endogenous insertion sequences. This system has the potential to advance the genetic manipulation of this important model organism.

scholarly journals Efficient Counterselection forMethylococcus capsulatus(Bath) by Using a MutatedpheSGene

2018 ◽  
Vol 84 (23) ◽  
Author(s):  
Masahito Ishikawa ◽  
Sho Yokoe ◽  
Souichiro Kato ◽  
Katsutoshi Hori
Keyword(s):  
Genetic Manipulation ◽  
Point Mutations ◽  
Trna Synthetase ◽  
Efficient Production ◽  
Gram Negative Bacteria ◽  
Methylococcus Capsulatus ◽  
Α Subunit ◽  
Gram Negative ◽  
Content Type ◽  
Research Fields

ABSTRACTMethylococcus capsulatus(Bath) is a representative gammaproteobacterial methanotroph that has been studied extensively in diverse research fields. ThesacBgene, which encodes levansucrase, causing cell death in the presence of sucrose, is widely used as a counterselectable marker for disruption of a target gene in Gram-negative bacteria. However,sacBis not applicable to all Gram-negative bacteria, and its efficiency for the counterselection ofM. capsulatus(Bath) is low. Here, we report the construction of an alternative counterselectable marker,pheS*, by introduction of two point mutations (A306G and T252A) into thepheSgene fromM. capsulatus(Bath), which encodes the α-subunit of phenylalanyl-tRNA synthetase. The transformant harboringpheS* on an expression plasmid showed sensitivity to 10 mMp-chloro-phenylalanine, whereas the transformant harboring an empty plasmid showed no sensitivity, indicating the availability ofpheS* as a counterselectable marker inM. capsulatus(Bath). To validate the utility of thepheS* marker in counterselection, we attempted to obtain an unmarked mutant ofxoxF, a gene encoding the major subunit of Xox methanol dehydrogenase, which we failed to obtain by counterselection using thesacBmarker. PCR, immunodetection using an anti-XoxF antiserum, and a cell growth assay in the absence of calcium demonstrated successful disruption of thexoxFgene inM. capsulatus(Bath). The difference in counterselection efficiencies of the markers indicated thatpheS* is more suitable thansacBfor counterselection inM. capsulatus(Bath). This study provides a new genetic tool enabling efficient counterselection inM. capsulatus(Bath).IMPORTANCEMethanotrophs have long been considered promising strains for biologically reducing methane from the environment and converting it into valuable products, because they can oxidize methane at ambient temperatures and pressures. Although several methodologies and tools for the genetic manipulation of methanotrophs have been developed, their mutagenic efficiency remains lower than that of tractable strains such asEscherichia coli. Therefore, further improvements are still desired. The significance of our study is that we increased the efficiency of counterselection inM. capsulatus(Bath) by employingpheS*, which was newly constructed as a counterselectable marker. This will allow for the efficient production of gene-disrupted and gene-integrated mutants ofM. capsulatus(Bath). We anticipate that this counterselection system will be utilized widely by the methanotroph research community, leading to improved productivity of methane-based bioproduction and new insights into methanotrophy.

scholarly journals An Effective Counterselection System for Listeria monocytogenes and Its Use To Characterize the Monocin Genomic Region of Strain 10403S

2016 ◽  
Vol 83 (6) ◽  
Author(s):  
Tal Argov ◽  
Lev Rabinovich ◽  
Nadejda Sigal ◽  
Anat A. Herskovits
Keyword(s):  
Listeria Monocytogenes ◽  
Point Mutations ◽  
Model Organism ◽  
Trna Synthetase ◽  
Genomic Region ◽  
Selection Marker ◽  
Content Type ◽  
Allelic Exchange ◽  
Suicide Vector ◽  
Screening Systems

