Clément, R. L.

Flatau, G. N.

Gauthier, M. J.

Mahdyoun, F.

OceanRep

Copyright ©Es gilt deutsches Urheberrecht.  Die Schrift darf zum eigenen Gebrauch kostenfrei heruntergeladen, konsumiert, gespeichert oder ausgedruckt, aber nicht im Internet bereitgestellt oder an Außenstehende weitergegeben werden ohne die schriftliche Einwilligung des Urheberrechtsinhabers. Es ist nicht gestattet, Kopien oder gedruckte Fassungen der freien Onlineversion zu veräußern. German copyright law applies.  The work or content may be downloaded, consumed, stored or printed for your own use but it may not be distributed via the internet or passed on to external parties without the formal permission of the copyright holders. It is prohibited to take money for copies or printed versions of the free online version. Kieler Meeresforsch., Sonderh. 8, 343-347. N. Kiel 1991 lnstitut National de la Santa et de la Recherche Medicale, U. 303 "Mer et Santa" 1 avenue Jean Lorrain, F-06300 Nice, France Introduction In some bacteria, the intracellular content of cadmium seems to depend on their resistance to the metal. In a strain of Staphylococcus aureus, a plasmid encoding for the resistance to S-lactams also provided resistance to zinc, cadmium, mer­cury, arsenic and lead (DYKE et al. 1970, EL-SOHL and EHRLICH 1982). In other strains of this species, resistance to cadmium can be afforded by an efflux system encoded by a resistance plasmid (TYNECKA et al. 198la, b) or by a chromosomic determinant (PERRY and SILVER 1982). Gram negative bacteria as well as Gram positive ones can develop a resistance to metals (TREVORS et al. 1985). Except for some species however (MERGEA Y et al. 1985, HORITSU et al. 1986), resistance to cadmium is generally not due to plasmid markers, but rather to metal trapping mechanisms, which seem to be more frequent than efflux or exclusion mechanisms (HIGHAM et al. 1985). Exo­polymers, through their ability to adsorb cadmium, can be considered as the first step of different resistance processes (BITTON and FRIEHOFER 1978). Detoxica­tion of cadmium can be achieved through its complexation by specific proteins, whose synthesis is induced by cadmium (KHAZAELI and MITRA 1981, HIGHAM and SADLER 1984) or by its trapping as granules of sulfides or phosphates (AIKING et al. 1984). Those results come from investigations on terrestrial bacteria. But how is it with marine bacteria, which are Gram negative halophilic microorganisms, for which availability of cadmium largely depends on environmental parameters? This work deals with different mechanisms of resistance to cadmium exerted by a marine bacterial strain sensitive to cadmium (MIC, 10 mg/1) and able to take up as much as 0.15 % of cadmium (w/w) dissolved in a saline solution. Material and methods The test strain was collected in a non polluted sediment in the Bay of Ville­franche-sur-Mer (Alpes-Maritimes, France). It belonged to the Pseudomonada­ceae. It was obligate aerobe, halophilic, and constituted by l x 2-3 µm motile rods. Its growth was inhibited by 10 mg Cd/1 (as chloride). Cells were, mass-produced by incubating Marine Agar (Difeo Lab. Mich.) plates for 18 h at 27 °C after inoculation with a previous culture incubated in Marine Broth (Difeo) for 7 h at 27 °C. Mureinoplasts and protoplasts were prepared as previously described (FLAT AU et al. 1986). 343 Cells were adapted by step by step cultures of the strain in progressively in­creasing concentration of cadmium dissolved in MB (FLAT AU et aL 1987a). Sulfides were analysed by the method of KING and MORRIS (1967). The role of the different cellular envelopes in the repartition of Cd was invest'i­gated by preparing mureinoplasts and protoplasts from previously intact cells ex­posed to 1 mg Cd/1 dissolved in a saline solution (SM) (FLA TAU et al. 1987b). The role of glucose was studied by adding this carbohydrate to the SM (final concentration 2 g/1) in which were suspended cells of the test strain. Results and discussion Influence of glucose: When cells were exposed for 3 h to 1 mg Cd/1 dissolved in SM, cells took up 0,96 ± 0.04 mg Cd/g (dry wt) (n = 4). When SM was supple­mented with glucose the uptake decreased to 0. 