Numerical Taxonomy of Moderately Halophilic Gram-negative Rods Isolated from the Salar de Atacama, Chile

System. Appl. Microbiol. 14,275-281 (1991) © Gustav Fischer Verlag, StuttgartlNew York

Numerical Taxonomy of Moderately Halophilic Gram-negative Rods Isolated· from the Salar de Atacama, Chile B. PRADO\ A. DEL MORAe, E. QUESADA2 , R. RiOS\ M. MONTEOLIVA-SANCHEZ 2 , V. CAMPOS\ and A. RAMOS-CORMENZANA h 1 2

Laboratory of Microbiology, Instinlte of Biology, Faculty of Basic and Mathematic Sciencies, Catholic University of Valparaiso, Valparaiso, Chile Department of Microbiology, Faculty of Pharmacy, University of Granada, Granada, Spain Received October 5, 1990

Summary A total of 161 moderately halophilic Gram-negative rods isolated from The Salar de Atacama, Northern Chile, were examined for 119 phenotypic characters including morphological, physiological, biochemical, nutritional and antimicrobial susceptibility tests. The results together with those from reference strains, were subjected to numerical analysis, using both, the Simple Matching (SSM) and Jaccard (SJ) coefficients and clustered by the unweighted pair group method of association (UPGMA). Using the SSM coefficient and UPGMA clustering, five phenons were obtained at a 70% similarity level. Representative strains from each phenon were chosen to determine DNA base composition and cellular morphology by electron microscopy. Stritins in phenon A were assigned to the genus Vibrio. Strains in the other four phenons could not be assigned to any recognised taxon but their similarities to the genera Acinetobacter (phenon B), Marinomonas (phenon C) and Alteromonas (phenons D and E) are discussed.

Key words: Numerical taxonomy - Moderately Halophilic Bacteria - Solar salterns - Saline soils

Introduction Moderately halophilic bacteria are those which grow optimally in media containing 3-15% NaCl (Kushner and Kamekura, 1988). Most are eubacteria and are widely distributed in different natural habitats: solar salterns, hypersaline lakes, saline soils and seawater (Rodriguez-Valera, 1988). Taxonomically, they constitute a heterogeneous group which includes both Gram-negative and Gram~posi­ tive bacteria (Ventosa, 1988). The Salar de Atacama is located in Northern Chile (Second Region, Antofagasta) and covers an area of 300 km 2 • It is the largest salt deposit in the country. Due to its location, in the Atacama desert at 2700 m above sea level, it has unique environmental conditions (Moraga et aI., 1974) and its microbial communities have never been studied before. The purpose of this work was to study 161 moderately halophilic Gram-negative rods isolated from this environment to define their taxonomic position rela* Corresponding author

tive to previously described species. We selected the Gramnegative rods, because they constitute the most abundant group within the heterotrophic moderately halophilic strains previously isolated from this habitat (Prado et aI., unpublished data).

Materials and Methods Isolation and maintenance of strains. Bacteria were isolated from samples taken in September 1984 and October 1985 at three different sites in the Salar de Atacama: Laguna Tevenquiche (centre Salar), Rio Salado (Northern Salar) and soils near Tilopozo (Southern Salar). Samples were taken, from water, soils and sediments at all three sites, having a wide variety of salt concentrations (3-10% total salts). Methods for isolation and selection of strains have been described previously (Quesada et aI., 1983, Ventosa et aI., 1982). The 161 moderately halophilic Gram-negative rods selected were maintained on agar slants of MH medium (Quesada et aI., 1983) with 10% (w/v) marine salts

