Pseudonocardia hispaniensis sp. nov., a novel actinomycete isolated from industrial wastewater activated sludge.

A novel actinomycete, designated PA3(T), was isolated from an oil refinery wastewater treatment plant, located in Palos de la Frontera, Huelva, Spain, and characterized taxonomically by using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate formed a distinct subclade in the Pseudonocardia tree together with Pseudonocardia asaccharolytica DSM 44247(T). The chemotaxonomic properties of the isolate, for example, the presence of MK-8 (H(4)) as the predominant menaquinone and iso-C(16:0) as the major fatty acid, are consistent with its classification in the genus Pseudonocardia. DNA:DNA pairing experiments between the isolate and the type strain of P. asaccharolytica DSM 44247(T) showed that they belonged to separate genomic species. The two strains were readily distinguished using a combination of phenotypic properties. Consequently, it is proposed that isolate PA3(T) represents a novel species for which the name Pseudonocardia hispaniensis sp. nov. is proposed. The type strain is PA3(T) (= CCM 8391(T) = CECT 8030(T)).

Thalassobius mediterraneus gen. nov., sp. nov., and reclassification of Ruegeria gelatinovorans as Thalassobius gelatinovorus comb. nov.

A Gram-negative, slightly halophilic, non-pigmented, strictly aerobic, chemo-organotrophic bacterium was isolated from sea water off the western Mediterranean coast near Valencia (Spain). This strain was able to grow on several organic acids and amino acids added to a minimal medium as carbon sources, but used few carbohydrates or yielded slight growth when sugars were used. Phylogenetic analysis based on an almost complete 16S rRNA gene sequence revealed that strain XSM19T was a member of the Roseobacter group within the 'Alphaproteobacteria', with its closest phylogenetic neighbour being Ruegeria gelatinovorans (97.6 % sequence similarity). Following a polyphasic approach, it was concluded that strain XSM19T represents a new genus and novel species, for which the name Thalassobius mediterraneus sp. nov. is proposed. The type strain is XSM19T (=CECT 5383T=CIP 108400T=CCUG 49438T). It is also proposed that R. gelatinovorans (Rüger & Höfle 1992) Uchino et al. 1999 is reclassified as Thalassobius gelatinovorus comb. nov.

Thalassobacter stenotrophicus Macian et al. 2005 is a later synonym of Jannaschia cystaugens Adachi et al. 2004, with emended description of the genus Thalassobacter.

The type strains of Jannaschia cystaugens (LMG 22015(T)) and Thalassobacter stenotrophicus (CECT 5294(T)) were analysed by means of genomic DNA-DNA hybridization, comparison of 16S rRNA gene sequences and phenotypic properties determined under the same methodological conditions. J. cystaugens LMG 22015(T) showed DNA-DNA relatedness levels of 72% when hybridized with the genomic DNA of T. stenotrophicus CECT 5294(T). Sequence comparisons revealed that the 16S rRNA genes of the two strains had a similarity of 99.8%. The cellular fatty acid and polar lipid compositions of the two strains and their DNA mol% G+C contents were almost identical. Bacteriochlorophyll a (Bchl a) and polyhydroxybutyrate were produced by both strains under the same culture conditions. Their closest phylogenetic neighbours were Jannaschia helgolandensis and Jannaschia rubra; however, the low sequence similarity values (95.7-95.9%) and several important differences in phenotypic traits (ionic requirements, Bchl a production and polar lipids) support the distinction between the genera Thalassobacter and Jannaschia. Thus, we propose the unification of J. cystaugens (LMG 22015(T)) and T. stenotrophicus (CECT 5294(T)) as Thalassobacter stenotrophicus (type strain, CECT 5294(T)=DSM 16310(T)). An emended description of the genus Thalassobacter is also presented.

Nereida ignava gen. nov., sp. nov., a novel aerobic marine alpha-proteobacterium that is closely related to uncultured Prionitis (alga) gall symbionts.

A Gram-negative, slightly halophilic, non-pigmented, strictly aerobic, chemo-organotrophic bacterium was isolated from Mediterranean sea water off the Spanish coast near Valencia. This strain was poorly reactive, being unable to grow in most carbon sources analysed in minimal medium. However, good growth was observed when more complex media and longer incubation times were used. Phylogenetic analysis based on an almost complete 16S rRNA gene sequence placed strain 2SM4(T) within the Roseobacter group, in the vicinity of uncultured bacteria described as gall symbionts of several species of the red alga Prionitis. Sequence similarity values between strain 2SM4(T) and the closest neighbouring species were below 95.0 %. The cellular fatty acid composition of the Mediterranean strain confirmed its position within the 'Alphaproteobacteria', sharing 18 : 1omega7c as the major cellular fatty acid. The phylogenetic distance from any taxon with a validly published name and also a number of distinguishing features support the designation of strain 2SM4(T) as representing a novel genus and species, for which the name Nereida ignava gen. nov., sp. nov. is proposed. The type strain is 2SM4(T) (=CECT 5292(T)=DSM 16309(T)=CIP 108404(T)=CCUG 49433(T)).

