Archaeal Biodiversity in Crystallizer Ponds from a Solar Saltern: Culture versus PCR

The culturable haloarchaeal diversity in a crystallizer pond from a solar saltern has been analyzed and compared with the biodiversity directly retrieved by analysis of rRNA genes amplified from the environment. Two different sets of culture conditions have been assayed: solid medium with yeast extract as carbon source and liquid media with either yeast extract or a mixture of fishmeal, Spirulina sp., and Artemia salina. Seventeen colonies grown on plates with yeast extract incubated at 30°C were analyzed by 16S rDNA partial sequencing. Sixteen were closely related to haloarchaea of the genus Halorubrum; 13 of them to Halorubrum coriense, a haloarchaeon isolated from a solar saltern pond in Australia, which had not been previously isolated from the pond analyzed in this study; and one to Haloarcula marismortui. Liquid cultures were analyzed by ribosomal internal spacer analysis (RISA) and partial sequencing of the 16SrRNA genes. A total of 18 sequences were analyzed, 15 corresponding to RISA bands obtained from cultures, and 3 from the environmental sample used as inoculum. Thirteen sequences obtained from cultures were related to several Halorubrum species, and 2 to Haloarcula. One of the clones obtained directly from the environmental sample was distantly related to a Natronobacterium, whereas two were related to SPhT, the phylotype most frequently retrieved from this environment by culture independent techniques. Our results show an extremely low diversity for the haloarchaea retrieved by cultivation even when modifications to the standard technique are introduced.     

Haloarchaeal diversity in 23, 121 and 419 MYA salts

DNA was extracted from surface-sterilized salt of different geological ages (23, 121, 419 million years of age, MYA) to investigate haloarchaeal diversity. Only Haloarcula and Halorubrum DNA was found in 23 MYA salt. Older crystals contained unclassified groups and Halobacterium . The older crystals yielded a unique 55-bp insert within the 16S rRNA V2 region. The secondary structure of the V2 region completely differed from that in haloarchaea of modern environments. The DNA demonstrates that unknown haloarchaea and the Halobacterium were key components in ancient hypersaline environments. Halorubrum and Haloarcula appear to be a dominant group in relatively modern hypersaline habitats.     

Lateral gene transfer occurring in haloarchaea: an interpretative imitation study

Lateral gene transfer (LGT) plays an important role in the molecular evolution of haloarchaea. Polyethylene glycol-mediated LGT in haloarchaea has been demonstrated in the laboratory, yet few explanations have been put forward for the apparently common, natural occurrence of plentiful plasmids within haloarchaeal cells. In this study, LGT was induced in two genera of haloarchaea, Haloferax and Halorubrum, by modification of salt concentration of media-a factor that may vary naturally in native haloarchaeal habitat. Minimal growth salt concentrations (MGSCs) of four strains of haloarchaea from these two genera were established, and transformations using two circular double-stranded DNAs (dsDNAs), pSY1 and pWL102, were then produced in media at strain-appropriate MGSCs. The four strains of haloarchaea were transformed successfully by both kinds of dsDNAs with an efficiency of 10(2)-10(3) transformants per microgram dsDNA. The transformation under reduced salt concentration may be an imitation of natural LGT of dsDNA into haloarchaea when salinity in normally hypersaline environments is altered by sudden introduction of fresh water-for example, by rainfall, snow-melt, or flooding-providing a reasonable interpretation for haloarchaea being naturally richer in plasmids than any other known organisms.     

Microbial ecology of an Antarctic hypersaline lake: genomic assessment of ecophysiology among dominant haloarchaea