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(Rodriguez-Valera et aI., 1981) contammg the following nutrients: yeast extract (Difco), 10 gil; proteose-peptone no. 3 (Difco) 5 gil and glucose, 1 gil. This medium was solidified with Baero-agar (Difco), 20 gil. The pH was adjusted to 7.2 with IN NaOH. Reference strains. The following reference strains were used Chtomohalobacter marismortui ATCC 1705 6T, Deleya halophila CCM 3662', Halomonas elongata ATCC 33173 7 , Halomonas halmophila CCM 2833 T, "Pseudomonas halosaccharolytica" CCM 2851, Vibrio costicola NCMB 701T, and Volcaniella eurihalina ATCC 49366T. Quotation marks indicate that the name is not in the Approved Lists of Bacterial Names nor in subsequent supplements (Skerman et aI., 1980; Moore et aI., 1985). Characterization of the isolates. One hundred and nineteen phenotypic characters including morphological, physiological, biochemical, nutritional and antimicrobial susceptibility tests, were determined for each strain (see Table 1). Details of the procedures have been previously described (Quesada et aI., 1984, 1987: Ventosa et aI., 1982). Numerical analysis. 101 characters were selected for numerical analysis. Positive and negative results were coded as 1 and 0, respectively; non-comparable or missing data were coded as 9. Strain similarities were estimated with both Simple Matching (SSM), (Sokal and Michener, 1958) and Jaccard (Sj) Uaccard, 1908) coefficients and clustering was achieved by the unweighted pari-group method of association (UPGMA) (Sneath and Sokal, 1973). The cophenetic correlation was also obtained for each

PERCENT

50 I

60

70

SIMILARITY 80

90

method (Sneath and Sokal, 1973). The test error was estimated by examining 20 strains in duplicate (Sneath and Johnson, 1972). The computation was performed by the MINT program (Rohlf, 1985), using an Eclipse model MV/10000 computer at the Computer Centre, University of Granada, Granada, Spain. Electron microscopy. Transmission electron microscopy was used to examine cell morphology of representative strains from each phenon. Bacteria were grown to mid-log phase on the surface of MH Agar plates covered with MH liquid medium. Samples from the liquid cultures were negatively stained with a 2% (w/v) phosphotungstic acid solution (pH 7.0). Observations were made in a EM 10C/CR high resolution transmission electron microscope at 80 Kw. DNA base composition. The control strains used for the DNA composition were Escherichia coli LAM 1264 (K-12), Vibrio costicola NCMB 701 T and Bacillus subtilis ATCC 6051. Five determinations were carried out on each control and on representative strains from each phenon. Exponentially growing cells were ruptured by freeze-thawing and DNA was isolated by the phenol extraction method of Saito and Miura (1963). The DNA base composition was determined essentially as described by Tamaoka and Komagata (1984). RNA was hydrolysed with a mixture of ribonuclease T I (Sigma) and ribonuclease A (Sigma). Purified DNA was suspended in distilled water (lmg/ml), the solution heated at 100°C for 5 min and then cooled rapidly in an ice bath. The denaturated DNA solution (lOIlI) was mixed with 10 fLl of nuclease PI solution and incubated at 50°C for 1 h. lOfLl of bacterial alkaline phosphatase (Sigma) (2.4 units/ml in 0.1 M

PHENON 100

STRAIN

DESIGNATION

A

(47 STRAINS)

B

(5

C

(83

STRAINS)

D

(9

STRAINS)

E

(17

STRAINS)

STRAINS)

HALOMONAS HALMOPHILA COlI 2833T HALCMONAS ELONGATA ATCC 33173T VCl.CANIELLA EURIHALINA ATec 49366T DELEYA HAWffllLA CCM 3662T QjROMJHAlJ)fJACTffi MARlSMJRTUI /lTCC 17056T T

PSEUDOMONAS HALOSACCHAROLYT/CA CCM285I VIBRIO COST/COLA NCMB 70lT

50

60

70

80

90

100

Fig. 1. Simplified dendrogram showing the clustering of strains into five phenons based on the SSM coefficient and unweighted average linkage clustering (UPGMA), for 161 Gram-negative moderately halophilic rods isolated from Salar de Atacama (Chile) and seven reference strains.

