![Biosynthesis of ectoines in C. salexigens. (A) Proposed pathway for the synthesis of ectoine and hydroxyectoine. Only the biosynthetic route from aspartate semialdehyde to ectoine has been elucidated at the biochemical level in the closely related H. elongata [31,32]. The genes for the three enzymes involved in ectoine synthesis, DA acetyltransferase, L-DA transaminase, and ectoine synthase, are designated ectA, ectB, and ectC, respectively. The genes encoding the ectoine hydroxylase and the two enzymes of the proposed alternative pathway for hydroxyectoine synthesis have not been identified. The crossed lines indicate the steps blocked in the salt-sensitive mutants. (B) Genetic organization of the C. salexigens ectABC region shown below a restriction map of pDE9, a pVK102-derived cosmid clone from the wild-type gene bank. The thick line in pDE9 corresponds to the 2869-bp nucleotide sequence deposited in the EMBL Data Library (accession number AJ011103). The 650-bp BglII fragment containing part of ectB used as a probe is depicted. The region containing the transposon Tn1732 plus flanking DNA in mutant CHR64 is shown at the bottom. The sites of Tn1732 insertion in mutants CHR63 [10] and CHR64 (this work) are indicated by inverted lollipops. Restriction sites are as follows: A, ApaI; Bg, BglII; C, ClaI; E, EcoRI; H, HindIII; P, PstI; Pv, PvuI; S, SalI; X, XhoI.](https://www.researchgate.net/profile/Joaquin-Nieto-2/publication/7606932/figure/fig2/AS:634470122131456@1528280873530/Biosynthesis-of-ectoines-in-C-salexigens-A-Proposed-pathway-for-the-synthesis-of_Q320.jpg)
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Contribution of chemical changes in membrane lipids to the osmoadaptation of the halophilic bacterium Chromohalobacter salexigens
Abstract and Figures
The long-term response of the broad-salt growing halophile Chromohalobacter salexigens DSM 3043T to salt stress has been investigated with respect to adaptive changes in membrane lipid composition. This study included the wild-type and three salt-sensitive, ectoine-deficient strains: CHR62 (ectA::Tn1732, unable to grow above 0.75 M NaCl), CHR63 (ectC::Tn1732, unable to grow above 1.5 M NaCl), and CHR64, which was able to grow in minimal medium M63 up to 2.5 M NaCl, but its growth was slower than the wild-type strain at salinities above 1.5 M NaCl. This mutant accumulated ectoine and hydroxyectoine as major compatible solutes, but also the ectoine precursor, N-gamma-acetyldiaminobutyric acid, and was found to be affected in the ectoine synthase gene ectC. The main phospholipids of the wild-type strain were phosphatidylethanolamine, phosphatidylglycerol (PG), and cardiolipin (CL). Major fatty acids were detected as 16:0, 18:1, and 16:1, including significant amounts of cyc-19:0, and cyc-17:0. CL and cyclopropane fatty acids (CFA) levels were elevated when the wild-type strain was grown at high salinity (2.5 M NaCl). Membranes of the most salt-sensitive trains CHR62 and CHR63, but not of the less salt-sensitive strain CHR64, contained lower levels of CL. The proportion of cyc-19:0 in CHR64 was three-fold (at 2.0M NaCl) and 2.5-fold (at 2.5 M NaCl) lower than that of the wild type, suggesting that this mutant has a limited capacity to incorporate CFA into phospholipids at high salt. The addition of 1 mM ectoine to cultures of the wild-type strain increased the ratio PG/CL from 1.8 to 3.3 at 0.75 M NaCl, and from 1 to 6.5 at 2.5 M NaCl, and led to a slight decrease in CFA content. Addition of 1 mM ectoine to the mutants restored the steady-state levels of CL and CFA found in the wild-type strain supplemented with ectoine. These findings suggest that exogenous ectoine might attenuate the osmostress response involving changes in membrane lipids.
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... Trehalose is also a compatible solute that can be synthesized by C. salexigens as a protectant against salinity and temperature (Reina-Bueno et al., 2012). Another adaptation strategy used by C. salexigens DSM 3043 to deal with varying salt concentrations is the chemical change in membrane lipids and fatty acid composition, increasing the levels of cardiolipin and converting unsaturated fatty acids to saturated cyclopropane fatty acids when growing at high salinity (Vargas et al., 2005). A similar behavior has been observed in a C. israelensis strain where the degree of unsaturation decreased in the presence of higher salinities (Mutnuri et al., 2005). ...
