Microbial diversity and activity in Southern California salterns and bitterns: analogues for remnant ocean worlds

Concurrent osmotic and chaotropic stress make MgCl₂-rich brines extremely inhospitable environments. Understanding the limits of life in these brines is essential to the search for extraterrestrial life on contemporary and relict ocean worlds, like Mars, which could host similar environments. We sequenced environmental 16S rRNA genes and quantified microbial activity across a broad range of salinity and chaotropicity at a Mars-analogue salt harvesting facility in Southern California, where seawater is evaporated in a series of ponds ranging from kosmotropic NaCl brines to highly chaotropic MgCl₂ brines. Within NaCl brines, we observed a proliferation of specialized halophilic Euryarchaeota, which corresponded closely with the dominant taxa found in salterns around the world. These communities were characterized by very slow growth rates and high biomass accumulation. As salinity and chaotropicity increased, we found that the MgCl₂-rich brines eventually exceeded the limits of microbial activity. We found evidence that exogenous genetic material is preserved in these chaotropic brines, producing an unexpected increase in diversity in the presumably sterile MgCl₂-saturated brines. Because of their high potential for biomarker preservation, chaotropic brines could therefore serve as repositories of genetic biomarkers from nearby environments (both on Earth and beyond) making them prime targets for future life-detection missions.     

Denitrifying haloarchaea within the genus Haloferax display divergent respiratory phenotypes,with implications for their release of nitrogenous gases

Haloarchaea are extremophiles, generally thriving at high temperatures and salt concentrations, thus, with limited access to oxygen. As a strategy to maintain a respiratory metabolism, many halophilic archaea are capable of denitrification. Among them are members of the genus Haloferax, which are abundant in saline/hypersaline environments. Three reported haloarchaeal denitrifiers, Haloferax mediterranei, Haloferax denitrificans and Haloferax volcanii, were characterized with respect to their denitrification phenotype. A semi-automatic incubation system was used to monitor the depletion of electron acceptors and accumulation of gaseous intermediates in batch cultures under a range of conditions. Out of the species tested, only H. mediterranei was able to consistently reduce all available N-oxyanions to N₂, while the other two released significant amounts of NO and N₂O, which affect tropospheric and stratospheric chemistries respectively. The prevalence and magnitude of hypersaline ecosystems are on the rise due to climate change and anthropogenic activity. Thus, the biology of halophilic denitrifiers is inherently interesting, due to their contribution to the global nitrogen cycle, and potential application in bioremediation. This work is the first detailed physiological study of denitrification in haloarchaea, and as such a seed for our understanding of the drivers of nitrogen turnover in hypersaline systems.     

NO3 −/NO2 − assimilation in halophilic archaea: physiological analysis, nasA and nasD expressions

The haloarchaeon Haloferax mediterranei is able to assimilate nitrate or nitrite using the assimilatory nitrate pathway. An assimilatory nitrate reductase (Nas) and an assimilatory nitrite reductase (NiR) catalyze the first and second reactions, respectively. The genes involved in this process are transcribed as two messengers, one polycistronic (nasABC; nasA encodes Nas) and one monocistronic (nasD; codes for NiR). Here we report the Hfx mediterranei growth as well as the Nas and NiR activities in presence of high nitrate, nitrite and salt concentrations, using different approaches such as physiological experiments and enzymatic activities assays. The nasA and nasD expression profiles are also analysed by real-time quantitative PCR. The results presented reveal that the assimilatory nitrate/nitrite pathway in Hfx mediterranei takes place even if the salt concentration is higher than those usually present in the environments where this microorganism inhabits. This haloarchaeon grows in presence of 2 M nitrate or 50 mM nitrite, which are the highest nitrate and nitrite concentrations described from a prokaryotic microorganism. Therefore, it could be attractive for bioremediation applications in sewage plants where high salt, nitrate and nitrite concentrations are detected in wastewaters and brines.     

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.     

