International Journal of Systematic Bacteriology (1 48, 1 187-1 196 Printed in Great Britain

~~ 998),

Proposal of a new halobacterial gen. nov., with two Natrinema pellirubrum norn. nov. and Natrinerna pallidum norn. nov.

Terry J. McGenity,’e2 Renia T. Gemmel12tand William D. Grant2

Author for correspondence : Terry J. McGenity. Tel : + 44 1 18 93 1 67 13. Fax : + 44 1 18 93 1 0279. e-mail: t.j.mcgenity(u reading.ac.uk

Postgraduate Research A phylogenetic analysis of 69 halobacterial 16s rRNA gene sequences has been Institute for carried out, integrating data from new isolates, previously described Sedimentology, University of Reading, Whiteknights, halobacteria and cloned sequences from uncultivated halobacteria. PO Box 227, Reading RG6 Halobacterium halobdum NCIMB 777, Halobacterium trapanicum NCIMB 784 and 6AB, UK Halobacterium salinarium NCIMB 786, together with several other strains Department of (strains T5.7, L11 and Halobacterium trapanicum NCIMB 767) constitute a Microbiology and distinct lineage with at least 98.2 O/O sequence similarity. These strains have Immunology, University of Leicester, University Road, been incorrectly assigned to the genus Halobacterium. Therefore, based on a Leicester LE1 9HN, UK variety of taxonomic criteria, it is proposed that Halobacterium salinarium NCIMB 786 is renamed as Natrinema pellirubrum nom. nov., the type species of the new genus Natrinema gen. nov., and that Halobacterium halobium NCIMB 777 and Halobacterium trapanicum NCIMB 784 are renamed as a single species, Natrinema pallidum nom. nov. It was notable that halobacteria closely related to the proposed new genus have been isolated from relatively low-salt environments.

Keywords: Natrinema gen. nov., halobacteria, hypersaline, halophile, phylogeny

INTRODUCTION mediated ion pumps (Ventosa & Nieto, 1995); (3) they have been isolated from ancient salt deposits on Earth, The term ‘ halobacteria’ refers to the red-pigmented, promoting suggestions of great longevity, thus extremely halophilic , members of the family influencing the choice of sites for life-detection experi- Halobacterinceae (Grant & Larsen, 19894. Halo- ments on other planets such as Mars (Grant, 1995). bacteria arc the most halophilic organisms known, and dominate in hypersaline environments in which the The of the halobacteria was last compre- salt concentration exceeds 250 g (Rodriguez-Valera hensively revised in 1989 (Grant & Larsen, 1989a) 1-1 when six genera were validly described. Many halo- et al., 198 1 1. Often, they appear at such high levels that they impart a red colour to hypersaline brines. bacteria that do not precisely match existing taxa have been described recently; this, together with re- Earlier this century, most studies on halobacteria assessment of existing taxa using phylogenetic concentrated on those proteolytic strains that spoiled methods, has led to the taxonomy of the group being in salted fish. meat and hides (Harrison & Kennedy, a state of flux. For example, Kamekura et al. (1997), 1922). However, there is now growing interest in the on the basis of 16s rRNA gene sequence comparisons, halobacteria for several reasons, for example : (1) they showed that the haloalkaliphiles are much more are easy to cultivate and so make ideal systems for diverse than had previously been imagined. In all, investigating the molecular genetics of archaea (Robb there are presently 10 formally described genera of et al., 1995) ; (2) they have biotechnological potential, halobacteria (Oren et a/., 1997; Kamekura et al., particularly with respect to polymers and light- 1997), and it is clear that halobacterial diversity extends beyond these 10, particularly when uncul- ...... , . .. . ., ., . . . . ., .. . .. , ...... t Present address: Oxford Centre for Environmental Biotechnology, tivated halobacteria are considered. For example, Institute of Virology and Environmental Microbiology, Mansfield Road, Benlloch et al. (1 995) described two 16s rRNA clones Oxford OX1 3SR. UK. from a crystallizer pond which formed a distinct

~ 00761 0 1998lUMS 1187 T. J. McGenity, R. T.Gemmel1 and W. D. Grant

Table 1. Sources of halobacteria and their 165 rRNA sequences

Strains sequenced as part of this study are in bold. Type species are indicated by [TI. T, Type strain. Culture collections: ACAM, Australian Collection of Antarctic Microorganisms, Tasmania, Australia ; ATCC, American Type Culture Collection, Rockville, MD, USA ; DSM, DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany; JCM, Japanese Collection of Microorganisms, RIKEN, Japan; NCIMB, National Collection of Industrial and Marine Bacteria, Aberdeen, UK; N RC, National Research Council, Ottawa, Canada ; VKM, All-Union Collection of Microorganisms, Puschino, Russia. Accession numbers are for the 16s rRNA sequences in databases; references describe the 16s rRNA sequences; numbering of region sequenced is according to E. coli (Brosius er nl., 1978).

