M. Carmen Márquez

Moderately halophilic gram-positive bacterial diversity in hypersaline environments

Abstract 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.

Phylogenetic relationships within the family Halomonadaceae based on comparative 23S and 16S rRNA gene sequence analysis

A phylogenetic study of the family Halomonadaceae was carried out based on complete 16S rRNA and 23S rRNA gene sequences. Several 16S rRNA genes of type strains were resequenced, and 28 new sequences of the 23S rRNA gene were obtained. Currently, the family includes nine genera (Carnimonas, Chromohalobacter, Cobetia, Halomonas, Halotalea, Kushneria, Modicisalibacter, Salinicola and Zymobacter). These genera are phylogenetically coherent except Halomonas, which is polyphyletic. This genus comprises two clearly distinguished clusters: group 1 includes Halomonas elongata (the type species) and the species Halomonas eurihalina, H. caseinilytica, H. halmophila, H. sabkhae, H. almeriensis, H. halophila, H. salina, H. organivorans, H. koreensis, H. maura and H. nitroreducens. Group 2 comprises the species Halomonas aquamarina, H. meridiana, H. axialensis, H. magadiensis, H. hydrothermalis, H. alkaliphila, H. venusta, H. boliviensis, H. neptunia, H. variabilis, H. sulfidaeris, H. subterranea, H. janggokensis, H. gomseomensis, H. arcis and H. subglaciescola. Halomonas salaria forms a cluster with Chromohalobacter salarius and the recently described genus Salinicola, and their taxonomic affiliation requires further study. More than 20 Halomonas species are phylogenetically not within the core constituted by the Halomonas sensu stricto cluster (group 1) or group 2 and, since their positions on the different phylogenetic trees are not stable, they cannot be recognized as additional groups either. In general, there is excellent agreement between the phylogenies based on the two rRNA gene sequences, but the 23S rRNA gene showed higher resolution in the differentiation of species of the family Halomonadaceae.

Halophilic and Halotolerant Micro-Organisms from Soils

Hypersaline environments are extreme habitats in which several other factors, in addition to high salt content, may limit the growth of organisms. These additional factors include temperature, pH, pressure, oxygen, nutrient availability, and solar radiations (Rodriguez-Valera 1988). Hypersaline environments comprise hypersaline waters and soils . Hypersaline waters are defined as those environments that have higher concentrations of salts than seawater (Rodriguez-Valera 1988). However, depending on their origin, the salt composition may differ from that of seawater and on that basis hypersaline water habitats are categorized as thalassohaline , when the relative amounts of the different inorganic salts are approximately equal to those present in seawater, or as athalassohaline , if the proportions of the different salts are markedly different from those of seawater. The later environments are more heterogeneous and may have very different origins. Examples of thalassohaline water habitats, which are typically chroride types, are the Great Salt Lake or the solar salterns used for the industrial production of marine salt by evaporation of seawater; among the athalassohaline waters are the Dead Sea, the Wadi Natrun, Lake Magadi, and several other soda lakes. In contrast to the hypersaline waters, the hypersaline soils are not well defined and in fact there is no clear definition of a saline or hypersaline soil. They are widely represented in our planet. Because most soils contain small amounts of soluble salts, a soil would be considered as hypersaline when its salt concentration is above a certain threshold (Rodriguez-Valera 1988). According to Kaurichev (1980), soils containing more than 0.2% (w/v) soluble salt should be considered as saline soils. Micro-organisms show quite different responses to salt . According to the particular salt concentration required for their optimal growth, several physiological groups of micro-organisms are considered : (i) nonhalophiles require less than 1%

Taxonomic study of the genus Salinicola: transfer of Halomonas salaria and Chromohalobacter salarius to the genus Salinicola as Salinicola salarius comb. nov. and Salinicola halophilus nom. nov., respectively

We have carried out a polyphasic taxonomic characterization of the type strains of the species with the recently validated name Salinicola socius, together with two species that were phylogenetically closely related, Halomonas salaria and Chromohalobacter salarius. 16S rRNA gene sequence analyses showed that they constituted a coherent cluster, with sequence similarities between 98.7 and 97.7 %. We have determined the almost complete 23S rRNA gene sequences of these three type strains, and the percentage of similarity between them was 99.2-97.6 %. Phylogenetic trees based on the 16S rRNA and 23S rRNA gene sequences, obtained by using three different algorithms, were consistent and showed that these three species constituted a cluster separated from the other species of the genera of the family Halomonadaceae, supporting their placement in a single genus. All three species have ubiquinone 9 as the major respiratory quinone, and showed similar fatty acid and polar lipid profiles. The level of DNA-DNA hybridization between Salinicola socius DSM 19940(T), Halomonas salaria DSM 18044(T) and Chromohalobacter salarius CECT 5903(T) was 41-21 %, indicating that they are different species of the genus Salinicola. A comparative phenotypic study of these strains following the proposed minimal standards for describing new taxa of the family Halomonadaceae has been carried out. The phenotypic data are consistent with the placement of these three species in a single genus and support their differentiation at the species level. On the basis of these data we have emended the description of the species Salinicola socius and we propose to transfer the species Halomonas salaria and Chromohalobacter salarius to the genus Salinicola, as Salinicola salarius comb. nov. (type strain M27(T) =KCTC 12664(T) =DSM 18044(T)) and Salinicola halophilus nom. nov. (type strain CG4.1(T) =CECT 5903(T) =LMG 23626(T)), respectively.