Deep Lake in Antarctica is a cold, hypersaline system where four types of haloarchaea representing distinct genera comprise >70% of the lake community: strain tADL ∼44%, strain DL31 ∼18%, Halorubrum lacusprofundi ∼10% and strain DL1 ∼0.3%. By performing comparative genomics, growth substrate assays, and analyses of distribution by lake depth, size partitioning and lake nutrient composition, we were able to infer important metabolic traits and ecophysiological characteristics of the four Antarctic haloarchaea that contribute to their hierarchical persistence and coexistence in Deep Lake. tADL is characterized by a capacity for motility via flagella (archaella) and gas vesicles, a highly saccharolytic metabolism, a preference for glycerol, and photoheterotrophic growth. In contrast, DL31 has a metabolism specialized in processing proteins and peptides, and appears to prefer an association with particulate organic matter, while lacking the genomic potential for motility. H. lacusprofundi is the least specialized, displaying a genomic potential for the utilization of diverse organic substrates. The least abundant species, DL1, is characterized by a preference for catabolism of amino acids, and is the only one species that lacks genes needed for glycerol degradation. Despite the four haloarchaea being distributed throughout the water column, our analyses describe a range of distinctive features, including preferences for substrates that are indicative of ecological niche partitioning. The individual characteristics could be responsible for shaping the composition of the haloarchaeal community throughout the lake by enabling selection of ecotypes and maintaining sympatric speciation.     

Purification and biological characterization of halocin C8, a novel peptide antibiotic from<Emphasis Type="Italic"> Halobacterium</Emphasis> strain AS7092

Halocins are bacteriocin-like proteins or peptides produced by many species of the family Halobacteriaceae. Halocin C8, excreted by the Halobacterium strain AS7092, is a single 6.3-kDa polypeptide with an isoelectric point of 4.4, which is sensitive to proteinase K but not to trypsin. Halocin C8 is quite stable, as it can be desalted, boiled, frozen, subjected to organic solvents, and stored in culture supernatant at 4°C or in dH₂O at –20°C for more than 1 year without losing activity. The purification of this halocin was achieved by combination of tangential flow filtration (TFF), Sephadex G50 and DEAE-sepharose chromatography. The N-terminal amino acid sequence was also determined by Edman degradation. Halocin C8 appeared to have a very wide activity spectrum, including most haloarchaea and even some haloalkaliphilic rods. When a sensitive strain of Halorubrum saccharovorum was exposed to halocin C8, the treated cells swelled at the initial stage, the cell wall appeared to be nicked and the cytoplasm was then extruded out, and the whole cell was eventually completely lysed. These results indicate that halocin C8 is a novel microhalocin and its primary target might be located in the cell wall of the sensitive cells.Communicated by W.D. Grant     

Biofilm formation by haloarchaea

A fluorescence‐based live‐cell adhesion assay was used to examine biofilm formation by 20 different haloarchaea, including species of Halobacterium, Haloferax and Halorubrum, as well as novel natural isolates from an Antarctic salt lake. Thirteen of the 20 tested strains significantly adhered (P‐value < 0.05) to a plastic surface. Examination of adherent cell layers on glass surfaces by differential interference contrast, fluorescence and confocal microscopy showed two types of biofilm structures. Carpet‐like, multi‐layered biofilms containing micro‐ and macrocolonies (up to 50 μm in height) were formed by strains of Halobacterium salinarum and the Antarctic isolate t‐ADL strain DL24. The second type of biofilm, characterized by large aggregates of cells adhering to surfaces, was formed by Haloferax volcanii DSM 3757^T and Halorubrum lacusprofundi DL28. Staining of the biofilms formed by the strongly adhesive haloarchaeal strains revealed the presence of extracellular polymers, such as eDNA and glycoconjugates, substances previously shown to stabilize bacterial biofilms. For Hbt. salinarum DSM 3754^T and Hfx. volcanii DSM 3757^T, cells adhered within 1 day of culture and remained viable for at least 2 months in mature biofilms. Adherent cells of Hbt. salinarum DSM 3754^T showed several types of cellular appendages that could be involved in the initial attachment. Our results show that biofilm formation occurs in a surprisingly wide variety of haloarchaeal species.     

