Numerical taxonomy of moderately halophilic Gram-negative bacteria from hypersaline soils

Jou nal o Gene al Mic obiology (1983), 129, 2649-2657. P in ed in G ea B i ain 2649 Nume ical Taxonomy o Mode a ely Halophilic G am-nega i e Bac e ia om Hype saline Soils By E. QUESADA,' A. VENTOSA,'? F. RODRIGUEZ-VALERA,* L. MEGIAS3 AND A. RAMOS-CORMENZANA'* I Depa men o Mic obiology, Facul y o Pha macy, Uni e si y o G anada, G anada, Spain Depa men o Mic obiology, Uni e si y o Alican e, Alican e, Spain Depa men o Ana omy, Facul y o Medicine, Uni e si y o G anada, G anada, Spain (Recei ed 15 No embe I982 ; e ised 26 Janua y 1983) ~ ~ ~ ~ ~ A o al o 132 mode a ely halophilic bac e ia we e isola ed om hype saline soils wi h a C1- con en be ween 2-36 and 12.72% (w/ ) loca ed nea Alican e (S.E. Spain) and examined o 98 pheno ypic cha ac e is ics including hei esponse o cy ological, physiological, biochemical and nu i ional es s. They we e submi ed o a nume ical analysis oge he wi h six e e ence s ains using bo h simple ma ching (SsM) and Jacca d (S,) coe icien s, and clus e analysis was ca ied ou by he unweigh ed pai g oup me hod o associa ion (UPGMA), single linkage and comple e linkage. Wi h he S, coe icien and UPGMA clus e ing, eigh phenons we e ob ained a he 65% simila i y le el. F om each phenon ep esen a i e s ains we e chosen o he de e mina ion o DNA base composi ion and o elec on mic oscopy. Bac e ia belonging o phenons D, E, and F we e assigned o he genus Alcaligenes. Phenon G included 27 s ains assigned o Acine obac e , bu he high G + C composi ion (58.9 mol%) o a ep esen a i e s ain o his phenon sugges s ha i may ep esen a new axon. Phenons A, B, and C we e designa ed Fla obac e ium and phenon H was Pseudomonas. The bac e ia ound in hese en i onmen s a e no ela ed o hose om hype saline wa e s o no mal soils. INTRODUCTION Mode a ely halophilic bac e ia ha e been de ined as hose showing op imal g ow h be ween 3 and 15% (w/ ) NaCl (Kushne , 1978). Al hough e y ew g oups o mode a e halophiles ha e p e iously been desc ibed (La sen, 1962; Kushne , 1978), we ecen ly showed ha a a ie y o axonomic g oups had mode a ely halophilic ep esen a i es in hype saline wa e s (Ven osa e al., 1982) and soils (Quesada e al., 1982). The lack o sys ema ic s udies o isola es om na u al sou ces has esul ed in a sca ci y o axonomic in o ma ion conce ning mode a e halophiles. Mos o he desc ip ions e e o o ganisms isola ed om sal ed ood (Buchanan & Gibbons, 1974) o ound as acciden al labo a o y con aminan s (No i sky & Kushne , 1976). In hype saline wa e s we ound mos o he gene a ha can be isola ed om seawa e : Vib io was he mos abundan , ollowed by Fla obac e ium, Alcaligenes, Al e omonas and Ch omobac e - ium (Ven osa e al., 1982). All hese bac e ia we e mode a e halophiles ha ing, in addi ion o a high (5-10%, w/ ) sal equi emen o op imal g ow h, a minimal equi emen o 2-5% (w/ ) NaCl in mos cases. In hype saline soils, howe e , al hough many halo ole an G am-posi i e bac e ia we e ound, mos o he mode a e halophiles we e G am-nega i e ods. These also equi ed a high (abou 5-10%, w/ ) sal concen a ion o op imal g ow h, bu a lowe minimal sal concen a ion (0-5-2 %, w/ ) suppo ed g ow h. These esul s p obably e lec he ecological di e ences be ween wa e and soil en i onmen s, wa e being ela i ely homogeneous and cons an whe eas soil is he e ogeneous and mo e a ec ed by ac o s such as ain all, which may elimina e o e -specialized o ganisms (Quesada e al., 1982). P esen add ess : Depa men o Mic obiology, Facul y o Pha macy, Uni e si y o Se illa, Se illa, Spain. 