PLASMIDS FOR HEAVY-METAL RESISTANCE IN ALCALIGENES-EUTROPHUS CH34 - MECHANISMS AND APPLICATIONS

Alcaligenes eutrophus CH34 is the main representative of a group of st rongly related strains (mostly facultative chemolithotrophs) that are well adapted to environments containing high levels of heavy metals. I t harbors the megaplasmids pMOL28 and pMOL30 which carry resistance de terminants to Co2+, Ni2+, CrO42-, Hg2+, Tl+, Cd2+, Cu2+, and Zn2+. Amo ng the best characterized determinants are the cnr operon (resistance to Co, Ni) on pMOL28 and the czc operon on PMOL30 (resistance to Co, C d and Zn). Although the two systems reveal a significant degree of ami no acid similarity in the structural genes, the regulation of the oper ons is different. The resistance mechanism in both cases is based on e fflux. The efflux mechanism leads to a pH increase outside of the cyto plasmic membrane. Metals are sequestered from the external medium thro ugh the bioprecipitation of metal carbonates formed in the saturated z one around the cell. This latter phenomenon can be exploited in biorea ctors designed to remove metals from effluents. The bacteria are immob ilized on composite membranes in a continuous tubular membrane reactor (CTMR). The effluent continuously circulates through the intertubular space, while the external surface of the tubes is in contact with the growth medium. Metal crystals are eventually removed by the effluent stream and collected on a glass bead column. The system has been appli ed to effluents containing Cd, Zn, Co, Ni and Cu. By introducing catab olic plasmids involved in the aerobic degradation of PCBs and 2,4-D in to metal-resistant A. eutrophus strains, the application range was wid ened to include effluents polluted with both organic and inorganic sub stances. Biosensors have been developed which are based on the fusion of genes induced by metals to a reporter system, the lux operon of Vib rio fischeri. Bacterial luciferases produce light through the oxidatio n of fatty aldehydes. The gene fusions are useful both for the study o f regulatory genes and for the determination of heavy metal concentrat ions in the environment.