云南黑井古盐矿可培养极端嗜盐古菌初步研究*

从云南禄丰县黑井古镇古盐矿采集30多个盐土样品,用6种极端嗜盐古菌的培养基进行分离,共挑选出425株嗜盐菌。经过盐浓度耐受等实验筛选并去除可能重复菌株后共有79株极端嗜盐菌,选出15株进行了16S rRNA基因序列测定,结果显示,其中11株为极端嗜盐古菌。对这11株菌进行初步系统发育分析发现,它们广泛分布在极端嗜盐古菌科至少4个不同属中,其中16S rRNA基因和已有效发表种间的序列相似性在97％以上的有6株,分布在Halorubrum,Natronococcus,Natrialba,Halalkalicoccus4个属中;序列相似性低于97％的有5株：菌株YIM-ARC 0032,YIM-ARC 0036,YIM-ARC 0037,YIM-ARC 0050,它们的分类地位有待进一步确定。实验初步显示出了云南黑井盐矿极端嗜盐古菌的多样性和丰富度,值得深入研究。     

Combined use of cultivation-dependent and cultivation-independent methods indicates that members of most haloarchaeal groups in an Australian crystallizer pond are cultivable

Haloarchaea are the dominant microbial flora in hypersaline waters with near-saturating salt levels. The haloarchaeal diversity of an Australian saltern crystallizer pond was examined by use of a library of PCR-amplified 16S rRNA genes and by cultivation. High viable counts (10(6) CFU/ml) were obtained on solid media. Long incubation times (> or =8 weeks) appeared to be more important than the medium composition for maximizing viable counts and diversity. Of 66 isolates examined, all belonged to the family Halobacteriaceae, including members related to species of the genera Haloferax, Halorubrum, and Natronomonas. In addition, isolates belonging to a novel group (the ADL group), previously detected only as 16S rRNA genes in an Antarctic hypersaline lake (Deep Lake), were cultivated for the first time. The 16S rRNA gene library identified the following five main groups: Halorubrum groups 1 and 2 (49%), the SHOW (square haloarchaea of Walsby) group (33%), the ADL group (16%), and the Natronomonas group (2%). There were two significant differences between the organisms detected in cultivation and 16S rRNA sequence results. Firstly, Haloferax spp. were frequently isolated on plates (15% of all isolates) but were not detected in the 16S rRNA sequences. Control experiments indicated that a bias against Haloferax sequences in the generation of the 16S rRNA gene library was unlikely, suggesting that Haloferax spp. readily form colonies, even though they were not a dominant group. Secondly, while the 16S rRNA gene library identified the SHOW group as a major component of the microbial community, no isolates of this group were obtained. This inability to culture members of the SHOW group remains an outstanding problem in studying the ecology of hypersaline environments.     

Halophilic archaea in the human intestinal mucosa

The human gastrointestinal tract microbiota, despite its key roles in health and disease, remains a diverse, variable and poorly understood entity. Current surveys reveal a multitude of undefined bacterial taxa and a low diversity of methanogenic archaea. In an analysis of the microbiota in colonic mucosal biopsies from patients with inflammatory bowel disease we found 16S rDNA sequences representing a phylogenetically rich diversity of halophilic archaea from the Halobacteriaceae (haloarchaea), including novel phylotypes. As the human colon is not considered a salty environment and haloarchaea are described as extreme halophiles, we evaluated and further discarded the possibility that these sequences originated from pre‐colonoscopy saline lavage solutions. Furthermore, aerobic enrichment cultures prepared from a patient biopsy at low salinity (2.5% NaCl) yielded haloarchaeal sequence types. Microscopic observation after fluorescence in situ hybridization provided evidence of the presence of viable archaeal cells in these cultures. These results prove the survival of haloarchaea in the digestive system and suggest that they may be members of the mucosal microbiota, even if present in low numbers in comparison with methanogenic archaea. Investigation of a potential physiological basis of this association may lead to new insights into gastrointestinal health and disease.     

Metagenomic study of red biofilms from Diamante Lake reveals ancient arsenic bioenergetics in haloarchaea

Arsenic metabolism is proposed to be an ancient mechanism in microbial life. Different bacteria and archaea use detoxification processes to grow under high arsenic concentration. Some of them are also able to use arsenic as a bioenergetic substrate in either anaerobic arsenate respiration or chemolithotrophic growth on arsenite. However, among the archaea, bioenergetic arsenic metabolism has only been found in the Crenarchaeota phylum. Here we report the discovery of haloarchaea (Euryarchaeota phylum) biofilms forming under the extreme environmental conditions such as high salinity, pH and arsenic concentration at 4589 m above sea level inside a volcano crater in Diamante Lake, Argentina. Metagenomic analyses revealed a surprisingly high abundance of genes used for arsenite oxidation (aioBA) and respiratory arsenate reduction (arrCBA) suggesting that these haloarchaea use arsenic compounds as bioenergetics substrates. We showed that several haloarchaea species, not only from this study, have all genes required for these bioenergetic processes. The phylogenetic analysis of aioA showed that haloarchaea sequences cluster in a novel and monophyletic group, suggesting that the origin of arsenic metabolism in haloarchaea is ancient. Our results also suggest that arsenite chemolithotrophy likely emerged within the archaeal lineage. Our results give a broad new perspective on the haloarchaea metabolism and shed light on the evolutionary history of arsenic bioenergetics.     

