System. App\. Microbiol. 8, 75-79 (1986) Haloarcula hispanica spec. nov. and Haloferax gibbonsii spec. nov., Two New Species of Extremely Halophilic Archaebacteria GUADALUPE JUEZh, F. RODRIGUEZ-VALERA I, ANTONIO VENTOSA2, and DONN J. KUSHNER 3 1 Departamento de Microbiologia, Facultad de Medicina, Universidad de Alicante, Alicante, Spain 2 Departamento de Microbiologia, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain 3 Biology Department, University of Ottawa, Ottawa, Ontario, Canada KIN 6N5 Received May 18, 1985 Summary After an extensive taxonomic study for extremely halophilic rods (Torreblanca et al., 1986), several groups appeared which did not fit with any previously described species of Halobacteria. In the present work two strains, Y27 (= ATCC 33960) and MA2.38 (= ATCC 33959), representatives of the rwo most distinct new groups, were studied in detail, and two new species are proposed. Key words: Haloferax gibbonsii - Haloarcula hispanica - Halobacteria - Archaebacteria Introduction In a previous work (Torreblanca et al., 1986) we studied a large number of extremely halophilic rods, members of the Halobacteriaceae, with regard to several phenotypic features which were analyzed by numerical taxonomy. The polar lipid composition of some representatives was also studied. These results together with those of other authors on genotypic analysis (Ross and Grant, 1985) led us to propose the grouping of the non-alkaliphilic halobacteria into three genera: Halobacterium, Haloarcu- La and Haloferax. Within each genus there were groups of strains which did not fit any of the previously described species. In the present work we have studied in detail one representative of each of the two most distinct groups. Both have proved to be sufficiently different to be considered new species. One was included in the genus Haloferax, for which we propose the species designation of Haloferax gibbonsii. The other group studied, also has characteristics that are different enough for it to be considered a new species of Haloarcula and the name H. hispanica is proposed. Materials and Methods Source of the microorganisms Strains were isolated from a solar saltern near Alicante (Spain) by means of enrichments in media of different complexity (Tor- • To whom offprint requests should be sent. reblanca et al., 1986). Strains Y27 and MA2.38 were studied as representatives of the phenons F (12 strains) and K (17 strains) respectively (Torreblanca et al., 1986). Susceptibility to antimicrobial agents The following antibiotics were tested: ampicillin (10 l1g/ml), bacitracin (40 l1g/ml), carbenicillin (50 Itg/ml), chloramphenicol (30 Itg/ml), cycloserine (30 Ilg/ml) , erythromycin (15 Itg/ml), gen- tamycin (10 Ilglml) , gramicidin (30 Ilg/ml), kanamycin (30 ltg/ ml), neomycin (30 I1glml ), penicillin (10 IU/ ml), polymyxin (300 IU/ml), streptomycin (10 Ilg/ml), and tetracycline (30 ltg/mI) . HgClz and novobiocin were tested at different concentrations: 1.25,2.5,5, 10,20 or 40 l1g/ml. Analysis of cell envelopes Harvested cells from 5000 ml of culture were resuspended in 50 ml of 25% (w/v) NaCl and broken by decompression in a pressure chamber (Parr Instrument Co.) at HIOO MPa of pure argon. The treatment was repeated until the suspension became very viscous (usually 3-4 times). This thick suspension was di- luted ten times with 25% (w/v) NaCI to reduce viscosity and envelopes were prepared by the method of Steens land and Larsen (1969). Salt-free dry weight was determined by drying at 105 °C to constant weight and subtracting the ash remaining after heating at 650°C for 4 h. Protein content was evaluated by the method of Lowry et al. (1951), using bovine serum albumin (Sigma) as standard. Neutral hexoses were determined by the anthrone as-