Base sequence homology and renaturation studies of the deoxyribonucleic acid of extremely halophilic bacteria

The genetic relatedness among various strains of halophilic bacteria has been assessed by deoxyribonucleic acid-deoxyribonucleic acid (DNA-DNA) duplex formation and ribosomal ribonucleic acid (RNA) hybridization. All of the strains of extremely halophilic rods are closely related, and the extent of divergence of base sequence is similar for the major and minor DNA components. Parallel experiments with ribosomal RNA revealed a relationship between the extremely halophilic rods and cocci and a more distant relationship to moderate halophiles and to a photosynthetic extreme halophile. Renaturation studies of halophile DNA exclude the possibility that the satellite DNA represents multiple copies of a small episomal element. The kinetics of DNA renaturation show that the genome size of the extreme and moderate halophiles is similar to that of Escherichia coli.     

Characteristics of the heterotrophic bacterial populations in hypersaline environments of different salt concentrations

Solar salterns, based on a multi-pond system, give a discontinuous gradient of salt concentrations. The heterotrophic bacterial populations of ponds containing from 10% salt to saturation have been studied. Saltern samples were spread on agar plates containing different media for halophilic bacteria and one medium made with water of the pond plus nutrients. Replica plating was done to determine the salt range for growth of the colonies. We studied 150 strains to determine the salt spectra of growth, the morphology, and nutrient requirements. The following conclusions were reached: (a) In salt concentrations above 10% (total salts), most bacteria are halophilic and few are halotolerant; (b) the two types of halophilic bacteria, moderate and extreme, show different distributions; in these ponds a narrow overlap exists between 25% and 32% salts with moderate halophiles predominating below this interval and extreme halophiles above it; (c) the populations of moderate halophiles are highly heterogeneous, and the salt concentration of their habitat affects their taxonomic composition, salt range for growth, and nutrient requirements. The population composition of extreme halophiles is less affected by the salt concentrations at which these bacteria are found.     

Box-shaped halophilic bacteria

Three morphologically similar strains of halophilic, box-shaped procaryotes have been isolated from brines collected in the Sinai, Baja California (Mexico), and southern California (United States). Although the isolates in their morphology resemble Walsby's square bacteria, which are a dominant morphological type in the Red Sea and Baja California brines, they are probably not identical to them. The cells show the general characteristics of extreme halophiles and archaebacteria. They contain pigments similar to bacteriorhodopsin which apparently mediate light-driven ion translocation and photophosphorylation.     

Metagenomic cloning and characterization of Na+ transporters from Huamachi Salt Lake in China

Moderately halophilic bacteria are a kind of extreme environment microorganism that can tolerate moderate salt concentrations ranging from 0.5 M to 2.5 M. Here, via a metagenomic library screen, we identified four putative Na⁺ transporters, designated H7-Nha, H16-Mppe, H19-Cap and H35-Mrp, from moderately halophilic community in the hypersaline soil of Huamachi Salt Lake, China. Functional complementation observed in a Na⁺(Ca²⁺)/H⁺ antiporter-defective Escherichia coli mutant (KNabc) suggests that the four putative Na⁺ transporters could confer cells a capacity of Na⁺ resistance probably by enhancing Na⁺ or Ca²⁺ efflux, but not Li⁺ or K⁺ exchange. Blastp analysis of the deduced amino-acid sequences indicates that H7-Nha has 71% identity to the NhaG Na⁺/H⁺ antiporter of Bacillus subtilis, while H19-Cap shows 99% identity to Enterobacter cloacae Ca²⁺ antiporter. Interestingly, H16-Mppe shares 59% identity to the metallophosphoesterase of Bacillus cellulosilyticus and H35-Mrp shows 68% identity to multidrug resistance protein of Lysinibacillus sphaericus. This is the first report that predicts a potential role of metallophosphoesterase in Na⁺ resistance in halophilic bacteria. Furthermore, everted membrane vesicles prepared from E. coli cells harboring H7-Nha exhibit Na⁺/H⁺ antiporter activity, but not Li⁺ (K⁺)/H⁺ antiporter activity, confirming that H7-Nha supports Na⁺ resistance mainly via Na⁺/H⁺ antiport. Our report also demonstrates that metagenomic library screen is a convenient and effective way to explore more novel types of Na⁺ transporters.     

