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Journal of Bacteriology, February 2001, p. 1184-1194, Vol. 183, No. 4
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.4.1184-1194.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Elaine E. Vaughan,1,2,* Patrick T. C. van den Bogaard,1 Pasquale Catzeddu,1,dagger Oscar P. Kuipers,1,Dagger and Willem M. de Vos1,2

Wageningen Centre for Food Sciences, NIZO Food Research, 6718 ZB Ede,1 and, Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 CT Wageningen,2 The Netherlands

Received 16 August 2000/Accepted 16 November 2000

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.

* Corresponding author. Mailing address: Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands. Phone: 31 317 483113. Fax: 31 317 483829. E-mail: elaine.vaughan{at}algemeen.micr.wau.nl.

dagger Present address: Porto Conte Ricerche, Santa Maria La Palma (SS), Italy.

Dagger Present address: Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9750 AA Haren, The Netherlands.

Journal of Bacteriology, February 2001, p. 1184-1194, Vol. 183, No. 4
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.4.1184-1194.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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