0022-1287/83/OOOl-0909 $02.00 0 1983 SGM 2650 E. QUESADA AND OTHERS The pu pose o his wo k was o compa e mode a e halophiles om soils wi h hose isola ed om wa e s, and also o p o ide a mo e comple e axonomy o his ecological g oup. Using nume ical me hods, we ha e s udied 132 andomly-chosen new isola es o mode a e halophiles, oge he wi h some e e ence s ains om cul u e collec ions. METHODS Zsola ion and main enance o s ains. The mic o-o ganisms we e isola ed om hype saline soils nea Alican e (S.E. Spain) which ha e been desc ibed p e iously in physical and chemical e ms (Quesada e al., 1982). Samples we e aken as be o e a egula in e als om Janua y 1980 o Ap il 1981 (12 samplings om i e di e en si es). The C1- con en o he soils (de e mined using AgNOJ was in he ange 2.36 o 12.72%. Isola ion medium con ained (%, w/ ): yeas ex ac (Di co), 1 ; p o eose-pep one no. 3 (Di co), 0.5; glucose, 0.1 ; bac o-aga (Di co), 2. This medium was supplemen ed wi h a balanced mix u e o sea sal s gi ing inal concen a ions o 0.5, 5, 10, 20, o 25% (w/ ). The sal s and p opo ions used we e as in e apo a ed seawa e acco ding o Subo (Quesada e al., 1982). One g am o each soil sample was suspended in 10 ml o a sal solu ion simila o hose used o he cul u e media, en old dilu ions we e made (always keeping he same balanced sal concen a ions) and 0.1 ml o each dilu ion was pla ed on he isola ion medium o he same sal concen a ion. A e incuba ion a 32 "C o 15 d in sealed plas ic bags, h ee o i e colonies pe pla e we e andomly selec ed and subcul u ed on he same medium un il pu e. A o al o 132 isola es o G am-nega i e ods ha we e mode a ely halophilic, i.e. able o g ow in media con aining 2-20% (w/ ) sal s ( he sal g ow h ange de e mined as p e iously: Ven osa e al., 1982), we e selec ed o his s udy. Re e ence s ains. 'Ch omobac e ium ma ismo ui' ATCC 17056, 'Pseudomonas halosaccha oly ica' CCM 285 1 and Vib io cos icola NCMB 701 we e included. Quo a ion ma ks indica e species no in he App o ed Lis s o Bac e ial Names (Ske man e al., 1980). Re e ence s ains om a p e ious wo k on hype saline wa e s (Ven osa e al., 1982) we e included: Alcaligenes sp. A-336, Al e omonas sp. A-387 and Fla obac e ium sp. A-101. Main enance medium. The s ains we e main ained on slan s o medium sui able o mode a e halophiles (MH medium) con aining 10% (w/ ) ma ine s?l s. The inal composi ion o his medium was (%, w/ ): NaCl, 8-1 ; MgC12, 0.7; MgS04, 0.96; CaCl,, 0.036; KCI, 0-2; NaHCO,, 0.006; NaB , 0.0026 (Rod iguez-Vale a e al., 1980) supplemen ed wi h 0-5 % (w/ ) p o eose-pep one no. 3 (Di co), 1 % (w/ ) yeas ex ac (Di co) and 0.1 % (w/ ) glucose. This was solidi ied wi h bac o-aga (Di co) 2.0% (w/ ) when necessa y. Cha ac e iza ion o isola es. The pheno ypic cha ac e is ics (98) examined a e gi en in Table 1. Mos o he es s ha e been desc ibed elsewhe e (Ven osa e al., 1982); de ails o he emainde a e gi en below. Incuba ion was a 32 "C o up o 7 d in sealed con aine