Microbial life at high salt concentrations: phylogenetic and metabolic diversity

Halophiles are found in all three domains of life. Within the Bacteria we know halophiles within the phyla Cyanobacteria, Proteobacteria, Firmicutes, Actinobacteria, Spirochaetes, and Bacteroidetes. Within the Archaea the most salt-requiring microorganisms are found in the class Halobacteria. Halobacterium and most of its relatives require over 100–150 g/l salt for growth and structural stability. Also within the order Methanococci we encounter halophilic species. Halophiles and non-halophilic relatives are often found together in the phylogenetic tree, and many genera, families and orders have representatives with greatly different salt requirement and tolerance. A few phylogenetically coherent groups consist of halophiles only: the order Halobacteriales, family Halobacteriaceae (Euryarchaeota) and the anaerobic fermentative bacteria of the order Halanaerobiales (Firmicutes). The family Halomonadaceae (Gammaproteobacteria) almost exclusively contains halophiles. Halophilic microorganisms use two strategies to balance their cytoplasm osmotically with their medium. The first involves accumulation of molar concentrations of KCl. This strategy requires adaptation of the intracellular enzymatic machinery, as proteins should maintain their proper conformation and activity at near-saturating salt concentrations. The proteome of such organisms is highly acidic, and most proteins denature when suspended in low salt. Such microorganisms generally cannot survive in low salt media. The second strategy is to exclude salt from the cytoplasm and to synthesize and/or accumulate organic 'compatible' solutes that do not interfere with enzymatic activity. Few adaptations of the cells' proteome are needed, and organisms using the 'organic-solutes-in strategy' often adapt to a surprisingly broad salt concentration range. Most halophilic Bacteria, but also the halophilic methanogenic Archaea use such organic solutes. A variety of such solutes are known, including glycine betaine, ectoine and other amino acid derivatives, sugars and sugar alcohols. The 'high-salt-in strategy' is not limited to the Halobacteriaceae. The Halanaerobiales (Firmicutes) also accumulate salt rather than organic solutes. A third, phylogenetically unrelated organism accumulates KCl: the red extremely halophilic Salinibacter (Bacteroidetes), recently isolated from saltern crystallizer brines. Analysis of its genome showed many points of resemblance with the Halobacteriaceae, probably resulting from extensive horizontal gene transfer. The case of Salinibacter shows that more unusual types of halophiles may be waiting to be discovered.     

Survival of extremely and moderately halophilic isolates of Tunisian solar salterns after UV-B or oxidative stress

Adaptation to a solar saltern environment requires mechanisms providing tolerance not only to salinity but also to UV radiation (UVR) and to reactive oxygen species (ROS). We cultivated prokaryote halophiles from two different salinity ponds: the concentrator M1 pond (240 g·L(-1) NaCl) and the crystallizer TS pond (380 g·L(-1) NaCl). We then estimated UV-B and hydrogen peroxide resistance according to the optimal salt concentration for growth of the isolates. We observed a higher biodiversity of bacterial isolates in M1 than in TS. All strains isolated from TS appeared to be extremely halophilic Archaea from the genus Halorubrum. Culturable strains isolated from M1 included extremely halophilic Archaea (genera Haloferax, Halobacterium, Haloterrigena, and Halorubrum) and moderately halophilic Bacteria (genera Halovibrio and Salicola). We also found that archaeal strains were more resistant than bacterial strains to exposure to ROS and UV-B. All organisms tested were more resistant to UV-B exposure at the optimum NaCl concentration for their growth, which is not always the case for H(2)O(2). Finally, if these results are extended to other prokaryotes present in a solar saltern, we could speculate that UVR has greater impact than ROS on the control of prokaryote biodiversity in a solar saltern.     

Biodiversity of poly-extremophilic Bacteria: Does combining the extremes of high salt, alkaline pH and elevated temperature approach a physico-chemical boundary for life?

