A New Family,Alteromonadaceae fam. nov., Including Marine Proteobacteria of the Genera Alteromonas,Pseudoalteromonas, Idiomarina,and Colwellia

The taxonomic positions of the marine genera Alteromonas, Pseudoalteromonas, Idiomarina, and Colwellia within the gamma subclass of the class Proteobacteria were specified on the basis of their phenotypic, genotypic, and phylogenetic characteristics. Gram-negative aerobic bacteria of the genera Alteromonas, Pseudoalteromonas, and Idiomarina and facultatively anaerobic bacteria of the genus Colwellia were found to form a phylogenetic cluster with a 16S rRNA sequence homology of 90% or higher. The characteristics of these genera presented in this paper allow their reliable taxonomic identification. Based on the analysis of our experimental data and analyses available in the literature, we propose to combine the genera Alteromonas, Pseudoalteromonas, Idiomarina, and Colwellia into a new family, Alteromonadaceae fam. nov., with the type genus Alteromonas.     

Diversity and association of psychrophilic bacteria in Antarctic sea ice.

The bacterial populations associated with sea ice sampled from Antarctic coastal areas were investigated by use of a phenotypic approach and a phylogenetic approach based on genes encoding 16S rRNA (16S rDNA). The diversity of bacteria associated with sea ice was also compared with the bacterial diversity of seawater underlying sea ice. Psychrophilic (optimal growth temperature, < or = 15 degrees C; no growth occurring at 20 degrees C) bacterial diversity was found to be significantly enriched in sea ice samples possessing platelet and bottom ice diatom assemblages, with 2 to 9 distinct (average, 5.6 +/- 1.8) psychrophilic taxa isolated per sample. Substantially fewer psychrophilic isolates were recovered from ice cores with a low or negligible population of ice diatoms or from under-ice seawater samples (less than one distinct taxon isolated per sample). In addition, psychrophilic taxa that were isolated from under-ice seawater samples were in general phylogenetically distinct from psychrophilic taxa isolated from sea ice cores. The taxonomic distributions of psychrotrophic bacterial isolates (optimal growth temperature, > 20 degrees C; growth can occur at approximately 4 degrees C) isolated from sea ice cores and under-ice seawater were quite similar. Overall, bacterial isolates from Antarctic sea ice were found to belong to four phylogenetic groups, the alpha and gamma subdivisions of the Proteobacteria, the gram-positive branch, and the Flexibacter-Bacteroides-Cytophaga phylum. Most of the sea ice strains examined appeared to be novel taxa based on phylogenetic comparisons, with 45% of the strains being psychrophilic. 16S rDNA sequence analysis revealed that psychrophilic strains belonged to the genera Colwellia, Shewanella, Marinobacter, Planococcus, and novel phylogenetic lineages adjacent to Colwellia and Alteromonas and within the Flexibacter-Bacteroides-Cytophaga phylum. Psychrotrophic strains were found to be members of the genera Pseudoalteromonas, Psychrobacter, Halomonas, Pseudomonas, Hyphomonas, Sphingomonas, Arthrobacter, Planococcus, and Halobacillus. From this survey, it is proposed that ice diatom assemblages provide niches conducive to the proliferation of a diverse array of psychrophilic bacterial species.     

Lipid composition of novel Shewanella species isolated from far Eastern seas

A comparative study of the lipid composition of 26 strains (including type strains) of marine Gammaproteobacteria belonging to the genera Shewanella, Alteromonas, Pseudoalteromonas, Marinobacterium, Microbulbifer, and Marinobacter was carried out. The bacteria exhibited genus-specific profiles of ubiquinones, phospholipids, and fatty acids, which can serve as reliable chemotaxonomic markers for tentative identification of new isolates. The studied species of the genus Shewanella were distinguished by the presence of two types of isoprenoid quinones, namely, ubiquinones Q-7 and Q-8 and menaquinones MK-7 and MMK-7; five phospholipids typical of this genus, namely, phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), lyso-PE, and acyl-PG; and the fatty acids 15:0, 16:0, 16:1 (n-7), 17:1 (n-8), i-13:0, and i-15:0. The high level of branched fatty acids (38-45%) and the presence of eicosapentaenoic acid (4%) may serve as criteria for the identification of this genus. Unlike Shewanella spp., bacteria of the other genera contained a single type of isoprenoid quinone: Q-8 (Alteromonas, Pseudoalteromonas, Marinobacterium, and Microbulbifer) or Q-9 (Marinobacter). The phospholipid compositions of these bacteria were restricted to three components: two major phospholipids (PE and PG) and a minor phospholipid, bisphosphatidic acid (Alteromonas and Pseudoalteromonas) or DPG (Marinobacterium, Microbulbifer, and Marinobacter). The bacteria exhibited genus-specific profiles of fatty acids.     

