Biodiversity of Moderately Halophilic Bacteria in Hypersaline Habitats in Egypt

J. Gen. Appl. Microbiol., 52, 63–72 (2006)

Full Paper

Biodiversity of moderately halophilic bacteria in hypersaline habitats in Egypt

Hanan Ghozlan,* Hisham Deif, Rania Abu Kandil, and Soraya Sabry

Department of Botany, Faculty of Science, University of Alexandria, Moharrem Bey, Egypt

(Received January 19, 2005; Accepted November 24, 2005)

Screening bacteria from different saline environments in Alexandria. Egypt, lead to the isolation of 76 Gram-negative and 14 Gram-positive moderately halophilic bacteria. The isolates were characterized taxonomically for a total of 155 features. These results were analyzed by numerical techniques using simple matching coefficient (SSM) and the clustering was achieved by the un- weighed pair-group method of association (UPGMA). At 75% similarity level the Gram-negative bacteria were clustered in 7 phena in addition to one single isolate, whereas 4 phena represented the Gram-positive. Based on phenotypic characteristics, it is suggested that the Gram- negative bacteria belong to the genera Pseudoalteromonas, Flavobacterium, Chromohalobacter, Halomonas and Salegentibacter, in addition to the non-identified single isolate. The Gram-positive bacteria are proposed to belong to the genera Halobacillus, Salinicoccus, Staphylococcus and Tetragenococcus. This study provides the first publication on the biodiversity of moderately halophilic bacteria in saline environments in Alexandria, Egypt.

Key Words——moderate halophiles; numerical taxonomy; saline environments

Introduction physiological adaptation to highly saline concentrations and their ecology (Martinez-Canovas et al., 2004; Moderately halophilic bacteria are microorganisms Tokunaga et al., 2004; Ventosa et al., 1998a, b). that can grow optimally in media containing between Hypersaline environments in Egypt are neglected 3% and 15% (w/v) salt (Lichﬁeld, 2002). Because of hunting grounds for potential interesting new types of the wide range of salinity in which this group of mi- moderately halophilic bcteria. It was thus aimed in this croorganisms can grow optimally, they are widely dis- paper to explore the biodiversity of this group of bacte- tributed in different saline habitats such as hypersaline ria in different saline habitats in Egypt since to our lakes, desert and saline soils, saltern ponds, salt knowledge such study has not been carried out. mines, salted foods and others (Lichﬁeld and Gillevet, 2002; Ventosa et al., 1998a, b). Materials and Methods The aspects that attracted the interest of re- searchers recently were mainly those related to their Study area and analysis of samples. Water and soil samples were collected from three sites representing different saline environments. These are the * Address reprint requests to: Dr. Hanan A. Ghozlan, Depart- coasted salt marshes and the solar salterns (40 km ment of Botany & Microbiology, Faculty of Science, University of Alexandria, Moharrem Bey, Egypt. west of Alexandria) and the salt lakes (21 km west of Tel: 0020–12–2491034 Fax: 0020–3–4240959 Alexandria). E-mail: [email protected] Samples collected in sterile bottles were sent to the 64 GHOZLAN et al. Vol. 52 laboratory, and examined within 2 to 3 h of collection. Halophilic medium (HM) adopted after Ventosa et al. (1982) was used for the isolation and contained in g%:

NaCl, 17.8; MgSO4 ·H2O, 0.1; CaCl2 ·2H2O, 0.036;

