Galactose-fermenting mutants of <i>Streptococcus thermophilus</i>

Benateya, Amina; Bracquart, Patrice; Linden, G.

doi:10.1139/m91-020

Cited by 13 publications

(7 citation statements)

References 16 publications

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“…In the work reported here, we demonstrated that S. thermophilus SMQ-301 was able to grow on galactose when complemented with the S. salivarius galK gene cloned on a lowcopy plasmid. Our results established that the inability of strain SMQ-301 to grow on galactose resulted from low galactokinase activity, a phenotypic defect that has previously been reported in several other S. thermophilus strains (6,20,35). As galactose metabolism also requires the galE and galT gene products, our results also confirmed that the S. thermophilus gal promoter was functional, as previously demonstrated by determinations of enzyme activities (10,30,35,40) and transcriptional studies (40).…”

Section: Discussionsupporting

confidence: 88%

“…Previous studies conducted with spontaneous or chemically induced S. thermophilus Gal ϩ mutants revealed that these strains still excreted galactose during growth on lactose (6,20,35,37). However, as the genotypes of these mutant strains were unknown, it was not possible to determine whether their phenotype resulted solely from an increase in galK expression.…”

Section: Discussionmentioning

confidence: 95%

“…This may account, at least in part, for the inability of S. thermophilus to grow on galactose (6,19,29). This hypothesis is reinforced by the observation that spontaneous S. thermophilus Gal ϩ mutants possess higher galactokinase activity than the wild-type strains (6,19,35). It is noteworthy that these mutants are unstable and lose their Gal ϩ phenotype under normal growth conditions in milk (35).…”

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confidence: 97%

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Characterization of a Galactokinase-Positive Recombinant Strain of Streptococcus thermophilus

Vaillancourt

LeMay

Lamoureux

et al. 2004

Appl Environ Microbiol

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The lactic acid bacterium Streptococcus thermophilus is widely used by the dairy industry for its ability to transform lactose, the primary sugar found in milk, into lactic acid. Unlike the phylogenetically related species Streptococcus salivarius, S. thermophilus is unable to metabolize and grow on galactose and thus releases substantial amounts of this hexose into the external medium during growth on lactose. This metabolic property may result from the inability of S. thermophilus to synthesize galactokinase, an enzyme of the Leloir pathway that phosphorylates intracellular galactose to generate galactose-1-phosphate. In this work, we report the complementation of Gal ؊ strain S. thermophilus SMQ-301 with S. salivarius galK, the gene that codes for galactokinase, and the characterization of recombinant strain SMQ-301K01. The recombinant strain, which was obtained by transformation of strain SMQ-301 with pTRKL2TK, a plasmid bearing S. salivarius galK, grew on galactose with a generation time of 55 min, which was almost double the generation time on lactose. Data confirmed that (i) the ability of SMQ-301K01 to grow on galactose resulted from the expression of S. salivarius galK and (ii) transcription of the plasmid-borne galK gene did not require GalR, a transcriptional regulator of the gal and lac operons, and did not interfere with the transcription of these operons. Unexpectedly, recombinant strain SMQ-301K01 still expelled galactose during growth on lactose, but only when the amount of the disaccharide in the medium exceeded 0.05%. Thus, unlike S. salivarius, the ability to metabolize galactose was not sufficient for S. thermophilus to simultaneously metabolize the glucose and galactose moieties of lactose. Nevertheless, during growth in milk and under time-temperature conditions that simulated those used to produce mozzarella cheese, the recombinant Gal ؉ strain grew and produced acid more rapidly than the Gal ؊ wild-type strain.Streptococcus thermophilus is widely used in yoghurt fermentation and cheese making. Specific S. thermophilus strains may also have the potential to improve human health as probiotics. For instance, they may interfere with the adhesion of indigenous cariogenic bacteria to teeth (9) and may prolong the useful life of indwelling voice prostheses by preventing colonization by Candida spp. (8).The ability of S. thermophilus to rapidly take up sugars from the environment is a prerequisite for these applications. Unlike several other lactic acid bacteria, S. thermophilus only uses a few sugars, with most strains showing a marked preference for saccharose and lactose, while glucose and fructose are more slowly fermented, if they are fermented at all (15,19). S. thermophilus takes up lactose via the membrane protein LacS, a member of the glycoside-pentoside-hexuronide:cation symporter family (29). Inside the cell, the disaccharide is hydrolyzed into glucose and galactose by the enzyme ␤-galactosidase. While all strains metabolize glucose via the EmbdenMeyerhof-Parnas pathway, most are unabl...