s; all media con ained 10% (w/ ) ma ine sal s and had hei pH adjus ed o 7.2 wi h 1 M-KOH (Rod iguez-Vale a e al., 1980). The abili y o g ow anae obically was e alua ed on pla es o MH medium which we e s eaked and incuba ed in ja s wi h he Gas Pak Anae obic Sys em (BBL). The appea ance o g ow h was checked a e 15 d incuba ion. The abili y o g ow in na ow Weinbe g ubes o liquid MH medium con aining glucose and phenol ed (0.001 %, w/ ) was de e mined by apid inocula ion in o he boiled and cooled medium. The ubes we e sealed wi h s e ile aga plugs and also co e ed wi h liquid pa a in. Phospha ase ac i i y was es ed by adding 1 % (w/ ) aqueous phenolph halein diphospha e solu ion o MH medium. Elec on mic oscopy. Rep esen a i e s ains om each phenon we e g own o mid-exponen ial phase on su ace MH pla e cul u es co e ed wi h liquid medium. Samples o he liquid cul u es we e nega i ely s ained wi h a 2% (w/ ) solu ion o phospho ungs ic acid (pH 7.0) and examined in a Ca l Zeiss EM-9s-2 ansmission elec on mic oscope. DNA composi ion. Exponen ial-phase cells o ep esen a i e s ains om each phenon we e up u ed, and he DNA was pu i ied using he me hod o Ma mu (1961). The mol % G + C con en was de e mined om he midpoin o he he mal dena u a ion p o ile (Ma mu & Do y, 1962). Nume ical analysis. Taxonomic cha ac e s we e coded in a bina y o m o he p esence/absence ype. S ain simila i ies we e es ima ed wi h bo h simple ma ching (SsM) (Sokal & Michene , 1958) and Jacca d (S,) (Jacca d, 1908) coe icien s, and clus e analysis was ca ied ou using he unweigh ed pai g oup me hod o associa ion (UPGMA), single linkage and comple e linkage (Snea h & Sokal, 1973). Tes e o was es ima ed in eigh epea ed s ains (Snea h & Johnson, 1972). Cophene ic co ela ion was also e alua ed (Snea h & Sokal, 1973). These compu a ions we e pe o med using he MINT p og am o D F. J. Rol o he Depa men o Ecology and E olu ion, S a e Uni e si y o New Yo k a S ony B ook, N.Y., U.S.A., using a Uni ac 1108 compu e in he Compu e Cen e o he Uni e si y o G anada, Spain. RESULTS Mo phological and halophilic cha ac e is ics All he s ains we e G am-nega i e ods and obliga e ae obes. Conspicuous polyhyd oxy- bu y a e inclusions we e obse ed in he majo i y o s ains. Taxonomy o bac e ia om hype saline soils 265 1 All 132 s ains g ew op imally in 5-10% (w/ ) sal s media and a e he e o e mode a e halophiles (Kushne , 1978). Mos equi ed a leas 2% (w/ ) sal s o g ow h (Table 1). The de ailed esul s o all he physiological, biochemical and nu i ional es s a e summa ized in Table 1. Nume ical analysis The esul s o he nume ical s udy o he cha ac e is ics o he s ains g ouped by means o he SJ coe icien and UPGMA clus e ing yielded he dend og am shown in Fig. 1. Simple ma ching coe icien (SsM) and single linkage and comple e linkage algo i hms we e also used, bu clus e composi ion was no ma kedly a ec ed by ei he he coe icien o he clus e ing me hod. Cophene ic alues we e 0.9726 and 0-8236 o he SJ and S,, coe icien s, espec i ely. The es ima ed es e o was less han 3.0%, which would no signi ican ly a ec he clus e analysis. The majo i y o he s ains we e g ouped in o eigh phenons a a 65% simila i y le el. Ele en s ains clus e ed sepa a ely, i e o which we e e e ence s ains. Table 1 shows he