KCl, 0.2; NaHCO3, 0.006; NaBr, 0.023; FeCl3 ·6H2O, traces; bacto-peptone, 0.5; yeast extract, 1.0; glucose, 0.1; agar, 2.0. Agar plates were inoculated with 1 ml of water or soil suspension samples or an appropriate di- lution of the sample and incubated at 30°C for up to 7 days. Samples were analyzed for salinity and major cation (Mg2, Na, K) contents according to Ramos-Cor- menzana (1989). Isolates representing different morphotypes were selected and successively streaked on HM to ensure purity. Selected strains were kept on agar slants of maintenance halophilic media containing 10% (w/v) total marine salts; supplemented with bacto- Fig. 1. Simplified dendrogram showing the clustering of peptone, 0.5; yeast extract, 1.0; glucose, 0.1 and agar, strains into 7 phena based on SSM coefficient and UPGMA 2%. analysis, for 76 moderately halophilic Gram-negative bacteria. Characterization and numerical analysis. The se- * No.Number of strains. # Ph.Phenon. lected strains were phenotypically characterized as previously described (Ventosa et al., 1982). All tests total isolates were Gram-negative, whereas the Gram- were carried out in cultures incubated at 30°C for up to positive (14 isolates) formed only 15.5% of the total. 7 days. A total of 155 characteristics were used in the Characteristics of the Gram-negative isolates are listed numerical analysis. Positive and negative results were in Table 1. Analysis of 155 characteristics using a SSM coded as 1 and 0, respectively. Strain similarities were coefficient with UPGMA clustering yielded the dendo- estimated using the simple matching coefficient (SSM) gram in Fig. 1. Data show that the majority of the (Sokal and Michenu, 1985) and the clustering was strains were grouped at the 75% similarity level into achieved by the unweighted pair-group method of as- seven phena. Only one strain, isolated from the salt sociation (UPGMA) (Sneath and Sokal, 1973). The lakes, formed a single separate cluster. The estimated test error was estimated by examining 10 strains in du- error was less than 3%. plicate (Sneath and Johnson, 1972). The computations Phenon A. The 6 strains grouped in phenon A were performed by using the SYSTAT-PC program V.7 possess several features that are common to mem- (Wilkinson et al., 1992) on an IBM computer. bers of genus Pseudoalteromonas. It is thus proposed that the strains are different species of this genus. The Results and Discussion data in Table 1 show that all of the strains are long motile rods. They require up to 20% NaCl for growth Increased attention has been given in the last few and tolerate up to 30% salinity. Colonies are round years to moderately halophilic bacteria. Several stud- convex and produce brown pigments. They utilize a ies have been conducted on their ecology, taxonomy wide range of compounds as a carbon source. and phylogeny as well as their biotechnological appli- Phenon B. The orange pigmented bacteria com- cations (Ramos-Cormenzana, 1991; Sanchez-Porro et prising phenon B (six strains) show phenotypic fea- al., 2003; Ventosa et al., 1998a, b). tures closely related to members of genus Flavobac- In the present study, a total of 90 strains chosen to terium. Flavobacterium is placed in the Flexibacer- represent different morphotypes were purified and Bacteroides-Flavobacterium phylum a (Woese et al., characterized with 155 tests. 1990). Flavobacterium is the type genus of the family Flavobacteriaceae which also encompasses several Gram-negative isolates other genera. The majority (76 isolates) representing 84.5% of the Data show that the strains are long rods with gliding 2006 Biodiversity of moderately halophilic bacteria in hypersaline habitats in Egypt 65

Table 1. Frequencies of positive characteristics found in the seven phena (expressed as a percentage of the total scored by each group for the given test).

ABCDEFG 664 62723 3

Salt concentration 10% 100 100 100 100 100 100 100 20% 100 100 100 100 100 100 100 30% 17 0 25 17 47 0 0 40% 0 0 25 17 67 0 0 Growth at pH 5 83 83 0 0 100 96 0 pH 9 83 67 100 100 85 57 100 Temperature 10°C 17 83 25 100 93 48 100 Temperature 40°C 0 0 0 0 81 52 0 Biochemical tests Oxidase 100 100 75 100 91 84 100 Methyl red 50 0 0 0 0 0 17 Voges-Proskauer 50 0 0 0 0 0 0

H2S production 83 50 75 83 93 43 0 Nitrate reductase 0 0 25 0 85 74 0 Nitrite reductase 0 0 0 0 85 74 0 Utilization of organic compounds as sole source of carbon and energy Arabinose 0 83 100 50 85 96 100 Mannose 100 100 0 100 85 100 100 Rafﬁnose 17 0 0 17 74 48 100 Rhamnose 83 33 0 17 85 43 100 Ribose 83 100 0 67 85 100 100 Xylose 0 83 100 67 85 100 100 Trehalose 83 100 75 100 100 96 0 Fructose 67 17 50 0 7 52 0 Galactose 67 100 50 67 100 100 100 Sucrose 0 100 100 83 96 100 100 Lactose 0 0 0 0 74 26 100 Maltose 100 100 100 100 100 83 100 Cellobiose 0 17 0 17 100 74 100 Glycerol 33 83 0 100 93 48 100 Mannitol 100 83 75 100 96 96 100 Lactate 33 83 25 100 78 48 100 Utilization of amino acids as sole source of carbon, nitrogen and energy L-Alanine 83 0 0 100 100 100 100 L-Phenylalanine 100 67 0 100 93 91 100 L-Arginine 100 0 100 100 100 100 100 L-Asparagine 33 67 25 100 93 48 100 L-Tyrosine 0 0 0 33 81 70 67 L-Glutamine 33 83 25 100 93 48 100 L-Glutamic acid 0 0 0 0 0 4 0 L-Cystine 0 0 0 33 0 9 33 L-Cysteine 0 0 0 33 11 13 33 L-Proline 33 100 100 100 100 100 100 L-Histidine 0 50 0 33 74 100 67 66 GHOZLAN et al. Vol. 52

