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

Abstract

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...

Molecular and Biochemical Analysis of the Galactose Phenotype of Dairy Streptococcus thermophilus Strains Reveals Four Different Fermentation Profiles

“…Mutations to a galactose-positive phenotype do not result in constitutive expression of the gal genes, which strongly suggests that S. thermophilus was Gal positive and became Gal negative in the recent past (30). Currently, the Gal-negative phenotype has been ascribed to a defect in the induction mechanism of GalK, which seems to be the rate-limiting enzyme of the Leloir pathway (2).…”

“…However, under appropriate selective conditions, such as limited lactose and excess galactose concentrations, Gal-positive derivatives of S. thermophilus can be obtained which ferment galactose and possess Leloir enzyme activities (11,14,22). It has been shown that the Gal-negative S. thermophilus strain CNRZ 302 contains structurally intact genes for the Leloir pathway enzymes, but that they are weakly transcribed, while independently isolated Gal-positive mutants contain up mutations in the gal promoter (30). The function of GalR has been determined as a transcriptional activator of both the gal and lac operons, which negatively regulates its own expression (30).…”

“…The galR gene is divergently transcribed and the gal operon consists of galK, galT, and galE. A polycistronic mRNA transcript galKTE has been reported in two S. thermophilus strains, namely CNRZ 302 and SMQ-301 (28,30). The galM gene has its own promoter (30).…”

Discovering lactic acid bacteria by genomics

“…Although a moderate thermophile, it is phylogenetically related to the more mesophilic lactococci and has a comparable low G+C ratio (40%). Genes coding for metabolic pathways involved in sugar catabolism (Poolman et al 1989;Vaughan et al 2001), protein and peptide utilization (Fernandez-Espla et al 2000;Garault et al 2002), polysaccharide production (Almirón-Roig et al 2000), the stress response system (Perrin et al 1999), and phage resistance mechanisms (Solow & Somkuti 2000;Burrus 2001) have been sequenced and characterized. Although most strains do not harbor plasmids, other mobile elements have been reported (Guedon et.…”

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

“…The glucose moiety is further metabolized via glycolysis, whereas the galactose moiety follows different pathways depending on the particular LAB. While some thermophilic strains of LAB (e.g., Lactobacillus bulgaricus and Streptococcus thermophilus) are known to release the galactose moiety of lactose into the medium, other LAB (e.g., Lactobacillus helveticus, Leuconostoc lactis and Streptococcus salivarius) metabolize this saccharide via the Leloir pathway (de Vos, 1996;Poolman, 1993;Vaughan et al, 2001). This pathway was one of the first central metabolic pathways to be discovered, by L. F. Leloir and coworkers in the early 1950s.…”