ABSTRACT Construction of Listeria monocytogenes mutants by allelic exchange has been laborious and time-consuming due to lack of proficient selection markers for the final recombination event, that is, a marker conveying substance sensitivity to the bacteria bearing it, enabling the exclusion of merodiploids and selection for plasmid loss. In order to address this issue, we engineered a counterselection marker based on a mutated phenylalanyl-tRNA synthetase gene (pheS*). This mutation renders the phenylalanine-binding site of the enzyme more promiscuous and allows the binding of the toxic p-chloro-phenylalanine analog (p-Cl-phe) as a substrate. When pheS* is introduced into L. monocytogenes and highly expressed under control of a constitutively active promoter, the bacteria become sensitive to p-Cl-phe supplemented in the medium. This enabled us to utilize pheS* as a negative selection marker and generate a novel, efficient suicide vector for allelic exchange in L. monocytogenes. We used this vector to investigate the monocin genomic region in L. monocytogenes strain 10403S by constructing deletion mutants of the region. We have found this region to be active and to cause bacterial lysis upon mitomycin C treatment. The future applications of such an effective counterselection system, which does not require any background genomic alterations, are vast, as it can be modularly used in various selection systems (e.g., genetic screens). We expect this counterselection marker to be a valuable genetic tool in research on L. monocytogenes. IMPORTANCE L. monocytogenes is an opportunistic intracellular pathogen and a widely studied model organism. An efficient counterselection marker is a long-standing need in Listeria research for improving the ability to design and perform various genetic manipulations and screening systems for different purposes. We report the construction and utilization of an efficient suicide vector for allelic exchange which can be conjugated, leaves no marker in the bacterial chromosome, and does not require the use of sometimes leaky inducible promoters. This highly efficient genome editing tool for L. monocytogenes will allow for rapid sequential mutagenesis, introduction of point mutations, and design of screening systems. We anticipate that it will be extensively used by the research community and yield novel insights into the diverse fields studied using this model organism.

scholarly journals Applications of Bacillus subtilis Spores in Biotechnology and Advanced Materials

2020 ◽  
Vol 86 (17) ◽  
Author(s):  
Xiaopei Zhang ◽  
Amal Al-Dossary ◽  
Myer Hussain ◽  
Peter Setlow ◽  
Jiahe Li
Keyword(s):  
Bacillus Subtilis ◽  
Large Scale ◽  
Genetic Manipulation ◽  
Industrial Applications ◽  
Advanced Materials ◽  
Content Type ◽  
Interdisciplinary Approaches ◽  
Food Shortages ◽  
Material Sciences ◽  
Basic Studies

ABSTRACT The bacterium Bacillus subtilis has long been an important subject for basic studies. However, this organism has also had industrial applications due to its easy genetic manipulation, favorable culturing characteristics for large‐scale fermentation, superior capacity for protein secretion, and generally recognized as safe (GRAS) status. In addition, as the metabolically dormant form of B. subtilis, its spores have attracted great interest due to their extreme resistance to many environmental stresses, which makes spores a novel platform for a variety of applications. In this review, we summarize both conventional and emerging applications of B. subtilis spores, with a focus on how their unique characteristics have led to innovative applications in many areas of technology, including generation of stable and recyclable enzymes, synthetic biology, drug delivery, and material sciences. Ultimately, this review hopes to inspire the scientific community to leverage interdisciplinary approaches using spores to address global concerns about food shortages, environmental protection, and health care.

scholarly journals Large-Scale Identification of AbaR-Type Genomic Islands in Acinetobacter baumannii Reveals Diverse Insertion Sites and Clonal Lineage-Specific Antimicrobial Resistance Gene Profiles

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
Dexi Bi ◽  
Ruting Xie ◽  
Jiayi Zheng ◽  
Huiqiong Yang ◽  
Xingchen Zhu ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Gene ◽  
Large Scale ◽  
Genomic Islands ◽  
Clonal Lineage ◽  
Content Type ◽  
Gene Profiles ◽  
Antimicrobial Resistance Genes ◽  
Antimicrobial Resistance Gene ◽  
Insertion Sites