Bacterial microorganisms that grow optimally at Na⁺ concentrations of 1.7 M, or the equivalent of 10% (w/v) NaCl, and greater are considered to be extreme halophiles. This review focuses on the correlation between the extent of alkaline pH and elevated temperature optima and the extent of salt tolerance of extremely halophilic eubacteria; the focus is on those with alkaline pH optima, above 8.5, and elevated temperature optima, above 50°C. If all three conditions are required for optimal growth, these microorganisms are termed "poly-extremophiles". However, only a very few extreme halophiles able to grow optimally under alkaline conditions as well as at elevated temperatures have been isolated so far. Therefore the question is: do the combined extreme growth conditions of the recently isolated poly-extremophiles, i.e., anaerobic halophilic alkalithermophiles, approach a physico-chemical boundary for life? These poly-extremophiles are of interest, as their adaptive mechanisms give insight into organisms' abilities to survive in environments which were previously considered prohibitive to life, as well as to possible properties of early evolutionary and extraterrestrial life forms.     

Isolation and Characterization of Moderately Halophilic Bacteria from Tunisian Solar Saltern

Bacterial screenings from solar saltern in Sfax (Tunisia) lead to the isolation of 40 moderately halophilic bacteria which were able to grow optimally in media with 5–15% of salt. These isolates were phylogenetically characterized using 16S rRNA gene sequencing. Two groups were identified including 36 strains of Gamma-Proteobacteria (90%) and 4 strains of Firmicutes (10%). The Gamma-Proteobacteria group consisted of several subgroups of the Halomonadaceae (52.5%), the Vibrionaceae (15%), the Alteromonadaceae (10%), the Idiomarinaceae (7.5%), and the Alcanivoracaceae (5%). Moreover, three novel species: 183ZD08, 191ZA02, and 191ZA09 were found, show <97% sequence similarity of the 16S rRNA sequences while compared to previously published cultivated species. Most of these strains (70%) were able to produce hydrolases: amylases, proteases, phosphatases, and DNAases. Over the isolates, 60% produced phosphatases, 15.0% proteases, 12.5% amylases and DNAases equally. This study showed that the solar saltern of Sfax is an optimal environment for halophilic bacterial growth, where diverse viable bacterial communities are available and may have many industrial applications.     

Temperature and pH optima of extremely halophilic archaea: a mini-review

Archaeal microorganisms that grow optimally at Na⁺ concentrations of 1.7 M, or the equivalent of 10% (w/v) NaCl, and greater are considered to be extreme halophiles. This review encompasses extremely halophilic archaea and their growth characteristics with respect to the correlation between the extent of alkaline pH and elevated temperature optima and the extent of salt tolerance. The focus is on poly-extremophiles, i.e., taxa growing optimally at a Na⁺ concentration at or above 1.7 M (approximately 10% w/v NaCl); alkaline pH, at or above 8.5; and elevated temperature optima, at or above 50°C. So far, only a very few extreme halophiles that are able to grow optimally under alkaline conditions as well as at elevated temperatures have been isolated. The distribution of extremely halophilic archaea growing optimally at 3.4 M Na⁺ (approximately 20% w/v NaCl) is bifurcated with respect to pH optima, either they are neutrophilic, with a pH_opt of approximately 7, or strongly alkaliphilic, with pH_opt at or above 8.5. Amongst these extreme halophiles which have elevated pH optima, only four taxa have an optimum temperature above 50°C: Haloarcula quadrata (52°C), Haloferax elongans (53°C), Haloferax mediterranei (51°C) and Natronolimnobius ‘aegyptiacus’ (55°C).     

Natronobiforma cellulositropha gen. nov., sp. nov., a novel haloalkaliphilic member of the family Natrialbaceae (class Halobacteria) from hypersaline alkaline lakes

Six strains of extremely halophilic and alkaliphilic euryarchaea were enriched and isolated in pure culture from surface brines and sediments of hypersaline alkaline lakes in various geographical locations with various forms of insoluble cellulose as growth substrate. The cells are mostly flat motile rods with a thin monolayer cell wall while growing on cellobiose. In contrast, the cells growing with cellulose are mostly nonmotile cocci covered with a thick external EPS layer. The isolates, designated AArcel, are obligate aerobic heterotrophs with a narrow substrate spectrum. All strains can use insoluble celluloses, cellobiose, a few soluble glucans and xylan as their carbon and energy source. They are extreme halophiles, growing within the range from 2.5 to 4.8 M total Na⁺ (optimum at 4 M) and obligate alkaliphiles, with the pH range for growth from 7.5 to 9.9 (optimum at 8.5–9). The core archaeal lipids of strain AArcel5^T were dominated by C₂₀–C₂₀ dialkyl glycerol ether (DGE) (i.e. archaeol) and C₂₀–C₂₅ DGE in nearly equal proportion. The 16S rRNA gene analysis indicated that all six isolates belong to a single genomic species mostly related to the genera Saliphagus-Natribaculum-Halovarius. Taking together a substantial phenotypic difference of the new isolates from the closest relatives and the phylogenetic distance, it is concluded that the AArcel group represents a novel genus-level branch within the family Natrialbaceae for which the name Natronobiforma cellulositropha gen. nov., sp. nov. is proposed with AArcel5^T as the type strain (JCM 31939^T = UNIQEM U972^T).     

