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Applied and Environmental Microbiology, April 2002, p. 1541-1547, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.1541-1547.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Cloning of rel from Listeria monocytogenes as an Osmotolerance Involvement Gene

Yumiko Okada,1* Sou-ichi Makino,2 Toru Tobe,3,{dagger} Nobuhiko Okada,4 and Shouji Yamazaki1

Department of Veterinary Public Health, The National Institute of Public Health, Tokyo 108-8638,1 Department of Veterinary Microbiology, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080-8555,2 Division of Bacterial Infection, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639,3 Department of Microbiology, School of Pharmaceutical Sciences, Kitasato University, Tokyo 108-8641, Japan4

Received 1 October 2001/ Accepted 31 January 2002

Transposon insertional mutants of Listeria monocytogenes were constructed to identify genes involved in osmotolerance, and one mutant that showed reduced growth under high osmotic pressure was obtained. The cloned gene from the transposon insertion site of the mutant, named rel, was 2,214 bp in length and had very high homology to relA of Bacillus subtilis, which encodes guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively designated (p)ppGpp] synthetase during stringent response. The mutant showed a deficiency in (p)ppGpp accumulation. In the parental strain, the amount of intracellular (p)ppGpp was not increased after an osmotic upshift but was slightly decreased compared with the level before the upward shift. The reduced osmotolerance of the mutant was restored to a level almost equal to that of the parent strain when the chromosomal region that included rel of L. monocytogenes was introduced into the mutant. After exposure to methyl glucoside, the rel mutant accumulated (p)ppGpp at a higher level than the basal level and partially restored the ability to grow in NaCl-supplemented brain heart infusion broth. The mutant was found to grow in chemically defined minimal medium supplemented with glycine betaine or carnitine, so-called compatible solutes, and 4% NaCl. Our results suggest that the appropriate intracellular concentration of (p)ppGpp is essential for full osmotolerance in L. monocytogenes and that its mechanism is different from that for the accumulation of compatible solutes.

* Corresponding author. Mailing address: Department of Veterinary Public Health, The National Institute of Public Health, Tokyo 108-8638, Japan. Phone: 81-3-3441-7111. Fax: 81-3-3446-4314. E-mail: okada{at}iph.go.jp.

{dagger} Present address: Division of Applied Bacteriology, Osaka University, Graduate School of Medicine, Osaka 565-0871, Japan.

Applied and Environmental Microbiology, April 2002, p. 1541-1547, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.1541-1547.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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