Sequence-Defined Transposon Mutant Library of Burkholderia thailandensis

Gallagher, Larry A.; Ramage, Elizabeth; Patrapuvich, Rapatbhorn; Weiss, Eli; Brittnacher, Mitch; Manoil, Colin

doi:10.1128/mbio.00604-13

Cited by 91 publications

(103 citation statements)

References 51 publications

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“…malL-lacZ is not expressed under standard growth conditions and served as a negative control. A lacZ reporter in btaK, which is quorum-sensing-regulated and expressed at high cell densities, provided a positive control (Methods) (21)(22)(23). A number of growth parameters, including temperature, growth media, agitation rates, total volume, and initial inoculum were assessed and provided an optimized, robust screening assay with a Z′ value of 0.51 in a 384-well format (SI Appendix, Table S1).…”

Section: Resultsmentioning

confidence: 99%

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High-throughput platform for the discovery of elicitors of silent bacterial gene clusters

Seyedsayamdost

2014

Proc. Natl. Acad. Sci. U.S.A.

248

235

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Over the past decade, bacterial genome sequences have revealed an immense reservoir of biosynthetic gene clusters, sets of contiguous genes that have the potential to produce drugs or drug-like molecules. However, the majority of these gene clusters appear to be inactive for unknown reasons prompting terms such as "cryptic" or "silent" to describe them. Because natural products have been a major source of therapeutic molecules, methods that rationally activate these silent clusters would have a profound impact on drug discovery. Herein, a new strategy is outlined for awakening silent gene clusters using small molecule elicitors. In this method, a genetic reporter construct affords a facile read-out for activation of the silent cluster of interest, while high-throughput screening of small molecule libraries provides potential inducers. This approach was applied to two cryptic gene clusters in the pathogenic model Burkholderia thailandensis. The results not only demonstrate a prominent activation of these two clusters, but also reveal that the majority of elicitors are themselves antibiotics, most in common clinical use. Antibiotics, which kill B. thailandensis at high concentrations, act as inducers of secondary metabolism at low concentrations. One of these antibiotics, trimethoprim, served as a global activator of secondary metabolism by inducing at least five biosynthetic pathways. Further application of this strategy promises to uncover the regulatory networks that activate silent gene clusters while at the same time providing access to the vast array of cryptic molecules found in bacteria.natural products discovery | cryptic metabolites | malleilactone

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Section: Resultsmentioning

confidence: 99%

“…However, the elicitors that lead to activation of this silent virulence factor are not known. To monitor expression of this cluster, a translational lacZ fusion to malL, a gene essential for the biosynthesis of 1, was used (hereafter malL-lacZ) (21). malL-lacZ is not expressed under standard growth conditions and served as a negative control.…”

Section: Resultsmentioning

confidence: 99%

High-throughput platform for the discovery of elicitors of silent bacterial gene clusters

Seyedsayamdost

2014

Proc. Natl. Acad. Sci. U.S.A.

248

235

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“…We used B. thailandensis strain E264 (14) and E. coli strain DH5␣ or MG4 for genetic manipulations and recombinant DNA expression, respectively (see Table S1 in the supplemental material). Our B. thailandensis malA-lacZ chromosomal insertion mutant (BT01447) was from a sequence-defined transposon mutant library (15). This mutant has a transposon insertion in the malA coding sequence after bp 5704 (out of 8,379) relative to the predicted malA translational start site.…”

Section: Methodsmentioning

confidence: 99%

“…The pUC18-mini-Tn7T derivatives were introduced into B. thailandensis strains with the helper plasmid pTNS2 by electroporation, as previously described (19). We used PCR to verify insertion into the attn7 site near glmS1 as previously described (15). Where indicated, we removed the trimethoprim resistance marker dhfrIIb from pUC18-mini-Tn7T derivatives by using plasmid pFLPe2, as described previously (20).…”

Section: Methodsmentioning

confidence: 99%

A Burkholderia thailandensis Acyl-Homoserine Lactone-Independent Orphan LuxR Homolog That Activates Production of the Cytotoxin Malleilactone