Cultivation of particle-associated heterotrophic bacteria during a spring phytoplankton bloom in the North Sea

Seawater contains free-living and particle-attached bacteria. Only a small fraction is cultivable on plates. As free-living and particle-associated bacteria differ in their physiological traits, their cultivability on plates may coincide with particle association. Using filtration and Imhoff sedimentation cones, particles were collected during a spring phytoplankton bloom off Helgoland (North Sea) in order to obtain particle-associated bacteria as inocula. Direct dilution plating resulted in 526 strains from 3 µm filtration retentates and 597 strains from settled particles. Motile Gammaproteobacteria from the genera Pseudoalteromonas, Shewanella, Psychrobacter, Vibrio and Colwellia, as well as particle-attached Flavobacteriia affiliating with the genera Tenacibaculum and Gramella, were frequently isolated. As a result, a diverse collection comprised of 266 strains was deposited. Two strains were most likely to represent novel genera and 78 strains were probably novel species. Recently, a high-throughput cultivation study from the same site using seawater as an inoculum had retrieved 271 operational phylogenetic units (OPUs) that represented 88% of the 4136 characterized strains at the species level. A comparison of 16S rRNA gene sequences revealed that the collection obtained matched 104 of the 271 seawater OPUs at the species level and an additional 113 at the genus level. This large overlap indicated a significant contribution of particle-associated bacteria to the cultivable microbiome from seawater. The presence of 49 genera not identified in the larger seawater study suggested that sample fractionation was an efficient strategy to cultivate rare members of the planktonic microbiome. The diverse collection of heterotrophic bacteria retrieved in this study will be a rich source for future studies on the biology of particle-associated bacteria.     

Comparative study of microbial communities from cultured and natural population of the mussel Mytilus trossulus in Peter the Great bay

The 525 strains of heterotrophic bacteria isolated from natural and cultured populations of the mussel Mytilus trossulus and the surrounding seawater were identified to a genus level on the basis of phenotypic analysis and the fatty acid composition of cell lipids. Gram-negative isolates were dominated by six genera of the family Enterobacteriaceae and by the genera Pseudoalteromonas, Vibrio, Photobacterium, Cytophaga/Flavobacterium/Bacteroides, Pseudomonas, and Moraxella, Gram-positive isolates were mainly represented by the genus Streptomyces. The taxonomic compositions of natural and cultured populations of the mussel M. trossulus in Peter the Great Bay were similar.     

Are readily culturable bacteria in coastal North Sea waters suppressed by selective grazing mortality?

We studied the growth of six culturable bacterial lineages from coastal North Sea picoplankton in environmental samples under different incubation conditions. The grazing pressure of heterotrophic nanoflagellates (HNF) was reduced either by double prefiltration through 0.8- micro m-pore-size filters or by 10-fold dilutions with 0.2- micro m (pore-size) prefiltered seawater. We hypothesized that those gamma-proteobacterial genera that are rapidly enriched would also be most strongly affected by HNF regrowth. In the absence of HNF, the mean protein content per bacterial cell increased in both treatments compared to environmental samples, whereas the opposite trend was found in incubations of unaltered seawater. Significant responses to the experimental manipulations were observed in Alteromonas, Pseudoalteromonas, and Vibrio populations. No treatment-specific effects could be detected for members of the Roseobacter group, the Cytophaga latercula-C. marinoflava lineage, or the NOR5 clade. Statistical analysis confirmed a transient increase in the proportions of Alteromonas, Pseudoalteromonas, and Vibrio cells at reduced HNF densities only, followed by an overproportional decline during the phase of HNF regrowth. Cells from these genera were significantly larger than the community average in the dilution treatments, and changes in their relative abundances were negatively correlated with HNF densities. Our findings suggest that bacteria affiliated with frequently isolated genera such as Alteromonas, Pseudoalteromonas, and Vibrio might be rare in coastal North Sea picoplankton because their rapid growth response to changing environmental conditions is counterbalanced by a higher grazing mortality.     

