Molecular and genetic characterization of lactose-metabolic genes of Streptococcus cremoris

Inamine, Julia M.; Lee, L N; LeBlanc, Donald J.

doi:10.1128/jb.167.3.855-862.1986

Cited by 34 publications

(16 citation statements)

References 27 publications

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“…The results (Table 3) show that high levels of activity were obtained with strains MG5267 and NZ5267-G32 during growth on lactose. During growth on glucose, these two strains showed approximately 10-fold lower activities, in agreement with the induction of lactose-hydrolyzing enzymes during growth on lactose shown previously (8,21,47). During growth on glucose, background levels of activity in strains NZ5267-E32, NZ5267-G40, and NZ5267-G42 were similar to the activity obtained with strain MG1363.…”

Section: Resultssupporting

confidence: 64%

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

1993

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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...

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Section: Resultssupporting

confidence: 64%

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

1993

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“…lactis subsp. cremoris (Inamine et al, 1986) in E. coli. However, since cloning systems for these lactic acid bacteria have only recently been developed, studies on the expression of the lacG genes have been confined to heterologous hosts (LeBlanc, 1987;de Vos, 1987).…”

Section: W M D E Vos a N D M J Gassonmentioning

confidence: 99%

Structure and Expression of the Lactococcus lactis Gene for Phospho- -galactosidase (lacG) in Escherichia coli and L. lactis

Vos

Gasson

1989

Microbiology

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The Lactococcus lactis subsp. lactis 712 lacG gene encoding phospho-beta-galactosidase was isolated from the lactose mini-plasmid pMG820 and cloned and expressed in Escherichia coli and L. lactis. The low phospho-beta-galactosidase activity in L. lactis transformed with high-copy-number plasmids containing the lacG gene contrasted with the high activity found in L. lactis containing the original, low-copy-number lactose plasmid pMG820, and indicated that the original lactose promoter was absent from the cloned DNA. In E. coli the phospho-beta-galactosidase could be overproduced using the strong inducible lambda PL promoter, which allowed a rapid purification of the active enzyme. The complete nucleotide sequence of the L. lactis lacG gene and its surrounding regions was determined. The deduced amino acid sequence was confirmed by comparison with the amino acid composition of the purified phospho-beta-galactosidase and its amino-terminal sequence. This also allowed the exact positioning of the lacG gene and identification of its characteristic Gram-positive translation initiation signals. The homologous expression data and the sequence organization of the L. lactis lacG gene indicate that the gene is organized into a large lactose operon which contains an intergenic promoter located in an inverted repeat immediately preceding the lacG gene. The organization and sequence of the L. lactis lacG gene were compared with those of the highly homologous lacG gene from Staphylococcus aureus. A remarkable bias for leucine codons was observed in the lacG genes of these two species. Heterogramic homology was observed between the deduced amino acid sequence of the L. lactis phospho-beta-galactosidase, that of the functionally analogous E. coli phospho-beta-glucosidase, and that of an Agrobacterium beta-glucosidase (cellobiase).

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“…The higher concentration of galactose compared to glucose may be due to preferences in glucose consumption by the starter culture. Although, L. lactis and S. thermophilus are able to synthesise GOS through the enzyme b-galactosidase (Inamine et al, 1986;Martini et al, 1991), no GOS was detected in the starting whey.…”

Section: Source Of Caprine Milk Oligosaccharidesmentioning

confidence: 67%

Composition and enrichment of caprine milk oligosaccharides from New Zealand Saanen goat cheese whey

Thum

Cookson

McNabb

et al. 2015

Journal of Food Composition and Analysis

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Molecular and genetic characterization of lactose-metabolic genes of Streptococcus cremoris

Cited by 34 publications

References 27 publications

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

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

Structure and Expression of the Lactococcus lactis Gene for Phospho- -galactosidase (lacG) in Escherichia coli and L. lactis

Composition and enrichment of caprine milk oligosaccharides from New Zealand Saanen goat cheese whey

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