ABSTRACT AbaR-type genomic islands (AbaRs) are important elements responsible for antimicrobial resistance in Acinetobacter baumannii. This study performed a large-scale identification of AbaRs to understand their distribution and compositions of antimicrobial resistance genes. We identified 2.89-kb left-end and 1.87-kb right-end conserved sequences (CSs) and developed a bioinformatics approach to identify AbaRs, using the CSs as signatures, in 3,148 publicly available genomes. AbaRs were prevalent in A. baumannii, being found in 2,091 genomes. They were sparse in other Acinetobacter species and confined only to this genus. Results from 111 complete genomes showed that over 85% of AbaRs resided on chromosomes. The external flanks adjacent to the inverted repeats available in all identified CSs were mapped to an AbaR-free chromosome or searched in the NCBI database for empty loci to define insertion sites. Surprisingly, 84 insertion sites with diverse origins were revealed, including 51 scattered on the chromosome, 20 plasmid borne, 12 located on prophages, transposons, ISAba1, complex AbaRs, and genomic islands of other types, and one uncharacterized, and some were strongly associated with clonal lineages. Finally, we found 994 antimicrobial resistance genes covering 28 unique genes from 70.9% (299/422) of intact AbaRs currently available. The resistance gene profiles displayed an apparent clonal lineage-specific pattern, highlighting the distinct features of AbaRs in global clone 1 (GC1) and GC2. The tet(B) gene was highly specific to the AbaRs in GC2. In conclusion, AbaRs have diverse insertion sites on the chromosome and mobile genetic elements (MGEs) and display distinct antimicrobial resistance gene profiles in different clonal lineages.

scholarly journals Proteome Analyses of Strains ATCC 51142 and PCC 7822 of the Diazotrophic Cyanobacterium Cyanothece sp. under Culture Conditions Resulting in Enhanced H2Production

2012 ◽  
Vol 79 (4) ◽  
pp. 1070-1077 ◽  
Author(s):  
Uma K. Aryal ◽  
Stephen J. Callister ◽  
Sujata Mishra ◽  
Xiaohui Zhang ◽  
Janani I. Shutthanandan ◽  
...  
Keyword(s):  
Large Scale ◽  
Genetic Manipulation ◽  
Nitrogenase Activity ◽  
Calvin Cycle ◽  
Proteome Analysis ◽  
Culture Conditions ◽  
Pentose Phosphate ◽  
Diurnal Changes ◽  
Content Type ◽  
Diurnal Cycles

ABSTRACTCultures of the cyanobacterial genusCyanothecehave been shown to produce high levels of biohydrogen. These strains are diazotrophic and undergo pronounced diurnal cycles when grown under N2-fixing conditions in light-dark cycles. We seek to better understand the way in which proteins respond to these diurnal changes, and we performed quantitative proteome analysis ofCyanothecesp. strains ATCC 51142 and PCC 7822 grown under 8 different nutritional conditions. Nitrogenase expression was limited to N2-fixing conditions, and in the absence of glycerol, nitrogenase gene expression was linked to the dark period. However, glycerol induced expression of nitrogenase during part of the light period, together with cytochromecoxidase (Cox), glycogen phosphorylase (Glp), and glycolytic and pentose phosphate pathway (PPP) enzymes. This indicated that nitrogenase expression in the light was facilitated via higher levels of respiration and glycogen breakdown. Key enzymes of the Calvin cycle were inhibited inCyanotheceATCC 51142 in the presence of glycerol under H2-producing conditions, suggesting a competition between these sources of carbon. However, inCyanothecePCC 7822, the Calvin cycle still played a role in cofactor recycling during H2production. Our data comprise the first comprehensive profiling of proteome changes inCyanothecePCC 7822 and allow an in-depth comparative analysis of major physiological and biochemical processes that influence H2production in both strains. Our results revealed many previously uncharacterized proteins that may play a role in nitrogenase activity and in other metabolic pathways and may provide suitable targets for genetic manipulation that would lead to improvement of large-scale H2production.

scholarly journals Genomic Characterization of a Pattern D Streptococcus pyogenesemm53Isolate Reveals a Genetic Rationale for Invasive Skin Tropicity

2016 ◽  
Vol 198 (12) ◽  
pp. 1712-1724 ◽  
Author(s):  
Yun-Juan Bao ◽  
Zhong Liang ◽  
Jeffrey A. Mayfield ◽  
Deborah L. Donahue ◽  
Katelyn E. Carothers ◽  
...  
Keyword(s):  
Large Scale ◽  
Point Mutations ◽  
Signal Peptides ◽  
Nucleotide Polymorphisms ◽  
Circular Chromosome ◽  
Content Type ◽  
Group A ◽  
Hyaluronic Acid Capsule ◽  
Type 3 ◽  
Genome Scale

