Using PATIMDB to Create Bacterial Transposon Insertion Mutant Libraries

The transition to reproduction is a crucial step in the life cycle of any organism. In Arabidopsis thaliana the establishment of reproductive growth can be divided into two phases: Firstly, cauline leaves with axillary meristems are formed and internode elongation begins. Secondly, lateral meristems develop into flowers with defined organs. Floral shoots are usually determinate and suppress the development of lateral shoots. Here, we describe a transposon insertion mutant in the Nossen accession with defects in floral development and growth. Most strikingly is the outgrowth of stems from the axillary bracts of the primary flower carrying secondary flowers. Therefore, we named this mutant flower-in-flower (fif). However, the transposon insertion in the annotated gene is not the cause for the fif phenotype. By means of classical and genome sequencing-based mapping, the mutation responsible for the fif phenotype was found to be in the LEAFY gene. The mutation, a G-to-A exchange in the second exon of LEAFY, creates a novel lfy allele and results in a cysteine-to-tyrosine exchange in the α1-helix of LEAFY’s DNA-binding domain. This exchange abolishes target DNA-binding, whereas subcellular localization and homomerization are not affected. To explain the strong fif phenotype against these molecular findings, several hypotheses are discussed.

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Effect of Metabolic Imbalance on Expression of Type III Secretion Genes in Pseudomonas aeruginosa

Infection and Immunity ◽

10.1128/iai.72.3.1383-1390.2004 ◽

2004 ◽

Vol 72 (3) ◽

pp. 1383-1390 ◽

Cited By ~ 84

Author(s):

Arne Rietsch ◽

Matthew C. Wolfgang ◽

John J. Mekalanos

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Cell Contact ◽

Insertion Mutant ◽

Insertion Mutation ◽

Type Iii ◽

Transposon Insertion ◽

Metabolic Imbalance ◽

Cell Expression

ABSTRACT The type III secretion system is a dedicated machinery used by many pathogens to deliver toxins directly into the cytoplasm of a target cell. Expression and secretion of the type III effectors are triggered by cell contact. In Pseudomonas aeruginosa and Yersinia spp., expression can be triggered in vitro by removing calcium from the medium. The mechanism underlying either mode of regulation is unclear. Here we characterize a transposon insertion mutant of P. aeruginosa PAO1 that displays a marked defect in cytotoxicity. The insertion is located upstream of several genes involved in histidine utilization and impedes the ability of PAO1 to intoxicate eukaryotic cells effectively in a type III-dependent fashion. This inhibition depends on the presence of histidine in the medium and appears to depend on the excessive uptake and catabolism of histidine. The defect in cytotoxicity is mirrored by a decrease in exoS expression. Other parameters such as growth or piliation are unaffected. The cytotoxicity defect is partially complemented by an insertion mutation in cbrA that also causes overexpression of cbrB. The cbrAB two-component system has been implicated in sensing and responding to a carbon-nitrogen imbalance. Taken together, these results suggest that the metabolic state of the cell influences expression of the type III regulon.

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Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis

mBio ◽

10.1128/mbio.02133-16 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 200

Author(s):

Michael A. DeJesus ◽

Elias R. Gerrick ◽

Weizhen Xu ◽

Sae Woong Park ◽

Jarukit E. Long ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Noncoding Rnas ◽

Open Reading Frames ◽

Insertion Mutant ◽

Statistical Interpretation ◽

Genomic Features ◽

Small Noncoding Rnas ◽

Transposon Insertion ◽

Biological Selection

ABSTRACT For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed. We sought to overcome this limitation by creating a completely saturated transposon library in Mycobacterium tuberculosis . In assessing the composition of this highly saturated library by deep sequencing, we discovered that a previously unknown sequence bias of the Himar1 element rendered approximately 9% of potential TA dinucleotide insertion sites less permissible for insertion. We used a hidden Markov model of essentiality that accounted for this unanticipated bias, allowing us to confidently evaluate the essentiality of features that contained as few as 2 TA sites, including open reading frames (ORF), experimentally identified noncoding RNAs, methylation sites, and promoters. In addition, several essential regions that did not correspond to known features were identified, suggesting uncharacterized functions that are necessary for growth. This work provides an authoritative catalog of essential regions of the M. tuberculosis genome and a statistical framework for applying saturating mutagenesis to other bacteria. IMPORTANCE Sequencing of transposon-insertion mutant libraries has become a widely used tool for probing the functions of genes under various conditions. The Himar1 transposon is generally believed to insert with equal probabilities at all TA dinucleotides, and therefore its absence in a mutant library is taken to indicate biological selection against the corresponding mutant. Through sequencing of a saturated Himar1 library, we found evidence that TA dinucleotides are not equally permissive for insertion. The insertion bias was observed in multiple prokaryotes and influences the statistical interpretation of transposon insertion (TnSeq) data and characterization of essential genomic regions. Using these insights, we analyzed a fully saturated TnSeq library for M. tuberculosis , enabling us to generate a comprehensive catalog of in vitro essentiality, including ORFs smaller than those found in any previous study, small (noncoding) RNAs (sRNAs), promoters, and other genomic features.