一平浪盐矿古老岩盐沉积中可培养细菌的系统发育多样性研究

运用纯培养法和基于16S rRNA基因序列的系统发育分析对云南省一平浪盐矿古老岩盐沉积中可培养细菌的多样性进行了研究。用补充0.5~3.5mol/L NaCl的MBA和ISP2琼脂培养基从卤水、岩盐和盐土样品中分离到38株细菌,用细菌通用引物进行16S rRNA基因扩增和序列测定,用相关软件进行序列相似性搜索、比对和系统发育分析。结果表明,38个分离菌株可分为31个物种,属于4个大的系统发育类群(Proteobacteria,Bacteroidetes,Firmicutes,Actinobacteria)、17个科、24个属。多数菌株属于Proteobacteria门(18株,47.3%;Gamma-Proteobacteria,31.5%;Alpha-Proteobacteria,15.8%)和Firmicutes门(13株,34.2%)。这些分离菌株中,至少有3个菌株可能代表3个不同属的3个新物种:Y3、Y15和Y25分别代表Idiomarina属、Salinicoccus属和Saccharospirillum属的新物种;而菌株Y21有可能代表Staphylococcaceae科的一个新属。从以上结果可以看出,一平浪盐矿古老岩盐沉积中存在较为丰富的微生物物种多样性和系统发育多样性,并且潜藏着新的微生物资源。     

Lipid Composition of Novel Shewanella Species Isolated from Far Eastern Seas

A comparative study of the lipid composition of 26 strains (including type strains) of marine Gammaproteobacteria belonging to the genera Shewanella, Alteromonas, Pseudoalteromonas, Marinobacterium, Microbulbifer, and Marinobacter was carried out. The bacteria exhibited genus-specific profiles of ubiquinones, phospholipids, and fatty acids, which can serve as reliable chemotaxonomic markers for tentative identification of new isolates. The studied species of the genus Shewanella were distinguished by the presence of two types of isoprenoid quinones, namely, ubiquinones Q-7 and Q-8 and menaquinones MK-7 and MMK-7; five phospholipids typical of this genus, namely, phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), lyso-PE, and acyl-PG; and the fatty acids [15:0, 16:0, 16:1 (n-7), 17:1 (n-8), i-13:0, and i-15:0]. The high level of branched fatty acids (38–45%) and the presence of eicosapentaenoic acid (4%) may serve as criteria for the identification of this genus. Unlike Shewanella spp., bacteria of the other genera contained a single type of isoprenoid quinone: Q-8 (Alteromonas, Pseudoalteromonas, Marinobacterium, and Microbulbifer) or Q-9 (Marinobacter). The phospholipid compositions of these bacteria were restricted to three components: two major phospholipids (PE and PG) and a minor phospholipid, bisphosphatidic acid (Alteromonas and Pseudoalteromonas) or DPG (Marinobacterium, Microbulbifer, and Marinobacter). The bacteria exhibited genus-specific profiles of fatty acids.     

Comparative Study of Microbial Communities from Cultured and Natural Populations of the Mussel Mytilus trossulus in Peter the Great Bay

The 525 strains of heterotrophic bacteria isolated from natural and cultured populations of the mussel Mytilus trossulus and the surrounding seawater were identified to a genus level on the basis of phenotypic analysis and the fatty acid composition of cell lipids. Gram-negative isolates were dominated by six genera of the family Enterobacteriaceae and by the genera Pseudoalteromonas, Vibrio, Photobacterium, Cytophaga/Flavobacterium/Bacteroides, Pseudomonas, and Moraxella. Gram-positive isolates were mainly represented by the genus Streptomyces. The taxonomic compositions of natural and cultured populations of the mussel M. trossulus in Peter the Great Bay were similar.     

Polyamine distribution patterns within the families Aeromonadaceae,Vibrionaceae, Pasteurellaceae,and Halomonadaceae,and related genera of the gamma subclass of the Proteobacteria