Moderately Halophilic Bacteria Tris-HCI buffer, pH 8.1) was then added to the sample and incubated at 37"C for 1 h. 5 ~tl of the resulting hydrolysate was applied to a reverse-phase HPLC column to determined the G+C content.

Results

Numerical analysis Using the SSM coefficient and UPGMA clustering, all the isolates were grouped into five phenons at 70% similarity level (%S). None of the reference strains clustered with the environmental isolates (Fig. 1). The cophenetic correlation value was 0.87 for the SSM coefficient and UPGMA clustering and the estimated test error was less than 3 % (Sneath and Johnson, 1972). The cluster composition was not markedly different when the Jaccard coefficient (SJ) and UPGMA clustering were used. All the strains were moderately halophilic Gram-negative rods with optimal growth at 7.5% (w/v) total salts and no growth at 0.5% (w/v) total salts; growth occurred at 7.5, 10, 15 and 20% (w/v) total salts; at pH 7, 8 and 9, and at 15, 25, 32 and 37°C. Catalase positive. None formed indole. All were phenylalanine deaminase negative and susceptible to ampicillin and chloramphenicol. Table 1 summarizes the phenotypic characteristics of strains included in each phenon. The most significant features of each phenon, based on those tests for which at least 80% of the strains were positive or negative, are described below. Phenon A: This phenon contained 47 strains related at 80% S; the highest similarity value in this study. All were curved rods, motile by means of a single polar flagellum. They accumulated poly-j3-hydroxybutyrate (PHB), were facultatively anaerobic, oxidase positive and grew between 3-25% (w/v) salts. Metabolism of carbohydrates was fermentative (acid was produced from most sugars tested). H 2 S was produced from cysteine. Nitrate or nitrite were not reduced; Tween 20, 80, were hydrolysed but not starch. Blood was haemolysed. They were phosphatase and methyl-red positive. Only sucrose, D-trehalose, DLglycerol, acetate, D-gluconate, DL-Iactate, pyruvate and succinate were used as sole sources of carbon and energy. L-alanine, L-asparagine, L-glutamic acid, L-histidine, Lornithine and L-serine were used as sole source of carbon, nitrogen and energy. Resistant to tetracycline. Strain CH906, taken as representative, had a mol% G+C content of 45.6. This phenon could be assigned to the genus Vibrio (Baumann et ai., 1984b; Garcia et ai., 1987). Phenon B: This phenon comprised 5 strains which clustered at 77% S. They were non-motile, short-rods and accumulated PHB. Oxidase negative and strict aerobes. They grew at 3-30% (w/v) salt, at pH 5-10 and at 5-45°C. Acid was produced from D-galactose, D-glucose, meso-inositol, lactose, D-mannitol and sucrose, but not from glycerol, maltose or rhamnose. H 2 S produced from cysteine. Nitrite not reduced. No hydrolysis of casein, starch, Tween 80 and Tween 20, DNA nor blood. They hydrolysed gelatin and ureet. Methyl-red and Voges-Proskauer were negative. They used a great variety of organic

