Affiliation:
1. Wageningen Centre for Food Sciences, NIZO Food Research, FNI Department, 6710 BA Ede, The Netherlands
Abstract
ABSTRACT
Lactococcus lactis
NZ9010 in which the
las
operon-encoded
ldh
gene was replaced with an erythromycin resistance gene cassette displayed a stable phenotype when grown under aerobic conditions, and its main end products of fermentation under these conditions were acetate and acetoin. However, under anaerobic conditions, the growth of these cells was strongly retarded while the main end products of fermentation were acetate and ethanol. Upon prolonged subculturing of this strain under anaerobic conditions, both the growth rate and the ability to produce lactate were recovered after a variable number of generations. This recovery was shown to be due to the transcriptional activation of a silent
ldhB
gene coding for an Ldh protein (LdhB) with kinetic parameters different from those of the native
las
operon-encoded Ldh protein. Nevertheless, cells producing LdhB produced mainly lactate as the end product of fermentation. The mechanism underlying the
ldhB
gene activation was primarily studied in a single-colony isolate of the recovered culture, designated
L
.
lactis
NZ9015. Integration of IS
981
in the upstream region of
ldhB
was responsible for transcription activation of the
ldhB
gene by generating an IS
981
-derived −35 promoter region at the correct spacing with a natively present −10 region. Subsequently, analysis of 10 independently isolated lactate-producing derivatives of
L
.
lactis
NZ9010 confirmed that the
ldhB
gene is transcribed in all of them. Moreover, characterization of the upstream region of the
ldhB
gene in these derivatives indicated that site-specific and directional IS
981
insertion represents the predominant mechanism of the observed recovery of the ability to produce lactate.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
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