ABSTRACTThe genome of an invasive skin-tropic strain (AP53) of serotype M53 group AStreptococcus pyogenes(GAS) is composed of a circular chromosome of 1,860,554 bp and carries genetic markers for infection at skin locales,viz.,emmgene family pattern D and FCT type 3. Through genome-scale comparisons of AP53 with other GAS genomes, we identified 596 candidate single-nucleotide polymorphisms (SNPs) that reveal a potential genetic basis for skin tropism. The genome of AP53 differed by ∼30 point mutations from a noninvasive pattern D serotype M53 strain (Alab49), 4 of which are located in virulence genes. One pseudogene, yielding an inactive sensor kinase (CovS−) of the two-component transcriptional regulator CovRS, a major determinant for invasiveness, severely attenuated the expression of the secreted cysteine protease SpeB and enhanced the expression of the hyaluronic acid capsule compared to the isogenic noninvasive AP53/CovS+strain. The collagen-binding protein transcriptsclBdiffered in the number of 5′-pentanucleotide repeats in the signal peptides of AP53 and Alab49 (9 versus 15), translating into different lengths of their signal peptides, which nonetheless maintained a full-length translatable coding frame. Furthermore, GAS strain AP53 acquired two phages that are absent in Alab49. One such phage (ΦAP53.2) contains the known virulence factor superantigen exotoxin gene tandemspeK-slaA. Overall, we conclude that this bacterium has evolved in multiple ways, including mutational variations of regulatory genes, short-tandem-repeat polymorphisms, large-scale genomic alterations, and acquisition of phages, all of which may be involved in shaping the adaptation of GAS in specific infectious environments and contribute to its enhanced virulence.IMPORTANCEInfectious strains ofS. pyogenes(GAS) are classified by their serotypes, relating to the surface M protein, theemm-like subfamily pattern, and their tropicity toward the nasopharynx and/or skin. It is generally agreed that M proteins from pattern D strains, which also directly bind human host plasminogen, are skin tropic. We have sequenced and characterized the genome of an invasive pattern D GAS strain (AP53) in comparison to a very similar strain (Alab49) that is noninvasive and developed a genomic rationale as to possible reasons for the skin tropicity of these two strains and the greater invasiveness of AP53.

scholarly journals Toward Improvement of Erythromycin A Production in an Industrial Saccharopolyspora erythraea Strain via Facilitation of Genetic Manipulation with an ArtificialattBSite for Specific Recombination

2011 ◽  
Vol 77 (21) ◽  
pp. 7508-7516 ◽  
Author(s):  
Jiequn Wu ◽  
Qinglin Zhang ◽  
Wei Deng ◽  
Jiangchao Qian ◽  
Siliang Zhang ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Large Scale ◽  
Genetic Manipulation ◽  
Saccharopolyspora Erythraea ◽  
Scale Production ◽  
Content Type ◽  
Large Scale Production ◽  
Endogenous Genes ◽  
Erythromycin A ◽  
Adenosylmethionine Synthetase

ABSTRACTLarge-scale production of erythromycin A (Er-A) relies on the organismSaccharopolyspora erythraea, in which lack of a typicalattBsite largely impedes the application of phage ΦC31 integrase-mediated recombination into site-specific engineering. We herein report construction of an artificialattBsite in an industrialS. erythraeastrain, HL3168 E3, in an effort to break the bottleneck previously encountered during genetic manipulation mainly from homologous or unpredictable nonspecific integration. Replacement of a cryptic gene,nrps1-1, with a cassette containing eightattBDNA sequences did not affect the high Er-producing ability, setting the stage for precisely engineering the industrial Er-producing strain for foreign DNA introduction with a reliable conjugation frequency. Transfer of either exogenous or endogenous genes of importance to Er-A biosynthesis, including theS-adenosylmethionine synthetase gene for positive regulation,vhbfor increasing the oxygen supply, and two tailoring genes,eryKanderyG, for optimizing the biotransformation at the late stage, was achieved by taking advantage of this facility, allowing systematic improvement of Er-A production as well as elimination of the by-products Er-B and Er-C in fermentation. The strategy developed here can generally be applicable to other strains that lack theattBsite.