Table 1. (Continued).

ABCDEFG 664 62723 3

L-Isoleucine 83 17 0 33 37 78 67 L-Leucine 0 0 0 83 100 91 100 L-Lysine 100 17 100 100 100 100 100 L-Ornithine 0 33 75 33 100 100 0 L-Serine 0 100 0 33 100 100 33 L-Tryptophan 0 83 0 100 89 100 33 L-Methionine 0 17 0 100 81 100 100 L-Threonine 0 0 100 0 33 0 0 L-Valine 100 17 0 100 85 100 100 Acid production from Glucose 100 100 75 50 100 91 100 Galactose 0 100 0 0 0 87 0 Maltose 100 17 0 0 0 4 0 Lactose 0 67 0 0 0 4 0 Sucrose 100 33 75 67 11 17 0 Mannitol 67 0 0 0 4 0 0 Degradation of Alginate 100 50 75 100 44 91 100 Aesculin 0 100 0 0 93 4 0 Chitin 100 0 0 0 0 0 0 Cellulose 50 50 0 17 0 13 100 Starch 0 50 0 0 0 4 100 Gelatine 67 100 50 0 7 0 33 Casein 100 0 0 0 0 0 100 DNA 100 33 0 33 4 30 33 Tween 20 33 0 0 17 26 13 0 Tween 40 0 67 0 0 67 22 0 Tween 80 50 0 0 50 15 9 100 Antibiotic sensitivity Penicillin G (10 units) 83 33 100 83 7 26 100 Polymyxin B (300 units) 100 83 50 67 93 70 67 Tetracycline (30 mg) 33 33 0 17 11 26 100 Ampicillin (10 mg) 83 100 100 100 30 96 100 Cephalothine (30 mg) 67 0 100 83 15 9 67 Chloramphenicol (30 mg) 83 83 100 100 100 96 100 Novobiocin (30 mg) 83 67 100 100 33 52 100