Specieslstrain Strain Isolated from : Accession Sequenced Reference no. region

ATCC 29605'" Dcad Sea KO042 1 18-1534 Gupta rt uI (1 983) ATCC 33500" Saltein (Alicmte, Spain) Dl 1107 18 1534 Kamekura & Seno (1992) ATCC 33959' Saltern (Alicmte, Spain) DI 3378 18-1 534 Kamekura & Seno ( 1993) ATCC 35960" Saltern ( BJJ?I.~.California) D14128 18-1534 Kamekura & Sen0 (1993) (Ad? 2) Saltern (Alic'inte. Spain) M33803 107-536 Holmes & Dyall-Smith ( 1990) (Aa2.2) Saltern (Alicante. Spain) M33804 675-924 Holmes & Dyall-Smith ( 1990) ( Aa2. 2) Saltern (Alicante. Spain) M33805 965- 1 5 1 6 Holmes & Dyall-Smith ( 1990) Strain EX Dcad Sea, 57-year-old sample U68540 1 8- I 534 Arahal Pt trl. (1996) Clone 2MT310 Salt marsh (Colne Point, UK) AF015988 18-1114 Munsoii rt a/. ( 1997) Clone 2MT103 Salt marsh (Colne Point. UK) AFO 15982 18-1114 Munson et nl. (1997) Clone 1MT315 Salt marsh (C'olne Point, UK) AFOl5964 18-1 I14 Munson t't (11. ( 1997) Holntrrculm I~dourcLrluiuNi.sniortis I [TI ATCC 29715' Salt flat (Death Valley. USA) UI 7593 18-1 534 Kamekura & Dyall-Smith ( 1995) Hrrlourcdu i~dlisninrtis2 (TI ATCC 29715' Salt flat (Death Valley. USA) D5085 1 18-1 534 Ihara rt ul. (1997) Hirlourcirlu niurisniortiri rrnA genc ( Ginzburg) Dead Sea X61688 18-1534 Mylvaganam & Dennis (1992) Haloarcula niurisniortui rrnB gene (Ginhurg) Dead Sea X6 1689 18-1534 Mylvaganam & Dennis (1992) ' H(rlotrrcdci siriuiicrisis ' major gene ATCC 33800' Red Sea sabkha Dl4129 18-1 534 Kamekura & Seno (1993) ' H~lo~ir~~uInsinaiicwsis ' minor gene ATCC 33800' Red Sea sabkha D14130 18-1534 Kamekura & Seno (1993) Huloarculu ,jupotiicu JCM 7785' Saltern soil (Noto Peninsula. Japan) D28872 18-1492 Takashina et ul. (1 994) Hulourcxlu Iiisjiuiii(z ATCC 33960' Saltern (Alicante, Spain) U68541 18-1534 Arahal cf a/. (1996) ' Hrrlomrciilcr uidinotisis' (B-2) Salt lake (Xinjiang. China) AB000563 18-1534 Unpublished Hdotrrcirln nrgui t incwsis JCM 9737' Salt flat (Saliiias Chicas, Argentina) D50849 18-1 534 lhara c't NI. (1997) Holourc~irluniirkolirrtci JCM 9738' Salt flat (Salinas Grandes. D50850 18-1 534 lhara ct 01. (1997) Argent i na ) Strain E? Dcad Sea. 57-year-old sample U68539 18-1 534 Arahal er d. (1996) Strain E I I Dead Sea. 57-year-old sample U68537 18-1534 Arahal r,t d. (1996) NATRO GROUP NNt1.0110(.0(.(.1iS N(itr[)iioi,o(,c,ri.soc,cirltu.c. [TI NClMB 2192'' Lake Magndi (Kenya) 228378 18-1 534 McGenity & Grant (1993) N(rtrorioc,oc,eLis rrt?iylo!l.ticrr.c.jlj~tii,ii,~ JCM 9655" Lake Magadi (Kenya) D43628 18-1 534 Kanai rt NI. (1 995) Strain 89M4 Lake Magadi (Kenya) x92172 36-1515 Duckworth rt ul. (1996) Strain 931LM4 Little Lake Magadi (Kenya) X92173 31-1515 Duckworth rt (11. (1996) Strain 86M4 Lake Magadi (Kenya) X92 175 32- 1 5 1 5 Duckworth cJt al. (1996) Nutrono hrr c. t i'r iun I

Natroiiohartcriuni gregorj'i[T NClMB 2189'' Lake Magadi saltern (Kenya) DX7970 1 8-1 534 Kamekura

Hrrlohuctoriimi trupanicxni NClMB 767 t NRC 34021i- Dl4125 18-1534 Kamekura & Seiio (1993) Strain L-ll (GSL-I I) Great Salt Lake (UT, USA) D14126 18-1 534 Kamekura & Dyall-Smith (1995) Strain T5.7 Former saltcrn (NE Thailand) A5002946 18-1 534 This study Strain 524 Saline lagoon (Crimea) AJ002952 278-500 This study

1188 International Journal of Systematic Bacteriology 48 New halobacterial genus Natrinema gen. nov. _____ ~~

Table I (cont.)

Specieslstrain Strain Isolated from : Accession Sequenced Reference no. region

NATRO 01JTLIERS Strain SKIS Former saltern (Roquetas de Mar. AJ002945 18-1 534 This study Spain) Little Lake Magadi (Kenya) X92 170 25-1515 Duckworth c't a/. (1996) Lake Magadi (Kenya) x92171 23-1 5 16 Duckworth et ul. (1996) Salt marsh (Colne Point, UK) AFO 15989 24-1 114 Munson st a/. (1997)

NCIMB 2081' Saltern (San Francisco, USA) X82 167 18-1 534 McGenity & Grant (1995) ACAM 34' Deep Lake (Antarctica) X82170 18-1 534 McGenity & Grant (1 995) ATCC 33755' Dead Sea X82169 18-1 534 McGenity & Grant (1995) NRC 34021 Trapani salt X82 168 18-1 534 McGenity & Grant (1995) JCM 9275T Saltern (Geelong. Australia) LO0922 18-1 534 Nuttall & Dyall-Smith (1993) VKM B-1733'" Sulphate saline soil (Turkmenia) D63572 18-1 534 Kamekura & Dyall-Smith (1995) - Great Salt Lake (UT, USA) A5002944 18-1 534 This study Saltern (Cabo de Gata, Spain) A5002943 18-1534 This study JCM 9060' Lake Magadi (Kenya) D87972 18-1534 Kamekura et a/. (1997)

DSM 671 Salted herring M38280 113-3534 Mankin et a/. (1985)

NRC 34001 Salted buffalo hide KO2971 18-1 534 Hui & Dennis (1985)

DSM 668 Salted fish X92978 18-927 Jurgens rt a/. (1997) Saltern D14127 18-1 534 Kamekura & Seno (1993) Dead Sea. 57-year-old sample U68538 18-1534 Arahal et a/. (1996) Humus layer of forest soil (Finland) X96686 18-927 Jurgens et a/. (1997) Humus layer of forest soil (Finland) X96687 18-927 Jurgens et al. (1997)