Jannaschia rubra sp. nov., a red-pigmented bacterium isolated from sea water.

A Gram-negative, slightly halophilic, strictly aerobic, chemo-organotrophic bacterium was isolated from Mediterranean sea water near Valencia (Spain). Comparison of the almost complete 16S rRNA gene sequence showed that strain 4SM3(T) belonged to the Roseobacter group, with Jannaschia helgolandensis as its closest relative, with a similarity of 98.7 %. DNA-DNA hybridization analysis showed that the Mediterranean isolate had a level of relatedness of less than 42 % with J. helgolandensis and therefore that it represented a novel species of the genus Jannaschia. Phenotypic characteristics gave further evidence that the two organisms are not related at the species level. Isolate 4SM3(T) grows on solid media as irregular pink-red colonies that penetrate into the agar. Cells are rods, motile by a tuft of polar flagella. The DNA base composition is 64.6 mol% G+C. Morphological, physiological and genotypic differences from related species support the description of a novel species, Jannaschia rubra sp. nov., with strain 4SM3(T) (=CECT 5088(T)=DSM 16279(T)) as the type strain.

Thalassobacter stenotrophicus gen. nov., sp. nov., a novel marine alpha-proteobacterium isolated from Mediterranean sea water.

A Gram-negative, slightly halophilic, strictly aerobic, chemo-organotrophic bacterium was isolated from Mediterranean sea water near Valencia (Spain). 16S rRNA gene sequence comparisons showed that the isolate represented a separate branch within the alpha-3 subclass of the Proteobacteria, now included within the order 'Rhodobacterales'. Jannaschia helgolandensis was the closest relative, but their low sequence similarity and other features indicated that they were not related at the genus level. Isolate 5SM22T produced bacteriochlorophyll a and grew on solid media as regular salmon-pink colonies. Cells are motile rods, with polar flagella. The DNA G+C content is 59.1 mol%. Morphological, physiological and genotypic differences from related, thus far known genera support the description of Thalassobacter stenotrophicus gen. nov., sp. nov. with strain 5SM22T (=CECT 5294T=DSM 16310T) as the type strain.

Marinobacter lipolyticus sp. nov., a novel moderate halophile with lipolytic activity.

In the course of a screening programme in hypersaline habitats of southern Spain to isolate halophilic bacteria that are able to produce different extracellular enzymes, a novel, moderately halophilic bacterium (strain SM19(T)) that displays lipolytic activity has been isolated and characterized. Strain SM19(T) is a Gram-negative rod that grows optimally in culture media that contain 7.5 % NaCl. The DNA G+C content was 57.0 mol%. According to phenotypic and genotypic data, this strain was assigned to the genus MARINOBACTER: However, 16S rDNA sequence similarity between strain SM19(T) and species of the genus Marinobacter was <96.7 %; this value is sufficiently low to propose its designation as a novel species. In addition, DNA-DNA hybridization with reference strains of close phylogenetic relatives was between 11 and 19 %. On the basis of these data, the inclusion of strain SM19(T) in the genus Marinobacter as a novel species is proposed, with the name Marinobacter lipolyticus sp. nov. The type strain of the novel species is SM19(T) (=DSM 15157(T)=NCIMB 13907(T)=CIP 107627(T)=CCM 7048(T)).

Transfer of Halomonas canadensis and Halomonas israelensis to the genus Chromohalobacter as Chromohalobacter canadensis comb. nov. and Chromohalobacter israelensis comb. nov..

16S rRNA gene sequence comparisons and DNA-DNA hybridization data support the conclusion that two species previously described as members of the genus Halomonas, Halomonas israelensis and Halomonas canadensis, should be placed in the genus Chromohalobacter. Both H. israelensis ATCC 43985T (= Ba1T) and H. canadensis ATCC 43984T (= NRCC 41227T) have been used extensively for physiological studies for many years; nevertheless, they were not named and classified taxonomically until quite recently. Their phenotypic resemblance (at least 65% Jaccard similarity) to some members of the genus Halomonas and the degree of DNA-DNA relatedness (lower than 60%) to other described species of this genus permitted the conclusion that they were distinct species belonging to the genus Halomonas. In this study, the 16S rDNA of both species has been sequenced completely and found to share higher similarity to the available sequences of the moderately halophilic bacterium Chromohalobacter marismortui than to sequences of members of the genus Halomonas. C. marismortui is the sole species of the genus Chromohalobacter, also included in the family Halomonadaceae, and shares many phenotypic features with H. canadensis and H. israelensis. It is proposed that the two species should be renamed as Chromohalobacter canadensis comb. nov. and Chromohalobacter israelensis comb. nov. An emended description of the genus Chromohalobacter is given in order to include the features of these two species.