Astaxanthin preparation by fermentation of esters from Haematococcus pluvialis algal extracts with Stenotrophomonas species

Natural astaxanthin (Ax) is an additive that is widely used because of its beneficial biochemical functions. However, the methods used to produce free Ax have drawbacks. Chemical saponification methods produce several by‐products, and lipase‐catalyzed hydrolysis methods are not cost effective. In this study, a bacterial strain of Stenotrophomonas sp. was selected to enzymatically catalyze the saponification of Ax esters to produce free all‐trans‐Ax. Through single‐factor experiments and a Box–Behnken design, the optimal fermentation conditions were determined as follows: a seed culture age of 37.79 h, an inoculum concentration of 5.92%, and an initial broth pH of 6.80. Under these conditions, a fermentation curve was drawn, and the optimal fermentation time was shown to be 60 h. At 60 h, the degradation rate of the Ax esters was 98.08%, and the yield of free all‐trans‐Ax was 50.130 μg/mL. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:649–656, 2016     

嗜盐古菌Halorubrum sp. CY的分离、鉴定及胞外淀粉酶特性初步研究

本研究从云南一平浪盐矿分离到一株产胞外淀粉酶的嗜盐古菌, 通过形态观察, 生理生化特性实验, 并结合16S rRNA序列分析, 初步鉴定为嗜盐古菌Halorubrum属, 命名为Halorubrum sp. CY。另外对该菌产生的胞外淀粉酶的性质进行初步研究, 结果显示其胞外淀粉酶发挥最大活性的pH值和温度分别为6.0和60oC, Zn2+、Cu2+、Al3+、SO32-对胞外淀粉酶的活性有抑制作用, 而Mn2+则有促进作用。     

3株禽滑液囊支原体分离株致病性的比较和评价

【背景】禽滑液囊支原体是感染鸡的一种重要病原体,给我国的家禽养殖业带来了严重的经济损失。【目的】评价从病鸡中分离到的3株禽滑液囊支原体的致病性,以期丰富不同区域来源的分离株对鸡致病性的认识。【方法】分别用分离株CHN-WF224-2016、CHN-BZJ2-2015、CHN-JNB19-2016感染商品肉鸡,并在攻毒后第10天和21天进行实验室解剖,通过气囊和足垫的临床病变、血清学检测结果及气管粘膜病理形态学改变来比较评价分离株的致病力。【结果】CHN-BZJ2-2015和CHN-WF224-2016分离株能引起明显足垫肿胀和关节滑膜炎,其中CHN-BZJ2-2015分离株致病力最强,感染21 d后的血清学检测和气管粘膜厚度与CHN-JNB19-2016分离株和MS-H疫苗株比较差异显著(P<0.05)。【结论】本研究表明我国目前流行的禽滑液囊支原体菌株其致病力存在明显差异,强调养殖场要积极控制和预防禽滑液囊支原体感染的重要性,同时为今后该疫苗的研发积累了实验数据。     

Application of attenuated Lactococcus garvieae strain lacking a virulence-associated capsule on its cell surface as a live vaccine in yellowtail Seriola quinqueradiata Temminck and Schlegel

An attenuated Lactococcus garvieae strain lacking a virulence‐associated capsule on its cell surface was evaluated for application as a live vaccine. The attenuated strain (MS93003A) was obtained from the parent strain (MS93003V), which produced a well‐developed capsule, by culturing on an agar medium supplemented with 2,3,5‐triphenyltetrazolium chloride. When live cells of L. garvieae (MS93003A) or formalin‐killed cells (MS93003A) were used as an injectable vaccine, protection against virulent L. garvieae (MS93003V) was conferred on Seriola quinqueradiata. The MS93003A cells did not recover their virulence even after in vivo passages in fish. MS93003A live cells also conferred long‐lasting protective immunity to S. quinqueradiata against virulent L. garvieae infection.     