ATCC 17082" Dead Sea X00662 18-1 534 Leffers & Garrctt (1984) NRC 16008 7 D1 I I06 18-1 534 Kamekura & Sen0 (1992) JCM 8979 + NRC 34021T D63786 18-1534 Kamekura et nl. (1997) DSM 8989' Rock salt (Bad Ischl salt mine, 228387 18-1 368 Denner c't a/. (1 994) Austria) Distinct Iine,ig-es Halohirt irl~inrgoniorrensr [TI DSM 9297" Dead Sea L37444 18-1534 Oren et al. (1995) AVatrononrtinm ( Natronohac trrium) JCM 8858'' Soda lake (Wadi Natrun, Egypt) D87971 18-1534 Kamekura rt ul. (1 997) phaicior11 [TI Clone HACl Saltern (Alicante. Spain) X8433 1 18-1 5 12 Benlloch et a/. (1995) Clone HAC 14 Saltern (Alicante. Spain) X84084 18-1 5 12 Benlloch rt a/. (1995) Clone 2M r16 Salt marsh (Colne Point. UK) AFO 15984 18-1 1 I4 Munson rt a/. (1997) Outgroup Mdliuno~irrilluni hiinguter DSM 864' Sewage sludge M60880 18-1534 Yang et al. (1 985)