motility, forming orange round convex colonies. They (McCammon and Bowman, 2000). grow optimally at 10% NaCl and tolerate up to 20%. Phena C and D. Strains comprising the two phena They grow at pH 5–8 and 10–30°C. These phenotypic C and D show phenotypic features (Table 1) clearly re- characteristics of the six isolates show some degree of lated to those of the genus Chromohalobacter (Arahal variability with known reported species. This degree of et al., 2001; Ventosa et al., 1989). Although members phenotypic diversity may be revealed as a result of the of this genus are described as extremely salt tolerant isolation and description of new species. A conse- microorganisms they should be considered as moder- quence of this diversity is that a degree of reliance on ately halophilic (Ventosa et al., 1998b), showing opti- phylogenetic and chemotaxonomic analysis is neces- mal growth at 7.5–10% (w/v) salt, rather than halotol- sary for the definitive identification of flavobacteria erant. The genus Chromohalobacter is phylogeneti- 2006 Biodiversity of moderately halophilic bacteria in hypersaline habitats in Egypt 67 cally closely related to Halomonas and includes three species, C. marismortui, C. israelensis and C. canadensis (Arahal et al., 2001). The biochemical and degrading patterns of the 10 strains included in phena C and D resemble the features of one of the three species and vary in others. It is thus concluded that they may belong to one of the species listed in the genus Chromohalobacter. Phena E and F. The phenotypic characteristics of the strains included in phena E and F are in agreement with the phenotypic heterogeneity reported for the species of the genus Halomonas. This genus was originally proposed to accommodate one species Halomonas elongata (Vreeland et al., 1980). It now contains a large number of recently discovered Fig. 2. Simplified dendrogram showing the clustering of species (Arahal et al., 2002; Duckworth et al., 2000; strains into 4 phena based on SSM coefficient and UPGMA Martinez-Canovas et al., 2004; Romanenko et al., analysis, for 14 moderately halophilic Gram-positive bacteria. 2002) as well as already known bacteria. A recent * No.=Number of strains. # Ph.Phenon. study (Arahal et al., 2002) shows that two phylogenetic groups can not be distinguished within the genus dase positive. Colonies are circular, convex, shiny and Halomonas in addition to six other species which can pigmented (yellow, orange, or beige). Growth occurs at not be assigned to either of the above-mentioned 10–20% NaCl, pH 9 and 10°C. groups. At present, this genus comprises two species, Sale- The 27 strains grouped in phenon E at 72% similar- gentibacter salegenis (formerly Flavobacterium sale- ity level show similarities in their phenotypic character- genes) (Dobson et al., 1993) and Salegentibacter istics (Table 1) to members of group 1 of the genus halothuriorum sp. nov. (Nedashkovskaya et al., 2004). Halomonas which comprises the type species Moderately halophilic or halotolerant Gram-positive Halomonas elongata and four other species. H. euri- rods or cocci have been commonly isolated from ma- halina; H. halomophila, H. halophila and H. salina rine environments and related regions or materials (Arahal et al., 2002). Cells are Gram-negative, non- (Arahal et al., 1999; Ventosa et al., 1998a; Waino et al., sporing long rods. Most of them grow in circular con- 1999). In the present study 14 Gram-positive moder- vex, cream colored mucoid colonies capable of grow- ately halophilic rods and cocci were isolated and taxo- ing in NaCl concentrations of 0.5–40.0%. Growth oc- nomically grouped at the 73% similarity level into 4 curs within the temperature range 15–40°C. The 23 phena (Fig. 2). Phenotypic characteristics of the strains grouped at 77% similarity level in phenon F are strains are given in Table 2. in agreement with the phenotypic heterogeneity re- The Gram-positive, endospore-forming rods are rep- ported for the species of group II of the genus resented by three strains forming phenon W. Most Halomonas. Group II is formed by the following Gram-positive or Gram-variable, endospore-forming species. H. aquamarina, H. meridiana, H. megadiensis, rods with moderately halophilic or halotolerant proper- H. variabilis, H. venusta, H. halodurans and H. sub- ties have previously been assigned to the genus Bacil- glaciescola (Arahal et al., 2002). lus. Recently, a reclassification was published and new Phenon G. Similarities in phenotypic characteris- genera and species were reported including Gra- tics of the three strains comprising phenon G support cilibacillus (Waino et al., 1999), Marinibacillus marinus their inclusion in the genus Salegentibacter. General (Yoon et al., 2001) and Halobacillus salinus (Yoon et characteristics reveal the resemblance of this group to al., 2003). The phenotypic characterization of the the Cytophaga-Flavobacterium-Bacteroides group cre- strains comprising phenon W are consistent with their ated by McCammon and Bowman (2000): Cells are assignment to the genus Halobacillus. They show Gram-negative, strictly aerobic, chemo-organotrophic, closely related characteristics to the recently published non-motile non-sporing long rods, catalase and oxi- Halobacillus salinus (Yoon et al., 2003). 68 GHOZLAN et al. Vol. 52

Table 2. Frequencies of positive characteristics found in the four phena (expressed as a percentage of the total scored by each group for the given test).

WX Y Z 36 32

Salt concentration 10% 100 100 100 100 20% 100 100 100 100 30% 0 50 100 0 40% 0 33 0 0 Growth at pH 5 0 0 100 100 Temperature 40°C 0 17 0 100 Biochemical tests Methyl red 0 0 100 100 Voges-Proskauer 0 0 0 100