277

compounds as sources of carbon and energy. Susceptible to erythromycin, penicillin and rifampycin but not to tetracycline. The G+C content of strain CH-210, chosen as representative of this phenon, was 50.8mol%. Strains from phenon B could be related to the genus Acinetobacter Uuni, 1984). Phenon C: The 83 strains included in this phenon clustered at 73%S. They were curved rods, motile by means of a single polar flagellum and accumulated PHB; oxidase negative and strictly aerobic. Growth occurred at 3-25% (w/v) salts, at pH 5-10 and at 5-40 0C. Acid was produced from D-galactose, but not from glycerol, meso-inositol, Dmannitol, rhamnose or sucrose. Urease was produced. The following tests were negative: nitrite reduction, casein, gelatin, starch and Tween 80 hydrolysis, DNase, haemolysis, methyl-red and Voges-Proskauer. They used D-fructose, D-galactose, D-mannose, DL-glycerol, citrate, fumarate, D-gluconate, DL-Iactate, pyruvate and succinate as sole sources of carbon and energy, and L-alanine, L-asparragine, L-aspartic acid, L-glutamic acid, L-ornithine and L-serine as sole source of carbon, nitrogen ad energy. They were susceptible to penicillin. The G+C content of strains CH-281, chosen as representative of this group, was 47.7 mol %. These strains could be related to the genus Marinomonas (Van Landschoot and De Ley, 1983). Phenon D: Nine strains clustered in this phenon at 71 %S. All were curved rods, motile by a single polar flagellum and accumulated PHB. Oxidase positive. They were strict aerobes growing at 5-25% (w/v) salts, at pH 6-10 and at 15-40°C. Acid was not produced from most sugars tested. They gave negative results in nitrite reduction, aesculin, casein and starch hydrolysis, methyl-red and Voges-Proskauer tests. Gelatin was hydrolysed. Few organic compounds were used as sole sources of carbon, nitrogen and energy. Susceptible to cephalotin, erythromycin, penicillin and rifampycin. The representative strain, CH-270, had a G+C mol% of 38.1. These strains resembled the genus Alteromonas (Baumann et ai., 1984a). Phenon E: This phenon contained 17 strains related at 73%S. All were curved rods, motile by a single polar flagellum and accumulated PHB. Oxidase positive. Strict aerobes. Acid was not produced from the sugars tested. The following tests were negative: H 2 S production from cysteine, nitrate and nitrite reduction, aesculin, casein and starch hydrolysis, methyl-red and Voges-Proskauer. They hydrolysed Tween 20. None of the substrates assayed as sole sources of carbon and energy or carbon, nitrogen and energy was used by the majority of the strains. Susceptible to rifampycin. The representative strain CH-I05 had a G+C mol% of 43.7. The members of this phenon could be related to the genus Alteromonas (Baumann et ai., 1984a). Discussion During the last few years several authors have studied the taxonomy of moderately halophilic bacteria isolated from different hypersaline environments and new species have been described (Ventosa, 1988; Ventosa et ai., 1989; Franzman et ai., 1988; Quesada et ai., 1990). Many of

278

B. Prado et al.

Phenon No. of strains

A 47

B 5

C 83

D 9

E 17

Morphology (rods)· Motility· Flagella arrangement* PHB* Pigmentation

curved 100 polar 100 cream

short 0

curved 100 polar 98 creamyellow

curved 100 polar 100 cream

curved 100 polar 100 cream

Salts: growth at % (w/v) 3 5 25 30

100 100 85 6

100 100 100 100

88 100 94 68

67 89 100 44

88 88 82 6

pH: growth at pH 5* pH6 pH 10

100 100 100

100 100 100

100 99

100 100

11

35 94 94

Temperature: growth at 5°C 40°C 45°C

100 100 74

100 100 100

88 96 60

56 100 67

82 88 65

Strict aerobes· Oxidase"

a

100

100

100

100 100

100 100

Acid production from: L-arabinose D-cellobiose D-galactose Glycerol * D-glucose* meso-inositol Lactose Maltose D-mannitol* Rhamnose Sucrose"

87 70 74 100 100 57 57 100 85 100 100

40 60 80 20 100 100 100

33 22 33 22 66 11 22 44

100

63 28 81 11 7 10 26 22 16 1 8

H 2 S production Nitrate reduction to nitrite Nitrite reduction to gas

100 11 2

80 60 20

78 26 7

66 33

a a a a a a a a a a a a 12 a

Degradation of: Aesculin Casein Gelatin Starch Tween 20 Tween 80 Urea DNA Blood

53 68 47 15 100 89 77 57 100

60

26 1 5

100

63 11 88 10 4

33 67 78 78 33

53 6 88 76 53 35 47

Phosphatase Methyl-red * Voges-Proskauer

100 89 66

60

18 7 0

67

59

100 cream

a

a a

100

a

80 20

a a

80 20

a a 0

92

a

a

11

0

a a a

11

a

a a

Utilization of organic compounds as sole source carbon and energy: Carbohydrates 31 67 49 60 Aesculin 77 33 L-arabinose 17 60 47 56 D-cellobiose 17 60 22 53 80 96 D-fructose 60 33 100 D-glucose 79 a 80 30 100 D-galactose 15 11 80 33 Imuline 36 22 6 40 Lactose

