Integration and gene replacement in the Lactococcus lactis lac operon: induction of a cryptic phospho-beta-glucosidase in LacG-deficient strains

Abstract

Insertions, replacement mutations, and deletions were introduced via single or double crossover recombination into the lacE (enzyme IIBMa) and lacG (phospho-1-galactosidase) genes of the Lactococcus lactis chromosomal lacABCDFEGX operon. LacG production was abolished in strains missing the lacG gene or carrying multicopy insertions in the lacE gene that affected expression of the lacG gene. However, these LacG-deficient strains could still ferment lactose slowly and were found to contain an enzymatic activity that hydrolyzed the chromogenic substrate o-nitrophenyl-1-D-galactopyranoside phosphate. Induction of this phospho-13-glycohydrolase activity coincided with the appearance of a new 55-kDa protein cross-reacting with anti-LacG antibodies that had a size similar to that of LacG but a higher isoelectric point (pI 5.2) and was not found in wild-type cells during growth on lactose. Since the phospho-13-glycohydrolase activity and this protein with a pI of 5.2 were highly induced in both mutant and wild-type cells during growth on cellobiose that is likely to be transported via a phosphoenolpyruvate-dependent phosphotransferase system, we propose that this induced activity is a phospho-13-glucosidase that also hydrolyzes lactose-6-phosphate.Site-specific integration of foreign DNA linked to chromosomal sequences by homologous recombination has been described for many microorganisms and used to generate insertion mutants (32,33), to map and clone genes (6,18,30), to introduce defined deletions (17, 34), and to stabilize and amplify important traits (25,38).Recently, integration systems have been developed for Lactococcus lactis, a gram-positive bacterium widely used in industrial food fermentations, whereby randomly cloned chromosomal DNA (26), prophage sequences (5), natural plasmid DNA (13, 36), transposon DNA (4, 19) and insertion elements (37, 40) were used to provide the required homology. In some cases, multicopy integration of sequences to a level of about 15 copies per chromosome has been obtained, but their stability was low in the absence of selective pressure (5,26). In addition, single crossover integration followed by loop-out deletion resulting in replacement recombination has been described for L. lactis (27).The primary function of lactococci is the fermentation of lactose into lactate. Lactose is the only fermentable sugar in industrial dairy fermentations, and it is transported via a lactose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) (8), in which the lactose-specific enzyme II"ac (LacE) and enzyme III'c (LacF) participate and the internalized lactose-6-phosphate is hydrolyzed by a phospho-p-galactosidase (LacG) (9). Recently, the complete nucleotide sequence and the functions of the various genes of the L. lactis lactose operon consisting of the genes of the tagatose 6-P pathway (49) and the genes encoding the lactose PTS (8) have been elucidated. The expression of the lac operon is repressed during growth on glucose and is regulated at the transcriptional level by the L...

Lactose and β-Glucosides Metabolism and Its Regulation in Lactococcus lactis: A Review

“…However, it was suggested that these PTSs are not specific for lactose, but rather for the translocation of other sugars (e.g., -glucosides), and lactose could be transported alternatively. This hypothesis was supported by observations suggesting that a putative P--glucosidase, involved in cellobiose hydrolysis, is probably also involved in lactose-6-P cleavage in L. lactis strain ATCC7962 (Simons et al, 1993). This seems reasonable, as according to http://www.tcdb.org/, PTS lactose transporters belong to the Lac family (TC No.…”

“…The existence in several lactococcal strains devoid of lac-plasmids of cryptic lactose transport and catabolism systems has already been suggested in earlier studies (Anderson & McKay, 1977;Cords & McKay, 1974;de Vos & Simons, 1988;Simons et al, 1993). The presence in L. lactis of chromosomally-encoded lactose permease has been proposed since introduction of the E. coli lacZ gene into a lactose-deficient L. lactis strain restored its ability to utilize lactose (de Vos & Simons, 1988).…”

Proteome Analyses of Heme-Dependent Respiration inLactococcus lactis: Involvement of the Proteolytic System

“…One such enzyme is the ␤-galactosidase present in strains growing in milk, where lactose is the main carbon source. Other phospho-␤-glucosidase activities have also been identified in lactic acid bacteria (31,48). This activity is usually detected in cells growing on ␤-glucosides like salicin, arbutin, and cellobiose but not on glucose, which suggests that the corresponding gene is controlled by carbon catabolite repression (31,48).…”

“…Other phospho-␤-glucosidase activities have also been identified in lactic acid bacteria (31,48). This activity is usually detected in cells growing on ␤-glucosides like salicin, arbutin, and cellobiose but not on glucose, which suggests that the corresponding gene is controlled by carbon catabolite repression (31,48). The presence of BglA under glucose fermentation growth but not in respiration conditions suggests that (i) BglA is probably not under carbon catabolite repression and (ii) BglA might have a role other than to degrade these sugars.…”

Alternative Lactose Catabolic Pathway in Lactococcus lactis IL1403

“…lac-PTS genes can also be located on chromosomes, as has been described for Streptococcus mutans (41) and the non-LAB Staphylococcus aureus (6,7,42). Moreover, there is some indirect evidence that additional lac-PTS genes can also be located in the genomes of several L. lactis strains (3,9,10,12,46).Besides the lac-PTS, there is only one other type of lactose transport system that has been described for L. lactis, namely, the lactose-H ϩ symport permease (26). Two other known types of lactose transport systems, the lactose-galactose antiporter and ABC protein-dependent lactose transporter, have been found in Streptococcus thermophilus (36,37,38) and in the non-LAB, gram-negative Agrobacterium radiobacter (57), respectively.…”