Road salt and organic additives affect mosquito growth and survival: an emerging problem in wetlands

The global increase in the application rate of road salts such as sodium chloride (NaCl) has led to concern about their negative effects on roadside habitats and freshwater ecosystems. To reduce the application rate of NaCl and minimize the ecological effects of road salts, transportation agencies are continuously seeking alternative salts such as magnesium chloride (MgCl₂) and organic additives such as beet juice and distillation byproducts. Yet, there is remarkably little information about how these road salt alternatives and additives affect aquatic communities, including their effects on mosquito populations. Nonetheless, understanding how anthropogenic factors such as road salts and salt additives affect mosquito populations could help minimize threats to human health, especially in urban environments. We used outdoor, freshwater mesocosms to experimentally investigate how the road salt MgCl₂ and two organic additives affect mosquito Culex restuans survival and emergence. Additionally, we measured changes to abiotic aspects of the environment that could affect mosquito larvae during development. We found that increased concentrations of MgCl₂ reduced mosquito survival while organic additives increased food resources that, in turn, reduced the average time to emergence for mosquitoes. Additionally, the organic additives reduced dissolved oxygen (DO) to hypoxic levels, which might negatively affect mosquito predators such as fish. In the absence of toxic concentrations of MgCl₂ or other salts, reduced predation coupled with the faster emergence times, means that organic additives, might increase mosquito population size. More comprehensive studies of multi‐trophic interactions in freshwater ecosystems should be conducted before agencies promote the application of alternative road salts and road salt additives.     

Nitrogen metabolism in haloarchaea

The nitrogen cycle (N-cycle), principally supported by prokaryotes, involves different redox reactions mainly focused on assimilatory purposes or respiratory processes for energy conservation. As the N-cycle has important environmental implications, this biogeochemical cycle has become a major research topic during the last few years. However, although N-cycle metabolic pathways have been studied extensively in Bacteria or Eukarya, relatively little is known in the Archaea. Halophilic Archaea are the predominant microorganisms in hot and hypersaline environments such as salted lakes, hot springs or salted ponds. Consequently, the denitrifying haloarchaea that sustain the nitrogen cycle under these conditions have emerged as an important target for research aimed at understanding microbial life in these extreme environments. The haloarchaeon Haloferax mediterranei was isolated 20 years ago from Santa Pola salted ponds (Alicante, Spain). It was described as a denitrifier and it is also able to grow using NO₃ ^-, NO₂ ^- or NH₄ ⁺ as inorganic nitrogen sources. This review summarizes the advances that have been made in understanding the N-cycle in halophilic archaea using Hfx mediterranei as a haloarchaeal model. The results obtained show that this microorganism could be very attractive for bioremediation applications in those areas where high salt, nitrate and nitrite concentrations are found in ground waters and soils.     

A CD study of the role of metal ions in the conformation of poly(L-lysine)

The effect of LiCl, NaCl, and CsCl as univalent salts, and of CaCl₂, ZnCl₂, and MgCl₂ as divalent salts, on the α and antiparallel β-sheet, and random conformations of poly(L-lysine) (PLL), in water at room temperature were examined by means of CD and compared quantitatively on the basis of elliptical strength at the maximal peak. Changes in the α-helical and antiparallel β-sheet helical conformations of PLL were markedly dependent on the salt concentrations of LiCl, NaCl, and CsCl, which induced decreases in negative intensity in that order. The CD spectrum of the random conformation, the most disordered form, displayed positive cotton effect in concentrations of these salts up to 3.0M and a negative peak in concentrations of 6.0M. The effect of these salts on the random conformation of PLL was stronger than that on the α- and β-conformations in higher concentrations. The CD spectrum of the random conformation in the presence of CaCl₂, ZnCl₂, and MgCl₂, on the other hand, showed negative cotton effect in salt concentrations as low as 3.0M. It was impossible, however, to measure the effect on α- and β-conformations of ZnCl₂ and MgCl₂ above concentrations of 10 mM because of a solubility problem with salts in alkaline solution.     

Growth Potential of Halophilic Bacteria Isolated from Solar Salt Environments: Carbon Sources and Salt Requirements

Eighteen strains of extremely halophilic bacteria and three strains of moderately halophilic bacteria were isolated from four different solar salt environments. Growth tests on carbohydrates, low-molecular-weight carboxylic acids, and complex medium demonstrated that the moderate halophiles and strains of the extreme halophiles Haloarcula and Halococcus grew on most of the substrates tested. Among the Halobacterium isolates were several metabolic groups: strains that grew on a broad range of substrates and strains that were essentially confined to either amino acid (peptone) or carbohydrate oxidation. One strain (WS-4) only grew well on pyruvate and acetate. Most strains of extreme halophiles grew by anaerobic fermentation and possibly by nitrate reduction. Tests of growth potential in natural saltern brines demonstrated that none of the halobacteria grew well in brines which harbor the densest populations of these bacteria in solar salterns. All grew best in brines which were unsaturated with NaCl. The high concentrations of Na⁺ and Mg²⁺ found in saltern crystallizer brines limited bacterial growth, but the concentrations of K⁺ found in these brines had little effect. MgSO₄ was relatively more inhibitory to the extreme halophiles than was MgCl₂, but the reverse was true for the moderate halophiles.     