scholarly journals Atovaquone-Proguanil Remains a Potential Stopgap Therapy for Multidrug-Resistant Plasmodium falciparum in Areas along the Thai-Cambodian Border

2015 ◽  
Vol 60 (3) ◽  
pp. 1896-1898 ◽  
Author(s):  
David L. Saunders ◽  
Suwanna Chaorattanakawee ◽  
Panita Gosi ◽  
Charlotte Lanteri ◽  
Sok Somethy ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Resistance Gene ◽  
Point Mutations ◽  
Multidrug Resistant ◽  
Blood Stage ◽  
Alternative Treatments ◽  
Content Type

Our recent report of dihydroartemisinin-piperaquine failure to treatPlasmodium falciparuminfections in Cambodia adds new urgency to the search for alternative treatments. Despite dihydroartemisinin-piperaquine failure, and higher piperaquine 50% inhibitory concentrations (IC50s) following reanalysis than those previously reported,P. falciparumremained sensitive to atovaquone (ATQ)in vitro. There were no point mutations in theP. falciparumcytochromebATQ resistance gene. Mefloquine, artemisinin, chloroquine, and quinine IC50s remained comparable to those from other recent reports. Atovaquone-proguanil may be a useful stopgap but remains susceptible to developing resistance when used as blood-stage therapy.

scholarly journals A Novel Tryptophanyl-tRNA Synthetase Gene Confers High-Level Resistance to Indolmycin

2009 ◽  
Vol 53 (9) ◽  
pp. 3972-3980 ◽  
Author(s):  
James J. Vecchione ◽  
Jason K. Sello
Keyword(s):  
Resistance Gene ◽  
Resistance Genes ◽  
Point Mutations ◽  
Trna Synthetase ◽  
Sensitive Strain ◽  
Antibacterial Drug ◽  
Gene Encoding ◽  
Trna Synthetases ◽  
High Level ◽  
Level Resistance

ABSTRACT Indolmycin, a potential antibacterial drug, competitively inhibits bacterial tryptophanyl-tRNA synthetases. An effort to identify indolmycin resistance genes led to the discovery of a gene encoding an indolmycin-resistant isoform of tryptophanyl-tRNA synthetase. Overexpression of this gene in an indolmycin-sensitive strain increased the indolmycin MIC 60-fold. Its transcription and distribution in various bacterial genera were assessed. The level of resistance conferred by this gene was compared to that of a known indolmycin resistance gene and to those of genes with resistance-conferring point mutations.

scholarly journals Interrogating Mitochondrial Biology and Disease Using CRISPR/Cas9 Gene Editing

Genes ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1604
Author(s):  
Jia Xin Tang ◽  
Angela Pyle ◽  
Robert W. Taylor ◽  
Monika Oláhová
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Gene Editing ◽  
Genetic Manipulation ◽  
Mitochondrial Diseases ◽  
Biological Research ◽  
Genetic Changes ◽  
Mitochondrial Proteome ◽  
A Genome ◽  
Indispensable Tool

Mitochondrial disease originates from genetic changes that impact human bodily functions by disrupting the mitochondrial oxidative phosphorylation system. MitoCarta is a curated and published inventory that sheds light on the mitochondrial proteome, but the function of some mitochondrially-localised proteins remains poorly characterised. Consequently, various gene editing systems have been employed to uncover the involvement of these proteins in mitochondrial biology and disease. CRISPR/Cas9 is an efficient, versatile, and highly accurate genome editing tool that was first introduced over a decade ago and has since become an indispensable tool for targeted genetic manipulation in biological research. The broad spectrum of CRISPR/Cas9 applications serves as an attractive and tractable system to study genes and pathways that are essential for the regulation and maintenance of mitochondrial health. It has opened possibilities of generating reliable cell and animal models of human disease, and with further exploitation of the technology, large-scale genomic screenings have uncovered a wealth of fundamental mechanistic insights. In this review, we describe the applications of CRISPR/Cas9 system as a genome editing tool to uncover new insights into pathomechanisms of mitochondrial diseases and/or biological processes involved in mitochondrial function.

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