Introduction of DNA into Actinomycetes by bacterial conjugation from E. coli—An evaluation of various transfer systems

Abstract

Gene transfer is a basic requirement for optimizing bioactive natural substances produced by an increasing number of industrially used microorganisms. We have analyzed quantitatively horizontal gene transfer from Escherichia coli to Actinomycetes. The efficiencies of DNA transfer of four different systems were compared that consist of conjugative and mobilizable plasmids with a broad-host range. Three novel binary vector set-ups were constructed based on: (i) the IncQ group of mobilizable plasmids (RSF1010), (ii) IncQ-like pTF-FC2 and (iii) pSB102 that belongs to a new class of broad-host-range plasmids. The established system based on the IncP␣ group of conjugative plasmids served as the reference. For all plasmids constructed, we confirmed the functional integrity of the selected transfer machineries by intrageneric matings between E. coli strains. We demonstrate that the transfer systems introduced in this study are efficient in mediating gene transfer from E. coli to Actinomycetes and are possible alternatives for gene transfer into Actinomycetes for which the IncP␣-based transfer system is not applicable. The use of plasmids that integrate into the recipients' chromosomes compared to that of plasmids replicating autonomously is shown to allow the access to a wider range of hosts.

Developing an efficient and reproducible conjugation-based gene transfer system for bifidobacteria

“…Efficiencies of 10 24 to 10 28 have been reported for Streptomyces (Blaesing et al, 2005;Mazodier et al, 1989) and 10 26 for Mycobacterium (Gormley & Davies, 1991 (Schäfer et al, 1994). From the initial conjugations into different bifidobacteria in this study, the highest conjugation efficiencies were obtained when the ratio of E. coli to bifidobacteria cells favoured E. coli (Table 3).…”

Identification of Type 3 Fimbriae in UropathogenicEscherichia coliReveals a Role in Biofilm Formation

“…Transposon mutagenesis was performed via filter paper bacterial conjugation (7,9). An overnight culture of the donor strain was concentrated 10-fold and left to stand at 37°C for 30 min to allow growth of the sex pili.…”

A Novel N,N-8-Amino-8-demethyl-d-riboflavin Dimethyltransferase (RosA) Catalyzing the Two Terminal Steps of Roseoflavin Biosynthesis in Streptomyces davawensis

“…A DNA fragment containing the putative ros gene cluster (sda77160, sda77170, sda77180, sda77190, sda77200, sda77210, rosA, sda77230, sda77240, and sda77250) and the natural putative promotor (see Fig. 3) was excised from plasmid FJ03 using restriction endonucleases and ligated to integrative Streptomyces plasmid pSET152 carrying no additional promoter sequence in front of the multiple cloning site (FJ04) (27). Expression experiments produced negative results, i.e.…”