70 ± 0.02 mg Cd/g (n = 4), cor­responding to a significant decrease of 27 % (p � 0.01) and suggesting an inhibi­tory effect due to the carbohydrate. Role of different envelopes of the cell wall: After 24 h exposure to l mg Cd/1 dissolved in SM cells had taken up 1.98 mg Cd/g. After the removal of the exo­polymer coating, cellular content of cadmium was l. 72 mg Cd/g, i.e. a decrease of 13 % (p � 0.05). Mureinoplasts contained only 1.14 mg Cd/g corresponding to a decrease of 42 % relative to intact cells (p � 0.05). However, the difference in Cd content between mureinoplasts and protoplasts was not significant (Fig. 1). EHOPOLYMERS - - -EHURNAL MEMBRANE -PEPTIDOGL YCRNE CYTOPLRSM IC MEMBRANE CYTOPLRSM w H 0 E c E L 100% w s H E M � D u R c B p E I R L N L 0 T p 0 p 87% 47 to 57% Fig. 1. Repartition of cadmium in the different layers of the cells (percentage relative to the whole cells). Cells exposed for 24 h to Cd were washed three times to eliminate exopolymers (washed cells). Mureinoplasts (mureinop) and pro-toplasts (protop) were prepared from these washed cells. Adaptation of cells to cadmium: The length of the lag phase was not correlated with cadmium concentration in the medium. During their adaptation, cells were less and less able to utilize citrate, or to acidify saccharose, glucose, maltose and starch. Partial loss of motility and decrease of the length of cells were ob­served at 50 mg Cd/1. Cd uptake from MB was roughly linear with Cd concen-344 trations in MB (r = 0.979) but the accumulation factor was a decreasing function of Cd concentration in MB (r = 0.979). Cellular trapping of cadmium: In the presence of cadmium, neither new cellular protein nor an increase in phosphate level was detected, suggesting that the metal did not induce any de nova protein synthesis and was not trapped as phos­phate. However, cellular sulfide content increased with increasing Cd concentra­tion in MB (Fig. 2), indicating a probable precipitation as sulfide in or on the cells. 0) 3 y = 0,044 + 0,258x R = 0,99 .......... 2 (/) 0) E Qllllllllll-���-.-�-,-�-.-�--.--�����������--1 0 2 4 6 mg Cd/ 1 8 10Fig. 2. Increase of sulphides in c.ells (mg S/g dry wt) with the increase of cad­mium in the medium (MB) (mg Cd/1). Three mechanisms providing a resistance to cadmium were observed: (i) the trapping of the metal by the exopolymers and the envelopes, at the level of the cell wall, (ii) an efflux mechanism and (iii) the detoxication of cadmium as sul­fide which may take place at the level of the periplasmic space, the cytoplasmic membrane or the cytoplasm. In this strain, cadmium transport was likely energy-dependent (FLAT AU et al. 1989). Enrichment of SM by glucose could have provided a carbon source but also a higher level of energy which could have triggered efflux mechanisms, as it was assumed for other bacteria (BR YNHILDSEN et al. 1988). But the most ef­ficient mechanism was probably the trapping of cadmium by exopolymers and outer membrane, since mureinoplasts contained roughly two times less cadmium than whole cells. As the cell wall adsorbs a large part of cadmium load and thus prevent its penetration into the deeper layers of the cell, it plays a leading part in the protection of cytoplasm against the entry of cadmium (and probably other metals) and its toxicity (FLAT AU et al. 1987b). When cadmium enters the peri­plasmic space or reaches the cell membrane or the cytoplasm, other mechanisms take place. Cells have to adapt to cadmium which can impair cellular metabo­lism. The fact that the initial characteristics of the strain were restored after one subculture in a Cd-free medium indicated a physiological adaptation to cad­mium rather than a selection of mutants (FLATAU et al. 1987a). The survival of cells with gradually increasing cellular content of cadmium suggests that they detoxify the metal by precipitating it as sulfide less soluble than as chloride, thus less available and less toxic. 