ea u es o he eigh phenons, and Table 2 summa izes hei di e en ial cha ac e is ics. Phenon A. This g oup included nine s ains which we e non-mo ile ods and oxidase posi i e. They educed ni a es bu no ni i es. Nei he p o eoly ic, lipoly ic no amyloly ic ac i i y was shown. The G + C con en o s ain F8-11, chosen as ep esen a i e o his g oup, was 694 mol%. These s ains could be included in he genus Fla obac e ium (Buchanan & Gibbons, 1974). Phenon B. This phenon, con aining only h ee s ains, was e y simila o phenon A (Table 1) bu hese s ains educed ni i es. S ain F8-6, aken as ep esen a i e, had a G + C con en o 61.8 molx. This phenon could also be placed in he genus Fla obac e ium (Buchanan & Gibbons, 1974). Phenon C. This g oup included h ee s ains which we e non-mo ile sho ods and oxidase posi i e. They educed ni a e and ni i e and u ilized a na ow ange o compounds as sole sou ce o ca bon and ene gy. S ain G-4 was selec ed as ep esen a i e o his g oup and had a G + C con en o 65.9mol%. These s ains may be placed in he genus Fla obac e ium (Buchanan & Gibbons, 1974). Phenon D. This comp ised 16 s ains ha clus e ed oge he because o hei simila biochemical and nu i ional cha ac e is ics ; howe e , some we e assigned o he genus Fla obac e ium and o he s o Alcaligenes. S ains G-23 and F8-10, ep esen a i es o each g oup, espec i ely, had G + C con en s o 65.4 and 69.2 mol%. Re e ence s ain Alcaligenes sp. A-336 appea ed in his phenon. Phenon E. Twen y- ou s ains clus e ed in his g oup; all we e s aigh , pe i ichously lagella ed ods. Mos o he s ains we e oxidase and ni a e nega i e. None possessed p o eoly ic o amyloly ic ac i i ies. Twel e o he s ains we e phospha ase posi i e, and his ea u e di e en ia ed wo subphenons ela ed a di e en simila i y le els. The wo ep esen a i e s ains selec ed, G-30 (phospha ase posi i e) and F9-4 (phospha ase nega i e), had G + C con en s o 66.7 and 67.4 mol%, espec i ely. Bo h subphenons can be assigned o Alcaligenes (Buchanan & Gibbons, 1974). Phenon F. The 40 s ains included he e we e long, mo ile ods wi h pe i ichous lagella ion (Fig. 1). They we e oxidase posi i e bu no p o eoly ic no lipoly ic. Some (30%) o he s ains hyd olysed u ea. S ain F5-7 had a G + C con en o 66.7 mol%. All membe s o his phenon we e assigned o he genus Alcaligenes (Buchanan & Gibbons, 1974). Phenon G. This g oup comp ised 27 s ains ha clus e ed a he 66% simila i y le el and cons i u ed a e y homogeneous g oup o p edominan ly sho ods, occasionally wi h la ge cu ed cells and ilamen s (some imes a he nume ous). Mos we e non-mo ile, oxidase nega i e, H2S p oduce s and educed ni a es bu no ni i es. All we e phospha ase posi i e. 2652 E. QUESADA AND OTHERS Pe cen age simila i y Phenon No. o s ains/ 0 20 40 60 80 100 I I I I I I1 I I 1 i I I I I I I I I ni I 11‘ I I 1 I I I 1 I I1 1 I I I A B C D E F G s ain designa ion 9 3 3 16 Fll-7 24 40 Flauobac e ium sp. A-101 27 ‘Ch o nobac e ium , ma ismo ui’ - / Al e omonas sp. A-387 -A - / F8-2 H4 F9- 1 G-l I -- - F 12-7 -- - __z - F8-4 -1 Vib io cos icola ‘Pseudomonas halosaccha oly ica’ Fig. 1. Simpli ied dend og am showing he clus e ing o s ains in o eigh phenons based on he S, coe icien and unweigh ed a e age linkage clus e ing (UPGMA), o 132 mode a ely halophilic G am-nega i