Microbial Enzymes and Biotransformations Microbial Enzymes and ...

Corynebacteria and Enzyme Production 117
plasts and electroporation of C. glutamicum with pUL609M is obtained with
high efficiency (10 6 transformants/µg DNA).
Shuttle E. coli-corynebacteria mobilizable vectors based on RP4 were initially
described by Schafer et al. (7). Plasmid pECM1 (Escherichia-corynebacteria-mobilizable)
resulted from the fusion of pSUP102 (mobilizable from E.
coli) with C. glutamicum plasmid pHM1519 (2). Later, Qian et al. (8) described
the construction of the mobilizable shuttle plasmids pXZ911, pBZ51, pBZ52,
which carry the Mob site (RP4) and replication origins of E. coli and
corynebacterial plasmids. These shuttle mobilizable vectors can be transferred
from E. coli to corynebacteria by conjugation.
Many of the suicide mobilizable vectors used in Gram-positive bacteria are also
based on the broad-host-range plasmid RP4 bearing the cis-acting DNA recognition
site for conjugative DNA transfer between bacterial cells (Mob site) and the
kanamycin resistance gene from Tn5 (9). Priefer et al. (10) developed the pSUP
series, broad-host-range vectors that are based on conventional E. coli vectors
(pBR325 and pACYC184) modified to include the mobilization and broad-hostrange
replication functions of the IncQ plasmid RSF1010. pSUP vectors are efficiently
mobilized by RP4 and are thus of particular interest for bacteria refractory
to transformation. Small mobilizable vectors based on the E. coli plasmids pK18
and pK19 have been described by Schafer et al. (11); these vectors combine the
useful properties of the pK plasmids (multiple cloning site, lacZ alpha fragment,
kanamycin resistance) with the broad-host-range transfer machinery of plasmid
RP4 and have been named pK18/19mob. Vectors pK18/19mob can be transferred
by RP4-mediated conjugation into a wide range of Gram-negative and Gram-positive
bacteria, including corynebacteria, and they behave as suicide vectors.
The literature contains only a few references to the construction of promoter
probe vectors for corynebacteria and the work described to date is based on the
promoterless kan gene from Tn5 (12), the cat gene from Tn9 (13,14), the β-glucuronidase
gene from E. coli (15), the α-amylase gene from Bacillus subtilis
(16) or from Streptomyces griseus (17), the β-galactosidase and permease genes
from E. coli (18), and the melC operon from Streptomyces glaucescens (19).
Some of these promoter-probe plasmids are monofunctional plasmids, whereas
others are bifunctional E. coli-corynebacteria plasmids.
1.2. Introduction of Exogenous DNA into Corynebacteria
All these vectors are monofunctional or bifunctional plasmids that must be
introduced into C. glutamicum protoplasts or electroporated into treated C. glutamicum
cells. Although protoplast transformation (20) and electroporation
(21,22) have been reported to be efficient methods for transforming coryneform
bacteria with plasmid DNA, conjugation is also a good method for introducing
plasmids into C. glutamicum (23,24).