Tolerance to Cadmium of Free-Living and Associated with Marine Animals and Eelgrass Marine Gamma-Proteobacteria Elena P. Ivanova, 1,2 Valerie V. Kurilenko, 1 Arcady V. Kurilenko, 3 Nataliya M. Gorshkova, 1 Felix N. Shubin, 4 Dan V. Nicolau, 2 Viktor P. Chelomin 3 1 Pacific Institute of Bio-organic Chemistry, Far-Eastern Branch of the Russian Academy of Sciences, pr. 100 let Vladivostoku, 159, 690022, Vladivostok, Russia 2 Industrial Research Institute Swinburne, Swinburne University of Technology, PO Box 218, Hawthorn, Vic 3122, Australia 3 Pacific Oceanological Institute, Far-Eastern Branch of the Russian Academy of Sciences, Baltiiskaya Str., 43, 690017, Vladivostok, Russia 4 Institute of Epidemiology and Microbiology, Sibirean Branch of the Russian Academy of Medicine, 690000 Vladivostok, Russia Received: 25 July 2001 / Accepted: 27 August 2001 Abstract. The tolerance to Cd 2 and possible mechanisms of Cd 2 detoxification by 178 free-living bacteria isolated from sea water, associated with marine animals (a mussel Crenomytilus grayanus, a scallop Patinopecten yessoensis), and eelgrass Zostera marina collected in The Sea of Japan and The Sea of Okhotsk have been studied. The concentrations of 25 and 50 mg Cd 2 /L were highly toxic and inhibited the growth from 54% to 78% of the total bacteria studied. The free-living bacteria isolated from seawater samples (up to 50%) were tolerant to high concentrations of cadmium. Marine gamma-proteobacteria tolerated Cd 2 by the activation of different detoxifying mechanisms. The strain Halomonas sp. KMM 734 isolated from seawater prevented the uptake of Cd 2 into bacterial cells. The chromosomal cadmium resistance system of Pseudoalteromonas citrea KMM 461 and Marinobacter sp. KMM 181 was found to be similar to class III metallothioneins (also known as phytochelatins). The presence of cadmium in the marine environment is of concern since this metal is highly toxic to most or- ganisms. However, some microorganisms survive in the presence of elevated levels of cadmium by employing detoxification mechanisms [2, 3, 5, 14, 15], including efflux “pumping” facilitated by efflux ATPases in Bacil- lus [12], Listeria [16], and Staphylococcus plasmids [4, 20], chemiosmotic cation-proton antiporters in Gram- negative soil bacteria [1], and metal-binding proteins that contain thiolate ligands [7]. Despite intensive investiga- tions of heavy metal resistance systems, including those for cadmium, the fundamental mechanism of bacterial tolerance or gene regulation at the molecular level is not understood [20]. In addition, little information is avail- able regarding evaluation of cadmium tolerance em- ployed by marine gamma-proteobacteria. The important role played by marine oxidative proteobacteria in the environment owing to their abun- dance and high metabolic activities makes them suit- able candidates for studies of the effects of cadmium and mechanisms of its detoxification. The present study focuses on the effects of Cd  on free-living bacteria, associated with marine invertebrates and ep- iphytes of eelgrass, and evaluation of possible mech- anisms of Cd  detoxification. Materials and Methods Bacterial strains and growth conditions. A total of 178 strains of marine bacteria that we used in this study were isolated from samples of sea water, marine animals (mussel Crenomytilus grayanus, scallop Patinopecten yessoensis), and eelgrass Zostera marina collected by scuba diving at a depth of 6–12 m in The Sea of Japan and The Sea of Okhotsk in 1988 –1998. The bacteria studied belong to the class Proteobacteria of the gamma-subclass, namely: Alteromonas, Pseudo- alteromonas, Marinobacter, Halomonas, Shewanella, to Flexibacter/ Bacteroides/Flavobacterium phylum, and some other genera as de- scribed elsewhere [9 –11; E. Ivanova, unpublished data]. In addition, several type strains of Alteromonas, Pseudoalteromonas were also included. All strains were routinely cultured on a medium that con- tained 0.2% (wt/vol) Bacto Peptone, 0.2% (wt/vol) casein hydrolysate, Bacto Yeast Extract, 0.1% (wt/vol) glucose, 0.002% (wt/vol) KH 2 PO 4 , 0.005% (wt/vol) MgSO 4 7H 2 O, 15% (wt/vol) of agar, 50% (vol/vol) of Correspondence to: E.P. Ivanova; email: eivanova@swin.edu.au CURRENT MICROBIOLOGY Vol. 44 (2002), pp. 357–362 DOI: 10.1007/s00284-001-0017-5 Current Microbiology An International Journal © Springer-Verlag New York Inc. 2002