... The complete genomes of the type strains of five species of this genus are not available, and thus, it is not possible to determine the phylogenomic relationship of all members of this genus by comparison of their genomes. The chemotaxonomic features (cellular fatty acids) of some species also support their coherent genus status (Mutnuri et al., 2005;Vargas et al., 2005;Peçonek et al., 2006;Sánchez-Porro et al., 2007). ...
Chro.mo.ha.lo.bac'ter. Gr. neut. n. chroma color; Gr. masc. n. hals halos the sea, salt; N.L. masc. n. bacter rod; N.L. masc. n. Chromohalobacter colored salt rod.
Proteobacteria / Gammaproteobacteria / Oceanospirillales / Halomonadaceae / Chromohalobacter
The genus Chromohalobacter is classified within the family Halomonadaceae and the order Oceanospirillales in the class Gammaproteobacteria. The cells are Gram‐stain‐negative, motile, and non‐endospore‐forming rods. Colonies are cream, yellow, white, brown, or black pigmented. Chemoorganotrophic. Strictly aerobic or facultatively anaerobic and catalase‐positive. Moderately halophilic. Optimal growth at 7.5–12.5% (w/v) NaCl, at pH 7.0–8.0 and 28–37°C. The predominant cellular fatty acids are C16:0, C19:0 cyclo ω8c, C18:1 ω7c, and C12:0 3‐OH. The predominant respiratory quinone is Q‐9. The DNA G + C content is 56.1–66.0 mol%. Currently, the genus includes eight species: Chromohalobacter marismortui (type species of the genus), Chromohalobacter beijerinckii, Chromohalobacter canadensis, Chromohalobacter israelensis, Chromohalobacter japonicus, Chromohalobacter nigrandesensis, Chromohalobacter salexigens, and Chromohalobacter sarecensis. The strains of these species were isolated from salt lakes, salterns, and other saline habitats or salted foods.
DNA G + C content (mol%): 56.1–66.0.
Type species: Chromohalobacter marismortui (ex Elazari‐Volcani 1940) Ventosa et al. 1989VP.
... A second, more accurate, strategy is to partially reformulate an experimentally-determined biomass composition by estimating the relative fraction of the biomass metabolites of the organism of interest that can be experimentally calculated. In this work, both biomass compositions were obtained by partially reformulating Escherichia coli biomass composition [25][26][27] by using previously published and experimental data from C. salexigens [7,24]. ...
... Appropriate amino acid molar ratio for both biomass reactions were calculated assuming that all proteins encoded by C. salexigens genome were expressed equally [26,28]. The proportion of total proteins in response to salinity was calculated based on previous results by our group [7] and the molar ratio of membrane phospholipids in response to salinity were previously determined by Vargas and co-workers [24]. The percentage of G+C in C. salexigens genome was experimentally estimated [1]. ...
Background
The halophilic bacterium Chromohalobacter salexigens is a natural producer of ectoines, compatible solutes with current and potential biotechnological applications. As production of ectoines is an osmoregulated process that draws away TCA intermediates, bacterial metabolism needs to be adapted to cope with salinity changes. To explore and use C. salexigens as cell factory for ectoine(s) production, a comprehensive knowledge at the systems level of its metabolism is essential. For this purpose, the construction of a robust and high-quality genome-based metabolic model of C. salexigens was approached. ResultsWe generated and validated a high quality genome-based C. salexigens metabolic model (iFP764). This comprised an exhaustive reconstruction process based on experimental information, analysis of genome sequence, manual re-annotation of metabolic genes, and in-depth refinement. The model included three compartments (periplasmic, cytoplasmic and external medium), and two salinity-specific biomass compositions, partially based on experimental results from C. salexigens. Using previous metabolic data as constraints, the metabolic model allowed us to simulate and analyse the metabolic osmoadaptation of C. salexigens under conditions for low and high production of ectoines. The iFP764 model was able to reproduce the major metabolic features of C. salexigens. Flux Balance Analysis (FBA) and Monte Carlo Random sampling analysis showed salinity-specific essential metabolic genes and different distribution of fluxes and variation in the patterns of correlation of reaction sets belonging to central C and N metabolism, in response to salinity. Some of them were related to bioenergetics or production of reducing equivalents, and probably related to demand for ectoines. Ectoines metabolic reactions were distributed according to its correlation in four modules. Interestingly, the four modules were independent both at low and high salinity conditions, as they did not correlate to each other, and they were not correlated with other subsystems. Conclusions
Our validated model is one of the most complete curated networks of halophilic bacteria. It is a powerful tool to simulate and explore C. salexigens metabolism at low and high salinity conditions, driving to low and high production of ectoines. In addition, it can be useful to optimize the metabolism of other halophilic bacteria for metabolite production.
