IS981-mediated adaptive evolution recovers lactate production by ldhB transcription activation in a lactate dehydrogenase-deficient strain of Lactococcus lactis

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 IS981 in the upstream region of ldhB was responsible for transcription activation of the ldhB gene by generating an IS981-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 IS981 insertion represents the predominant mechanism of the observed recovery of the ability to produce lactate.

FIG. 1.

Recovery of lactate production in ldh-deficient L. lactis NZ9010. L. lactis NZ9010 was subcultured anaerobically in GM17 for 7 days with 100-fold dilution (approximately 6.7 generations) each day. Lactate production relative to glucose consumption is shown (black bars). Recovery of lactate production occurs after a varying number of generations in independent NZ9010 cultures.

FIG. 2.

Enzyme kinetics of LdhB and las operon-encoded Ldh. Affinity constants were determined by measuring NADH consumption as a function of the concentration of the activator FBP (A) and the cofactor NADH (B) with crude cell extracts of L. lactis NZ9000 and NZ9015, respectively. Activity curves of Ldh (filled dots) and LdhB are shown (filled triangles). The inset in panel A shows NADH consumption at low concentrations of FBP for las operon-encoded Ldh.

FIG. 3.

Northern blot detection of ldh- and ldhB-specific transcripts in L. lactis NZ9000 and derivatives thereof. Total RNAs isolated from L. lactis NZ9000 grown under aerobic and anaerobic conditions, L. lactis NZ9010 grown under aerobic conditions, and L. lactis NZ9015 grown under aerobic and anaerobic conditions (lanes 1 to 5, respectively) were size fractionated on a formaldehyde denaturing 1% agarose gel and transferred to a membrane. ldh- and ldhB-specific mRNAs were detected with radioactively labeled ldh and ldhB gene probes (panels A and B, respectively). A 0.24- to 9.49-kb RNA marker (lane M) was used to determine the mRNA sizes.

FIG. 4.

L. lactis ldhB promoter region amplification. The rlrD-ldhB intergenic region was amplified by PCR with rlrD forward and ldhB reverse primers on chromosomal DNAs of L. lactis NZ9000, NZ9010, and NZ9015 (lanes 1 to 3, respectively). PCR products were size fractionated on a 1% agarose gel. BstEII-digested λ DNA was used as a reference (lane λ).

FIG. 5.

Schematic representation of the ldhB promoter region of L. lactis NZ9015. (A) Insertion position of IS981 (indicated by a filled triangle). The rlrD, ldhB, and tra981 genes and the positions of the IS981 imperfect inverted repeats (IR) are indicated by white arrows. (B) Nucleotide sequence of the ldhB promoter region of L. lactis NZ9015 from the rlrD stop codon to the ldhB start codon (light gray background). The upright sequence represents the native promoter region as it is also present in strains NZ9000 and NZ9010. Underlining arrows indicate the rlrD terminator. The IS981-originated sequence is in italics. The IS981 imperfect inverted repeats are underlined. The −35 and −10 regions are in bold. The vertical arrowhead indicates the ldhB transcription start site determined in L lactis NZ9015. nt, nucleotides.

FIG. 6.

Schematic representation of mutations in the ldhB promoter regions of ldhB-expressing NZ9010 derivatives without IS981 integration. The nucleotide sequence of the ldhB promoter region of L. lactis NZ9010, from the rlrD stop codon to the ldhB start codon (light gray background), is shown. Mutations detected in the same region in five independent cultures that displayed ldhB expression are indicated (numbered 1 to 5) and include nucleotide substitutions (arrowheads) or insertions (light gray triangles). Cultures: 1, ATAAATTCA inserted after A91, A222G, T223A; 2, A187G; 3, G77A; 4, T131G, T205C; 5, A69G.