*Although ,111 strains listed under this heading probably belong to the newly proposed genus Natrinema, there are insufficient taxonomic data to formally reclassify some of these strains. t Derived from Halorubrum trapanicunz NRC 34021 (see text for details). phylogenetic group. Also, Munson et al. (1997) Halobacterium halobium NCIMB 777 and Halo- obtained several halobacterial clones from salt bacterium salinarium NCIMB 786, which are closely marshes with a maximum salinity of 0-8 M. This was related to each other and distinct from other halo- particularly surprising as no halobacterium has been bacteria (Ross & Grant, 1985; Ross et al., 1985; Grant shown to grow at this relatively low salinity. One of the & Larsen, 1989a). Grant & Ross (1986) originally most intriguing of all uncultivated micro-organisms is suggested the genus name 'Halonema' for these three Walsby's square gas-vacuolate halobacterium ; it is species, although this was never validly published. It assumed to be halobacterial on the basis of habitat has recently become clear that many other halo- (Walsby, I980), cell wall ultrastructure (Grant & bacterial isolates may be related to these species Larsen, 1989b) and polar lipid analysis of a hypersaline (Kamekura & Dyall-Smith, 1995), and other members lake in which it was the dominant micro-organism of the group are frequently isolated from hypersaline (Oren et d.,1996). However, its closest affiliation environments. remains unresolved. In this paper we have carried out a phylogenetic Several halobacteria have been misclassified for many analysis of halobacterial 16s rRNA gene sequences, years, e.g. Halobacterium trapanicum NCIMB 184, integrating data from new isolates, previously de-

~~~~ International Journal of Systematic Bacteriology 48 1189 T. J. McGenity, R. T.Gemmel1 and W. D. Grant scribed halobacteria and cloned sequences from un- were scanned and analysed using the GelCompar software cultivated halobacteria. On the basis of these and other package (version 3.1b, Applied Maths). taxonomic data we propose a new genus, Natrinema Restriction endonuclease digestion and 165 rDNA probing gen. nov., to accommodate Halobacterium trapanicum (ribotyping). DNA extracts (see earlier) were digested over- NCIMB 784, Halobacterium halobium NCIMB 777 night with the restriction endonucleases EcoRI, MI, KpnI and Halobacterium salinarium NCIMB 786. and XhoI at 37 "C and TaqI at 65 "C. DNA fragments were separated by electrophoresis on a 1 % (w/v) agarose gel at 50 V for 2.5 h. Fragments containing 16s rRNA genes were METHODS detected by blotting onto Hybond-N membrane and hybridizing with a digoxygenin-labelled halobacterial 16s Sources of strains. 16s rRNA sequence information was rRNA gene probe prepared by PCR (Boehringer Mannheim determined from seven halobacterial strains and compared PCR digoxygenin labelling mix), followed by colorimetric with 62 previously published 16s rRNA sequences (Table 1). detection (Boehringer Mannheim digoxygenin nucleic acid Halobacterium trapanicum NCIMB 784, Halobacterium detection kit), using the protocol recommended by the halobium NCIMB 777 and Halobacterium salinarium manufacturer. NCIMB 786 were obtained from culture collections as Antibiotic sensitivities. Whatman Antibiotic Assay discs indicated in Table 1. Strains T5.7, SR1.5, GSL5.48 and impregnated with the particular antibiotic were placed on SC1.2 were isolated as part of this study. HM agar plates previously spread with 150 ml of late Sampling and isolation of new strains. Sources of new strains exponential phase cultures of Halobacterium halobium are given in Table 1. Strains were isolated on plates of NCIMB 777 and Halobacterium salinarium NCIMB 786. halophile medium (HM) (Norton & Grant, 1988) with Resistances and zones of inhibition were recorded after 4 added starch (1 YO,w/v) (HMS) at different temperatures: and 6 d incubation at the appropriate temperature. Anti- T5.7 (30 "C), SRl.5 and SCl.2 (40 "C) and GSL5.48 (50 "C). biotics used were as follows: amphotericin B (350 mg), ampicillin (25 mg), anisomycin (5 mg), bacitracin (5 U), DNA extraction, PCR and sequencing 16s rDNA. Strains were carbenicillin (100 mg), cephaloridine (25 mg), chloram- grown in HMS broth and incubated at 37 "C. DNA was phenicol (400 U), colistin sulphate (10 mg), erythromycin extracted using the guanidinium thiocyanate method of (50 mg), flavomycin (350 mg), gentamicin (10 mg), keto- Pitcher et al. (1989) except that cells were not pretreated with conazole (100 mg), micoazole (100 mg), novobiocin (10 mg), lysozyme and Sarkosyl was not included in the lysis reagent. 0/129 (vibriostatic agent, 100 mg), polymyxin B (300 U), rifampicin (50 mg), streptomycin (25 mg), sulpha- The gene(s) encoding 16s rRNA were amplified using the met hoxazole/ trime thoprim (25 mg), sulp hafurazole PCR method of Embley (1991), as modified by McGenity & (500 mg) and tetracycline (50 mg). Grant (1993). The forward amplification primer was 27F, TCCGGTTGATCCTGCCGGAG (positions 8-27) and the Nitrate and nitrite reduction. Test tubes containing liquid reverse amplification primer was 1525R, AAGGAGGT- HM supplemented with either 0.1 YO(w/v) KNO, or KNO, GATCCAGCC (positions 1541-1525). PCR product was were inoculated and incubated at 37 "C. Nitrate reduction to sequenced either manually (McGenity & Grant, 1993) or nitrite was detected by withdrawing 0.5 ml samples period- automatically on an ABI 373A automated sequencer, as ically and adding naphthylamine and sulphanilic acid described in the ABI protocol for Taq Dye Deoxy Ter- reagents as described by Smibert & Krieg (1981). A minator Cycle Sequencing kit. purple-pink colour indicated the reduction of nitrate to nitrite but no reduction of nitrite. The presence of nitrates Sequence alignment and analyses. Sixty-nine 16s rDNA was detected using the same reagents followed by addition of sequences (Table 1) were aligned manually, taking account a small amount of zinc dust. A purple-pink colour indicated of secondary structure, using the program DNADoc. the presence of nitrate. Programs in the PHYLIP package (Felsenstein, 1993) were used throughout. All sites were included in the analyses and Utilization of carbon sources. Flasks containing 50 ml liquid given equal weight (positions 18-1 