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Applied and Environmental Microbiology, September 1999, p. 4002-4007, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

D-Lactate Dehydrogenase Gene (ldhD) Inactivation and Resulting Metabolic Effects in the Lactobacillus johnsonii Strains La1 and N312

Luciane Lapierre, Jacques-Edouard Germond, Andreas Ott, Michele Delley, and Beat Mollet*

Nestlé Research Center, Nestlé Ltd., Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland

Received 5 March 1999/Accepted 27 June 1999

Lactobacillus johnsonii La1, a probiotic bacterium with demonstrated health effects, grows in milk, where it ferments lactose to D- and L-lactate in a 60:40% ratio. The D-lactate dehydrogenase (D-LDH) gene (ldhD) of this strain was isolated, and an in vitro-truncated copy of that gene was used to inactivate the genomic copy in two strains, La1 and N312, by gene replacement. For that, an 8-bp deletion was generated within the cloned ldhD gene to inactivate its function. The plasmid containing the altered ldhD was transferred to L. johnsonii via conjugative comobilization with Lactococcus lactis carrying pAMbeta 1. Crossover integrations of the plasmid at the genomic ldhD site were selected, and appropriate resolution of the cointegrate structures resulted in mutants that had lost the plasmid and in which the original ldhD was replaced by the truncated copy. These mutants completely lacked D-LDH activity. Nevertheless, the lower remaining L-LDH activity of the cells was sufficient to reroute most of the accumulating pyruvate to L-lactate. Only a marginal increase in production of the secondary end products acetaldehyde, diacetyl, and acetoin was observed. It can be concluded that in L. johnsonii D- and L-LDH are present in substantial excess for their role to eliminate pyruvate and regenerate NAD+ and that accumulated pyruvate is therefore not easily redirected in high amounts to secondary metabolic routes.

* Corresponding author. Mailing address: Nestlé Research Center, Nestlé Ltd., Vers-Chez-les-Blanc, P.O. Box 44, 1000 Lausanne 26, Switzerland. Phone: 41 (21) 7858930. Fax: 41 (21) 7858925. E-mail: beat.mollet{at}rdls.nestle.com.

Applied and Environmental Microbiology, September 1999, p. 4002-4007, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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