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Section: Discussionsupporting

confidence: 88%

Section: Discussionmentioning

confidence: 95%

mentioning

confidence: 97%

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Characterization of a Galactokinase-Positive Recombinant Strain of Streptococcus thermophilus

Vaillancourt

LeMay

Lamoureux

et al. 2004

Appl Environ Microbiol

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“…1998), and that some Strep. thermophilus strains can utilize galactose (Benateya et al . 1991), it is not clear that these enzymes are functional in strains of Lact.…”

Section: Discussionmentioning

confidence: 99%

Exopolysaccharide-producing strains of thermophilic lactic acid bacteria cluster into groups according to their EPS structure

Marshall

Laws

et al. 2001

Lett Appl Microbiol

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Aims: To compare galactose-negative strains of Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus isolated from fermented milk products and known to produce exopolysaccharides (EPSs). Methods and Results:The structures of the EPSs were determined using nuclear magnetic resonance (NMR) and their genetic relationships determined using restriction endonuclease analysis (REA) and random ampli®cation of polymorphic DNA (RAPD). Similar groupings were apparent by REA and RAPD, and each group produced an EPS with a particular subunit structure. Conclusions: Although none of the strains assimilated galactose, all inserted a high proportion of galactose into their EPS when grown in skimmed milk, and fell into three distinct groups. Signi®cance and Impact of the Study: This information should help in an understanding of genetic exchanges in lactic acid bacteria.

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“…The present study was undertaken to gain insight into the presence and regulation of the gal genes of S. thermophilus and the mechanism by which the genes, in particular the galK gene, are prevented from being expressed. Here we describe the characterization of the gal operon, consisting of the galK, galT and galE genes, and its promoter from S. thermophilus CNRZ 302, for which galactose-fermenting (Gal ϩ ) revertants have been reported (5). A regulatory gene, galR, was identified that is divergently transcribed from the gal operon.…”

mentioning

confidence: 99%

Activation of Silent gal Genes in the lac-gal Regulon of Streptococcus thermophilus

Vaughan

Bogaard²,

Catzeddu³

et al. 2001

J Bacteriol

107

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Streptococcus thermophilus strain CNRZ 302 is unable to ferment galactose, neither that generated intracellularly by lactose hydrolysis nor the free sugar. Nevertheless, sequence analysis and complementation studies with Escherichia coli demonstrated that strain CNRZ 302 contained structurally intact genes for the Leloir pathway enzymes. These were organized into an operon in the order galKTE, which was preceded by a divergently transcribed regulator gene, galR, and followed by a galM gene and the lactose operon lacSZ. Results of Northern blot analysis showed that the structural gal genes were transcribed weakly, and only in medium containing lactose, by strain CNRZ 302. However, in a spontaneous galactose-fermenting mutant, designated NZ302G, the galKTE genes were well expressed in cells grown on lactose or galactose. In both CNRZ 302 and the Gal ؉ mutant NZ302G, the transcription of the galR gene was induced by growth on lactose. Disruption of galR indicated that it functioned as a transcriptional activator of both the gal and lac operons while negatively regulating its own expression. Sequence analysis of the gal promoter regions of NZ302G and nine other independently isolated Gal ؉ mutants of CNRZ 302 revealed mutations at three positions in the galK promoter region, which included substitutions at positions ؊9 and ؊15 as well as a single-base-pair insertion at position ؊37 with respect to the main transcription initiation point. Galactokinase activity measurements and analysis of gusA reporter gene fusions in strains containing the mutated promoters suggested that they were gal promoter-up mutations. We propose that poor expression of the gal genes in the galactose-negative S. thermophilus CNRZ 302 is caused by naturally occurring mutations in the galK promoter.After its discovery almost 40 years ago, the Escherichia coli lactose operon, encoding enzymes of lactose metabolism, became the first model for gene regulation (reviewed in reference 4). The key component of this system is the lac repressor (LacI), the product of the lacI gene. The lac operon contains a primary operator (O 1 ), which is the major element of repression by LacI, and two pseudo-operators, which enhance repressor binding to O 1 by cooperativity. Control of the lac operon also involves activation by the cyclic AMP receptor protein.Many other paradigm systems of negative control have since been described, including GalR, one of the two repressors of the gal regulon encoding enzymes of galactose transport and metabolism in E. coli. Regulation of the gal regulon is mediated through GalR, GalS (Gal isorepressor), and the cyclic AMP receptor protein. GalR and GalS negatively regulate transcription of the two promoters of the gal operon, although GalS is not as efficient as GalR (57).The bioconversion of lactose, which is the primary carbon and energy source in milk, into lactic acid is an essential process in industrial dairy fermentations carried out by lactic acid bacteria. Genetic studies of the metabolic pathways for lactose utilization i...

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Galactose-fermenting mutants of Streptococcus thermophilus

Cited by 13 publications

References 16 publications

Characterization of a Galactokinase-Positive Recombinant Strain of Streptococcus thermophilus

Characterization of a Galactokinase-Positive Recombinant Strain of Streptococcus thermophilus

Exopolysaccharide-producing strains of thermophilic lactic acid bacteria cluster into groups according to their EPS structure

Activation of Silent gal Genes in the lac-gal Regulon of Streptococcus thermophilus

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