... Proper fluidity of the membrane is essential for the mobility and functionality of embedded proteins and lipids, diffusion of proteins and other molecules across the membrane, and the appropriate separation of membranes during cell division. It has been reported that bacterial membrane composition changes during acid shock (Chang and Cronan, 1999;Kim et al., 2005), osmolarity fluctuations (Vargas et al., 2005), freeze-drying (Muñoz- Rojas et al., 2006), and exposure to suboptimal growth temperatures (Guerzoni et al., 2001). ...
Adhesion has been regarded as one of the basic features of probiotics. The aim of this study was to investigate the influence of acid stress on the functional properties, such as hydrophobicity, adhesion to HeLa cells, and composition of membrane fatty acids, of Lactobacillus probiotics strains. Two strains of Lacto-bacillus casei were used. Adhesion on polystyrene, hy-drophobicity, epithelial cells adhesion, and fatty acids analysis were evaluated. Our results showed that the membrane properties such as hydrophobicity and fatty acid composition of stressed strains were significantly changed with different pH values. However, we found that acid stress caused a change in the proportions of unsaturated and saturated fatty acid. The ratio of saturated fatty acid to unsaturated fatty acids observed in acid-stressed Lactobacillus casei cells was significantly higher than the ration in control cells. In addition, we observed a significant decrease in the adhesion ability of these strains to HeLa cells and to a polystyrene surface at low pH. The present finding could first add new insight about the acid stress adaptation and, thus, enable new strategies to be developed aimed at improving the industrial performance of this species under acid stress. Second, no relationship was observed between changes in membrane composition and fluidity induced by acid treatment and adhesion to biotic and abiotic surfaces. In fact, the decrease of cell surface hydrophobicity and the adhesion ability to abiotic surface and the increase of the capacity of adhesion to biotic surface demonstrate that adhesive characteristics will have little relevance in probiotic strain-screening procedures.
... The 15-methylpalmitic acid and 14-methylpalmitic acid are the iso-and ante-iso-branched fatty acids found in Grampositive bacteria (Oren 2012). From experiments on saltstress-effect on lipid composition of various haloalkaliphilic bacteria (Vargas et al. 2005b), it was found that the membrane fluidity between the environmental factors (temperature and pH) and cell response (phospholipids and fatty acids composition) play an important role. ...
The haloalkaliphilics are an important subset of extremophiles that grow in salt [upto 33% (wt/vol) NaCl] and alkaline pH (> 9). They are found in hypersaline environments especially in the brines in arid, coastal and deep sea locations, and in alkaline environments, such as soda soils, lakes and deserts. Some authors have described haloalkaliphilic bacteria as moderate halophilic bacteria, but the molecular and classical studies revealed that they belong to moderately to extremely halophilic bacteria and archaea. Organic solutes, such as glycine, betaine and other amino acid derivatives, sugars such as, sucrose and trehalose, and sugar alcohols present in the haloalkaliphilics help for their osmoadaptation, and also serve as stabilizers. Haloalkalphilics secrete exoenzymes like proteases, amylases, xylanases, cellulases and peroxidases which have potential industrial applications. They also produce bacteriorhodopsin, compatible solutes, pigments, biopolymers, secondary metabolites like biosurfactants, polyhydroxyalkanoate (PHA) and exopolysaccharides and antimicrobial/anticancer compounds. They have unique metabolic pathways which can be used to treat industrial pollutants, heavy metals and waste water.
... In fact, proper fluidity of the membrane is essential for the mobility and functionality of embedded proteins and lipids, diffusion of proteins and other molecules across the membrane and the appropriate separation of membranes during cell division. It has been reported that bacterial membrane composition changes during acid shock (Chang and Cronan 1999;Kim et al. 2005), osmolarity fluctuations (Vargas et al. 2005), freeze-drying (Muñoz- Rojas et al. 2006), and exposure to suboptimal growth temperatures (Guerzoni et al. 2001). ...