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Generation of mariner-based transposon insertion mutant library of Bacillus sphaericus 2297 and investigation of genes involved in sporulation and mosquito-larvicidal crystal protein synthesis

FEMS Microbiology Letters ◽

10.1111/j.1574-6968.2012.02539.x ◽

2012 ◽

Vol 330 (2) ◽

pp. 105-112 ◽

Cited By ~ 9

Author(s):

Yiming Wu ◽

Xiaomin Hu ◽

Yong Ge ◽

Dasheng Zheng ◽

Zhiming Yuan

Keyword(s):

Protein Synthesis ◽

Bacillus Sphaericus ◽

Crystal Protein ◽

Insertion Mutant ◽

Mutant Library ◽

Transposon Insertion

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Genes Contributing to Staphylococcus aureus Fitness in Abscess- and Infection-Related Ecologies

mBio ◽

10.1128/mbio.01729-14 ◽

2014 ◽

Vol 5 (5) ◽

Cited By ~ 76

Author(s):

Michael D. Valentino ◽

Lucy Foulston ◽

Ama Sadaka ◽

Veronica N. Kos ◽

Regis A. Villet ◽

...

Keyword(s):

Staphylococcus Aureus ◽

New Drugs ◽

Insertion Mutant ◽

Mutant Library ◽

Entire Genome ◽

Antibiotic Resistant ◽

Content Type ◽

Transposon Insertion ◽

Genes Encoding ◽

Novel Targets

ABSTRACTStaphylococcus aureusis a leading cause of both community- and hospital-acquired infections that are increasingly antibiotic resistant. The emergence ofS. aureusresistance to even last-line antibiotics heightens the need for the development of new drugs with novel targets. We generated a highly saturated transposon insertion mutant library in the genome ofS. aureusand used Tn-seq analysis to probe the entire genome, with unprecedented resolution and sensitivity, for genes of importance in infection. We further identified genes contributing to fitness in various infected compartments (blood and ocular fluids) and compared them to genes required for growth in rich medium. This resulted in the identification of 426 genes that were important forS. aureusfitness during growth in infection models, including 71 genes that could be considered essential for survival specifically during infection. These findings highlight novel as well as previously known genes encoding virulence traits and metabolic pathways important forS. aureusproliferation at sites of infection, which may represent new therapeutic targets.IMPORTANCEStaphylococcus aureuscontinues to be a leading cause of antibiotic-resistant community and nosocomial infection. With the bacterium’s acquisition of resistance to methicillin and, more recently, vancomycin, the need for the development of new drugs with novel targets is urgent. Applying a highly saturated Tn-seq mutant library to analyze fitness and growth requirements in a murine abscess and in various infection-relevant fluids, we identifiedS. aureustraits that enable it to survive and proliferate during infection. This identifies potential new targeting opportunities for the development of novel therapeutics.

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Construction and Characterization of Transposon Insertion Mutations in Corynebacterium diphtheriae That Affect Expression of the Diphtheria Toxin Repressor (DtxR)

Journal of Bacteriology ◽

10.1128/jb.184.20.5723-5732.2002 ◽

2002 ◽

Vol 184 (20) ◽

pp. 5723-5732 ◽

Cited By ~ 32

Author(s):

Diana Marra Oram ◽

Ana Avdalovic ◽

Randall K. Holmes

Keyword(s):

Oxidative Stress ◽

Diphtheria Toxin ◽

Corynebacterium Diphtheriae ◽

Insertion Mutant ◽

Toxin Gene ◽

Wild Type ◽

Transposon Insertion ◽

Diphtheria Toxin Repressor ◽

Mutant Strains

ABSTRACT Transcription of the bacteriophage-borne diphtheria toxin gene tox is negatively regulated, in response to intracellular Fe2+ concentration, by the chromosomally encoded diphtheria toxin repressor (DtxR). Due to a scarcity of tools, genetic analysis of Corynebacterium diphtheriae has primarily relied on analysis of chemically induced and spontaneously occurring mutants and on the results of experiments with C. diphtheriae genes cloned in Escherichia coli or analyzed in vitro. We modified a Tn5-based mutagenesis technique for use with C. diphtheriae, and we used it to construct the first transposon insertion libraries in the chromosome of this gram-positive pathogen. We isolated two insertions that affected expression of DtxR, one 121 bp upstream of dtxR and the other within an essential region of the dtxR coding sequence, indicating for the first time that dtxR is a dispensable gene in C. diphtheriae. Both mutant strains secrete diphtheria toxin when grown in medium containing sufficient iron to repress secretion of diphtheria toxin by wild-type C. diphtheriae. The upstream insertion mutant still produces DtxR in decreased amounts and regulates siderophore secretion in response to iron in a manner similar to its wild-type parent. The mutant containing the transposon insertion within dtxR does not produce DtxR and overproduces siderophore in the presence of iron. Differences in the ability of the two mutant strains to survive oxidative stress also indicated that the upstream insertion retained slight DtxR activity, whereas the insertion within dtxR abolished DtxR activity. This is the first evidence that DtxR plays a role in protecting the cell from oxidative stress.