Chromohalobacter salexigens sp. nov., a moderately halophilic species that includes Halomonas elongata DSM 3043 and ATCC 33174.

Two strains that were originally isolated and characterized as members of the moderately halophilic species Halomonas elongata, strains DSM 3043 (= 1H11) and ATCC 33174 (= 1H15), were studied in detail. Their complete 16S rRNA sequences were determined and, when compared to sequences available from the databases, they showed a close phylogenetic relationship to Chromohalobacter marismortui. In addition, DNA-DNA hybridization experiments showed that both strains are members of the same species, but their DNA relatedness to the type strains of Halomonas elongata, ATCC 33173T, and Chromohalobacter marismortui, ATCC 17056T, is very low. Phenotypically, the two strains showed very similar features, related to those of Chromohalobacter, but clear differences were found between these two strains and Chromohalobacter marismortui. On the basis of these data, it is proposed that Halomonas elongata DSM 3043 and ATCC 33174 should be included in a new species of the genus Chromohalobacter, Chromohalobacter salexigens sp. nov. The type strain is DSM 3043T (= ATCC BAA-138T = CECT 5384T = CCM4921T = CIP106854T = NCIMB 13768T).

Taxonomic analysis of extremely halophilic archaea isolated from 56-years-old dead sea brine samples.

A taxonomic study comprising both phenotypic and genotypic characterization, has been carried out on a total of 158 extremely halophilic aerobic archaeal strains. These strains were isolated from enrichments prepared from Dead Sea water samples dating from 1936 that were collected by B. E. Volcani for the demonstration of microbial life in the Dead Sea. The isolates were examined for 126 morphological, physiological, biochemical and nutritional tests. Numerical analysis of the data, by using the S(J) coefficient and UPGMA clustering method, showed that the isolates clustered into six phenons. Twenty-two out of the 158 strains used in this study were characterized previously (ARAHAL et al., 1996) and were placed into five phenotypic groups. The genotypic study included both the determination of the guanineplus-cytosine content of the DNA and DNA-DNA hybridization studies. For this purpose, representative strains from the six phenons were chosen. These groups were found to represent some members of three different genera - Haloarcula (phenons A, B, and C), Haloferax (phenons D and E) and Halobacterium (phenon F) - of the family Halobacteriaceae, some of them never reported to occur in the Dead Sea, such as Haloarcula hispanica, while Haloferax volcanii (phenons D and E) was described in the Dead Sea by studies carried out several decades later than Volcani's work.

Reclassification of Bacillus marismortui as Salibacillus marismortui comb. nov.

Recently, the features of a group of strains isolated from Dead Sea enrichments obtained in 1936 by one of us (B. E. Volcani) were described. They were gram-positive, moderately halophilic, spore-forming rods, and were placed in a new species, Bacillus marismortui. At the same time, the new genus Salibacillus was proposed for the halophilic species Bacillus salexigens. B. marismortui and Salibacillus salexigens have similar phenotypic characteristics and the same peptidoglycan type. Phylogenetic analysis based on 16S rRNA sequence comparisons showed that they are sufficiently closely related (96.6% similarity) as to warrant placement in the same genus. However, DNA-DNA hybridization experiments showed that they constitute two separate species (41% DNA similarity). Therefore the reclassification of Bacillus marismortui as Salibacillus marismortui comb. nov. is proposed.

Bacillus marismortui sp. nov., a new moderately halophilic species from the Dead Sea.

A group of 91 moderately halophilic, Gram-positive, rod-shaped strains were isolated from enrichments prepared from Dead Sea water samples collected 57 years ago. These strains were examined for 117 morphological, physiological, biochemical, nutritional and antibiotic susceptibility characteristics. All strains formed endospores and were motile, strictly aerobic and positive for catalase and oxidase. They grew in media containing 5-25% (w/v) total salts, showing optimal growth at 10% (w/v). Eighteen strains were chosen as representative isolates and were studied in more detail. All these strains had mesodiaminopimelic acid in the cell wall and a DNA G + C content of 39.0-42.8 mol%; they constitute a group with levels of DNA-DNA similarity of 70-100%. The sequences of the 16S rRNA genes of three representative strains (strains 123T, 557 and 832) were almost identical (99.9%), and placed the strains in the low G + C content Gram-positive bacteria. On the basis of their features, these isolates should be regarded as members of a new species of the genus Bacillus, for which the name Bacillus marismortui sp. nov. is proposed. The type strain is strain 123T (= DSM 12325T = ATCC 700626T = CIP 105609T = CECT 5066T).

Moderately halophilic gram-positive bacterial diversity in hypersaline environments.