H2S production 100 0 100 0 Phosphatase production 100 50 0 100 Utilization of organic compounds as sole source of carbon and energy Rafﬁnose 100 100 0 0 Rhamnose 100 83 100 100 Xylose 67 67 0 100 Cellobiose 67 83 33 100 Galactose 100 83 100 100 Sucrose 100 67 100 0 Lactose 100 50 100 0 Maltose 100 100 100 0 Glycerol 100 83 100 100 Sorbitol 0 67 0 0 Adonitol 100 83 0 100 Dulcitol 0 17 0 0 Lactate 100 100 0 0 Utilization of amino acids as sole source of carbon, nitrogen and energy L-Alanine 100 83 100 0 L-Arginine 100 100 100 0 L-Asparagine 100 100 100 0 L-Aspartic acid 0 0 100 0 L-Tyrosine 0 67 100 0 L-Glutamine 100 100 100 0 L-Glutamic acid 0 17 100 100 L-Cystine 0 83 0 0 L-Cysteine 0 67 100 0 L-Proline 100 83 100 0 L-Histidine 0 83 100 0 L-Isoleucine 0 50 0 0 L-Leucine 0 17 100 100 L-Lysine 100 100 0 0 L-Ornithine 0 50 100 100 L-Serine 0 50 100 0 L-Tryptophan 100 83 100 100 L-Valine 100 100 100 0 2006 Biodiversity of moderately halophilic bacteria in hypersaline habitats in Egypt 69

Table 2. (Continued).

WX Y Z 36 32

L-Methionine 0 100 100 100 Degradation of Alginate 100 67 0 0 Aesculin 100 17 0 100 Cellulose 0 33 0 0 Starch 100 0 0 0 Gelatin 100 83 0 0 Protein 0 83 0 0 Urea 0 0 100 0 Tween 20 100 67 0 100 Tween 40 100 0 100 100 Tween 80 0 17 0 0 Antibiotic sensitivity Penicillin G (10 units) 0 100 100 100 Polymyxin B (300 units) 0 67 0 0 Streptomycin (10 mg) 100 50 0 0 Tetracycline (30 mg) 100 100 100 0 Erythromycin (15 mg) 100 67 100 0 Neomycin (30 mg) 0 100 0 0 Kanamycin (30 mg) 100 83 0 0 Gentamicin (30 zg) 0 50 0 0

Table 3. Sources of isolates in each cluster shown as the percentage isolated from each sampling site.

Phenon

Location Site Sample ABCDEFGWXYZ 66462723 3 3 6 3 2

Coastal salt 1 Soil 0 0 0 0 0 0 0 0 0 0 100 Solar salterns 2 Water 0 0 0 0 15 0 0 0 0 0 0 3 Soil 0 17 100 0 63 0 0 0 0 0 0 Salt lakes 4 Water 0 0 0 0 0 0 0 100 0 100 0 5Water 100 83 0 99 15 100 100 0 100 0 0

Phena X, Y and Z comprising Gram-positive, non- Salinococcus. Three species are known for the genus, sporulating cocci. Seven species are now currently S. roseus (Ventosa et al., 1990), S. hispanicus (Ven- recognized in the moderately halophilic cocci (Ventosa tosa et al., 1992) and the moderate alkaliphile S. alka- et al., 1998a). The majority of the Gram-positive cocci liphilus (Zhang et al., 2002). (7 strains) are grouped in phenon X. Cells are cocci Phenon Y contains three strains that show high simi- occurring singly, in pairs, tetrads or clumps. En- larity to the genus Staphylococcus. Cells are arranged dospores are not formed. Colonies are round, smooth in irregular aggregates and form orange ﬂat colonies. and slightly convex with red color and non-diffusible They grow at pH 5–8 and 10–30°C. Although no pigments; the morphological, physiological and bio- strains were reported to be moderate halophiles, chemical characteristics of these strains are consistent species of Staphylococcus are known to be tolerant to with their assignment as members of the genus, up to 20% of NaCl concentration. 70 GHOZLAN et al. Vol. 52

Table 4. Physicochemical characteristics of studied samples.

Location Site Sample Temp. %Sa %Mgb %Na %K

Coastal salt marshes 1 Soil 30.0 13.5 4.4 6.5 0.9 Solar salterns 2 Water 27.6 36.4 10.2 14.3 1.4 3 Soil 27.3 24.6 6.9 12.0 0.7 Salt lakes 4 Water (edges) 32.2 36.8 13.9 11.0 1.6 5Water 32.0 23.2 6.0 12.4 0.7

a % Salt content represents the salt content as % TDS and % TSS. b Mg, Na, K and Cl ions represent cation and anion concentrations calculated as % (w/v) for water samples and as % (w/w) for soil or soil samples.