Halocins: are they involved in the competition between halobacteria in saltern ponds?

Many representatives of the family Halobacteriaceae ("halobacteria") excrete halophilic bacteriocins (halocins) that inhibit the growth of other halobacteria. In spite of the fact that halocin production is widespread among the Halobacteriaceae, no information is available on their ecological significance. To test whether halocins may play a role in the interspecies competition between dif-ferent types of halobacteria in saltern crystallizer ponds inhabited by dense communities of these red halophiles, we assayed for halocins active against a variety of halobacteria in salterns from different locations worldwide. Detection of halocin activity was based on the inhibition of growth of indicator organisms on agar plates, the decreased incorporation of radiolabeled substrates, and microscopic examinations. No halocin activity was detected in any of the brines examined, in spite of the fact that halocin production was demonstrated in cultures of most microorganisms isolated from these brines. Thus, the contribution of halocins in the competition between different halobacteria in hypersaline aquatic environments is probably negligible. Received: July 29, 1999 / Accepted: October 18, 1999     

The use of protein synthesis inhibitors in the estimation of the contribution of halophilic archaebacteria to bacterial activity in hypersaline environments

Abstract Antibiotics affecting protein synthesis were used to differentiate between the activity of different groups of organisms (halophilic archaebacteria, eubacteria and eukaryotes) in water samples from hypersaline ecosystems. Anisomycin (inhibiting both archaebacterial halophiles and eukaryotes) can be used to quantitate the contribution of the archaebacterial halophiles to amino acid incorporation by the microbial community, when cycloheximide (inhibiting eukaryotic protein synthesis, but not affecting halobacteria) is used as a control. Both in saltern ponds at salinities above 300 g/1 and in Dead Sea surface water more than 95% of the amino acid incorporation activity was abolished by anisomycin, but not by cycloheximide. Inhibition by anisomycin was well correlated with inhibition by low concentrations of bile salts, which specifically affect bacteria of the Halobacterium group. Chloramphenicol (an inhibitor of eubacterial protein synthesis) quantitatively inhibited amino acid incorporation in saltern brines of relatively low salinity, but also caused significant (28–42%) inhibition at high salinities. Erythromycin was also found valuable in the estimation of activities of the different bacterial groups.     

Osmoadaptation in Representatives of Haloalkaliphilic Bacteria from Soda Lakes

The adaptation of microorganisms to life in brines allows two strategies: the accumulation of organic osmoregulators in the cell (as in many moderate halophiles, halomonads in particular) or the accumulation of inorganic ions at extremely high intracellular concentrations (as, for example, in haloanaerobes). To reveal the regularities of osmoregulation in haloalkaliphiles developing in soda lakes, Halomonas campisalis Z-7398-2 and Halomonas sp. AIR-2 were chosen as representatives of halomonads, and Natroniella acetigena, as a representative of haloanaerobes. It was established that, in alkaliphilic halomonads, the intracellular concentrations of inorganic ions are insufficient for counterbalancing the environmental osmotic pressure and balance is attained due to the accumulation of organic osmoregulators, such as ectoine and betaine. On the contrary, the alkaliphilic haloanaerobe N. acetigena employs K⁺, Na⁺, and Cl^? ions for osmoregulation. High intracellular salt concentrations increasing with the content of Na⁺ in the medium were revealed in this organism. At a concentration of 1.91 M Na⁺ in the medium, N. acetigena accumulated 0.83 M K⁺, 0.91 M Na⁺, and 0.29 M Cl^? in cells, and, with an increase in the Na⁺ content in the medium to 2.59 M, it accumulated 0.94 M K⁺, 1.98 M Na⁺, and 0.89 M Cl^?, which counterbalanced the external osmotic pressure and provided for cell turgor. Thus, it was shown that alkaliphilic microorganisms use osmoregulation strategies similar to those of halophiles and these mechanisms are independent of the mechanism of pH homeostasis.     