6 12

a

0

24 24 29 24 59 6 6 6

Table 1. The frequencies of positive characters found in the phenons (expressed as a percentage of the total scored to each group for the given test)

279

Moderately Halophilic Bacteria Table 1. Continued

Phenon No. of strains

A 47

B 5

C 83

D 9

E 17

Maltose D-mannose L-raffinose L-rhamnose D-salicine Starch D-sorbose Sucrose D-trehalose D-xylose

70 60 9 57 17 47 6 85 91 6

40 100 40 20 100 20 100 60 80 100

67 81 9 51 47 19 19 63 63 73

0 44 33 0 100 44 67 11 0 33

6 29 29 18 47 18 6 18 18 24

Alcohols: Adonitol Dulcitol * DL-glycerol meso-inositol D-mannitol D-sorbitol

0 0 100 0 34 4

60 80 20 40 100 40

17 85 12 61 48

0 100 56 78 0 44

18 24 24 6 18 12

100 2 6 15 34 72 94 9 94 2 2 100

80 100 0 100 20 100 100 80 100 100 60 100 100 100 100

73 17 4 94 23 96 94 27 97 36 9 97 51 95 18

89 100 0 78 56 56 100 89 67 100 33 100 67 67 100

24 0 0 6 6 18 12 12 12 12 0 53 35 24 12

Carboxylic acids: Acetate * Benzoate * Caprylate Citrate Formate Fumarate D-gluconate* Hippurate DL-lactate DL-malate* Oxalate Pyruvate Propionate Succinate D-tartrate *

11

87 0

Utilization of amino acids as sole source of carbon, L-alanine 100 100 L-arginine 34 80 L-asparragine 100 100 L-aspartic acid 26 60 L-cysteine 19 0 L-glutamic acid 100 80 L-histidine* 100 20 L-isoleucine 2 0 L-leucine 2 0 L-lysine 9 0 L-ornithine 83 0 L-serine* 100 0 L-tryptophan * 2 0 L-valine 6 0 Susceptibility to Cephalothin Erythromycin Penicillin Rifampycin Tetracycline

36 28 40 49 19

40 100 100 100 0

11

nitrogen and energy: 95 33 77 33 92 33 22 88 2 11 44 95 0 13 9 0 12 33 21 44 80 44 90 22 1 0 10 0 18 74 88 73 15

100 100 100 100 33

35 24 18 18 0 35 24 6 6 18 29 24 0 12 59 76 76 82 29

All strains were Gram-negative, catalase positive. They grow at 7.5, 10, 15 and 20% (w/v) salts (optimal growth at 7.5%), at pH 7, 8, 9 and at 15,25,32 and 37"C. Incapable to grow at 0.5% (w/v) salts. None formed Indole. All were phenylalanine deaminase negative. They were sensible to ampicillin and cloramphenicol. * Characteristics differentiating the strains of the five phenons.