The two strains grouped in phenon Z show pheno- edges (site 4) represents hypersaline environments typic characteristics (Table 3) closely related to genus (36% salinity). The moderately halophilic bacteria ob- Tetragenococcus. Only one species is recongnized, T. tained from the water of solar salterns were assigned muriaticus (Satomi et al., 1997). to the Gram-negative genus Halomonas. The great Environments of moderate salinity (13.5–25%) are salt lake yielded a number of new species belonging to represented in this study by the two soils of coastal family Halomonadaceae (Dobson and Franzmann, salt marshes and solar salterns (sites 1 and 3) and the 1996; Fendrich, 1988). However, the edges of salt water of a salt lake (site 5). The characteristics of each lakes yielded only Gram-positive species belonging to site are shown in Table 4. the genera Halobacillus and Staphylococcus. Although The soil habitat is inherently inhomogenous, and it the Gram-positive endospore-forming rods were previ- can be expected that a wide range of salinities might ously isolated from hyper saline environments (Spring be present in any one saline soil (Grant, 1991). Saline et al., 1996), this is the first report on the isolation of soils appear to yield mostly halotolerant rather than Staphylococcus from such an environment. halophilic microorganisms, presumably reflecting adaptation to periodic episodes of relatively high dilu- References tion (Quesada et al., 1982). Further studies (Quesada Arahal, D., Garcia, M., Ludwig, W., Schleifer, K., and Ventosa, et al., 1983) showed that species distribution reflects A. (2001) Transfer of Halomonas canadensis and the ecological difference between water and soil envi- Halomonas israelensis to the genus Chromohalobacter ronments, with water being relatively homogenous and as Chromohalobacter canadensis comb. nov. and constant. Chrornohalobacter israelensis comb. nov. Int. J. Syst. Evol. In the present study, the soil of the coastal salt Microbiol., 51, 1441–1448. marsh yielded moderate halophilic Gram-positive cocci Arahal, D., Ludwig, W., Schleifer, K., and Ventosa, A. (2002) Phylogeny of the family Halomonadaceae based on 23S assigned to the genus Tetragenococcus. Many halotol- and 16S rDNA sequence analyses. Int. J. Syst. Evol. Mi- erant Gram-negative bacteria were isolated from crobiol., 52, 241–249. saline soil near Alicante, Spain (Queseda et al., 1985). Arahal, D., Marquez, M., Volcani, B., Schleifer, K., and Ventosa, On the other hand, saline soils of solar salterns were A. (1999) Bacillus marismortui sp. nov., a new moderate characterized by 24% salt content and comparatively halophilic species from the Dead Sea. Int. J. Syst. Bacte- higher concentrations of Na, Mg, Na and K than riol., 49, 521—530. coastal salt marshes. Numerical taxonomic study Dobson, S. J., Colwell, R. R., McMeekin, T. A., and Franzmann, revealed that the dominant types of moderately P. D. (1993) Direct sequencing of the polymerase chain re- halophilic bacteria encountered in this habitat are action-amplified 16S rDNA gene of Flavobacterium gond- wanense sp. nov. and Flavobacterium salegens sp. nov., Gram-negative ones belonging to the genera two new species from a hypersaline Antarctic lake. Int. J. Flavobacterium, Chromohalobacter and Halomonas. Syst. Bacteriol., 43, 77–83. Similar results were recorded by Queseda et al. (1985) Dobson, S. J. and Franzmann, P. D. (1996) Unification of the in their study in saline soil near Alicante, Spain. genera Deleya (Baumann et al., 1983). Halomonas (Vree- The water of solar saltern (site 2) and salt lakes land et al., 1980), and Halovibrio (Fendrich, 1988) and the 2006 Biodiversity of moderately halophilic bacteria in hypersaline habitats in Egypt 71

