A plasmid RK2-based broad-host-range cloning vector useful for transfer of metagenomic libraries to a variety of bacterial species

Abstract

The majority of microorganisms in natural environments are difficult to cultivate, but their genes can be studied via metagenome libraries. To enhance the chances that these genes become expressed we here report the construction of a broad-host-range plasmid vector (pRS44) for fosmid and bacterial artificial chromosome (BAC) cloning. pRS44 can be efficiently transferred to numerous hosts by conjugation. It replicates in such hosts via the plasmid RK2 origin of replication, while in Escherichia coli it replicates via the plasmid F origin. The vector was found to be remarkably stable due to the insertion of an additional stability element (parDE). The copy number of pRS44 is adjustable, allowing for easy modifications of gene expression levels. A fosmid metagenomic library consisting of 20 000 clones and BAC clones with insert sizes up to 200 kb were constructed. The 16S rRNA gene analysis of the fosmid library DNA confirmed that it represents a variety of microbial species. The entire fosmid library and the selected BAC clones were transferred to Pseudomonas fluorescens and Xanthomonas campestris (fosmids only), and heterologous proteins from the fosmid library were confirmed to be expressed in P. fluorescens. To our knowledge no other reported vector system has a comparable potential for functional screening across species barriers.

The art and design of functional metagenomic screens

“…The application of automation for readout not only decreases labour costs, but increases reproducibility and comparability between samples. Pseudomonas putida g-proteobacteria negative conjugation pigmentation [35 ] Xanthomonas campestris g-proteobacteria negative conjugation [54 ] To overcome potential problems associated with intracellular accumulation of enzyme activity, cell lysis can be applied in microtiter plates to increase assay sensitivity [11,37]. Common cell lysis methods use enzymes or detergents (for visualized experiment see [38 ]).…”

Tapping uncultured microorganisms through metagenomics for drug discovery

“…1, N-acyltyrosine; 2, isocyanide functionalized indole; 3, indigo; 4, turbomycin A; 5, cyanobactinpatellamide D; 6, nocardamine and cyanobactin patellamide A (Banik and Brady, 2010). metagenomic libraries in diverse bacteria have the potential to expand the number and type of bioactive compounds that can be discovered from metagenomic analysis (Aakvik et al, 2009;Craig et al, 2009 and.…”

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

“…To overcome this, fertility (F)-factor-based origin of replication from Escherichia coli was developed as a single-copy origin of replication for fosmid, cosmid ) and BAC coli and transferred to other hosts such as Streptomyces lividans and Pseudomonas putida for functional screening of natural products leading to drug discovery (Courtois, Cappellano et al 2003, Martinez, Kolvek et al 2004. A plasmid RK2-based broad-host range vector (pRS44/pTA44) was used to transfer metagenomic libraries to a variety of bacterial species like E. coli, Pseudomonas fluorescens and Xanthomonas compestris (Aakvik, Degnes et al 2009). In a more recent and comprehensive study, broad-hostrange cosmid environmental DNA libraries were screened in parallel for small molecules across six different proteobacterial hosts (Craig, Chang et al 2010).…”