In vitro salt and thermal tolerance of fungal endophytes of Nicotiana spp. growing in arid regions of north-western Australia

Abstract

Endophytic fungal strains isolated from indigenous Nicotiana plants naturally growing in dry and hot regions of north-western Australia were characterised based on their tolerance to salinity and temperature. Sixty-eight fungal isolates were tested on eight levels (0.5 M, 1.0 M, 1.5 M, 2.0 M, 2.5 M, 3.0 M, 3.5 M and 4.0 M) of five different of salts solutions NaCl, KCl, MgCl₂, CaCl₂ and MgSO₄ and at various temperatures (25–50?°C). The salt adaptation test indicated that the fungal strains namely Aspergillus niger (E-202), A. ochraceous-A (E-134), Aurantiporus sp. (E-135), Cladosporium halotolerance (E-128), Pleurostomophora richardsiae (E-13) and Trichoderma sp. (E-185.1) were tolerant to higher concentrations of various salts. The most growth-limiting salt turned out to be MgCl₂ followed by the chaotrope CaCl₂. Responses to temperature tolerance revealed that most fungi tested could grow at 30?°C. About 50% all the fungi did not show any growth when the temperature was raised above 30?°C. When the temperature was raised up to 50?°C all the fungi failed to grow but the fungus Rasamsonia piperina (E-172). Endophyte strains identified could be promising candidates for future research in investigating the fungus–plant interactions and their roles in plant adaptation to inhospitable environments.     

Nitrate and nitrite removal from salted water by Haloferax mediterranei

Haloferax mediterranei is a denitrifying halophilic archaeon, able to assimilate nitrate or nitrite in the presence of oxygen by the assimilatory nitrate pathway. It can also grow in the presence of high nitrate or nitrite concentrations under anoxic conditions, using both nitrogen species as electron acceptors. In this study, the ability of H. mediterranei to remove high nitrate and nitrite concentrations from culture media has been demonstrated. This suggests that this haloarchaeon could be applied in water bioremediation processes to repair damage caused by anthropogenic activities. This could be beneficial in regions such as Comunidad Valenciana or Murcia (Spain), where the water tables contain high nitrate and nitrite concentrations due to fertiliser addition, and high salt concentrations due to marine intrusions.     

Ribulose bisphosphate carboxylase activity in halophilic Archaebacteria

Among the several strains of halobacteria grown heterotrophically, ribulose bisphosphate carboxylase activity was detected in those which accumulate poly (-hydroxybutyrate), viz. Haloferax mediterranei, Haloferax volcanii and Halobacterium marismortui. In H. mediterranei, the activity was present in cell extracts prepared after growth on a variety of carbohydrates. The ribulose bisphosphate carboxylase activity in H. mediterranei was inhibited by carboxyarabinitol bisphosphate, and the enzyme cross-reacted with antibodies raised against the spinach enzyme. CO₂ fixation by cell extract was stimulated by the addition of ATP and NADH. Preliminary data suggested that hydrogen could be a possible reductant.Abbreviations RuBP ribulose bisphosphate - Ru5P ribulose 5-phosphate - R5P ribose 5-phosphate - CABP carboxyarabinitol bisphosphate - PHB poly (-hydroxybutyrate) - DTT dithiothreitol     

Glucosylglycerol-phosphate synthase: target for ion-mediated regulation of osmolyte synthesis in the cyanobacterium Synechocystis sp. strain PCC 6803

The response of cyanobacteria to a changing osmotic environment includes the accumulation of organic osmolytes such as glucosylglycerol. The activation of the enzymes involved in glucosylglycerol synthesis [glucosylglycerol-phosphate synthase (GGPS) and glucosylglycerol-phosphate phosphatase (GGPP)] in Synechocystis sp. strain PCC 6803 by various salts and salt concentrations was investigated in vitro. GGPS seemed to be the target for salt-mediated regulation of glucosylglycerol synthesis in vitro. GGPS activation was dependent on the concentration of NaCl, and a sigmoidal plot was obtained. Sensitivity to NaCl was markedly enhanced by low Mg⁺² concentrations (optimal at 4 mM), but Mg²⁺ was not absolutely necessary for the Na⁺ stimulation. As in the case of NaCl, other salts (including MgCl₂) stimulated GGPS. The relative order of GGPS activation in the presence of chloride by the cations at constant ionic strength was Li⁺ > Na⁺ > K⁺, Mg²⁺ Mn²⁺. No absolute dependence on ionic strength was observed in Mg²⁺/Na⁺-exchange experiments. The degree of activation by ions at various concentrations was positively related to the increasing destabilizing properties of the cations according to the Hofmeister rule, where chaotropic cations are most efficient. Cations were responsible for activation since chaotropic anions counteracted the activating effect of cations. Received: 10 August 1998 / Accepted: 11 November 1998     

