Heterologous recognition in vivo of promoter sequences from the Streptomyces coelicolor dagA gene

Abstract

The Streptomyces coelicolor dagA gene that codes for an extracellular agarase was cloned in the closely related bacterium S. lividans and transferred to the distantly related low G+C Gram-positive bacterium Bacillus subtilis and to the far more distantly related Gram-negative bacterium Escherichia coli. S1 nuclease mapping experiments identified a putative fifth promoter from which transcription of the dagA gene can take place, and accurately mapped the transcription termination site. The transcription terminator was specific for the Streptomyces strains and could terminate transcription initiated by promoters other than those of dagA. The agarase gene is efficiently transcribed in B. subtilis and E. coli, although pulse-chase experiments failed to detect the synthesis of agarase in these two bacteria.

The Bacillus subtilis 168 csn gene encodes a chitosanase with similar properties to a Streptomyces enzyme

“…Escherichia coli MC1061 (hsdR2 mcrB1 araD139 ∆(araABC-leu)7679 ∆lacX74 galU galK rpsL thi) was used as a host for plasmid propagation. Plasmid pNR2 (Parro & Mellado, 1993) was used to propagate csn in high copy number. Plasmid pUCAT194 is a pUC19 derivative carrying the EcoRI fragment from pZA327 (a gift from J. C. Alonso) which contains the chloramphenicol resistance gene (cat) and was used as a vector for csn disruption.…”

Four genes encoding different type I signal peptidases are organized in a cluster in Streptomyces lividans TK21 The GenBank accession number for the sequence data reported in this paper is Z86111.

“…The amplification ended with a 10 min incubation at 72 mC. Preparations of total RNA from bacterial cultures and S1 nuclease protection experiments were done as described previously (Parro & Mellado, 1993, except that for labelling of the TaqI 3h ends [α-$#P]dCTP was used instead of [α-$#P]dATP. Probes for S1 nuclease mapping are indicated in Fig.…”

Effects of phosphate limitation on agarase production byStreptomyces lividansTK21

“…S. lividans strain TK21 [16], an agarase non‐producing strain, was used as the host for the high copy number plasmid, pAGAs1, encoding dagA and thiostrepton resistance [30]. General procedures for the growth and manipulation of Streptomyces were as described previously [17].…”