534, Escherichia coli HM supplemented with 1 Y (w/v) of the carbon source under test (glucose, fructose, lactose, mannitol, ribose, numbering). The nucleotide substitution model of Jukes & sucrose, xylose) were inoculated with 0.5 ml liquid culture in Cantor (1969) was used to obtain distance matrices, and exponential phase and incubated with shaking. Growth rate, phylogenetic trees were constructed from the pairwise measured by optical density at 600 nm, or final yield of cells distances using the least-squares algorithm of Fitch & was compared with a control culture. Margoliash (1967). Confidence limits for evolutionary distances were calculated from 100 bootstrapped alignments Sulphide production. The production of sulphide from (Felsenstein, 1985). cysteine, already present in HM, was detected by insertion of paper impregnated with lead acetate into the mouth of a Whole-cell protein profiles. Colonies were taken directly tube containing the culture. A blackening of the paper from HMS agar plates and whole-cell proteins extracted by indicated sulphide production. boiling in SDS-PAGE sample buffer (Laemmli, 1970) for 15 min. The denaturing gel electrophoresis method was based lndole production. Production of indole from tryptophan on that of Laemmli (1970), using the Bio-Rad Protean 11 during growth was detected by shaking 1 ml liquid culture slab cell kit at a constant current of 30 mA per gel, for about with an equal volume of Kovacs' reagent (Kovacs, 1956). A 6 h. Proteins were stained with 0.5% (w/v) Coomassie red-mauve colour indicated the presence of indole. brilliant blue and destained in an aqueous solution of 25 YO Starch hydrolysis. Solid medium supplemented with 1 Oh (v/v) propan-2-01 and 10% (v/v) glacial acetic acid. Gels (w/v) soluble starch was streaked with the organism under were preserved by drying in an aqueous solution of 20% test. After 10-14 d growth, the plate was flooded with iodine (v/v) ethanol and 10% (v/v) glycerol, using a procedure solution. Clear zones around colonies were indicative of recommended by the NOVEX system (R&D Systems). Gels starch hydrolysis.

~~ 1190 International Journal of Systematic Bacteriology 48 New halobacterial genus Natrinema gen. nov.

Gelatin liquefaction. A sterile charcoal-gelatin disc (Oxoid) the genus Haloferax, which forms a tight cluster, has was added to 10 ml liquid HM in a universal vial. After more than 98 % similarity between cultivated strains inoculation and subsequent growth of the test strain, the (Fig. I), which is equivalent to the similarity between culture was examined for release of charcoal granules, Halobacterium trapanicum NCIMB 767 and indicative of gelatin liquefaction. ‘ Natrinema’ spp. However, to date there are insufficient taxonomic data on these strains to warrant RESULTS AND DISCUSSION renaming them. 165 rRNA sequence analysis The relationship between the newly isolated strain, SRl.5, and the genus Natrinema is less clear-cut, Halobacteria constitute a monophyletic group, but the because it has 96% sequence similarity with most distantly related species have 83.2 YO sequence spp. If strain SR1.5 were included with similarity, indicating that the halobacteria are diverse ‘Nutrinema’ ‘ Natrinema’ it would still form a more tightly clustered (Fig. 1). Different models of nucleotide substitution genus than some others, such as (93% (Jukes & Cantor, 1969; Kimura, 1980) and treeing Natrialba similarity), Halorubrum (92.8 YO) and Haloarcula methods (Fitch & Margoliash, 1967; Saitou & Nei, (89.4 Yo). 1987) were used in the phylogenetic analyses. No difference in branching order was seen with the two The specific epithet ‘ trapanicum ’ appears four times in models of nucleotide substitution. Differences Fig. 1. Halobacterium trapanicum NCIMB 767 and the resulting from the two treeing methods were restricted halococcus, Halobacterium trapanicum JCM 8979, are to nodes near the base of the tree, where there is great supposedly derived from Halorubrum trapanicum uncertainty, and a few other nodes where bootstrap NRC 34021 (Kamekura et al., 1997), indicating values were less than 75% (Fig. 1). Notably, the problems of transfer between culture collections in the branching order in the cluster containing ‘ Nutrinema’ past. The situation is currently being clarified by the was unaffected by the different methods. deposition of Halorubrum trapanicum NCIMB 13488, a neotype of the original NRC 34021 strain (Grant We have designated the rather diffuse cluster of et al., 1998). Halobacterium trapanicum NCIMB 784 is halo bacteria that includes Natronobacterium gregoryi, distinct and not derived from any of the other strains. as the Natro group (Fig. 1 and Table 1). Repre- sentatives of the Natro group are more than 89.5% similar in 16s rRNA sequence. There are presently Salt tolerance of ’Natrinerna‘ spp. and related three genera in the group, but there should undoubt- halobacteria edly be more. Strain T5.7, which is very closely related to Most of the halobacteria in the Natro cluster are ‘Nutrinema’ (Fig. I), was isolated from a relatively alkaliphilcs, but Natrialba asiatica, plus repre- low-salt environment (Table 1). However, it is notable sentatives of the cluster including Halobacterium that the related strain SR1.5 was also isolated from a halobium NCIMB 777 and Halobacterium trapanicum disused saltern with a very low NaCl concentration of NCIMB 767, and strain SR1.5 (denoted by a dashed 0.2 M, and clone 2MT320 originated from a salt marsh line around Natrinema in Fig. I), are neutrophilic. This (0.8 M NaCl). Both of these are phylogenetically same group, excepting strain SR1.5, also has in similar to ‘Nutrinema’ spp. (Fig. 1). Indeed, common C,,C,, and C,,C,, core lipids, (Ross & Grant, Formisano (1 962) noted that Halobacterium 1985; Ross et al., 1985; Kamekura & Dyall-Smith, trapanicum NCIMB 784 not only survived, but grew 1995). moderately, at 0-7 M NaCl, which is at odds with our finding of a requirement of 1.8 M NaCl for growth (see Halobacterium trapanicum NCIMB 784, Halo- species description). The ability to withstand low salt bac teriuvlz halo bium NCI M B 77 7, Halo bac ter ium concentrations may be lost upon repeated subculture salinariuni NCIMB 786, strain T5.7, Halobacterium of laboratory strains at high salt