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Development of a genetic transformation system for an alga-lysing bacterium.

ABSTRACT: Four marine bacteria, Alteromonas sp. strains A27, A28, A29, and A30, that lyse the diatom Skeletonema costatum NIES-324 were isolated from coastal seawater samples. They were also able to lyse the diatoms Thalassiosira sp. and Eucampia zodiacs and the raphidophycean flagellate Chattonella antiqua. Cryptic indigenous plasmids, designated pAS28 and pAS29, were detected in Alteromonas sp. strains A28 and A29, respectively. These plasmids appeared to be similar based on size and restriction site analysis. A shuttle vector that replicates in Escherichia coli and Alteromonas sp. strain A28 was constructed by fusing pAS28 and E. coli vector pCRIIc. The 16-kbp chimeric plasmid, designated pASS1, had the ability to transform strain A28 at a frequency of 10(6) transformants per microg of DNA. Deletion analysis of pASS1 showed that the 4.7-kb EcoRI-HindIII region of pAS28 was essential for plasmid maintenance in strain A28. This EcoRI-HindIII fragment contained an open reading frame which appeared to encode a 708-amino-acid protein.

SUBMITTER: Kato J

PROVIDER: S-EPMC106278 | biostudies-literature | 1998 Jun

REPOSITORIES: biostudies-literature

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Development of a genetic transformation system for an alga-lysing bacterium.

Kato J J Amie J J Murata Y Y Kuroda A A Mitsutani A A Ohtake H H

Applied and environmental microbiology 19980601 6

Four marine bacteria, Alteromonas sp. strains A27, A28, A29, and A30, that lyse the diatom Skeletonema costatum NIES-324 were isolated from coastal seawater samples. They were also able to lyse the diatoms Thalassiosira sp. and Eucampia zodiacs and the raphidophycean flagellate Chattonella antiqua. Cryptic indigenous plasmids, designated pAS28 and pAS29, were detected in Alteromonas sp. strains A28 and A29, respectively. These plasmids appeared to be similar based on size and restriction site an ...[more]

PMID: 9603814

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Project description:BackgroundThe plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize the infection process at the molecular biological level, no genetic transformation protocol has yet been established for F. avenaceum. In the current study, it is shown that F. avenaceum can be efficiently transformed by Agrobacterium tumefaciens mediated transformation. In addition, an efficient and versatile single step vector construction strategy relying on Uracil Specific Excision Reagent (USER) Fusion cloning, is developed.ResultsThe new vector construction system, termed USER-Brick, is based on a limited number of PCR amplified vector fragments (core USER-Bricks) which are combined with PCR generated fragments from the gene of interest. The system was found to have an assembly efficiency of 97% with up to six DNA fragments, based on the construction of 55 vectors targeting different polyketide synthase (PKS) and PKS associated transcription factor encoding genes in F. avenaceum. Subsequently, the ΔFaPKS3 vector was used for optimizing A. tumefaciens mediated transformation (ATMT) of F. avenaceum with respect to six variables. Acetosyringone concentration, co-culturing time, co-culturing temperature and fungal inoculum were found to significantly impact the transformation frequency. Following optimization, an average of 140 transformants per 106 macroconidia was obtained in experiments aimed at introducing targeted genome modifications. Targeted deletion of FaPKS6 (FA08709.2) in F. avenaceum showed that this gene is essential for biosynthesis of the polyketide/nonribosomal compound fusaristatin A.ConclusionThe new USER-Brick system is highly versatile by allowing for the reuse of a common set of building blocks to accommodate seven different types of genome modifications. New USER-Bricks with additional functionality can easily be added to the system by future users. The optimized protocol for ATMT of F. avenaceum represents the first reported targeted genome modification by double homologous recombination of this plant pathogen and will allow for future characterization of this fungus. Functional linkage of FaPKS6 to the production of the mycotoxin fusaristatin A serves as a first testimony to this.

Dataset Information

Development of a genetic transformation system for an alga-lysing bacterium.

Publications

Development of a genetic transformation system for an alga-lysing bacterium.

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