... The bacterial response to salinity might include morphological, physiological and biochemical changes, and induced defensive mechanisms (Rothschild & Mancinelli, 2001;DasSarma &Aora, 2002 andLe Borgne et al., 2008). Different microorganisms have the ability to adapt or tolerate the salinity stress by many ways such accumulating osmolytes (Zahran et al., 1992 andSagot et al., 2010), amino acid substitutions (Lanyi, 1974) and cell membrane lipids modification (Kaneda, 1991 andVargas et al., 2005). Especially, an important adaptive response of bacterial cells to salt stress is that the modifications in their fatty acids and lipids (Ventosa et al., 1998;Nicolaus et al., 2001;Kaye &Baross, 2004 andDe Carvalho et al., 2014). ...
Since salinity is one of the major abiotic stresses, bacteria totally have different adaptive or tolerant mechanisms that respond to salinity stress. Multiwavelength UV-Vis spectroscopy used to estimate the bacterial growth under different sodium chloride concentrations. Absorbance ratio of 280nm to 260nm (A280/A260) varied considerably according to the salt concentration.
This showed some metabolic activity changes as a part of the adaptive response that allows Halomonas alkaliphila to face salinity stress changes. Here, adaptive changes of fatty-acid composition of H. alkaliphila YHSA35 because of different sodium chloride concentrations were determined. In this work, fatty acids methyl esters (FAME) composition analysis was achieved and estimated the presence of thirty-four fatty acids in H. alikaliphila YHSA35 cells. Quantitatively changes were found within the level of saturated fatty acids; Caproic, Lauric, Undecanoic, Myristic, Palmitic, Heptadecanoic and in unsaturated fatty acids; Oleic, cis-11- Eicosenoic, Erucic. In high salt concentration, unsaturated fatty acids synthesis rate is reduced, resulting in an accumulation of palmitic acid. In conclusion, levels of saturated fatty acid profile changed in H. alkaliphila YHSA35 because of salinity stress that may modulate the membrane lipid viciousness for adaptation and best cellular perform.
... Another common approach to modify the cardiolipin content is to culture bacteria under high salt concentrations or to add exogeneous cardiolipin during the bacterial growth. Increase in cardiolipin content under conditions of high salinity has been largely reported both in Gram-positive bacteria including B. subtilis 61,62 and S. aureus 63 and in Gram-negative bacteria including R. sphaeroides 64 , C. salexigens 65 , and E. coli 66 . Unfortunately these approaches also show drawbacks since increase in NaCl concentrations could affect the behavior of free and/or bound diNn to membrane components favoring aggregation in solution and/or proximity of bound diNn in the membrane, respectively. ...
Some bacterial proteins involved in cell division and oxidative phosphorylation are tightly bound to cardiolipin. Cardiolipin is a non-bilayer anionic phospholipid found in bacterial inner membrane. It forms lipid microdomains located at the cell poles and division plane. Mechanisms by which microdomains are affected by membrane-acting antibiotics and the impact of these alterations on membrane properties and protein functions remain unclear. In this study, we demonstrated cardiolipin relocation and clustering as a result of exposure to a cardiolipin-acting amphiphilic aminoglycoside antibiotic, the 3′,6-dinonyl neamine. Changes in the biophysical properties of the bacterial membrane of P. aeruginosa, including decreased fluidity and increased permeability, were observed. Cardiolipin-interacting proteins and functions regulated by cardiolipin were impacted by the amphiphilic aminoglycoside as we demonstrated an inhibition of respiratory chain and changes in bacterial shape. The latter effect was characterized by the loss of bacterial rod shape through a decrease in length and increase in curvature. It resulted from the effect on MreB, a cardiolipin dependent cytoskeleton protein as well as a direct effect of 3′,6-dinonyl neamine on cardiolipin. These results shed light on how targeting cardiolipin microdomains may be of great interest for developing new antibacterial therapies.
... The phenomena that increasing the salt concentration in growth media may lead to a reduced inhibitory effect of some antibiotics to moderate halophiles had been reported previously Nieto et al. 1993), although the resistance mechanism was exactly unclear. There were some evidences indicated that several antibiotics could react with ions under high salinity, which may affect its permeability across the membrane (Piekarski et al. 2009), and the chemical changes in membrane lipids in response to the high salt concentration may alter the membrane permeability toward antibiotics (Ventosa and Nieto 2005). The resistance of C. salexigens ZW4-1 toward kanamycin will result in predominantly false positives clones following conjugation. ...