e bac e ia isola ed om hype saline soils and six e e ence s ains. ' Taxonomy o bac e ia om hype saline soils 2653 Table 1. F equencies o posi i e cha ac e s ound in he eigh phenons, exp essed as a pe cen age o he o al sco ed o each g oup o he gi en es Phenon . . . A 9 No. o s ains . . . Mo phology Long ods Coccobacilla y Colony pigmen C eam Yellowish O ange Mo ili y Sal s: g ow h a % (w/ ) 0.5 2 15 20 25 30 PH 5 PH 8 PH 9 pH 10 5 "C 15 "C 45 "C Galac ose Glucose Lac ose Mal ose Manni ol Suc ose Xylose pH: g ow h a Tempe a u e : g ow h a Acid p oduc ion om Oxidase H,S p oduc ion Ni a e educ ion o ni i e Ni i e educ ion o gaseous compounds Casein hyd olysis Gela in hyd olysis S a ch hyd olysis Tween 80 hyd olysis U ea hyd olysis Me hyl ed Voges-P oskaue DNAase Phospha ase U iliza ion o o ganic compounds as sole sou ce o ca bon and ene gy: Ca bohyd a es Aesculin L-A abinose D-Cellobiose D-F uc ose D-Galac ose Lac ose Mal ose D-Mannose L-Ra inose L-Rhamnose Salicin L-So bose 44 56 66 34 0 0 0 100 90 50 11 11 0 89 54 22 33 89 11 11 77 0 22 0 22 0 89 33 100 0 0 0 0 0 0 0 0 0 0 88 55 11 100 66 33 100 55 0 0 0 33 B 3 0 100 33 66 0 0 0 100 1 00 100 0 0 0 100 100 66 0 100 0 33 0 0 0 33 33 0 100 33 100 100 0 0 0 0 0 0 0 0 0 0 0 0 100 33 0 66 0 0 0 0 0 C 3 0 100 0 100 0 0 0 100 100 1 00 33 0 33 100 100 100 100 100 0 0 33 0 100 0 0 33 100 0 100 100 0 0 0 0 0 0 0 0 0 0 0 0 66 0 0 100 33 0 0 0 0 D 16 63 37 44 56 0 56 12 100 100 94 50 25 0 100 100 75 50 94 56 12 87 0 68 48 75 0 100 0 69 62 0 0 6 6 0 0 0 0 0 6 0 0 94 31 0 I00 50 0 12 0 0 E 24 96 4 88 12 0 96 0 100 96 96 83 40 0 100 96 88 50 100 50 75 50 0 25 4 16 12 20 76 20 0 0 0 0 0 0 4 4 0 54 16 80 20 100 100 0 92 92 0 54 20 4 F 40 100 0 78 22 0 95 10 100 100 1 00 93 93 47 100 93 88 10 100 25 93 93 2 83 10 88 22 80 80 65 2 0 0 2 0 30 0 0 0 0 78 98 90 100 98 10 90 90 2 15 85 0 G 27 4 96 67 7 26 22 85 96 100 96 89 89 41 100 100 96 70 100 48 4 7 0 0 7 0 4 4 89 93 0 4 26 0 0 0 0 0 4 100 100 93 1 00 100 100 100 100 100 93 89 96 37 H 4 100 0 0 100 0 100 50 100 75 50 0 0 0 100 100 25 0 100 0 75 100 0 75 0 0 75 100 50 100 25 0 0 100 100 0 0 0 0 0 0 0 0 100 0 0 100 75 0 0 0 0 2654 E. QUESADA AND OTHERS Table 1 (con inued) Phenon . . . No. o s ains . . . S a ch Suc ose D-T ehalose D-x ylose Alcohols Adoni ol Dulci ol DL-Glyce ol meso-Inosi o1 D-Manni ol D-So bi ol Ace a e Benzoa e Cap yla e Ci a e Fo ma e Fuma a e D-Glucona e Hippu a e DL-Lac a e DL-Maia e Oxala e Py u a e P opiona e Succina e D-Ta a e Ca boxylic acids U iliza ion o amino acids as sole sou ce o ca bon, ni ogen and ene gy : L-Alanine L-A ginine L- Aspa agine L-Aspa ic acid L-Glu amic acid L-His idine L-Isoleucine L-Leucine L-Lysine L-O ni hine L-Se ine L-T yp ophan L-Valine A 9 55 77 77 88 88 0 88 88 44 88 88 0 0 88 100 100 100 66 100 100 0 100 88 100 11 100 88 100 100 100 88 33 33 11 11 100 0 0 B 3 0 100 66 100 0 0 66 66 33 100 100 0 0 100 100 100 100 66 100 100 66 100 100 100 66 100 66 100 100 100 0 66 100 0 0 100 33 33 C 3 0 66 100 33 0 0 100 0 0 66 66 0 0 100 100 100 66 0 66 66 0 66 66 100 0 100 66 100 100 100 0 0 0 0 66 100 0 100 D 16 0 100 100 31 0 0 87 31 44 44 100 6 0 94 100 100 75 56 94 100 100 100 94 87 19 100 94 100 94 100 81 56 81 56 81 100 6 25 E 24 32 92 100 60 0 0 96 64 50 54 96 28 0 100 100 100 100 66 88 100 