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Comprehensive MALDI-TOF Biotyping of the Non-Redundant Harvard Pseudomonas aeruginosa PA14 Transposon Insertion Mutant Library

PLoS ONE ◽

10.1371/journal.pone.0117144 ◽

2015 ◽

Vol 10 (2) ◽

pp. e0117144 ◽

Cited By ~ 5

Author(s):

Tonio Oumeraci ◽

Vanessa Jensen ◽

Steven R. Talbot ◽

Winfried Hofmann ◽

Markus Kostrzewa ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Insertion Mutant ◽

Mutant Library ◽

Maldi Tof ◽

Transposon Insertion

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Degradation of Alkyl Methyl Ketones by Pseudomonas veronii MEK700

Journal of Bacteriology ◽

10.1128/jb.01279-06 ◽

2007 ◽

Vol 189 (10) ◽

pp. 3759-3767 ◽

Cited By ~ 55

Author(s):

Christina Onaca ◽

Martin Kieninger ◽

Karl-H. Engesser ◽

Josef Altenbuchner

Keyword(s):

Ethyl Acetate ◽

Methyl Ketone ◽

Glycogen Synthase ◽

Short Chain ◽

Insertion Mutant ◽

Methyl Ketones ◽

Monooxygenase Activity ◽

Transposon Insertion ◽

Waste Air ◽

Pseudomonas Veronii

ABSTRACT Pseudomonas veronii MEK700 was isolated from a biotrickling filter cleaning 2-butanone-loaded waste air. The strain is able to grow on 2-butanone and 2-hexanol. The genes for degradation of short chain alkyl methyl ketones were identified by transposon mutagenesis using a newly designed transposon, mini-Tn5495, and cloned in Escherichia coli. DNA sequence analysis of a 15-kb fragment revealed three genes involved in methyl ketone degradation. The deduced amino acid sequence of the first gene, mekA, had high similarity to Baeyer-Villiger monooxygenases; the protein of the second gene, mekB, had similarity to homoserine acetyltransferases; the third gene, mekR, encoded a putative transcriptional activator of the AraC/XylS family. The three genes were located between two gene groups: one comprising a putative phosphoenolpyruvate synthase and glycogen synthase, and the other eight genes for the subunits of an ATPase. Inactivation of mekA and mekB by insertion of the mini-transposon abolished growth of P. veronii MEK700 on 2-butanone and 2-hexanol. The involvement of mekR in methyl ketone degradation was observed by heterologous expression of mekA and mekB in Pseudomonas putida. A fragment containing mekA and mekB on a plasmid was not sufficient to allow P. putida KT2440 to grow on 2-butanone. Not until all three genes were assembled in the recombinant P. putida was it able to use 2-butanone as carbon source. The Baeyer-Villiger monooxygenase activity of MekA was clearly demonstrated by incubating a mekB transposon insertion mutant of P. veronii with 2-butanone. Hereby, ethyl acetate was accumulated. To our knowledge, this is the first time that ethyl acetate by gas chromatographic analysis has been definitely demonstrated to be an intermediate of MEK degradation. The mekB-encoded protein was heterologously expressed in E. coli and purified by immobilized metal affinity chromatography. The protein exhibited high esterase activity towards short chain esters like ethyl acetate and 4-nitrophenyl acetate.

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Yersinia enterocolitica ClpB Affects Levels of Invasin and Motility

Journal of Bacteriology ◽

10.1128/jb.182.19.5563-5571.2000 ◽

2000 ◽

Vol 182 (19) ◽

pp. 5563-5571 ◽

Cited By ~ 16

Author(s):

Julie L. Badger ◽

Briana M. Young ◽

Andrew J. Darwin ◽

Virginia L. Miller

Keyword(s):

Yersinia Enterocolitica ◽

Insertion Mutant ◽

Gene Transcript ◽

Null Mutant ◽

Early Stationary Phase ◽

Flagellin Gene ◽

Transposon Insertion ◽

Isolation And Characterization ◽

Mutant Phenotypes

ABSTRACT Expression of the Yersinia enterocolitica inv gene is dependent on growth phase and temperature. inv is maximally expressed at 23°C in late-exponential- to early-stationary-phase cultures. We previously reported the isolation of a Y. enterocolitica mutant (JB1A8v) that shows a decrease in invasin levels yet is hypermotile when grown at 23°C. JB1A8v has a transposon insertion within uvrC. Described here is the isolation and characterization of a clone that suppresses these mutant phenotypes of the uvrC mutant JB1A8v. This suppressing clone encodes ClpB (a Clp ATPase homologue). The Y. enterocolitica ClpB homologue is 30 to 40% identical to the ClpB proteins from various bacteria but is 80% identical to one of the two ClpB homologues ofYersinia pestis. AclpB::TnMax2 insertion mutant (JB69Qv) was constructed and determined to be deficient in invasin production and nonmotile when grown at 23°C. Analysis ofinv and fleB (flagellin gene) transcript levels in JB69Qv suggested that ClpB has both transcriptional and posttranscriptional effects. In contrast, a clpB null mutant, BY1v, had no effect on invasin levels or motility. A model accounting for these observations is presented.

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