Cloning and partial characterization of the putative rifamycin biosynthetic gene cluster from the actinomycete Amycolatopsis mediterranei DSM 46095

The actinomycete Amycolatopsis mediterranei produces the commercially and medically important polyketide antibiotic rifamycin, which is widely used against mycobacterial infections. The rifamycin biosynthetic (rif) gene cluster has been isolated, cloned and characterized from A. mediterranei S699 and A. mediterranei LBGA 3136. However, there are several other strains of A. mediterranei which also produce rifamycins. In order to detect the variability in the rif gene cluster among these strains, several strains were screened by PCR amplification using oligonucleotide primers based on the published DNA sequence of the rif gene cluster and by using dEBS II (second component of deoxy-erythronolide biosynthase gene) as a gene probe. Out of eight strains of A. mediterranei selected for the study, seven of them showed the expected amplification of the DNA fragments whereas the amplified DNA pattern was different in strain A. mediterranei DSM 46095. This strain also showed striking differences in the banding pattern obtained after hybridization of its genomic DNA against the dEBS II probe. Initial cloning and characterization of the 4-kb DNA fragment from the strain DSM 46095, representing a part of the putative rifamycin biosynthetic cluster, revealed nearly 10% and 8% differences in the DNA and amino acid sequence, respectively, as compared to that of A. mediterranei S699 and A. mediterranei LBGA 3136. The entire rif gene cluster was later cloned on two cosmids from A. mediterranei DSM 46095. Based on the partial sequence analysis of the cluster and sequence comparison with the published sequence, it was deduced that among eight strains of A. mediterranei, only A. mediterranei DSM 46095 carries a novel rifamycin biosynthetic gene cluster.     

地中海富盐菌中非编码RNA的鉴定与分析

【目的】通过转录组高通量测序技术(即RNA-seq),结合生物信息学分析和分子生物学方法,在组学水平鉴定极端嗜盐菌中可能的非编码RNA(nc RNA)。【方法】将培养至对数中期的地中海富盐菌在不同盐浓度下处理30分钟,提取RNA,进行链特异的转录组测序和5′端区分的转录组测序,通过生物信息学分析在全基因组范围内鉴定nc RNA,预测其转录边界;然后通过Northern blot和环化RNA反转录聚合酶链式反应(CR-RT-PCR)对部分预测的nc RNA进行实验验证。【结果】比较两种RNA-seq技术在不同培养条件下的RNA测序结果和对转录单元的精细分析,共鉴定到105个高可信度的nc RNA,并发现4个在不同盐度下表达差异较大的nc RNA,通过Northern blot和CR-RT-PCR验证了inc RNA1436和inc RNA1903的表达情况、转录本、转录起始位点及终止位点等。【结论】首次在组学水平鉴定了地中海富盐菌中的nc RNA,不同盐浓度刺激下部分nc RNA的转录差异暗示其有可能参与地中海富盐菌对盐胁迫的适应,高可信度nc RNA的组学发现为今后全面开展嗜盐古菌nc RNA的功能机制研究提供了基础数据及重要的切入点。     

DNA-DNA homology studies among strains ofHaloferax and other halobacteria

Deoxyribonucleic acid (DNA)-DNA hybridization was used to determine the relationships of the type strains of the three species of the genusHaloferax to 21 halobacterial strains representing isolates from hypersaline habitats and collection strains. Two genotypic groups with high DNA homology were obtained, belonging to the speciesHaloferax gibbonsii andHaloferax mediterranei. The highest DNA relatedness (74%) was obtained in theHalobacterium salinarium group.Halobacterium denitrificans showed very low DNA homology with the other halobacteria investigated. The G+C content of the eight strains ofHaloferax tested ranged between 59.1 and 65.5 mol%.     

Glucose transport into the extremely halophilic archaebacteria

Penetration of glucose into cells of several extremely halophilic archaebacteria of the Halobacterium and Haloferax genera (Halobacterium saccharovorum and Halobacterium salinarium, Haloferax volcanii and Haloferax mediterranei) has been studied. Some characteristics of transport systems of carbohydrate-utilizing halobacteria Halobacterium saccharovorum, Haloferax mediterranei and Haloferax volcanii (pH and temperature optima, stereospecificity, kinetic parameters) have been determined. Inability of H. salinarium cells for active glucose transport has been shown. The dependence of glucose transport on the Na⁺ ions gradient (on the whole cells and membrane vesicles) has been demonstrated. Cells or membrane vesicles of carbohydrate-utilizing halobacteria grown in media containing this sugar indicated the activation of glucose transport, whereas cells grown in media without sugars did not. This fact has allowed us to conclude that corresponding transport systems are inducible.     