species Paracoccus halodenitrificans (Robinson and Gib- 289–309. bons 1952) into a single genus Halomonas, and placement Ramos-Cormenzana, A. (1991) Halophilic organisms and the of the genus Zymobacter into a family Halomonadaceae. environment. In General and Applied Aspects of Halophilic Int. J. Syst. Bacteriol., 46, 550—558. Microorganisms, ed. by Rodriguez-Valera, F., Plenum Duckworth, A., Grant, W., Jones, B., Meijer, D., Marquez, M., Press, New York, pp. 15–24. and Ventosa, A. (2000) Halomonas magadii sp. nov., a new Romanenko, L. A., Schumann, P., Rohde, M., Mikhailov, V. V., member of the genus Halomonas, isolated from a soda and Stackebrandt, E. (2002) Halomonas halocynthiae sp. lake of the East African Rift Valley. Extremophiles, 4, nov., isolated from the marine ascidian Halocynthia auran- 53–60. tium. Int. J. Syst. Evol. Microbiol., 52, 1767–1772. Fendrich, C. (1988) Halovibrio variabilis gen. nov. sp. nov., Sanchez-Porro, C., Martin, S., Mellado, E., and Ventosa, A. Pseudomonas halophila sp. nov. and a new halophilic aero- (2003) Diversity of moderately halophilic bacteria producing bic coccoid eubacterium from Great Salt Lake, Utah, USA. extracellular hydrolytic enzymes. J. Appl. Microbiol., 94, Syst. Appl. Microbiol., 11, 36–43. 295–300. Grant, W. D. (1991) General view of halophiles. In Superbugs, Satomi, M., Kimura, B., Mizoi, M., Sato, T., and Fujii, T. (1997) Microorganisms in Extreme Environments, ed. by Tetragenococcus muriaticus sp. nov., a new moderately Horikoshi, K. and Grant, W. D., Japan Scientific Societies halophilic lactic acid bacterium isolated from fermented Press, Tokyo, pp. 15–37. squid liver sauce. Int. J. Syst. Bacteriol., 47, 832–836. Lichfield, C. D. (2002) Halophiles. J. Ind. Microbiol. Biotechnol., Sneath, P. and Johnson, R. (1972) The influence on numerical 28, 21–22. taxonomic similarities of errors in microbiological tests. J. Lichfield, C. D. and Gillevet, P. M. (2002) Microbial diversity and Gen. Microbiol., 72, 377–392. complexity in hypersaline environments. A preliminary as- Sneath, P. and Sokal, R. (1973) Numerical Taxonomy, the Prin- sessment. J. Ind. Microbiol. Biotechnol., 28, 48–55. cipals and Practice of Numerical Classification, W. H. Free- Martinez-Canovas, M., Queseda, E., Llamas, L., and Bejar, V. man, San Francisco, pp. 62–76. (2004) Halomonas ventosae sp. nov., a moderately Sokal, R. and Michenu, C. (1985) A statistical method for evalu- halophilic denitrifying, exopolysaccharide-producing bac- ating systematic relationships. Kansa. Univ. Sci. Bull., 38, terium. Int. J. Syst. Evol. Microbiol., 54, 733–737. 1409–1438. McCammon, S. and Bowman, J. (2000) Taxonomy of Antarctic Spring, S., Ludwig, W., Marquez, M. C., Ventosa, A., and Flavobacterium species: description of Flavobacterium Schleifer, K.-H. (1996) Halobacillus gen. nov., with descrip- gillisiae sp. nov., Flavobacterium tegetincola sp. nov. and tions of Halobacillus litoralis sp. nov. and Halobacillus Flavobacterium xanthum sp. nov., nom. rev. and reclassifi- trueperi sp. nov., and transfer of Sporosarcina halophila to cation of [Flavobacterium] salegens as Salegentibacter Halobacillus halophilus comb. nov. Int. J. Syst. Bacteriol., salegens gen. nov., comb. nov. Int. J. Syst. Evol. Microbiol., 46, 492–496. 50, 1055–1063. Tokunaga, H., Mitsuo, K., Ichinose, S., Omori, A., Ventosa, A., Nedashkovskaya, O., Suzuki, M., Vancanneyt, M., Cleenwerck, Nakae, T., and Tokunaga, M. (2004) Salt inducible mul- I., Zhukova, N., Vysotskii, M., Mikhailov, V., and Swings, J. tidrug efflux pump protein in the moderately halophilic bac- (2004) Salegentibacter holothuriorum sp. nov., isolated terium Chromohalobacter sp. Appl. Environ. Microbiol., 70, from the edible holothurian Apostichopus japonicus. Int. J. 4424–4431. Syst. Evol. Microbiol., 54, 1107–1110. Ventosa, A., Gutierrez, M., Garcia, M., and Ruiz-Berraquero, F. Quesada, E., Bejar, V., Valderrama, M. J., Ventosa, A., and (1989) Classification of “Chromobacterium marismortui” in Ramos-Cormenzana, A. (1985) Isolation and characteriza- a new genus Chromohalobacter gen. nov., as Chromo- tion of moderately halophilic nonmotile rods from different halobacter marismortui comb. nov., nom. Rev. Int. J. Syst. saline habitats. Microbiologia SEM, 1, 89–96. Bacteriol., 39, 382–386. Quesada, E., Ventosa, A., Rodriguez-Valera, F., Megias, L., and Ventosa, A., Marquez, M., Garabito, M., and Arahal, D. (1998a) Ramos-Cormenzana, A. (1983) Numerical taxonomy of Moderately halophilic Gram-positive bacterial diversity in moderately halophilic Gram-negative bacteria from hyper- hypersaline environments. Extremophiles, 2, 297–304. saline soils. J. Gen. Microbiol., 129, 2649–2657. Ventosa, A., Marquez, M., Ruiz-Berraquero, F., and Kocur, M. Quesada, E., Ventosa, A., Rodriguez-Valera, F., and Ramos- (1990) Salinococcus roseus gen. nov., a new moderately Comenzana, A. (1982) Types and properties of some bac- halophilic Gram-positive coccus. Syst. Appl. Microbiol., 13, teria isolated from hypersaline soils. J. Appl. Microbiol., 53, 29–33. 153–161. Ventosa, A., Marquez, M., Weiss, N., and Tindall, B. (1992) Ramos-Cormenzana, A. (1989) Ecological distribution and Transfer of Marinococcus hispanicus to the genus biotechnological potential of halophilic microorganisms. In Salinococcus as Salinococcus hispamicus comb. nov. Syst. Microbiology of Extreme Environments and Its Potential for Appl. Microbiol., 15, 530–534. Biotechnology, ed. by Da Costa, M. S., Duarte, J. C., and Ventosa, A., Nieto, J., and Oren, A. (1998b) Biology of moder- Williams, R. A. D., Elsevier Applied Science, London, pp. ately halophilic aerobic bacteria. Microbiol. Mol. Biol., 62, 72 GHOZLAN et al. Vol. 52