Polyamines of the four families and the five related genera within the gamma subclass of the class Proteobacteria were analyzed by HPLC with the objective of developing a chemotaxonomic system. The production of putrescine, diaminopropane, cadaverine, and agmatine are not exactly correlated to the phylogenetic genospecies within 36 strains of the genus Aeromonas (the family Aeromonadaceae) lacking in triamines. The occurrence of norspermidine was limited but not ubiquitous within the family Vibrionaceae, including 20 strains of Vibrio, Listonella, Photobacterium, and Salinivibrio. Spermidine was not substituted for the absence of norspermidine in the family. Agmatine was detected only in Photobacterium. Salinivibrio and some strains of Vibrio were devoid of polyamines. Vibrio ("Moritella") marinus contained cadaverine. Within the family Pasteurellaceae, Haemophilus contained cadaverine only and Actinobacillus contained no polyamine. Halomonas, Chromohalobacter, and Zymobacter, belonging to the family Halomonadaceae, ubiquitously contained spermidine and sporadically cadaverine and agmatine. Shewanella contained putrescine and cadaverine; Alteromonas macleodii, putrescine, 2-hydroxyputrescine, cadaverine, 2-hydroxyspermidine, and spermidine; Pseudoalteromonas, putrescine, cadaverine, and spermidine; Marinobacter, spermidine; and Marinomonas, putrescine and spermidine. Their polyamine profiles serve as a chemotaxonomic marker within the gamma subclass.     

Effect of Bacteria Associated with the Green Alga Ulva reticulata on Marine Micro- and Macrofouling

The green alga Ulva reticulata (Forsskal) is often free from biofouling in Hong Kong waters. An early study indicated that bioactive substances from this alga inhibit settlement of the polychaete Hydroides elegans (Haswell). It is also predicted that epibiotic bacteria protect this alga from micro- and macrofouling. In this study, bacterial strains from the surface of U. reticulata were isolated and their inhibitive activities on micro- and macrofouling assayed. The strains were identified by 16S rRNA analysis as belonging to the genera Alteromonas , Pseudoalteromonas and Vibrio . There was no significant effect of these strains or their extracts (aqueous and ethanol) on the growth of five Vibrio strains isolated from natural biofilm. Two bacterial strains ( Alteromonas sp. and Vibrio sp. 3) were non-toxic to the benthic diatom Nitzschia paleacea (Grunow) while the other five strains caused a low level of mortality. No one bacterial strain was toxic to the larvae of H. elegans . Aqueous extract of one of the isolated bacterial species, i.e. Vibrio sp. 2, significantly ( p <0.00001) inhibited the settlement and metamorphosis of H. elegans larvae. The putative antifouling compounds have a molecular weight of >100 kD. On the other hand, biofilm of Pseudoalteromonas sp. 2 and aqueous extract of Vibrio sp. 2 suppressed the settlement of larvae induced by 3-isobutyl-1-methylxanthine (IBMX). Other epibiotic bacteria and their extracts had neither inhibitive nor inductive effects on larval settlement of H. elegans . The results indicate that the antifouling mechanism of U. reticulata may be dependent not only on materials from the macroalga itself but also on the epibiotic bacteria on the algal surface.     

深海细菌的分子鉴定分类

从东太平洋海底 5,30 0m深的沉积物中分离到一批嗜冷细菌 ,克隆了其中 1 3株细菌的1 6SrRNA基因 ,通过其序列的测定和比较 ,对这 1 3株细菌进行了分子鉴定 ,结果表明它们分属于副球菌 ,假单胞菌 ,盐单胞菌和假交替单胞菌 4个不同的菌属 ,同时进行了系统发育分析。     

南海孔石藻可培养细菌多样性及代谢产物四溴吡咯研究

钙化藻和微生物在造礁石珊瑚幼虫的附着变态过程中扮演着重要角色,对珊瑚礁生态系统的健康具有重要意义。对南海珊瑚礁区钙化藻的共附生细菌进行分离培养,一方面有利于发掘南海珊瑚礁的微生物资源,另一方面有利于进行微生物与珊瑚、钙化藻等珊瑚礁框架生物的相互作用研究。本研究分离获得孔石藻(Porolithon onkodes)表面共附生细菌,基于16S rRNA基因序列进行系统发育分析,并通过高效液相色谱和质谱对分离得到的假交替单胞菌属菌株的代谢产物进行比较分析。从孔石藻分离获得的369株细菌菌株分别隶属于变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes)的5纲12目22科47属的97个种级类群。在属级水平上,假交替单胞菌属(Pseudoalteromonas)的菌株数量最多,分属于7个种。在假交替单胞菌属的代谢产物分析中,发现3株在系统发育树中聚为一簇的假交替单胞菌的代谢产物中存在四溴吡咯,而其余没有。研究结果不仅表明了钙化藻中含有丰富的可培养微生物和潜在新物种资源,还首次发现四溴吡咯在南海珊瑚礁区特定类群菌株的代谢产物中的存在,为珊瑚幼虫附着变态的化学信号研究提供基础。     