et al. 2015

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Burkholderia thailandensis has three acyl-homoserine lactone (AHL) LuxR-LuxI quorum-sensing circuits and two orphan LuxR homologs. Orphans are LuxR-type transcription factors that do not have cognate LuxI-type AHL synthases. One of the orphans, MalR, is genetically linked to the mal gene cluster, which encodes enzymes required for production of the cytotoxic polyketide malleilactone. Under normal laboratory conditions the mal gene cluster is silent; however, antibiotics like trimethoprim induce mal transcription. We show that trimethoprim-dependent induction of the mal genes requires MalR. MalR has all of the conserved amino acid residues characteristic of AHL-responsive LuxR homologs, but in B. thailandensis, MalR activation of malleilactone synthesis genes is not responsive to AHLs. MalR can activate transcription from the mal promoter in E. coli without addition of AHLs or trimethoprim. Expression of malR in B. thailandensis is induced by trimethoprim. Our data indicate that MalR binds to a lux box-like element in the mal promoter and activates transcription of the mal genes in an AHL-independent manner. Antibiotics like trimethoprim appear to activate mal gene expression indirectly by somehow activating malR expression. MalR activation of the mal genes represents an example of a LuxR homolog that is not a receptor for an AHL quorum-sensing signal. Our evidence is consistent with the idea that mal gene activation depends solely on sufficient transcription of the malR gene. A cyl-homoserine lactone (AHL) quorum-sensing systems are widespread among the Proteobacteria. AHLs are generated by members of the LuxI family of AHL synthases. Members of the LuxR protein family serve as cognate AHL-dependent transcription factors. Often, the genes targeted by LuxR family members have an 18-to 20-base inverted repeat in their promoter regions that shows conservation across species. These repeat elements are called lux box-like sequences and serve as binding sites for the transcription factors. Sequence identity in pairwise comparisons of LuxR polypeptides is rather limited (18 to 35%), but there is a conserved N-terminal AHL binding region and a conserved C-terminal DNA binding region where sequence identity is higher and where there are highly conserved residues (see references 1 and 2 for reviews).Burkholderia thailandensis has three pairs of luxI-luxR-type genes called btaI1-btaR1, btaI2-btaR2, and btaI3-btaR3. Each of the luxR homologs is linked to its cognate luxI homolog, as is the case for luxI-luxR-type gene pairs in many other bacteria. This species also has two additional luxR homologs, btaR4 (now called malR [see below]) and btaR5 (3). LuxR family members like MalR and BtaR5 are called orphans (4) or solos (5), because the genes encoding these polypeptides are not associated with a luxI homolog. Relatively few orphans have been characterized. Some orphans, like Pseudomonas aeruginosa QscR or Salmonella enterica serovar Typhimurium SdiA, show complete conservation of all of the invariant residues in the ...

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“…T26 insertions were favored over T101 insertions. Strains were cherry picked, re-colony purified, incubated, and stored in a manner similar to one previously described (54).…”

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confidence: 99%

Resources for Genetic and Genomic Analysis of Emerging Pathogen Acinetobacter baumannii

et al. 2015

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Acinetobacter baumannii is a Gram-negative bacterial pathogen notorious for causing serious nosocomial infections that resist antibiotic therapy. Research to identify factors responsible for the pathogen's success has been limited by the resources available for genome-scale experimental studies. This report describes the development of several such resources for A. baumannii strain AB5075, a recently characterized wound isolate that is multidrug resistant and displays robust virulence in animal models. We report the completion and annotation of the genome sequence, the construction of a comprehensive ordered transposon mutant library, the extension of high-coverage transposon mutant pool sequencing (Tn-seq) to the strain, and the identification of the genes essential for growth on nutrient-rich agar. These resources should facilitate large-scale genetic analysis of virulence, resistance, and other clinically relevant traits that make A. baumannii a formidable public health threat. IMPORTANCEAcinetobacter baumannii is one of six bacterial pathogens primarily responsible for antibiotic-resistant infections that have become the scourge of health care facilities worldwide. Eliminating such infections requires a deeper understanding of the factors that enable the pathogen to persist in hospital environments, establish infections, and resist antibiotics. We present a set of resources that should accelerate genome-scale genetic characterization of these traits for a reference isolate of A. baumannii that is highly virulent and representative of current outbreak strains.A cinetobacter baumannii is a Gram-negative opportunistic pathogen that causes infections with serious morbidity and mortality and is one of a group of six pathogens responsible for most multidrug-resistant (MDR) nosocomial infections (the ESKAPE pathogens, i.e., Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) (1, 2). The pathogen is infamous for its ability to persist in hospital settings, a feature that reflects its capacity for long-term survival on abiotic surfaces through resistance to desiccation and disinfectants (3).Genomic and molecular epidemiological studies of A. baumannii isolates have helped define the pathogen's global population structure, its antibiotic resistance gene repertoire, the size and content of its pangenome, and phylogenetic relationships among outbreak strains (3-6). Three primary clonal lineages (GC1 to GC3) appear responsible for the majority of hospital outbreaks globally (7). Although these lineages display restricted genetic diversity among core genes (7), the species' genome is actually quite dynamic. Strains display striking variability in accessory gene content (5, 8), including antibiotic resistance genes (9), even among related isolates of a single outbreak (10). This genomic variability presumably reflects the actions of transmissible plasmids, insertion elements, phage, integrons, natural transformation, and reco...

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Sequence-Defined Transposon Mutant Library of Burkholderia thailandensis

Cited by 91 publications

References 51 publications

High-throughput platform for the discovery of elicitors of silent bacterial gene clusters

High-throughput platform for the discovery of elicitors of silent bacterial gene clusters

A Burkholderia thailandensis Acyl-Homoserine Lactone-Independent Orphan LuxR Homolog That Activates Production of the Cytotoxin Malleilactone

Resources for Genetic and Genomic Analysis of Emerging Pathogen Acinetobacter baumannii

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