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Applied and Environmental Microbiology, February 2007, p. 897-905, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.01945-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Exopolysaccharide Expression in Lactococcus lactis subsp. cremoris Ropy352: Evidence for Novel Gene Organization{triangledown}

Eric P. Knoshaug,1,{dagger} Jeff A. Ahlgren,2,{ddagger} and Janine E. Trempy1*

Department of Microbiology, Oregon State University, Corvallis, Oregon 97331-3804,1 Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, USDA, Peoria, Illinois 616042

Received 16 August 2006/ Accepted 15 November 2006

Lactococcus lactis subsp. cremoris Ropy352 produces two distinct heteropolysaccharides, phenotypically described as ropy and mucoid, when cultured in nonfat milk. One exopolysaccharide precipitated with 50% ethanol as a series of elongated threads and was composed of glucose and galactose in a molar ratio of 3:2. The second exopolysaccharide precipitated with 75% ethanol as a fine flocculant and consisted of galactose, glucose, and mannose with a molar ratio of 67:21:12. A mutant strain, L. lactis subsp. cremoris EK240, lacking the ropy phenotype did not produce the exopolysaccharide that precipitated with 50% ethanol; however, it produced the exopolysaccharide that precipitated with 75% ethanol, indicating that the former exopolysaccharide is essential for the ropy phenotype. Cultures of L. lactis subsp. cremoris Ropy352 in 10% nonfat milk reached a viscosity of 25 Pa-s after 24 h, while those of the nonropy L. lactis subsp. cremoris EK240 mutant did not change. A mutation abolishing ropy exopolysaccharide expression mapped to a region on a plasmid containing two open reading frames, epsM and epsN, encoding novel glycosyltransferases bordered by ISS1 elements oriented in the same direction. Sequencing of this plasmid revealed two other regions involved in exopolysaccharide expression, an operon located between partial IS981 and IS982 elements, and an independent gene, epsU. Two and possibly three of these regions are involved in L. lactis subsp. cremoris Ropy352 exopolysaccharide expression and are arranged in a novel fashion different from that of typical lactococcal exopolysaccharide loci, and this provides genetic evidence for exopolysaccharide gene reorganization and evolution in Lactococcus.

* Corresponding author. Mailing address: Department of Microbiology, Nash Hall 220, Oregon State University, Corvallis, OR 97331-3804. Phone: (541) 737-4441. Fax: (541) 737-0496. E-mail: trempyj{at}oregonstate.edu.

{triangledown} Published ahead of print on 22 November 2006.

{dagger} Present address: National Renewable Energy Laboratory, National Bioenergy Center, Golden, CO 80401.

{ddagger} Present address: Wyatt Technology Corporation, Santa Barbara, CA 93117.

Applied and Environmental Microbiology, February 2007, p. 897-905, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.01945-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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