Cloning of the Lactococcus lactis adhE gene, encoding a multifunctional alcohol dehydrogenase, by complementation of a fermentative mutant of Escherichia coli

Abstract

The Lactococcus lactis adhE gene, which encodes a multifunctional alcohol dehydrogenase, has been cloned and characterized. A DNA fragment encoding the putative alcohol dehydrogenase domain of the AdhE protein was cloned by screening an L. lactis genomic library in a fermentative mutant of Escherichia coli and selecting for the ability to grow anaerobically. Further analysis of the clone obtained allowed the cloning of the entire adhE gene sequence. Analysis of adhE expression in L. lactis during anaerobiosis showed induction at the transcriptional level, especially in medium containing glucose. Constructed mutant strains produced reduced amounts of ethanol under anaerobic conditions. With the L. lactis gene as a probe, adhE homologs were found in other industrially relevant lactic acid bacteria.

FIG. 1

Genetic maps of the adhE chromosomal regions of L. lactis MG1363 and DB1341. Only relevant restriction sites are shown. E, EcoRI; H, HindIII; N, NsiI; Nd, NdeI; P, PstI; S, Sau3AI. The DNA fragments included in pC1 are shown, indicating the three different Sau3AI DNA fragments that were ligated together under library construction and that included an rRNA gene sequence (rRNA box in clone 1). The position of the consensus E. coli expression signals (Shine-Dalgarno sequence and promoter region) in pC1 is shown as a bent arrow (see also Fig. 2). The fragment used for gene inactivation of the MG1363 adhE gene is depicted as a filled box (plasmid construction pKAS15 [see Materials and Methods]). Putative transcriptional terminators for orfB and adhE are shown as open circles.

FIG. 2

Sequence of the L. lactis DB1341 adhE gene. The sequence begins upstream of orfB and includes the adhE gene and ca. 350 bp of sequence downstream of adhE. +1 mRNA refers to the adhE transcription start site. Ribosome binding sites (RBS) for orfB and adhE are shown in boldface type. Promoter regions, −35 and −10, are indicated with double underlines. Pairs of arrows depict putative transcriptional terminator sequences. The deduced protein sequences are shown in single-letter code. The region included in pC1 is shown (Clone-1 Start to Clone-1 Stop) above the sequence. Consensus E. coli expression signals (Shine-Dalgarno sequence [SD; boldface type] and −35 and −10 promoter regions [underlined]) just upstream of the ADH domain in pC1 [ADH-domain (clone-1)] are shown. An inverted triangle above the sequence depicts the position of gene inactivation in the adhE strain MGKAS15.

FIG. 3

Multiple alignment of AdhE proteins. The lactococcal AdhE sequence from strain DB1341 was aligned with the sequences of all AdhE entries in the databases by using the Clustal alignment of the MEGALIGN program (DNAstar, Lasergene). Shaded boxes show positions of residues identical to the consensus. Abbreviations: dbadhe, strain DB1341 AdhE; ecadhe, E. coli AdhE; caaad, C. acetobutylicum Aad; ehadhe, E. histolytica AdhE; gladhe, G. lamblia AdhE.

FIG. 4

Northern blot and primer extension analysis of the L. lactis adhE gene. (A) Northern blot analysis of total RNA from L. lactis MG1363 grown in fermentors. Lane 1, aerobic growth in GM17; lane 2, anaerobic growth in GM17; lane 3, aerobic growth in GalM17; lane 4, anaerobic growth in GalM17. The size of the adhE transcript is shown on the left in kilobases. (B and C) Primer extension carried out with primers mgadhe-PE1 (B) and mgadhe-PE2 (C) on total RNA of strain MG1363. Lanes 1 to 4, MG1363 grown as described for panel A. A sequence ladder was run, in each case, with the corresponding primer. The adhE transcription start site is indicated with a box in the sequence line. Extension products are indicated with arrows.

FIG. 5

Detection of an adhE homolog in other lactic acid bacteria. Southern blot analysis of genomic DNA digested with EcoRI by using the L. lactis adhE probe (Fig. 1). Lane 1, L. lactis MG1363; lane 2, S. thermophilus; lane 3, Leuconostoc mesenteroides; lane 4, Lactobacillus acidophilus. Bands sizes are shown.