Bacillus locisalis sp. nov., a new haloalkaliphilic species from hypersaline and alkaline lakes of China, Kenya and Tanzania.

A polyphasic taxonomic study was performed on seven Bacillus-like bacteria isolated from three hypersaline and alkaline lakes located in China, Kenya and Tanzania. All strains were moderately halophilic and alkaliphilic, Gram positive, motile rods. The DNA G+C content from the seven isolates ranged from 42.2 to 43.4mol% and their major fatty acid was anteiso-C(15:0). Strain CG1(T), selected as representative strain of the isolates, possesses meso-diaminopimelic acid in the cell wall peptidoglycan, MK-7 as the predominant menaquinone and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine as the major polar lipids. Comparative 16S rRNA gene sequence analysis indicated that the isolates belonged to the genus Bacillus. The seven isolates shared 97.7-99.9% 16S rRNA gene sequence similarity, and formed a branch that was distinct from the type strains of the recognized species of the genus Bacillus. They were most closely related to Bacillus agaradhaerens DSM 8721(T) (92.6-93.8% 16S rRNA sequence similarity). DNA-DNA hybridization values between the seven isolates were 85-100%. According to the polyphasic characterization, the strains represent a novel species, for which the name Bacillus locisalis sp. nov. is proposed. The type strain is CG1(T) (CCM 7370(T)=CECT 7152(T)=CGMCC 1.6286(T)=DSM 18085(T)).

Halophilic and Haloalkaliphilic, Aerobic Endospore-forming Bacteria in Soil

This chapter reviews the aerobic, endospore-forming moderately halophilic and haloalkaliphilic (and some halotolerant) bacteria that have been reported to be isolated from saline soils or sediment samples. These species belong to the family Bacillaceae, within the phylum Firmicutes, and are included in the following genera: Alkalibacillus, Bacillus, Filobacillus, Gracilibacillus, Halalkalibacillus, Halobacillus, Lentibacillus, Ornithinibacillus, Paraliobacillus, Salirhabdus, Salsuginibacillus, Tenuibacillus, Terribacillus, Thalassobacillus and Virgibacillus. Besides their taxonomic features, their ecological roles and biotechnological potential are reviewed.

Taxonomy of Halophilic Archaea and Bacteria

Microorganisms that inhabit hypersaline habitats are designated as halophiles and they are extremophilic organisms that must cope not only with the high ionic composition but also with other environmental factors such as alkaline pH values, low oxygen availability, high or low temperatures, presence of heavy metals and/or other toxic compounds, etc. They are normal inhabitants of natural saline environments such as saline lakes and soils, marine and inland salterns and several other hypersaline habitats. Besides, they are found in a variety of food products and several other saline derived goods. In this chapter we review the taxonomy of Halophilic Arcgaea abd Bacteria as well as the features and criteria which are used for taxonomic characterization. We emphasize those aspects that could be of interest for scientists for the correct characterization of these microorganisms.

The Hypersaline Lakes of Inner Mongolia: The MGAtech Project

Scientific cooperation between five institutions supported by the European Union and agencies in China and South Africa permitted the study of several Chinese extreme environments under the project MGATech during the period 2002–2006. This chapter summarizes the studies carried out in seven saline, and in some cases, also alkaline lakes in Inner Mongolia, China. These studies include the geographical description of the lakes and data concerning their chemical composition, as well as microbiological studies on six of the lakes focused on the description of the bacterial and archaeal diversity based on both culture-independent (molecular analysis techniques based on the direct isolation of DNA) and culture-dependent methods. Haloarchaeal viruses from one lake were also studied.

Halophilic Bacteria and Archaea as Producers of Lipolytic Enzymes

Among the extremophiles, halophiles constitute an interesting group of microorganisms as producers of lipolytic enzymes with potential biotechnological interest since these extremozymes are not only active in a wide range of salt concentrations but they are also tolerant to organic solvents and high temperature, offering new possibilities for different industrial processes operating at extreme conditions. Thus, although halophilic enzymes have not been studied in deep, if compared with their heat-stable counterparts, the unique characteristics exhibited by these enzymes have increased the interest in them and their biotechnological applications are likely to increase. In this chapter we review the diversity of halophilic microorganisms from saline and hypersaline environments producing lipolytic enzymes. We focus in the description of the most important characteristics of the lipolytic enzymes from halophilic bacteria and archaea, including a summary of the screening methods and the substrates used for detecting these enzymes. Moreover, we highlight the potential biotechnological applications of the characterized enzymes.