345 References AIKING, H., A. STIJNMAN, C. van GARDEREN, H. van HEERIKHUIZEN and J. van't RIET, 1984. Inorganic phosphate accumulation and cadmium detoxifi­cation in Klebsiella aerogenes NCTC 418 growing in continuous culture. Appl. Environ. Microbial. 47, 374-377. BITTON, G. and V. FRIEHOFER, 1978. Influence of extracellular polysaccharides on the toxicity of copper and cadmium on Klebsiella aerogenes. Microbial. Ecol. 4, 119-123. BR YNHILDSEN, L., B. V. LUNDGREN, B. ALLARD and T. ROSSWALL, 1988. Ef­fects of glucose concentrations on cadmium, copper, mercury and zinc to­xicity to a Klebsiella aerogenes sp. Appl. Environ. Microbial. 54, 689-693. DYKE, K.H.G., M.T. PARKER and M.H. RICHMOND, 1970. Penicillinase produc­tion and metal ion resistance in Staphylococcus aureus isolated from hospi­tal patients. J. Med. Microbial. 3, 125-136. EL SOHL, N. and S.D. EHRLICH, 1982. A small cadmium resistance plasmid iso­lated from Staphylococcus aureus. Plasmid 7, 77-84. FLATAU, G.N., R.L. CLEMENT and M.J. GAUTHIER, 1986. Influence of the cell envelope on the accumulation of cadmium by a marine pseudomonad. Microbios Lett. 32, 141-146. FLATAU, G.N., R.L. CLEMENT, M.J. GAUTHIER and F.M. LAUMOND, 1987a. Adaptation to cadmium in a sensitive marine pseudomonad. Environ. Tech­nol. Lett. 8, 641-646. FLATAU, G.N., R.L. CLEMENT, F. MAHDYOUN and M.J. GAUTHIER, 1987b. Repartition du cadmium dans les enveloppes cellulaires d'une bacterie ma­rine. Chemosphere 16, 2623-2626. FLATAU, G.N., R.L. CLEMENT, F. MAHDYOUN, G. LEBLANC and M.J. GAU­THIER, 1989. Role of transmembrane electrical potential on cadmium fixa­tion by a marine pseudomonad. Res. microbial. 140, 553-562. HIGHAM, D.P. and P.J. SADLER, 1984. Cadmium resistant Pseudomonas putidasynthesizes novel cadmium protein. Science 225, 1043-1045. HIGHAM, D.P., P.J. SADLER and M.W. SCAWEN, 1985. Effect of the cadmium on the morphology, membrane integrity and permeability of Pseudomonas putida. J. Gen. Microbial. 131, 2539-2544. HORITSU, H., K. YAMAMOTO, S. WACHI, K. KAWAI and A. FUKUCHI, 1986. Plasmid determined cadmium resistance in Pseudomonas putida GAM-1 iso­lated from soils. J. Bacterial. 165, 334-335. KHAZAELI, M.B. and R.S. MITRA, 1981. Cadmium binding component in Esche­richia coli during accomodation to low levels of this ion. Appl. Environ. Microbial. 41, 46-50. KING, T.E. and R.O. MORRlS, 1967. Determination of acid labile sulfide and sulfhydryl groups. Metods Enzymol. 10, 634-641. MERGEAY, M., D. NIES, H.G. SCHLEGEL, J. GERITS, P. CHARLES and F. van GISJSEGEM, 1985. Alcaligenes eutrophus CH34 is a facultative chemolitho­troph with plasmidbound resistance to heavy metals. J. Bacterial. 162, 328-334. 346 PERRY, R.D. and S. SILVER, 1982. Cadmium and manganese transport in Sta­phylococcus aureus membrane vesicles. J. Bacterial. 150, 973-976. TREVORS, T.J., K.M. DODIE and B.H. BELLIVEAU, 1985. Metal resistance in bacteria. FEMS Microbial. Rev. 32, 39-54. TYNECKA, Z., J. ZAJAC and z. GOS, 198la. Reduced cadmium transport by a resistance plasmid in Staphylococcus aureus. J. Bacterial. 147, 305-312. TYNECKA, Z., J. ZAJAC and Z. GOS, 198lb. Energy-dependent efflux of cad­mium coded by a plasmid resistance determinant in Staphylococcus aureus. J. Bacterial. 14 7, 313-319.347 

Resistance to cadmium in a marine bacterium

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Resistance to cadmium in a marine bacterium

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