concentrations. trapanicurn NCIMB 767 and strain L11 have more than 98.2 Yo 16s rRNA sequence similarity and share Large numbers of halobacteria were isolated from the similar unidentified glycolipids (Ross & Grant, 1985 ; disused salterns that gave rise to strains T5.7 and Ross et ul., 1985; Kamekura & Dyall-Smith, 1995). SR1.5. For example, more than 300 halobacterial These strains have been incorrectly assigned to the colonies per ml were isolated from the saltern in north- genus Hulobacterium and merit reclassification. We east Thailand (0.7 M NaCl), which had not been in use propose the genus name ‘Nutrinema’ for Halo- for about 10 years; and similarly large numbers were bacterium salinarium NCIMB 786, Halobacterium isolated from Roquetas de Mar (0.2 M NaCl) on HMS halobiurn NCIMB 777 and Halobacterium trapanicum at 40 “C. Several isolates resembling Halobacterium NCIMB 784. The basis for this reclassification is halobium NCIMB 777, based on polar lipid analysis, discussed at some length later in the paper. were obtained from soil close to the disused saltern in Thailand. It is probable that Halobacterium trapanicum NCIMB 767 (and strains L11 and T5.7) should belong to the The apparent survival of halobacteria in low salt newly proposed genus, particularly if a comparison is concentrations is not restricted to ‘ Nutrinema’-like made with other halobacterial genera. For example, strains. Isolations from low-salt environments include : __ International Journal of Systematic Bacteriology 48 T. J. McGenity, R. T.Gemmel1 and W. D. Grant

Huloarculu niukohatei JCM 9738 -'Huloarculu sinuiiensi3 ' ATCC 33800 (minor gene) -strain E 1 1 I00 'Lstrain E2 Hulourculu hispunica ATCC 33960 'Haloarculu aidinensis' B-2 HALOARCULA

loarcufa vulfisniortisATCC 297 15 no. 1 [TI urculu niarismortui Ginzburg (rrnB) Hulourculu juponicu JCM 7785 ~Hulourculuniurisniortui C inzburg (rrnA) clone 1MT315 clone 2MT103 %clone 2MT3 10 95 -Haloferux mediterrunei ATCC 33500 -strain E8 -9 -9 'Huloferux alicuntei' Aa2.2 LLHuloferux gibbonsii ATCC 33959 HALOFERAX 98 Hulofrrav volcunii ATCC 29605 [TI -Huloferax denifrijicans ATCC 35960 loyclone HAC 14 JI clone HAC 1 Hulobaculum goivrorrense DSM 9297 [TJ HALOBACULUM Halorubruni vucuolutum JCM 9060

HALORUBRUM

Hulorubruni saccharovorum NCIMB 208 I [T] clone 2MT16 Hulococcus sulifodinae DSM 8989 9 Hulococcus morrhuue NRC 16008 HALOCOCCUS

I strain Y 12 clone FFSB9 clone FFSB I2 HALOBACTERIUM LHalobucterium sulinurum DSM 668 Halobacteriurn salinuruni R 1 [TI Plfalobucterium salinuruni N RC 3400 1 strain 93dLM4

I

I - 8, 81 T5.7" Hb. salinarium NCIMB 786 (Natrinema pellirubrum [TI)" 6 Hb. trapanicum NCIMB 784 (Natrinema pallidum)" NATRINEMA Hb. halobium NCIMB 777 (Natrinema pallidum)" 7i 99strain L11 9 Hulobacferium trupunicum NCIMB 767 I00 l0O~Nutriulbuusiuticu JCM 9577 Nutriulbu usiuticu JCM 9576 [TI N ATRIi. ALB A h'utrialbu niagudii NClMB 2 I90 JI Nafronohacteriuni gregoryi NCIMB 21 89 [TI NATRONOBACTERIUM - Natronococcus aniylolyticus JCM 9655 -97 T NATRONOCOCCUS Natronococcus occultus NCIMB 2 I92 [TI JI iVatrononionasphuraonis JCM 8858 [TI NATRONOMONAS Mefhutiospirillumhungutei DSM 864 0.1 Knu

...... ,, ., ., ...... , ., ...... , ...... , . . . . ,. . , ...... , ., ...... , ...... , . , ...... , ,. , ...... , ...... , ., ...... ,...... , ...... Fig. 1. Phylogenetic tree showing the relationship between species of halobacteria and the position of the newly proposed genus 'Natrinerna' based on 165 rDNA sequences. On the right-hand side the extent of different genera is shown by the arrows. The dashed line around 'Natrinerna' covers those strains that upon further investigation may prove to belong to this genus (see text for discussion). The least-squares algorithm of Fitch & Margoliash (1967) was used to construct the tree from evolutionary distances (Jukes & Cantor, 1969) obtained from an alignment of 165 rDNA sequences (nucleotides 18-1534, E. coli numbering). Bootstrap percentages are indicated at the node, except when values were less than 75%. Strains sequenced as part of this study are indicated by an asterisk. Type species are indicated by [TI. The bar at the bottom indicates 0.1 K,, (10 nucleotide substitutions per 100 bases).

~~ 1192 International Journal of Systematic Bacteriology 48 New halobacterial genus Natrimwia gen. nov. ~ __ ~______

1 23456 1234

Fig. 3. Restriction endonuclease (SalI) digests, hybridized with a 165 rDNA-probe (ribotype), of DNA extracted from: Lanes: 1, strain T5.7; 2, Halobacterium halobium NClMB 777; 3, Halo bacterium trapa nicum NC I MB 784; 4, Ha lobacterium salinarium NCIMB 786.

~-~~__~~__ ~ ______

and other halobacteria in terms of the sensitivity of its protein synthesis system to different antibiotics. These misclassified halobacteria and strain T5.7 have fig. 2. SDS-PAGE gel of whole-cell proteins extracted and run many common properties. For example, they have as indicated in Methods. Lanes: 1, Halobacterium halobium similar whole-cell protein profiles (Fig. 2), with Halo- NCIMB 777; 2, Halobacterium trapmicum NClMB 784; 3, NCIM 784 and Halobacterium salinarium NClMB 786; 4, strain T5.7; 5, standard bacteriurn trapmicum B Halobac~criiinz strain for Gelcornpar system; 6, molecular mass standards. halobiuni NCIMB 777 being most similar (85% simi- ~~__~__larity calculated using the Pearson product-moment coefficient). These two strains were 84% similar to strain T5.7, and this cluster was 75% similar to clones related to Halofira.