Chromohalobacter salexigens DSM 3043 can grow over a wide range of salinity, which makes it as an excellent model organism for understanding the mechanism of prokaryotic osmoregulation. Functional analysis of C. salexigens genes is an essential way to reveal their roles in cellular osmoregulation. However, the lack of an effective markerless gene deletion system has prevented construction of multiple gene deletion mutants for the members in the genus. Here, we report the development of a markerless gene deletion system in C. salexigens using allelic exchange method. In this system, the in vitro mutant allele of target gene was inserted into a pK18mobsacB-based integrative vector pMDC21, which contained a chloramphenicol resistance cassette as the positive selection marker and a sacB gene from Bacillus subtilis as the counterselectable marker. To validate this system, two single-gene deletion mutants and a double-gene deletion mutant were constructed. In addition, our results showed that growth of the merodiploids and sucrose screening at 25 °C were more effective to decrease the occurrence of spontaneous sucrose resistance colonies than at higher temperature (30 or 37 °C), and growth of the merodiploids in mineral salt medium instead of the complex medium was critical to increase the recovery rate of deletion mutants.
... In fact, proper fluidity of the membrane is essential for the mobility and functionality of embedded proteins and lipids, diffusion of proteins and other molecules across the membrane and the appropriate separation of membranes during cell division. It has been reported that bacterial membrane composition changes during acid shock (Chang and Cronan 1999;Kim et al. 2005), osmolarity fluctuations (Vargas et al. 2005), freeze-drying (Muñoz- Rojas et al. 2006), and exposure to suboptimal growth temperatures (Guerzoni et al. 2001). ...
Adhesion has been regarded as one of the basic features of probiotics. We undertake this study in the aim to give new insight about the change in cellular physiological state under heat and acid treatments of Lactobacillus plantarum. Different cell properties have been investigated such as adhesive ability to abiotic surfaces, the cell surface hydrophobicity and the fatty acids profiles. The results of cell surface properties and Gas chromatography analysis demonstrated a modification in term adhesive ability and fatty acid (FA) composition of the tested strain under stressful conditions. In fact, after the exposure of the strain to heat and acid treatments, an increase in the hydrophobicity level and the adhesion capacity on HeLa cells was shown. Our findings revealed that high temperature and low pH change the fatty acids profiles of the treated cells, especially the proportions of unsaturated and saturated fatty acid. In this context, our data revealed that the unsaturated FA-to-saturated FA ratio was increased significantly (P < 0.05) for stressed strains compared with control cells. The results of the present finding suggest that the tested strain have suffered changes like the modifications on bacterial membrane as a cellular response to survive the hard environmental conditions, allowing them to withstand harsh conditions and sudden environmental changes to survive under.
Hypersaline environments with salt concentrations up to NaCl saturation are inhabited by a great diversity of microorganisms belonging to the three domains of life. They all must cope with the low water activity of their environment, but different strategies exist to provide osmotic balance of the cells' cytoplasm with the salinity of the medium. One option used by many halophilic Archaea and a few representatives of the Bacteria is to accumulate salts, mainly KCl and to adapt the entire intracellular machinery to function in the presence of molar concentrations of salts. A more widespread option is the synthesis or accumulation of organic osmotic, so-called compatible solutes. Here we review the mechanisms of osmotic adaptation in a number of model organisms, including the KCl accumulating Halobacterium salinarum (Archaea) and Salinibacter ruber (Bacteria), Halomonas elongata as a representative of the Bacteria that synthesize organic osmotic solutes, eukaryotic microorganisms including the unicellular green alga Dunaliella salina and the black yeasts Hortaea werneckii and the basidiomycetous Wallemia ichthyophaga, which use glycerol and other compatible solutes. The strategies used by these model organisms and by additional halophilic microorganisms presented are then compared to obtain an integrative picture of the adaptations to life at high salt concentrations in the microbial world.
