Construction of new cloning vectors for Brevibacterium lactofermentum.

Two plasmid cloning vectors (pULMJ55 and pULMJ95) were constructed for Brevibacterium lactofermentum using the origin of replication of the endogenous plasmid pBL1. Plasmid pULMJ55 is a replacement vector with transcriptional terminators from the B. lactofermentum trp operon flanking the BglII cloning sites. Religation of the BglII digested vector without insert creates a 376 bp perfect palindrome that is not tolerated in B. lactofermentum, giving positive selection for recombinant plasmids with inserts. Plasmid pULMJ95 contains the promoter-less alpha-amylase gene from Streptomyces griseus downstream of the trp terminator and is particularly suitable for the detection of promoters which are activated late during the growth phase. alpha-Amylase is secreted and its activity can be detected using simple plate tests.