504–544. Woese, C. R., Yang, D., Mandelco, L., and Stetter, K. O. (1990) Ventosa, A., Quesada, E., Rodriguez-Valera, F., Ruiz-Berra- The Flexibacter-Flavobacter connection. Syst. Appl. Micro- quere, F., and Ramos-Cormenzana, A. (1982) Numerical biol., 13, 161–165. taxonomy of moderate halophilic Gram-negative rods. J. Yoon, J.-H., Kang, K. H., and Park, Y.-H. (2003) Halobacillus Gen. Microbiol., 128, 1959–1968. salinus sp. nov., isolated from a salt lake on the coast of Vreeland, R. H., Litchﬁeld, C. D., Martin, E. L., and Elliot, E. the East Sea in Korea. Int. J. Syst. Evol. Microbiol., 53, (1980) Halomonas elongata, a new genus and species of 687–693. extremely salt-tolerant bacteria. Int. J. Syst. Bacteriol., 30, Yoon, J.-H., Weiss, N., Lee, K.-C., Lee, I.-S., Kang, K. H., and 485–495. Park, Y.-H. (2001) Jeotgalibacillus alimentarius gen. nov., Waino, M., Tindall, B. J., Schumann, P., and Ingvorsen, K. sp. nov., a novel bacterium isolated from jeotgal with L-ly- (1999) Gracilibacillus gen. nov., with description of Gra- sine in the cell wall, and reclassiﬁcation of Bacillus marinus cilibacillus halotolerans gen. nov., sp. nov., transfer of Rueger 1983 as Marinibacillus marinus gen. nov., comb. Bacillus dipsosauri to Gracilibacillus dipsosauri comb. nov., nov. Int. J. Syst. Evol. Microbiol., 51, 2087–2093. and Bacillus salexigens to the genus Salibacillus gen. nov., Zhang, W., Xue, Y., Ma, Y., Zhou, P., Venotsa, A., and Grant, W. as Salibacillus salexigens comb. nov. Int. J. Syst. Bacteriol., (2002) Salinicoccus alkaliphilus sp. nov., a novel alkaliphile 49, 821–831. and moderate halophile from Baer Soda Lake in Inner Wilkinson, L., Hill, M., Miceli, S., Birkenbeuel, G., and Vang, E. Mongolia autonomous region, China. Int. J. Syst. Evol. Mi- (1992) SYSTAT for Windows, Version 5 ed., Evanston, IL, crobiol., 52, 789–793. SYSTAT, Inc., Ill.