The moderately halophilic heterotrophic aerobic bacteria form a diverse group of microorganisms. The property of halophilism is widespread within the bacterial domain. Bacterial halophiles are abundant in environments such as salt lakes, saline soils, and salted food products. Most species keep their intracellular ionic concentrations at low levels while synthesizing or accumulating organic solutes to provide osmotic equilibrium of the cytoplasm with the surrounding medium. Complex mechanisms of adjustment of the intracellular environments and the properties of the cytoplasmic membrane enable rapid adaptation to changes in the salt concentration of the environment. Approaches to the study of genetic processes have recently been developed for several moderate halophiles, opening the way toward an understanding of haloadaptation at the molecular level. The new information obtained is also expected to contribute to the development of novel biotechnological uses for these organisms.
Strain CHR63 is a salt-sensitive mutant of the moderately halophilic wild-type strain Halomonas elongata DSM 3043 that is affected in the ectoine synthase gene (ectC). This strain accumulates large amounts of N gamma-acetyldiaminobutyrate (NADA), the precursor of ectoine (D. Canovas, C. Vargas, F. Iglesias-Guerra, L. N. Csonka, D. Rhodes, A. Ventosa, and J. J. Nieto, J. Biol. Chem. 272:25794-23801, 1997). Hydroxyectoine, ectoine, and glucosylglycerate were also identified by nuclear magnetic resonance (NMR) as cytoplasmic organic solutes in this mutant. Accumulation of NADA, hydroxyectoine, and ectoine was osmoregulated, whereas the levels of glucosylglycerate decreased at higher salinities. The effect of the growth stage on the accumulation of solutes was also investigated. NADA was purified from strain CHR63 and was shown to protect the thermolabile enzyme rabbit muscle lactate dehydrogenase against thermal inactivation. The stabilizing effect of NADA was greater than the stabilizing effect of ectoine or potassium diaminobutyrate. A H-1 NMR analysis of the solutes accumulated by the wild-type strain and mutants CHR62 (ectA::Tn1732) and CHR63 (ectC::Tn1732) indicated that H. elongata can synthesize hydroxyectoine by two different pathways-directly from ectoine or via an alternative pathway that converts NADA into hydroxyectoine without the involvement of ectoine.
The morphological, biochemical, and physiological characteristics of nine bacterial strains isolated from a solar salt facility located on Bonaire, Netherlands Antilles are described. The bacteria were gram-negative rods which produce white, opaque colonies on solid media. During the log phase of growth, the cultures consisted of single and paired cells with polar flagella predominating. Older cultures characteristically produced highly elongated, flexible rods. All of these strains reduced NO3 to NO2, grew anaerobically in the presence of NO3, and fermented glucose but oxidized sucrose, glycerol, mannose, and cellobiose. All strains were ornithine and lysine decarboxylase positive, catalase positive, and cytochrome oxidase negative. Eight of the nine strains grew in a complex Casamino Acids liquid medium containing from 0 to 32% (wt/vol) solar salt at temperatures from 23 to 37°C; the ninth strain was restricted in its growth to 0 to 20% solar salt. The guanine plus cytosine content of the deoxyribonucleic acid was 61 ± 1 mol%. This combination of morphology, salt tolerance, and guanine plus cytosine content supports the establishment of a new genus, Halomonas, in Family II (Vibrionaceae) of part 8, Gram-Negative Facultatively Anaerobic Bacteria, of Bergey's Manual (8th edition). The type species of this genus is H. elongata, the type strain of which is isolate 1H9 (= ATCC 33173). Strain 1H15 is regarded as belonging to a biovar of H. elongata on the basis of its production of lophotrichous cells and its inability to grow at 37°c in the presence of 32% solar salt.
The Halomonadaceae (Franzmann et al., 1988) presently contains representatives of two genera, the type genus Halomonas and at least one species of the genus Deleya (D. aesta) (see Chapter 168). The establishment of the family was suggested because Sab values obtained from 16S rRNA catalogs show that Halomonas and Deleya are phylogenetically isolated from all other major groups of the gamma subdivision of the Proteobacteria (Stackebrandt et al., 1988 Woese et al., 1985), forming an internally coherent cluster at an Sab of 0.60. Internally, the family contains two subgroups composed of the type species of Halomonas, H. elongata and Halomonas halmophilum (formerly Flavobacterium halmophilum). This cluster forms at an Sab of 0.66. The companion cluster contains H. subglaciescola and D. aesta and forms at an Sab of 0.67. The position of the other halomonads (Table 1) within the phylogeny is not presently known.