Production of poly-3-(hydroxybutyrate-co-hydroxyvalerate) by Haloferax mediterranei using rice-based ethanol stillage with simultaneous recovery and re-use of medium salts

Haloferax mediterranei holds promise for competitive industrial-scale production of polyhydroxyalkanoate (PHA) because cheap carbon sources can be used thus lowering production costs. Although high salt concentration in production medium permits a non-sterile, low-cost process, salt disposal after process completion is a problem as current environmental standards do not allow total dissolved solids (TDS) above 2000 mg/l in discharge water. As the first objective of this work, the waste product of rice-based ethanol industry, stillage, was used for the production of PHA by H. mediterranei in shake flasks. Utilization of raw stillage led to 71 ± 2 % (of dry cell weight) PHA accumulation and 16.42 ± 0.02 g/l PHA production. The product yield coefficient was 0.35 while 0.17 g/l h volumetric productivity was attained. Simultaneous reduction of BOD₅ and COD values of stillage by 83 % was accomplished. The PHA was isolated by osmotic lysis of cells, purification by sodium dodecyl sulfate and organic solvents. The biopolymer was identified as poly-3-(hydroxybutyrate-co-15.4 mol%-hydroxyvalerate) (PHBV). This first report on utilization of rice-based ethanol stillage for PHBV production by H. mediterranei is currently the most cost effective. As the second objective, directional properties of decanoic acid together with temperature dependence of water solubility in decanoic acid were applied for two-stage desalination of the spent stillage medium. We report for the first time, recovery and re-use of 96 % of the medium salts for PHA production thus removing the major bottleneck in the potential application of H. mediterranei for industrial production of PHBV. Final discharge water had TDS content of 670 mg/l.     

Efficient hydroxylamine mutagenesis ofHaloferax mediterranei and other extremely halophilic archaebacteria

The lethal and mutagenic effects of hydroxylamine onHaloferax mediterranei and five other extremely halophilic archaebacteria are described for the first time. Although previous studies have shown thatH. mediterranei was very resistant to the lethal action of other DNA-damaging agents, this strain was found to be relatively sensitive to hydroxylamine, but also more successfully mutated by the latter. The efficiency of the mutagenicity obtained with the hydroxylamine treatment was tested under a variety of conditions, and optimal procedures are described that yielded a number of useful auxotrophic mutant strains ofH. mediterranei. Likewise, a strong induced mutagenicity after hydroxylamine mutagenesis was achieved for the majority of the other archaebacteria tested.     

A halocin-H4 mutant Haloferax mediterranei strain retains the ability to inhibit growth of other halophilic archaea

Many members of the Halobacteriaceae were found to produce halocins, molecules that inhibit the growth of other halophilic archaea. Halocin H4 that is produced by Haloferax mediterranei and inhibits the growth of Halobacterium salinarum is one of the best studied halocins to date. The gene encoding this halocin had been previously identified as halH4, located on one of Hfx. mediterranei megaplasmids. We generated a mutant of the halH4 gene and examined the killing ability of the Haloferax mediterranei halH4 mutant with respect to both Halobacterium salinarum and Haloferax volcanii. We showed that both wild-type Hfx. mediterranei and the halH4 mutant strain efficiently inhibited the growth of both species, indicating halocin redundancy. Surprisingly, the halH4 deletion mutant exhibited faster growth in standard medium than the wild type, and is likely to have a better response to several nucleotides, which could explain this phenotype.     

Effects of Culture Conditions on Poly(β-Hydroxybutyric Acid) Production by Haloferax mediterranei

The halobacterium Haloferax mediterranei accumulates poly(β-hydroxybutyrate) (PHB) as intracellular granules. The conditions for PHB production in batch and continuous cultures have been studied and optimized. Phosphate limitation is essential for PHB accumulation in large quantities. Glucose and starch are the best carbon sources. With 2% starch, 0.00375% KH₂PO₄, and 0.2% NH₄Cl in batch culture, a production of ca. 6 g of PHB per liter was reached, being 60% of the total biomass dry weight, and giving a yield over the carbon source of 0.33 g/g. The PHB production in continuous cultures was stable over a 3-month period. Our results demonstrate that H. mediterranei is an interesting candidate for industrial production of biological polyesters.