~(Munson et al., 1997) and Halobacterium salinariuni NCIMB 786. It is without Halobactc riim (Jurgens ct a/.,1997); and strains doubt that Halobl-rct~rii~nitrupanict~ni NCIMB 784 related to Halococcus (Rodriguez-Valera c't a/.,1982). and Halohac~tcv-iunihalobiuni NCIM B 777 belong to It should be remembered that Halobacterium the same species as they have very similar whole-cell salinariirni NCI M B 786, Halobuc~terium lialobium protein profiles (Fig. 2) and identical ribotype patterns NCIM B '77 and Halohac'feriunz trapmicum NCIM B when digested with SalI (Fig. 3), TayI, EcoRI and 784 were isolated from salted hides and fish (Table l), Xliol (data not shown) and supported by their identical which implies that they were originally present in 16s rRNA sequence (Fig. 1) and polar lipids (Ross et saturated brines at the point of crystallization. Also, al., 1985). Zvyagintseva et al. (1995) isolated strain 524 from a Halobacterium trapanicuni NCIM B 784 and Halo- cyanobacterial community in a hypersaline lagoon. hactcriunz lzulobium NCIMB 777 have 52 % and 50 YO This strain is identical between bases 278-500 of 16s DNA homology, respectively, with Halobacterium rRNA to Halobacterium lzalobium NCIMB 777, il- salinariuni NCIMB 786 (Ross & Grant, 1985), indi- lustrating that halobacteria belonging to the proposed cating that the latter strain is a separate species of the genus ' A-citrinema' are widespread in hypersaline same genus. This is supported by other criteria, for environin en t s. example : (i) the ability of Halobac~teritim saliizarizinz NCIMB 786 to use ribose and its insensitivity to Chemotaxonomic and physiological characteristics of rihmpicin ; (ii) difference in ribotype patterns when 'Natrinerna' spp. and related strains digested with Sol1 (Fig. 3), KpnI and EcoRI (data not shown); (iii) differences in protein profiles (Fig. 2); and Ross & Grant (1985), on the basis of 16s rRNA-DNA (iv) differences in polar lipid composition - Halo- hybridization, demonstrated that Halohucteriunz bacteriuni salhiariui~i NCIMB 786 has an additional salinariiiii I NCIM B 786, Halobacterium halobium glycolipid and lacks phosphatidyl glycerosulphate and NCIMB 777 and Halohuctc~iunitrapunicum NCIMB one of three characteristic glycolipids that are present 784 were related to each other but unrelated to in Halohacteriim trupariic~unzNCI M B 784 and Halo- Halobuctuiutn sensu stricto. This was supported by bcicterium halobiuni NCIMB 777 (Ross ct a/.,1985). polar lipid analysis (Ross et a/.,1985). Sanz et al. (1993) also showed that Halobacterium l?alohiunz Members of the proposed genus ' Natrinenia', includ- NCIMB 777, which has three rRNA operons (Sanz et ing those not formally proposed here, have been a/.,1988). was quite different from Hulohactrriuni spp. isolated worldwide from a variety of habitats (Table

~~ __- ~~ ~~ ~__~- ~ International Journal of Systematic Bacteriology 48 T. J. McGenity, R. T.Gemmel1 and W. D. Grant

1). Some are involved in the spoilage of salted fish and from cysteine. Possesses C,,C,, and C,,C,, diether hides (Formisano, 1962), while others may produce core lipids and several unidentified glycolipids. lipids suitable for enhanced oil recovery (Post & Al- Possesses phospholipids : phosphatidyl glycerol, Harjan, 1988). It is clear that these strains represent a phosphatidyl glycerol methylphosphate, but only trace distinct and important group in the , amounts of phosphatidyl glycerosulphate. Sensitive to and so a reclassification of the adequately described anisomycin, bacitracin, novobiocin, vibriostatic agent strains is presented below. We propose that Halo- (0/ 129) and sulphamethoxazole/trimethoprim. Insen- bacterium salinarium NCIM B 786, Halobacterium sitive to ampicillin, chloramphenicol, ketoconzaole, trapanicum NCIMB 784 and Halobacterium hulobiurn flavomycin, rifampicin, streptomycin and tetracycline. NCIMB 777 are placed into the new genus Natrinemu Type strain: NCIMB 786. Comment: NCIMB 786 and, as other halobacteria have the specific epithets was originally described (but not formally proposed) ' snlinur(i)um ', ' trupanicum ' and ' Izalobium ', it has as Halobacterium cutirubrum subsp. proteolyticuvzz by been necessary to change these. Formisano (1962). However, since its deposition in the NCIMB it has been referred to as Halobacterium Description of Natrinema gen. nov. snlinar ium.

Natrinema gen. nov. (Na.tri.ne'ma. L. n. natrium Natrinema pallidurn nom. nov. sodium; Gr. n. nema a thread; M.L. n. Natrinemn the sodium thread, referring to the high sodium ion Nutrinema pallidum (pal.li.dum. L. fem. adj. pallidurn requirement, and the cell shape). pale ; the pale Natrinema). Cells rod-shaped 1-5 pm by 0.6-1.0 pm, but pleo- Cells are rod-shaped 1.5-6 pm by 0-7-1.0 pm but morphic in unfavourable conditions. Cells lyse in less become pleomorphic in unfavourable conditions. than 1.5 M NaCI. Colonies light orange-red or pale Requires at least 1.7 M NaCl for growth and cells lyse orange, 1-2 mm in diameter, smooth, circular, convex. in less than 1-5 M NaCl. Optimum NaCl concentration Gram-negative. Chemo-organotrophs. Strict aerobes. for growth 3.4-4.3 M. Cells contain only low levels of Nitrogen sources : Casamino acids. Carbon sources : carotenoids and hence colonies are pale orange, beige Casamino acids and certain sugars. Require at least or almost colourless. Colonies are circular, entire, 1.7 M NaCl for growth, optimum 3-4-4.3 M NaC1. convex, translucent, 1-2 mm in diameter. Isolated Optimum pH 7.0-76. Some strains grow slowly at pH from salted fish and hides (Formisano, 1962). Chemo- 8.6. Gelatin liquefied, starch not hydrolysed, sulphide organotrophic and strictly aerobic. Nitrogen sources : and indole not produced. Possesses menaquinones Casamino acids. Carbon sources : Casamino acids, MK-8 and MK-8(H2) (Collins et al., 1981) and both glucose, fructose, lactose but not ribose. Reduces C,,C,, and C,,C,, diether core lipids (Ross et a/.,1985; nitrates to nitrites and nitrites to an unknown end Ross & Grant, 1985). Possesses several unidentified product. Liquefies gelatin, does not produce sulphide glycolipids (Ross et al., 1985). Sensitive to anisomycin, from cysteine, indole or amylase. Possesses C,,C,, and bacitracin, novobiocin, vibriostatic agent (0/ 129) and C,,C,, diether core lipids (Ross et a/., 1985) and menaquinones MK-8 and MK-8(H2) (Collins et a/., sulphamethoxazole/trimetlioprim. G + C content of major DNA component is 69.9 mol YOand 60.0 mol % 198 1). Possesses phospholipids : phosphatidyl glycerol, for the minor component (Ross & Grant, 1985). p hos pha tidy1 glycerol me thylphospha te, significant Possesses a large plasmid of approximately 144 kbp amounts of phosphatidyl glycerosulphate and several (Ross & Grant, 1985). Type species: Nutrinema unidentified glycolipids (Ross et a/., 1985). Grows in pellirubrum nom. nov. pH range 6.0-8.4, optimum 7-2-7-6. Temperature optimum 37-40 "C. Sensitive to anisomycin, baci- Description of Natrinema pellirubrum nom. nov. tracin, novobiocin, vibriostatic agent (0/ 129) and sul- phamethoxazole/trimethoprim. Slightly sensitive to