Dataset Information

A Mini-ISY100 Transposon Delivery System Effective in ? Proteobacteria.

ABSTRACT: Transposons are invaluable biological tools for the genetic manipulation of microorganisms. ISY100 from Synechocystis sp. PCC6803 is a member of the Tc1/mariner/IS630 superfamily, and is characterized by high transposition efficiency and a strong preference for TA target sequences. In this paper, we describe the design and application of a mini-ISY100 suicide vector for the in vivo creation of stable random transposon insertion libraries. The system was successfully applied in seven species belonging to four different orders of ? proteobacteria. In all cases, delivery using conjugation consistently showed the highest transposition efficiency compared to chemical transformation or electroporation. We determined the frequency of transposon insertions in all the species and proved the utility of the system by identifying genes involved in colony coloration in Shewanella oneidensis. The ease and the efficiency of the protocol developed here allow the creation of complete knock-out libraries in an extensive range of host microorganisms in less than a week with no requirement for preparatory modification.

SUBMITTER: Conte E

PROVIDER: S-EPMC6400869 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

A Mini-ISY100 Transposon Delivery System Effective in γ Proteobacteria.

Conte Emanuele E Mende Linda L Grainge Ian I Colloms Sean D SD

Frontiers in microbiology 20190227

Transposons are invaluable biological tools for the genetic manipulation of microorganisms. ISY100 from Synechocystis sp. PCC6803 is a member of the Tc1/mariner/IS630 superfamily, and is characterized by high transposition efficiency and a strong preference for TA target sequences. In this paper, we describe the design and application of a mini-ISY100 suicide vector for the in vivo creation of stable random transposon insertion libraries. The system was successfully a ...[more]

PMID: 30873132

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Project description:Bacteria rapidly adapt to their environment by integrating external stimuli through diverse signal transduction systems. Pseudomonas aeruginosa, for example, senses surface-contact through the Wsp signal transduction system to trigger the production of cyclic di-GMP. Diverse mutations in wsp genes that manifest enhanced biofilm formation are frequently reported in clinical isolates of P. aeruginosa, and in biofilm studies of Pseudomonas spp. and Burkholderia cenocepacia. In contrast to the convergent phenotypes associated with comparable wsp mutations, we demonstrate that the Wsp system in B. cenocepacia does not impact intracellular cyclic di-GMP levels unlike that in Pseudomonas spp. Our current mechanistic understanding of the Wsp system is entirely based on the study of four Pseudomonas spp. and its phylogenetic distribution remains unknown. Here, we present a broad phylogenetic analysis to show that the Wsp system originated in the β-proteobacteria then horizontally transferred to Pseudomonas spp., the sole member of the γ-proteobacteria. Alignment of 794 independent Wsp systems with reported mutations from the literature identified key amino acid residues that fall within and outside annotated functional domains. Specific residues that are highly conserved but uniquely modified in B. cenocepacia likely define mechanistic differences among Wsp systems. We also find the greatest sequence variation in the extracellular sensory domain of WspA, indicating potential adaptations to diverse external stimuli beyond surface-contact sensing. This study emphasizes the need to better understand the breadth of functional diversity of the Wsp system as a major regulator of bacterial adaptation beyond B. cenocepacia and select Pseudomonas spp. Importance The Wsp signal transduction system serves as an important model system for studying how bacteria adapt to living in densely structured communities known as biofilms. Biofilms frequently cause chronic infections and environmental fouling, and they are very difficult to eradicate. In Pseudomonas aeruginosa, the Wsp system senses contact with a surface, which in turn activates specific genes that promote biofilm formation. We demonstrate that the Wsp system in Burkholderia cenocepacia regulates biofilm formation uniquely from that in Pseudomonas species. Furthermore, a broad phylogenetic analysis reveals the presence of the Wsp system in diverse bacterial species, and sequence analyses of 794 independent systems suggest that the core signaling components function similarly but with key differences that may alter what or how they sense. This study shows that Wsp systems are highly conserved and more broadly distributed than previously thought, and their unique differences likely reflect adaptations to distinct environments.

Dataset Information

A Mini-ISY100 Transposon Delivery System Effective in ? Proteobacteria.

Publications

A Mini-ISY100 Transposon Delivery System Effective in γ Proteobacteria.

Similar Datasets

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