Downloaded from www.microbiologyresearch.org by IP: 54.152.119.124 On: Thu, 20 Jul 2017 06:23:37 Microbiology (1996), 142, 1249-1 254 Printed in Great Britain Department of Plant Pathology and Microbiology, and Otto Warburg Center for Biotechnology in Agriculture, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel Biochemistry and Food Science, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 761 00, Israel 3 Division of Microbial and Molecular Ecology, institute of Life Sciences and Moshe Shilo Center for Marine Biogeochemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel * Department of The role of the outer membrane in formaldehyde tolerance in Escherichia coli VU3695 and Halornonas sp. MAC Malkit Azachi,’ Yigal Henis,’ Roni Shapka2and Aharon Oren3 Author for correspondence : Malkit Azachi. Tel : + 972 8 481 165. Fax : + 972 8 466794. e-mail : azachi@agri.huji.ac.il To investigate the mechanism of formaldehyde tolerance in Gram-negative bacteria, two formaldehyde-tolerant strains, Escherichia coli VU3695 and Halomonas sp. MAC (DSM 7328), and formaldehyde-sensitive revertants obtained by ethidium bromide or novobiocin treatment were studied. The presence of high levels of formaldehyde dehydrogenase activity alone proved insufficient to confer tolerance to high formaldehyde concentrations, as shown by the high activity displayed by formaldehyde-sensitive revertants of Halomonas MAC. Moreover, formaldehyde-tolerant strains also proved to be tolerant to high concentrations of acetaldehyde and glutaraldehyde, which are not oxidized by formaldehyde dehydrogenase. Treatment with sublethal concentrations of EDTA rendered the resistant strains highly sensitive to formaldehyde without affecting the activity of formaldehyde dehydrogenase. Comparison of the outer membrane proteins of formaldehyde-resistant strains with those of their sensitive revertants showed the presence of at least one additional high molecular mass protein in the tolerant strains. It is concluded that formaldehyde tolerance in the bacteria studied depends on the composition and structure of the outer membrane. 1 I Keywords : Escbericbia coli, Halomonas sp., formaldehyde, outer membrane, tolerance INTRODUCTION In recent years a number of reports have appeared on the occurrence of bacteria resistant to high concentrations of formaldehyde. Strains of Escherichia coli and Serratia marcescens tolerating formaldehyde concentrations of 500 and 200 p.p,m., respectively, have been isolated (Kaulfers & Marquardt, 1991 ; Kaulfers & Laufs, 1985). Likewise, highly formaldehyde-tolerant strains of Psezldomonasputida and Halamonas sp. have been characterized (Azachi et al., 1995; Kato et al., 1984, 1986). One of the factors that may play a role in conferring formaldehyde tolerance in bacteria is the presence of intracellular enzymes metabolizing formaldehyde. Thus, high levels of activity of an NAD- and glutathione- dependent formaldehyde dehydrogenase (EC 1 .2.1.1) were demonstrated in formaldehyde-resistant strains of E. coli, S. marcescens, and Halomonas sp. (Azachi et al., 1995; Kaulfers & Marquardt, 1991 ; Kaulfers & Laufs, 1985). Additional enzymes, such as formaldehyde dismutase (EC 1 .2.99.4) and dye-linked formaldehyde dehydrogenase (EC 1 .2.99.3) may be involved in the detoxification of formaldehyde in other micro-organisms (Kato et al., 1984, 1986, 1988). In certain bacteria at least, the property of formaldehyde tolerance may be plasmid-mediated (Kaulfers & Brandt, 1987; Kaulfers & Laufs, 1985). In the present work we investigated the possible role of the outer membrane of Gram-negative bacteria in for- maldehyde resistance. The outer membrane is the primary barrier that formaldehyde molecules encounter when interacting with these bacteria. The outer membrane has been shown to act as a molecular sieve which determines, mainly on the basis of molecular size, whether or not a substrate can enter the cell (Lieve, 1974; Nikaido & Vaara, 1985). To our knowledge, the role of the outer membrane in formaldehyde tolerance has not previously been investigated. We present evidence that the properties of the outer membrane are of prime importance in conferring formaldehyde tolerance in E. coli and in a Halomonas isolate. 0002-0498 0 1996 SGM 1249