Perchlorate and halophilic prokaryotes: implications for possible halophilic life on Mars

In view of the finding of perchlorate among the salts detected by the Phoenix Lander on Mars, we investigated the relationships of halophilic heterotrophic microorganisms (archaea of the family Halobacteriaceae and the bacterium Halomonas elongata) toward perchlorate. All strains tested grew well in NaCl-based media containing 0.4 M perchlorate, but at the highest perchlorate concentrations, tested cells were swollen or distorted. Some species (Haloferax mediterranei, Haloferax denitrificans, Haloferax gibbonsii, Haloarcula marismortui, Haloarcula vallismortis) could use perchlorate as an electron acceptor for anaerobic growth. Although perchlorate is highly oxidizing, its presence at a concentration of 0.2 M for up to 2 weeks did not negatively affect the ability of a yeast extract-based medium to support growth of the archaeon Halobacterium salinarum. These findings show that presence of perchlorate among the salts on Mars does not preclude the possibility of halophilic life. If indeed the liquid brines that may exist on Mars are inhabited by salt-requiring or salt-tolerant microorganisms similar to the halophiles on Earth, presence of perchlorate may even be stimulatory when it can serve as an electron acceptor for respiratory activity in the anaerobic Martian environment.     

江苏盐城盐场中度嗜盐菌的分离与鉴定

采用高盐的牛肉膏蛋白胨培养基(盐浓度为8%NaCl),研究江苏省盐城市盐场土壤里中度嗜盐菌的分布情况及种群特征。从盐城市的射阳、新滩、灌东三处盐场土壤中共采集和分离得到13株中度嗜盐菌。通过形态观察、生理生化分析、16S rRNA序列分析和系统进化分析等方法进行初步鉴定,结果表明:分离到的中度嗜盐菌分属3个属,Virgibacillus属4株、Halomonas属7株和Marinobacter属2株。研究结果揭示盐城市的盐场存在较为丰富的中度嗜盐菌,具有较高的研究和利用价值。     

Sambhar Salt Lake

The saline and alkaline brines from the Sambhar Salt Lake (SSL), both from the main lake and from the solar evaporation pans at Sambhar Salt Limited, Sambhar, Rajasthan, India, were studied with respect to their chemical composition and presence of red, extremely haloalkaliphilic archaebacteria. The brines had pH values of 9.5±0.2 and a total salt content ranging from 7% (w/v) to more than 30% (w/v). Sodium chloride, sodium carbonate, sodium bicarbonate and sodium sulphate were the principal salts present in these brines which lacked divalent cations (calcium and magnesium). Six strains of red, extremely haloalkaliphilic bacteria, designated SSL 1 to SSL 6, were isolated. All the isolates showed obligate requirements for sodium chloride (>15%, w/v) and high pH (>9.0). Magnesium ions were required in traces for maintaining morphological structure and pigmentation. All these strains possessed the diether core lipids, phosphatidylglycerol (PG), phosphatidylglycerophosphate (PGP), and bacterioruberins characteristic of halophilic archaebacteria. The strains were assigned to the newly proposed genus Natronobacterium.Part of the paper was presented by the authors at XIV International Congress of Microbiology 7–13 September 1986, Manchester, UK     

Diversity of Heterotrophic Halophilic Bacteria Isolated from Coastal Solar Salterns,Bulgaria and Their Ability to Synthesize Bioactive Molecules with Biotechnological Impact