Screening of marine bacteria for fucoidan hydrolases

Twenty-five strains of epiphytic marine bacteria isolated from the brown algae Fucus evanescens and Chorda filum and fifty-three bacteria isolated from the sea cucumber Apostichopus japonicus were screened for fucoidanases using fucoidans prepared from the brown algae F. evanescens, Laminaria cichorioides, and L. japonica. Eighteen bacterial epiphytes and thirty-eight bacterial isolates from the sea cucumber were found to contain fucoidanases, which were able to hydrolyze either all of the fucoidans studied or some of them. Bacteria of the genera Cytophaga and Alteromonas/Pseudoalteromonas exhibited the highest fucoidanase activities, which, however, did not exceed the activity of fucoidanases from the already known sources.     

Phylogenetic structure of the genera Flexibacter,Flexithrix, and Microscilla deduced from 16S rRNA sequence analysis

The 16S rDNA sequences of 40 strains of 17 species in the genus Flexibacter, 5 strains of 4 species in the genus Microscilla, and 1 strain of Flexithrix dorotheae, including all type strains of approved and validated species in these genera, were determined to reveal their phylogenetic relationships. The 16S rRNA sequence analysis demonstrated the extreme heterogeneity of the genera Flexibacter and Microscilla. The strains examined diverged into 24 distinct lines of descent (1 group included both flexibacteria and flexithrix, and 1 group included both flexibacteria and microscilla) that were remote from each other at the genus level or higher. Flexibacter strains were scattered across the cytophaga-flavobacteria-bacteroides phylum and divided into 20 phylogenetic groups, and the genus Microscilla was separated into 5 groups. Flexibacter flexilis, the type species of the genus Flexibacter, and Microscilla marina, the type species of the genus Microscilla, were isolated from other organisms in their respective genera. This means that each genus should be restricted to only the type species. Flexithrix dorotheae, the type species of the genus Flexithrix, clustered with Flexibacter aggregans. The heterogeneity was found not only within genera but also within species. Flexibacter aggregans, Flexibacter aurantiacus, Flexibacter flexilis, Flexibacter roseolus, Flexibacter tractuosus, and "Microscilla sericea" each contained phylogenetically distant strains. The taxonomic concept of the genera Flexibacter, Flexithrix, and Microscilla should be reorganized in accordance with the natural relationships revealed in this study.     

Rapid radiation and cryptic speciation in squat lobsters of the genus Munida (Crustacea, Decapoda) and related genera in the South West Pacific: molecular and morphological evidence

Squat lobsters (genus Munida and related genera) are among the most diverse taxa of western Pacific crustaceans, though several features of their biology and phylogenetic relationships are unknown. This paper reports an extensive phylogenetic analysis based on mitochondrial DNA sequences (cytochrome c oxidase subunit I and 16S rRNA) and the morphology of 72 species of 12 genera of western Pacific squat lobsters. Our phylogenetic reconstruction using molecular data supports the recent taxonomic splitting of the genus Munida into several genera. Excluding one species (M. callista), the monophyly of the genus Munida was supported by Bayesian analysis of the molecular data. Three moderately diverse genera (Onconida, Paramunida, and Raymunida) also appeared monophyletic, both according to morphological and molecular data, always with high support. However, other genera (Crosnierita and Agononida) seem to be para- or polyphyletic. Three new cryptic species were identified in the course of this study. It would appear that the evolution of this group was marked by rapid speciation and stasis, or certain constraints, in its morphological evolution.     

Morphological, physiological, and biochemical characteristics of some violet-pigmented bacteria isolated from seawater.

Sixteen violet-pigmented heterotrophic bacilli were isolated from Mediterranean coastal waters. Morphological and physiological studies showed that they have a number of characteristics specific to the genus Chromobacterium. However, the absence of catalase, the presence of oxidase, and, more especially, the low percentage of bases guanine and cytosine in their DNA exclude them from the genus. The specificity of some characters assigned to Chromobacterium can thus be discussed. Several features allow us to consider such bacteria as being related to the genus Alteromonas; their consistency within the 16 strains investigated leads us to consider them as a single species, for which the specific name Alteromonas luteo-violaceus (sp. nov.) is proposed.     