8 100 1 00 100 16 100 100 100 100 100 96 96 80 80 96 100 24 0 F 40 0 98 95 85 2 2 90 0 35 32 95 95 2 93 95 95 93 90 98 98 10 90 98 98 5 95 95 98 93 95 15 20 25 75 57 98 27 2 G 27 4 100 100 100 44 33 100 96 74 89 96 0 18 100 100 100 100 15 15 15 11 100 100 100 89 100 100 67 100 100 100 44 52 67 33 100 7 7 H 4 100 25 25 25 0 0 75 25 0 0 75 0 0 100 100 100 100 25 100 100 0 50 100 100 0 50 25 100 50 100 0 0 0 25 25 25 25 0 All s ains we e G am-nega i e, o med ca alase, we e s ic ae obes, g ew a 5 and 10% (w/ ) o al sal s, g ew a pH 6 and 7, g ew a 25 and 32 "C, and g ew on D-glucose as sole sou ce o ca bon and ene gy. None o med indol o g ew on inulin o L-cys eine. They we e e y e sa ile nu i ionally, and we e able o u ilize a e y high numbe o compounds as sole ca bon and ene gy sou ces. The s ains in his phenon we e able o g ow a low sal concen a ions (0.5 %, w/ ) al hough o op imal g ow h hey needed 5-10% (w/ ) o al sal s. S ain F2-12 had a G + C con en o 58.9 mol%. Wi h he excep ion o G + C alue hese s ains could be assigned o he genus Acine obac e (Buchanan & Gibbons, 1974). Phenon H. This phenon was composed o ou s ains, which we e s aigh ods mo ile by one o a ew pola lagella. Thin ilamen s we e common. They we e oxidase posi i e, educed ni a es and showed amyloly ic and lipoly ic ac i i ies. Few o ganic compounds we e used as sole ca bon and ene gy sou ces. The ep esen a i e s ain F12-1 had a G + C con en o 58.7 mol%, These ou s ains can be included in he genus Pseudomonas (Buchanan 8z Gibbons, 1974). Taxonomy o bac e ia om hype saline soils 2655 Table 2. Di e en ial cha ac e is ics o he eigh phenons o mode a ely halophilic G am-nega i e bac e ia om hype saline soils Phenon ... A B C D E F G H No. o s ains.. . 9 3 3 16 24 40 27 4 Mo phology Long ods Coccobacilla y Mo ili y Flagella G ow h a 0.5% (w/ ) sal concen a ion G ow h a 5 "C Oxidase H,S p oduc ion Ni a e educ ion o ni i e Ni i e educ ion o gaseous compounds S a ch hyd olysis Tween 80 hyd olysis Phospha ase U iliza ion o : DCellobiose Oxala e L-His idine L-Leucine - - - 3-+ 8++ 3 1- +++ -++ - - - 1- - 1 2- 8- - 3+- - 10 23 + 1+ 9 23 38 6 + -/pe . pe . pe . -/pe . pol. 4 23 2 2- 8 12 4 19 - + 5 - 32 1+ - 19 32 24 2 6 1-26- - - - 1- 1-+ 1- - -+ -13-+- - 536+- + 243- - 13g6+- 13 10 14 - - +, all s ains posi i e; -, all s ains nega i e. Numbe s indica e numbe o posi i e s ains; unde lined numbe s indica e ha he numbe ep esen s 80% o mo e o he s ains. pe ., pe i ichous lagella; pol., pola lagella. DISCUSSION The e a e ew published s udies o bac e ia om saline soils (Ce an es & Oli a es, 1976; Mahmoud e al., 1978), and axonomic desc ip ions o such o ganisms a e sca ce. Henis & E en (1963), wo king on a saline soil nea he Red Sea, concluded ha mo e colonies de eloped on media wi hou sal s han on media wi h 10% (w/ ) NaCl. These esul s di e om ou s, p obably because hese au ho s used media wi h NaCl as sole sal , while o he ions such as Mg2+ and K+ a e also equi ed o good g ow h o mode a ely halophilic bac e ia (Kushne , 1978). As in ou p e ious s udy on bac e ia om hype saline soils (Quesada e al., 1982), mos o he popula ion li ing in he soils s udied we e mode a e halophiles as de ined by Kushne (1978), since hey g ew op imally be ween 5 and 10% (w/ ) sal s. This