An extractive membrane biofilm reactor as alternative technology for the treatment of methyl tert‐butyl ether contaminated water

Among the strategies developed for contaminated groundwater bioremediation, those based on the use of bacteria adhering to inert supports and establishing biofilms have gained great importance in this field. Extractive membrane biofilm reactor (EMBFR) technology offers productive solutions for the removal of volatile and semi‐volatile compounds. EMBFR technology is based on the use of extractive semipermeable membranes through which contaminants migrate to the biological compartment in which microorganisms with pollutant biotransformation and/or mineralization capacities can grow, forming an active biofilm on the membrane surface. The objective of this study was to assess the use of three bacterial strains (Paenibacillus sp. SH7 CECT 8558, Agrobacterium sp. MS2 CECT 8557, and Rhodococcus ruber EE6 CECT 8612), as inoculum in a lab‐scale EMBFR running for 28 days under aerobic conditions to eliminate methyl tert‐butyl ether (MTBE) from water samples. Three different hydraulic retention times (1, 6, and 12 h) were employed. MTBE degradation values were determined daily by a gas GC‐MS technique, as well as suspended bacterial growth. The biofilm established by the bacterial strains on the semipermeable membrane was detected by Field‐Emission Scanning Electron Microscopy (FESEM) at the end of each experiment. The acute toxicity of the treated effluents and biomedium was determined by Microtox^© assay (EC₅₀).The results achieved from the MTBE degradation, biofilm formation, and toxicity analysis indicated that bacterial strains MS2 and EE6 were the best options as selective inoculum, although further research is needed, particularly with regard to their possible use as a mixed culture. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1238–1245, 2016     

Enantioselective Degradation and Chiral Stability of Metalaxyl‐M in Tomato Fruits

Metalaxyl is an important chiral acetanilide fungicide, and the activity almost entirely originates from the R‐enantiomer. Racemic metalaxyl has been gradually replaced by the enantiopure R‐enantiomer (metalaxyl‐M). In this study a chiral residue analysis method for metalaxyl and the metabolite metalaxyl acid was set up based on high‐performance liquid chromatography tandem mass spectroscopy (HPLC‐MS/MS). The enantioselective degradation and chiral stability of metalaxyl‐M in tomato fruits in two geographically distinct regions of China (Heilongjiang and Hunan Province) were evaluated and the enantioselectivity of metalaxyl acid was also investigated. Tomato plants grew under field conditions with a one‐time spray application of metalaxyl‐M wettable powder. It was found that R‐metalaxyl was not chirally stable and the inactive S‐metalaxyl was detected in tomato fruits. At day 40, S‐metalaxyl derived from R‐metalaxyl accounted for 32% and 26% of the total amount of metalaxyl, respectively. The metabolites R‐metalaxyl acid and S‐metalaxyl acid were both observed in tomato, and the ratio of S‐metalaxyl acid to the sum of S‐ and R‐metalaxyl acid was 36% and 28% at day 40, respectively. For both metalaxyl and metalaxyl acid, the half‐life of the S‐enantiomer was longer than the R‐enantiomer. The results indicated that the enantiomeric conversion should be considered in the bioactivity evaluation and environmental pollution assessment. Chirality 28:382–386, 2016. © 2016 Wiley Periodicals, Inc.     

A traditional Japanese-style salt field is a niche for haloarchaeal strains that can survive in 0.5% salt solution

Background

Most of the haloarchaeal strains have been isolated from hypersaline environments such as solar evaporation ponds, salt lakes, or salt deposits, and they, with some exceptions, lyse or lose viability in very low-salt concentrations. There are no salty environments suitable for the growth of haloarchaea in Japan. Although Natrialba asiatica and Haloarcula japonica were isolated many years ago, the question, "Are haloarchaea really thriving in natural environments of Japan?" has remained unanswered.

