Transposon insertion site sequencing (TIS) of Pseudomonas aeruginosa

2021 ◽  
Vol 59 (12) ◽  
pp. 1067-1074
Author(s):  
Hongbaek Cho
2020 ◽  
Vol 75 (9) ◽  
pp. 2516-2525
Author(s):  
A Keith Turner ◽  
Sabine E Eckert ◽  
Daniel J Turner ◽  
Muhammud Yasir ◽  
Mark A Webber ◽  
...  

Abstract Objectives A whole-genome screen at sub-gene resolution was performed to identify candidate loci that contribute to enhanced or diminished ciprofloxacin susceptibility in Salmonella enterica serovar Typhi. Methods A pool of over 1 million transposon insertion mutants of an S. Typhi Ty2 derivative were grown in a sub-MIC concentration of ciprofloxacin, or without ciprofloxacin. Transposon-directed insertion site sequencing (TraDIS) identified relative differences between the mutants that grew following the ciprofloxacin treatment compared with the untreated mutant pool, thereby indicating which mutations contribute to gain or loss of ciprofloxacin susceptibility. Results Approximately 88% of the S. Typhi strain’s 4895 annotated genes were assayed, and at least 116 were identified as contributing to gain or loss of ciprofloxacin susceptibility. Many of the identified genes are known to influence susceptibility to ciprofloxacin, thereby providing method validation. Genes were identified that were not known previously to be involved in susceptibility, and some of these had no previously known phenotype. Susceptibility to ciprofloxacin was enhanced by insertion mutations in genes coding for efflux, other surface-associated functions, DNA repair and expression regulation, including phoP, barA and marA. Insertion mutations that diminished susceptibility were predominantly in genes coding for surface polysaccharide biosynthesis and regulatory genes, including slyA, emrR, envZ and cpxR. Conclusions A genomics approach has identified novel contributors to gain or loss of ciprofloxacin susceptibility in S. Typhi, expanding our understanding of the impact of fluoroquinolones on bacteria and of mechanisms that may contribute to resistance. The data also demonstrate the power of the TraDIS technology for antibacterial research.


Author(s):  
Eliana Drenkard ◽  
Rhianna M. Hibbler ◽  
D. Alina Gutu ◽  
Alexander D. Eaton ◽  
Amy L. Silverio ◽  
...  

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Deisy Abril ◽  
Ricaurte Alejandro Marquez-Ortiz ◽  
Betsy Castro-Cardozo ◽  
José Ignacio Moncayo-Ortiz ◽  
Narda María Olarte Escobar ◽  
...  

2004 ◽  
Vol 72 (3) ◽  
pp. 1383-1390 ◽  
Author(s):  
Arne Rietsch ◽  
Matthew C. Wolfgang ◽  
John J. Mekalanos

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.


2004 ◽  
Vol 186 (14) ◽  
pp. 4730-4739 ◽  
Author(s):  
Andrea K. White ◽  
William W. Metcalf

ABSTRACT DNA sequencing and analysis of two distinct C—P lyase operons in Pseudomonas stutzeri WM88 were completed. The htxABCDEFGHIJKLMN operon encodes a hypophosphite-2-oxoglutarate dioxygenase (HtxA), whereas the predicted amino acid sequences of HtxB to HtxN are each homologous to the components of the Escherichia coli phn operon, which encodes C—P lyase, although homologs of E. coli phnF and phnO are absent. The genes in the htx operon are cotranscribed based on gene organization, and the presence of the intergenic sequences is verified by reverse transcription-PCR with total RNA. Deletion of the htx locus does not affect the ability of P. stutzeri to grow on phosphonates, indicating the presence of an additional C—P lyase pathway in this organism. To identify the genes comprising this pathway, a Δhtx strain was mutagenized and one mutant lacking the ability to grow on methylphosphonate as the sole P source was isolated. A ca.-10.6-kbp region surrounding the transposon insertion site of this mutant was sequenced, revealing 13 open reading frames, designated phnCDEFGHIJKLMNP, which were homologous to the E. coli phn genes. Deletion of both the htx and phn operons of P. stutzeri abolishes all growth on methylphosphonate and aminoethylphosphonate. Both operons individually support growth on methylphosphonate; however, the phn operon supports growth on aminoethylphosphonate and phosphite, as well. The substrate ranges of both C—P lyases are limited, as growth on other phosphonate compounds, including glyphosate and phenylphosphonate, was not observed.


2006 ◽  
Vol 74 (2) ◽  
pp. 931-939 ◽  
Author(s):  
P. Hudson ◽  
T. S. Gorton ◽  
L. Papazisi ◽  
K. Cecchini ◽  
S. Frasca ◽  
...  

ABSTRACT To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.


2005 ◽  
Vol 71 (7) ◽  
pp. 3389-3398 ◽  
Author(s):  
Gwénaëlle Choquet ◽  
Nathalie Jehan ◽  
Christine Pissavin ◽  
Carlos Blanco ◽  
Mohamed Jebbar

ABSTRACT The ability of Erwinia chrysanthemi to cope with environments of elevated osmolality is due in part to the transport and accumulation of osmoprotectants. In this study we have identified a high-affinity glycine betaine and choline transport system in E. chrysanthemi. By using a pool of Tn5-B21 ousA mutants, we isolated a mutant that could grow in the presence of a toxic analogue of glycine betaine (benzyl-glycine betaine) at high osmolalities. This mutant was impaired in its ability to transport all effective osmoprotectants in E. chrysanthemi. The DNA sequence of the regions flanking the transposon insertion site revealed three chromosomal genes (ousVWX) that encode components of an ABC-type transporter (OusB): OusV (ATPase), OusW (permease), and OusX (periplasmic binding protein). The OusB components showed a significant degree of sequence identity to components of ProU from Salmonella enterica serovar Typhimurium and Escherichia coli. OusB was found to restore the uptake of glycine betaine and choline through functional complementation of an E. coli mutant defective in both ProU and ProP osmoprotectant uptake systems. Competition experiments demonstrated that choline, dimethylsulfoniacetate, dimethylsulfoniopropionate, and ectoine were effective competitors for OusB-mediated betaine transport but that carnitine, pipecolate, and proline were not effective. In addition, the analysis of single and double mutants showed that OusA and OusB were the only osmoprotectant transporters operating in E. chrysanthemi.


2006 ◽  
Vol 103 (8) ◽  
pp. 2833-2838 ◽  
Author(s):  
N. T. Liberati ◽  
J. M. Urbach ◽  
S. Miyata ◽  
D. G. Lee ◽  
E. Drenkard ◽  
...  

2005 ◽  
Vol 49 (5) ◽  
pp. 1782-1786 ◽  
Author(s):  
Mara L. Sobel ◽  
Didier Hocquet ◽  
Lily Cao ◽  
Patrick Plesiat ◽  
Keith Poole

ABSTRACT Mutations in genes mexR and nalC have previously been shown to drive overexpression of the MexAB-OprM multidrug efflux system in Pseudomonas aeruginosa. A transposon insertion multidrug-resistant mutant of P. aeruginosa overproducing MexAB-OprM was disrupted in yet a third gene, PA3574, encoding a probable repressor of the TetR/AcrR family that we have dubbed NalD. Clinical strains overexpressing MexAB-OprM but lacking mutations in mexR or nalC were also shown to carry mutations in nalD. Moreover, the cloned nalD gene reduced the multidrug resistance and MexAB-OprM expression of the transposon mutant and clinical isolates, highlighting the significance of the nalD mutations vis-à-vis MexAB-OprM overexpression in these isolates.


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