was also he case o bac e ia om hype saline wa e s (Rod iguez-Vale a e al., 1981 ; Ven osa e al., 1982). Ne e heless, many bac e ia isola ed om hype saline soils we e able o g ow a low sal concen a ions, (abou 0.5%, w/ ), while bac e ia om hype saline wa e s could no . This was a common cha ac e o he G am-posi i e bac e ia desc ibed by Quesada e al. (1982); howe e , 31 o he G am-nega i e s ains included in his s udy also showed ole ance o low sal concen a ions. Al hough di e ences exis ed among he s ains included in phenons A, B and C, all can be conside ed as ep esen a i es o he genus FZa obac e ium, and he sepa a ion among hem p obably a ose om hei di e en capabili y o use o ganic compounds as sole ca bon and ene gy sou ces. Wi hin he genus Fla obac e ium, he only mode a ely halophilic species desc ibed o da e is F. halmephilum (Elaza i-Volcani, 1940). The s ains included in hese phenons show some esemblance he desc ip ion o his species, al hough hey di e by hei abili y o p oduce acids om suga s, hei p o eoly ic ac i i y and hei nu ien equi emen s. Besides, F. halmephilum has a G + C con en o 45.6 mol % and he e o e belongs o Sec ion I o his genus, while ou s ains, wi h a G + C con en in he ange 61.8-69.4 mol%, a e in Sec ion 11. Among he ma ine bac e ia belonging o he genus Fla obac e ium and included in he App o ed Lis s o Bac e ial Names (Ske man e al., 1980), F. oceanosedimen um is he only one ha esembles he s ains ha appea in his s udy, and i di e s om hem only by he p oduc ion o acids om some suga s, he abili y o educe ni a es, and sal esponse ; he 2656 E. QUESADA AND OTHERS G + C con en o F. oceanosedimen um is also e y simila (67.5 mol%) o ha o he s ains in phenons A, B and C. Holmes & Owen (1979, 1981) ha e p oposed a ede ini ion o he genus Fla obac e ium, es ic ing his genus o he species wi h low G + C con en (31-40 mol %). Since i is clea ha la obac e ia o high G + C con en a e easily isola ed om a wide a ie y o en i onmen s (Hayes & Mapp, 1981), mo e s udies a e necessa y o decide i hey could be accommoda ed in he genus Empedobac e as p oposed by McMeekin & Shewan (1978). The s ains included in phenons E and F we e assigned o he genus Alcaligenes. Phenon E was e y simila o he desc ip ion o he ma ine bac e ium A caligenes cupidus (Baumann e al., 1972) excep in he abili y o educe ni a e o ni i e and hei di e en sal ange o g ow h. Howe e , phenon F was composed o a la ge g oup o bac e ia wi h p ope ies e y di e en om hose o p e iously desc ibed species o Alcaligenes. Acco ding o he G + C con en , hey we e ela ed o he ma ine bac e ium A. paci icus (Baumann e al., 1972) bu he e we e many physiological, biochemical and nu i ional di e ences om his species. In ou opinion, hey could cons i u e a new axon no p e iously desc ibed. Howe e , mo e gene ic and molecula da a a e necessa y o suppo his sugges ion. The 27 s ains ha clus e ed in phenon G had mo phological, physiological, biochemical and nu i ional cha ac e is ics o he genus Acine obac e (Buchanan & Gibbons, 1974). Howe e , he G + C con en o he ep esen a i e s ain o his g oup was 58-9 mol%, whe eas he ange accep ed o his genus is 42-47 mol % (Hen iksen, 1973). Mode a ely halophilic bac e ia ha e no been desc ibed and accep ed as membe s o his genus. Onishi & Hidaka (1978) s udied he p ope ies o an amylase p oduced by a mode a ely halophilic Acine obac e sp. bu a desc ip ion o his s ain has no been published. I seems clea ha he s ains in phenon G ep esen a new g oup o mic o-o ganisms ha could be included in Acine obac e , bu he di e en G + C con en could indica e ha hey belong o no only a di e en species bu possibly a di e en genus. S udies a e in p og ess o de e mine i his di e ence in he G + C con en is also e lec ed by di e ences a he pheno ypic le el. Phenon H was included in he genus Pseudomonas, bu hese s ains we e no ela ed o he mode a e halophile P. halosaccha oly ica used a ma ke in his s udy (Fig. 1). The e we e di e ences in nu i ional e sa ili y as well as in amyloly ic, lipoly ic and p o eoly ic ac i i ies. Wi h espec o ma ine species o he genus Pseudomonas included in he App o ed Lis s (Ske man e al., 1980), hey could be ela ed o P. doudo o ii (Baumann e al., 1972), he di e ence being hei lipoly ic and amyloly ic ac i i ies, absen in his species. Compa ison wi h p e ious s udies (Ven osa e al., 1982) sugges s ha he bac e ial popula ions in he saline e es ial en i onmen s s udied he e show many di e ences om bac e ia om hype saline wa e s. In ac , he e e ence s ains om hype saline wa e s did no g oup wi h he s ains om he e es ial habi a . All he s ains s udied he e had a s ic ae obic me abolism, a p edominan ea u e ha we had al eady epo ed o bac e ia om hype saline soils: e y ew s ains we e acul a i ely anae obic (Quesada e al., 1982). This poin has been con i med by an independen s udy ca ied ou by D M. E. Rhodes-Robe s (pe sonal communica ion). Howe e , in hype saline wa e s acul a i e anae obes ep esen a majo pa o he popula ion (Ven osa e al., 1982). In aqueous saline en i onmen s Vib io spp. we e he mos abundan g oup o mode a ely halophilic bac e ia, whe eas his genus is e y sca ce in hype saline soils, which a e domina ed by he gene a Alcaligenes and Fla obac e ium. Pseuda nonas is a e y common inhabi an o no mal soils (Alexande , 1961), bu sca ce in he hype saline soils we s udied. Ne e heless, he mic o lo a o hese hype saline soils is much mo e simila axonomically o he mic o lo a o no mal soils (Alexande , 1961) han i is o he mic o lo a o hype saline wa e s. This sugges s ha he gene al ea u es o he en i onmen may be mo e impo an han indi idual ac o s such as high salini y in de e mining he mic o lo a o a pa icula habi a . The au ho s hank D A. Vila o he in aluable help wi h he compu a ion and he Compu e Cen e o he Uni e si y o G anada, Spain, o he use o i s compu e and acili ies. We a e g a e ul o D F. Ruiz-Be aque o o his ad ice du ing he cou se o his wo k, D C. Cal o o he G + C con en de e mina ion, J. Muiioz o his aid in he p epa a ion o he EM g ids, and especially o D M. E. Rhodes-Robe s (Depa men o Bo any and Taxonomy o bac e ia om hype saline soils 2657 Mic obiology, Uni e si y College o Wales, Abe ys wy h, U.K.), who kindly con i med some o he esul s o his wo k and ga e aluable ad ice and discussion. E. Q. g a e ully acknowledges he eceip o a ellowship om he Plan de Fo macibn del Pe sonal in es igado . REFERENCES ALEXANDER, M. (1961). Soil Mic obiology. Edi ed by J. Wiley. New Yo k: Blackwell. 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