Biodiversity of polycyclic aromatic hydrocarbon-degrading bacteria from deep sea sediments of the Middle Atlantic Ridge

The bacteria involved in the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in deep sea subsurface environments are largely unknown. In order to reveal their biodiversity, sediments from 2.2 m under the bottom surface at a water depth of 3542 m were sampled on the Middle Atlantic Ridge with a gravity column sampler. The sediments were promptly enriched with either crude oil or a mixture of PAHs (naphthalene, phenanthrene and pyrene) as the sole carbon source, and further enriched with the PAH mixture mentioned above in the lab. The resulting consortia were named C2CO and C2PPN respectively. Their bacterial composition was analysed with plate cultivation, PCR-DGGE and 16S rDNA library analysis. On plates, isolates belonging to Pseudoalteromonas , Halomonas , Marinobacter , Thalassospira and Tistrella dominated the culturable populations. With PCR-DGGE, five major bands closely related to Cycloclasticus , Alteromonas , Thalassospira , Alcanivorax and Rhodospirillaceae were detected in consortium C2CO, while only one major band of Cycloclasticus was detected in consortium C2PPN. In addition, the dynamics of community structure in response to aromatic substrate alterations were examined. As a result, three ribotypes of Cycloclasticus were detected by 16S rDNA library analysis, one which played a key role in phenanthrene degradation; two Alteromonas bacteria dominated the naphthalene reselected consortium. Although bacteria of the two genera grew as the main members of the communities, none of them were isolated, probably owing to their poor cultivability. These results confirm that bacteria of Cycloclasticus are important obligate PAH degraders in marine environments, and coexist with other degrading bacteria that inhabit the deep subsurface sediment of the Atlantic. This supports the view that PAH accumulation and bioattenuation occur in remote areas consistently and continuously.     

Diversity analysis of diazotrophic bacteria associated with the roots of tea (Camellia sinensis (L.) O. Kuntze)

The diversity elucidation by amplified ribosomal DNA restriction analysis and 16S rDNA sequencing of 96 associative diazotrophs, isolated from the feeder roots of tea on enriched nitrogen-free semisolid media, revealed the predominance of Gram-positive over Gram-negative bacteria within the Kangra valley in Himachal Pradesh, India. The Gram-positive bacteria observed belong to two taxonomic groupings; Firmicutes, including the genera Bacillus and Paenibacillus; and Actinobacteria, represented by the genus Microbacterium. The Gram-negative bacteria included alpha-Proteobacteria genera Brevundimonas, Rhizobium, and Mesorhizobium; gamma-Proteobacteria genera Pseudomonas and Stenotrophomonas; and beta-Proteobacteria genera Azospira, Burkholderia, Delftia, Herbaspirillum and Ralstonia. The low level of similarity of two isolates, with the type strains Paenibacillus xinjiangensis and Mesorhizobium albiziae, suggests the possibility of raising species novum. The bacterial strains of different phylogenetic groups exhibited distinct carbon-source utilization patterns and fatty acid methyl ester profiles. The strains differed in their nitrogenase activities with relatively high activity seen in the Gramnegative strains exhibiting the highest similarity to Azospira oryzae, Delftia lacustris and Herbaspirillum huttiense.     

Numerical taxonomy of heavy metal-tolerant nonhalophilic bacteria isolated from hypersaline environments.

A total of 232 metal-tolerant bacterial strains were isolated from water and sediment samples collected in different hypersaline environments located in Cádiz, Huelva and Morón de la Frontera (Spain). They were isolated on a medium containing mercury, chromium, cadmium, copper or zinc. These halotolerant isolates were analyzed by numerical taxonomy techniques by using the simple matching (SSM) and Jaccard (SJ) coefficients; clustering was achieved using the unweighted pair group method with averages (UPGMA) algorithm. At the 81% and 83% similarity level, different numbers of phenons were obtained for Gram-negative and Gram-positive halotolerant microorganisms. Most of the 48 Gram-negative metal-tolerant strains studied were grouped into nine phenons, representing the genera Pseudoalteromonas, Alteromonas, Xanthomonas, Pseudomonas, Alcaligenes and Enterobacteria. The 72 Gram-positive metal-tolerant strains grouped into eight phenons, with only 15 strains left unassigned. Most of the isolates were assigned to the genus Bacillus (seven phenons), and one phenon comprised microorganisms with phenotypic characteristics similar to those of the genus Celullomonas.