Investigations on the microbial life in several coastal solar salterns have revealed the presence of novel organisms and synthesis of unusual molecules active in extreme conditions which might be useful in different biotechnological industries. Biodiversity of heterotrophic aerobic bacteria isolated from two salterns, Pomorie salterns and Burgas salterns located at Burgas Bay, Black Sea coast, Bulgaria, as well as ability of the isolates to synthesize biotechnologically valuable compounds were investigated. The results revealed high taxonomic and metabolic bacterial diversity—we isolated 20 morphologically different moderately halophilic and two halotolerant strains affiliated with 11 species from eight genera referred to the phyla Proteobacteria, Firmicutes, and Actinobacteria. Gram-negative bacteria belonged to the genera Halomonas, Chromohalobacter, Salinivibrio, Cobetia, and Nesiotobacter, and gram-positive strains were representatives of the genera Virgibacillus, Salinicoccus, and Brevibacterium. All isolates were found to be alkalitolerant, and 41% of them were psychrotolerant. The strains degraded nine of the tested 18 substrates; polygalacturonase, catalase, phytase, and lipase producers were predominant. This is the first reported detection of xanthan lyase, gellan lyase, arabinase, and phytase activities in halophilic bacteria. Nine of the strains belonging to five different genera were found to produce exopolysaccharides (EPS). The highest level of EPS was observed in Chromohalobacter canadensis strain 28. More than a half of the strains displayed antimicrobial activity against one to five test bacteria and yeasts. The present study is the first report on halophilic bacteria isolated from salterns at the Black Sea coast indicating that the investigated area is an untapped resource of halophilic bacteria with biotechnological potential.     

Plasmids from Halomonas eurihalina, a microorganism which produces an exopolysaccharide of biotechnological interest

The moderately halophilic bacterium Halomonas eurihalina strain F2-7, able to produce an exopolysaccharide, was found to contain two plasmids named pVE1 and pVE2, of 8.1 and 5.8 kb respectively. We found no evidence for the involvement of these plasmids in the expression of the mucoid phenotype. Restriction maps of both plasmids were constructed. Southern hybridization revealed similarities between them but excluded the existence of sequences homologous to other plasmids isolated from the Halomonas species. Neither pVE1 nor pVE2 displayed any homology with other plasmids isolated from moderate halophiles. The occurrence of similar plasmids in other strains of Halomonas eurihalina, isolated from hypersaline soils, has been detected. These small plasmids may be useful for the development of cloning vectors for moderately halophilic bacteria.     

Interrelationships between <Emphasis Type="Italic">Dunaliella</Emphasis> and halophilic prokaryotes in saltern crystallizer ponds

Thanks to their often very high population densities and their simple community structure, saltern crystallizer ponds form ideal sites to study the behavior of halophilic microorganisms in their natural environment at saturating salt concentrations. The microbial community is dominated by square red halophilic Archaea, recently isolated and described as Haloquadratum walsbyi, extremely halophilic red rod-shaped Bacteria of the genus Salinibacter, and the unicellular green alga Dunaliella as the primary producer. We review here, the information available on the microbial community structure of the saltern crystallizer brines and the interrelationships between the main components of their biota. As Dunaliella produces massive amounts of glycerol to provide osmotic stabilization, glycerol is often postulated to be the most important source of organic carbon for the heterotrophic prokaryotes in hypersaline ecosystems. We assess here, the current evidence for the possible importance of glycerol and other carbon sources in the nutrition of the Archaea and the Bacteria, the relative contribution of halophilic Bacteria and Archaea to the heterotrophic activity in the brines, and other factors that determine the nature of the microbial communities that thrive in the salt-saturated brines of saltern crystallizer ponds. Three-letter abbreviations for names of genera of Halobacteriaceae conform the recommendations of the ICSP Subcommittee on the Taxonomy of Halobacteriaceae.     

Aminopeptidase Activity of Haloalkalophilic Bacteria of the Genus <Emphasis Type="Italic">Halomonas</Emphasis> Isolated from the Soda-Saline Lakes in the Badain Jaran Desert

Three strains of haloalkaliphilic bacteria were isolated from microbial mats of soda-saline lakes of the Badain Jaran Desert, Inner Mongolia (China). Based on the data on ribosomal phylogeny, they were identified as members of the genus Halomonas. These bacteria were moderate alkaliphiles and extreme halophiles. The peptidases secreted by these bacteria were shown to have narrow substrate specificity. They hydrolyzed proteins and para-nitroanilide substrates and showed maximal activity in the hydrolysis of L-leucyl-p-nitroanilides (LpNA). The maximum activity of the peptidases occurred at alkaline pH values (8–10) and elevated salinity (50–100 g/L); the enzymes were thermostable (up to 50°С). The results of inhibitor analysis and substrate specificity examination of extracellular enzymes indicated them to belong to the class of aminopeptidase-like metallopeptidases.