Moderately halophilic bacteria are microorganisms that grow optimally in media containing 3%-15% (w/v) salt. They are represented by a heterogeneous group of microorganisms included in many different genera. Gram-negative moderately halophilic bacteria have been studied in more detail, but studies on gram-positive species are more scarce. Recent studies carried out by our research group on gram-positive moderate halophiles have permitted clarifying their taxonomic and phylogenetic position and describing new species. Thus, we have isolated six strains from ponds of salterns that show phenotypic and genotypic characteristics similar to those of Nesterenkonia halobia (formerly Micrococcus halobius), a moderately halophilic gram-positive coccus that was described on the basis of a single strain. Our data demonstrate quite clearly that they are members of this species and contribute to a better description of these moderately halophilic cocci. Similarly, a study of a large number of gram-positive moderately halophilic rods that were able to produce endospores led us to describe a new species, designated Bacillus salexigens. Further, isolates grouped in other three phenons, obtained by numerical taxonomy analysis and showing phenotypic features quite similar to those of this species, represent different genomovars, with very low DNA-DNA homology. Although they might represent additional new species, it will be necessary to determine new phenotypic features to differentiate them from previously described Bacillus species. We have also studied the viability of some old enrichments provided by B.E. Volcani, which were set up in 1936. We isolated 31 gram-positive motile endospore-forming rods that, according to their phenotypic characteristics, could represent a new species of the genus Bacillus.

Polyphasic taxonomy of Nesterenkonia halobia.

A phenotypic study has been carried out on six moderately halophilic gram-positive nonmotile cocci isolated from ponds of a saltern located in Huelva, Spain. These strains were examined for 150 morphological, physiological, biochemical, and nutritional traits and showed phenotypic characteristics similar to those of Nesterenkonia halobia (formerly Micrococcus halobius). The guanine-plus-cytosine (G + C) content of their DNA ranged between 70 and 72 mol%, values quite similar to those described for N. halobia (71.5 mol%). The 16S rDNA sequence analysis of one representative isolate showed that it is phylogenetically quite close to N. halobia, within the high-G + C-content gram-positive branch. DNA-DNA hybridization experiments showed a high degree of homology (72 to 100%) among the six isolates and the type strain N. halobia ATCC 21727. All data demonstrate quite clearly that the six isolates are members of the species N. halobia. Since this species was described on the basis of a single strain isolated from unrefined solar salt, and its description is not complete (especially in the utilization of different compounds), our study contributes to a better description of the moderate halophile N. halobia.

Bacillus salexigens sp. nov., a new moderately halophilic Bacillus species.

Bacillus salexigens sp. nov. is proposed based on the characteristics of six moderately halophilic, grampositive, rod-shaped strains isolated from salterns and hypersaline soils located in different geographical areas of Spain. These strains were motile, formed endospores, were strictly aerobic, were catalase and oxidase positive, and contained peptidoglycan of the meso-diamlnopimelic acid type in their vegetative cell walls. The DNA base compositions of these strains ranged from 36.3 to 39.5 mol%, and these organisms constitute a homology group with levels of DNA-DNA homology ranging from 73 to 100%. The 16S rRNA sequence of strain C-20MoT, which was used as the representative strain of these isolates, groups with the 16S rRNA sequences of members of the genus Bacillus, and the highest level of similarity is 95.4%. The type strain is strain C-20Mo (= ATCC 700290 = DSM 11483 = CCM 4646).

Phylogenetic analyses of some extremely halophilic archaea isolated from Dead Sea water, determined on the basis of their 16S rRNA sequences.

Twenty-two extremely halophilic aerobic archaeal strains were isolated from enrichments prepared from Dead Sea water samples collected 57 years ago. The isolates were phenotypically clustered into five different groups, and a representative from each group was chosen for further study. Almost the entire sequences of the 16S rRNA genes of these representatives, and of Haloarcula hispanica ATCC 33960, were determined to establish their phylogenetic positions. The sequences of these strains were compared to previously published sequences of 27 reference halophilic archaea (members of the family Halobacteriaceae) and two other archaea, Methanobacterium formicicum DSM 1312 and Methanospirillum hungatei DSM 864. Phylogenetic analysis using approximately 1,400 base comparisons of 16S rRNA-encoding gene sequences demonstrated that the five isolates clustered closely to species belonging to three different genera--Haloferax, Halobacterium, and Haloarcula. Strains E1 and E8 were closely related and identified as members of the species Haloferax volcanii, and strain E12 was closely related and identified as a member of the species Halobacterium salinarum. However, strains E2 and E11 clustered in the Haloarcula branch with Haloarcula hispanica as the closest relative at 98.9 and 98.8% similarity, respectively. Strains E2 and E11 could represent two new species of the genus Haloarcula. However, because strains of these two new species were isolated from a single source, they will not be named until additional strains are isolated from other sources and fully characterized.