280

B. Prado et al.

these studies used numerical analysis of the phenotypic features of the strains, but classification, the isolates as members of previously described taxa has proved difficult because the chosen reference strains did not cluster with the phenons formed by the new isolates. . In our study, 70% of the moderately halophilic, Gramnegative rods studied had a respiratory type of metabolism and clustered in five phenons (A, B, C, D and E). The remainder (30%), all facultatively anaerobic strains, grouped in one phenon (A) (Fig. 1). The preponderance of aerobic strains in this habitat corresponds to the results of studies of other hypersaline environments (Rodriguez- Valera et al., 1986; Rodriguez-Valera, 1988). Halophilic eubacteria, with aerobic chemoorganotrophic metabolism, are a diverse group containing representatives of most of the genera found in the ocean and normal soils, while halophilic eubacteria with a fermentative metabolism are most usually identified as species of the genus Vibrio to which we have tentatively assigned the 47 fermentative strains (phenon A) (Baumann et al., 1984b). This genus contains the moderately halophilic species Vibrio costicola (Baumann et al., 1984a) the description of which has been emmended (Garcia et al., 1987). Phenon A strains resemble the new description of V. costicola. They differ from this species, however, in that no growth occurs at 0.5% (w/v) salts, acid is produced from L-arabinose, Tween 80 is hydrolysed, H 2 S is produced and neither DL-malate nor propionate are usually utilized as sole sOl}rces of carbon and energy. The strains included in phenon B resemble the genus Acinetobacter in their phenotypic features Uuni, 1984) although they differ from this genus in their salt requirements and, more importantly, in their G+C content of DNA (50.8) which is higher than that described for the genus (40-46 mol%; Juni, 1984; Bouvet and Grimont, 1986). Quesada et al. (1987) described a group of oxidase negative, non-motile, moderately halophilic Gram-negative strains which they tentatively assigned to the genus Acinetobacter. Very recently, it has been proposed that those strains be assigned to a new species, Volcaniella eurihalina (Quesada et al., 1990). Our isolated differ from V. eurihalina in their G+C content, capability to hydrolyse Tween 80 and susceptibility to penicillin. Nonmotile, moderately halophilic bacteria have been isolated frequently from different hypersaline habitats, where they always constitute a minor component (Quesada et al., 1982 1987; Rodriguez-Valera et al., 1985; RodriguezValera, 1988). Further study is required to determine the taxonomic position of all such bacteria. A large number of the isolates (51 %), were motile rods, oxidase negative and had a respiratory-type of metabolism; they grouped in phenon C (Fig. 1) and exhibited some properties of the genus Marinomonas (Van Landschoot and De Ley, 1983), especially those of the species Marinomonas vaga, which is oxidase negative and has a G+C content of 47.9 mol%. However, our strains differ from Marinomonas in their creamy-yellow pigmentation, their ability to accumulate PHB and to grow between 5 and 40°C. In addition, species of Marinomonas are considered slight halophiles (Kushner and Kamekura, 1988) in

contrast to our isolates from phenon C which have higher NaCl requirements for optimal growth (7.5% w/v). Although differences exist between phenons D and E, both phenons appear similar to the genus Alteromonas (Baumann et al., 1984a). The separation of these phenons probably arose because of their different capacities to use sugars' and other organic compounds as sole carbon and energy sources. Strains in phenons D and E differ from the description of the genus Alteromonas in their high salt requirement and in their capacity to accumulate PHB. While, phenons B, C, D and E exhibit some phenotypic features of the genera Acinetobacter (phenon B), Marinomonas (phenon C) and Alteromonas (phenons D and E), none can be classified with condifence in any of these genera. Nor do any of the phenons appear to be closely related to currently described moderately halophilic taxa. These results agree with the views of other authors who have described moderately halophilic bacteria as a very heterogeneous group. The different environmental conditions in each hypersaline habitat, drastically affect the types of halophilic bacteria present. There is also little doubt that the halophilic character is not confined to one phylogenetic group of eubacteria (Rodriguez-Valera et al., 1986; Javor, 1989; Kushner and Kamekura, 1988). Further studies are required to resolve the classification of such bacteria.

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Moderately Halophilic Bacteria

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Dr. A. Ramos-Cormenzana, Departmento de Microbiologia, Facultad de Farmacia, Campus Universitario de Carhlja, Granada, Spain