Nutrinema pellirubrum (pel.li.ru'brum. L. fem. n. pellis rifampicin. Insensitive to amphotericin B, ampicillin, a skin or hide; L. adj. ruber, rubrum red; pellirubrum chloramphenicol, erythromycin, flavomycin, strepto- the red-hided Nutrinema). mycin and tetracycline. Possesses a large plasmid of Cells rod-shaped 1-4 pm by 0.6-1.0 pm but pleo- about 144kbp (Ross & Grant, 1985). Type strain: morphic in unfavourable conditions. Cells contain NCIMB 777. Comment: NCIMB 777 was deposited in carotenoids, making colonies light red or orange. the culture collection as Halobacterium halobium by Isolated from salted hides. Cells lyse in less than 1.5 M D. J. Kushner. Strain NCIMB 784, deposited as Halo- NaC1. Growth in 2 M NaCI, optimum 3.4-4-3 M. bacterium trapanicum by Formisano (1962) is regarded Temperature range for growth 20-45 "C. pH range for as synonymous with NCIMB 777. growth 6-0-8.6, optimum 7.2-7.8. Chemo-organo- trophic. Strictly aerobic. Nitrogen sources : Casamino ACKNOWLEDGEMENTS acids. Carbon sources: Casamino acids and the sugars We are grateful to Dr M. J. Valderrama for providing fructose, glucose, lactose and ribose. Gelatinase-posi- samples from Spanish salterns and Dr I. S. Zvyagintseva for tive, nitrates reduced to nitrites, nitrites reduced but no providing strains. The sequence analyses benefited from the gas produced, end product unknown. Does not pro- use of the SEQNET facility. R.T.G. was supported by a duce indole, hydrolyse starch or produce sulphide NERC studentship.

1194 International Journal of Systematic Bacteriology 48 New halobacterial genus Nutrinemu gen. nov.

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International Journal of Systematic Bacteriology 48 1195 T. J. McGenity, R. T.Gemmel1 and W. D. Grant genus Halorubrum gen. nov., as Halorubrum saccharovorum Rodriguez-Valera, F., Ventosa, A., Quesada, E. & Ruiz-Berraquero, comb. nov., Halorubrum sodomense comb. nov., Halorubrum F. (1982). Some physiological features of a Halococcus sp. at low trapmicum comb. nov., and Halorubrum lucusprofundi comb. salt concentrations. FEMS Microbiol Lett 15, 249-252. nov. Syst Appl Microbiol 18, 237-243. Rodriguez-Valera, F., Ruiz-Berraquero, F. & Ramos-Cormenzana, Mankin, A. S., Kagramanova, V. K., Teterina, N. L., Rubtsov, A. (1981). Characteristics of the heterotrophic bacterial popu- P. M., Belova, E. N., Kopylov, A. M., Baratova, L. A. & Bogdanov, lations in hypersaline environments of different salt concen- A. A. (1985). The nucleotide sequence of the gene coding for the trations. Microb Ecol7, 235-243. 16s rRNA from the archaebacterium Halobacterium halobiunz. Ross, H. N. M. & Grant, W. D. (1985). Nucleic acid studies on Gene 37, 18 1-1 89. halophilic archaebacteria. J Gen Microbiol 131, 165-1 73. Munson, M. A., Nedwell, D. B. & Embley, T. M. (1997). Phylo- Ross, H. N. M., Grant, W. D. & Harris, 1. E. (1985). Lipids in genetic diversity of Archaea in sediment samples from a coastal archaebacterial taxonomy. In Chemical Methods in Bactwiul salt marsh. Appl Environ Microbiol63, 4729-4733. Systematics, pp. 289-300. Edited by M. Goodfellow & D. E. Mylvaganam, 5. & Dennis, P. P. (1992). Sequence heterogeneity Minnikin. London: Academic Press. between the two genes encoding 16s rRNA from the halophilic Saitou, N. & Nei, M. (1987). The neighbour-joining method: a archaebacterium Haloarcula marismortui. Gonetics 130, new method for reconstructing phylogenetic trees. Mol Biol 399-41 0. Evol4, 406-425. Norton, C. F. & Grant, W. D. (1988). Survival of halobacteria Sanz, 1. L., Marin, I., Urefia, D. 81 Amils, R. (1993). Functional within fluid inclusions in salt crystals. J Gerz hlicrohiol 134, analysis of seven ribosomal systems from extremely halophilic 1365-1373. archaea. Can J Microbiol39, 3 1 1-3 17. Nuttall, 5. D. & Dyall-Smith, M. L. (1993). Ch2, a novel halophilic Sanz, 1. L., Marin, I., Ramirez, L., Abad, 1. P., Smith, C. L. & Amils, archaeon from an Australian solar saltern. Int J Sj>stBacteriol R. (1988). Variable rRNA gene copies in extreme halobacteria. 43, 729-734. Nucleic Acids Res 16, 7827-7832. Oren, A., Gurevich, P., Gemmell, R. T. & Teske, A. (1995). Smibert, R. M. & Krieg, N. R. (1981). General characterization. In Halobaculuin gomorrense gen. nov., sp. nov., a novel extremely Manual qf Methods .for General Bacteriology, pp. 409-443. halophilic archaeon from the Dead Sea. Int J Sjlst Bacteriol45, Edited by P. Gerhardt, R. G. E. Murray, R. N. Costilow, 747-754. E. W. Nester, W. A. Wood, N. R. Krieg & G. B. Phillips. Oren, A., Duber, 5. & Ritter, 5. (1996). The polar lipid composition Washington, DC : American Society for Microbiology of Walsby's square bacterium. FEMS Microbiol Lett 138, Takashina, T., Otozai, K., Hamamoto, T. & Horikoshi, K. (1994). 135-140. Isolation of halophilic and halotolerant bacteria from a Oren, A., Ventosa, A. & Grant, W. D. (1997). Proposed minimal Japanese salt field and comparison of the partial 16s rRNA standards for description of new taxa in the order Halo- sequence of an extremely halophilic isolate with those of other hacterialt~s.Int J Syst Bacteriol47, 233-238. extreme halophiles. Biodivers Conserv 3, 632-642. Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid Ventosa, A. & Nieto, J. 1. (1995). Biotechnological applications extraction of bacterial genomic DNA with guanidium thio- and potentialities of halophilic microorganisms. World J cyanate. Lett Appl Microhiol8, 15 1-1 56. Microbiol Biotechml 11, 95-1 14. Post, F. 1. & Al-Harjan, F. A. (1988). Surface activity of halo- Walsby, A. E. (1980). A square bacterium. Nature 283, 3 14-3 15. bacteria and potential use in microbially enhanced oil recovery. Yang, D., Kaine, B. P. & Woese, C. R. (1985). The phylogeny of Syst Appl Microbiol 11, 97-101. archaebacteria. Syst Appl Microbiol6, 25 1-256. Robb, F. T., Place, A. R., Sowers, K. R., Schreier, H. J., DasSarma, 5. Zvyagintseva, 1. S., Gerasimenko, L. M., Kostrikana, N. A., & Fleischmann, E. M. (1995). Archaea: a Laboratory Manual- Bulygina, E. 5. & Zavarzin, G. A. (1995). Interaction of halo- Halophiles. Cold Spring Harbor, NY: Cold Spring Harbor bacteria and cyanobacteria in a halophilic cyanobacterial Laboratory. community. Mikrobiologiya 64, 252-258 (in Russian).

1196 lnterna tional Journal of Systematic Bacteriology 48