Results

Ten strains were isolated from a traditional Japanese-style salt field at Nie, Noto Peninsula, Japan by plating out the soil samples directly on agar plates containing 30% (w/v) salts and 0.5% yeast extract. They were most closely related to strains of three genera, Haladaptatus, Halococcus, and Halogeometricum. Survival rates in 3% and 0.5% SW (Salt Water, solutions containing salts in approximately the same proportions as found in seawater) solutions at 37°C differed considerably depending on the strains. Two strains belonging to Halogeometricum as well as the type strain Hgm. borinquense died and lysed immediately after suspension. Five strains that belonged to Halococcus and a strain that may be a member of Halogeometricum survived for 1–2 days in 0.5% SW solution. Two strains most closely related to Haladaptatus possessed extraordinary strong tolerance to low salt conditions. About 20 to 34% of the cells remained viable in 0.5% SW after 9 days incubation.

Conclusion

In this study we have demonstrated that haloarchaea are really thriving in the soil of Japanese-style salt field. The haloarchaeal cells, particularly the fragile strains are suggested to survive in the micropores of smaller size silt fraction, one of the components of soil. The inside of the silt particles is filled with concentrated salt solution and kept intact even upon suspension in rainwater. Possible origins of the haloarchaea isolated in this study are discussed.     

Toxicity of chlorpyrifos‐methyl to Sitophilus zeamais collected in Korea and biochemical differences

In this study, lethal concentration (LC₅₀) values of chlorpyrifos‐methyl (CPM) were determined for two Korean strains (CBNU and KNU) of Sitophilus zeamais. The two strains had similar susceptibilities (1.70 and 1.86 μg a.i./cm², respectively) to CPM. Carboxylesterase (CE) activity was twice as high in the CBNU strain as in the KNU strain. Lower acetylcholinesterase (AChE) activity was also noted in the latter; however, the activity of glutathione S‐transferase (GST) was twice as high as in the CBNU strain. Gel electrophoresis of CE of crude extracts from adults of the two strains of S. zeamais showed clearly different band patterns, with molecular weights of 60 kDa and 71 kDa in the CBNU and KNU strains, respectively. MALDI‐TOF MS/MS was used to profile small proteins (less than 10 kDa), with results indicating that 206 proteins are expressed differently in the two strains. The peak of interest of 2247.7 m/z was applied to TOF‐TOF MS and its de novo peptide sequence was identified as a tyrosine phosphatase fragment. Phospholipids from the two strains were analyzed and 34 phospholipids were found to be significantly different between strains. Results suggest that the two strains collected from Korea showed different biochemical results, presumably differences in insecticide selection by different living locations.     

新疆两盐湖可培养嗜盐古菌多样性研究

从新疆地区艾比盐湖和艾丁盐湖卤水及泥土样品中分离到86株嗜盐古菌。16S rRNA基因序列分析结果表明,分离自艾比湖的嗜盐古菌分别属于Haloarcula、Halobacterium、Halorubrum、Haloterrigena、Natrinema和Natronorubrum6个属的11个分类单元,而分离自艾丁湖的嗜盐古菌分别属于Haloarcula、Halobiforma、Halorubrum、Haloterrigena、Natrialba、Natrinema6个属的8个分类单元,这一结果表明艾比湖可培养嗜盐古菌生物多样性稍高于艾丁湖。基于16S rRNA基因序列的系统发育分析表明代表菌株ABH15应为Natronorubrum属的中性嗜盐古菌新种,代表菌株ABH07、ABH12、ABH17、ABH19、ABH51和AD30可能是Halobacterium、Halorubrum、Haloterrigena、Haloarcula的新成员。     

Extremely halophilic archaea and the issue of long-term microbial survival

Halophilic archaebacteria (haloarchaea) thrive in environments with salt concentrations approaching saturation, such as natural brines, the Dead Sea, alkaline salt lakes and marine solar salterns; they have also been isolated from rock salt of great geological age (195–250 million years). An overview of their taxonomy, including novel isolates from rock salt, is presented here; in addition, some of their unique characteristics and physiological adaptations to environments of low water activity are reviewed. The issue of extreme long-term microbial survival is considered and its implications for the search for extraterrestrial life. The development of detection methods for subterranean haloarchaea, which might also be applicable to samples from future missions to space, is presented.