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1.  Identification of polyhydroxyalkanoates in Halococcus and other haloarchaeal species 
Polyhydroxyalkanoates (PHAs) are accumulated in many prokaryotes. Several members of the Halobacteriaceae produce poly-3-hydroxybutyrate (PHB), but it is not known if this is a general property of the family. We evaluated identification methods for PHAs with 20 haloarchaeal species, three of them isolates from Permian salt. Staining with Sudan Black B, Nile Blue A, or Nile Red was applied to screen for the presence of PHAs. Transmission electron microscopy and 1H-nuclear magnetic resonance spectroscopy were used for visualization of PHB granules and chemical confirmation of PHAs in cell extracts, respectively. We report for the first time the production of PHAs by Halococcus sp. (Halococcus morrhuae DSM 1307T, Halococcus saccharolyticus DSM 5350T, Halococcus salifodinae DSM 8989T, Halococcus dombrowskii DSM 14522T, Halococcus hamelinensis JCM 12892T, Halococcus qingdaonensis JCM 13587T), Halorubrum sp. (Hrr. coriense DSM 10284T, Halorubrum chaoviator DSM 19316T, Hrr. chaoviator strains NaxosII and AUS-1), haloalkaliphiles (Natronobacterium gregoryi NCMB 2189T, Natronococcus occultus DSM 3396T) and Halobacterium noricense DSM 9758T. No PHB was detected in Halobacterium salinarum NRC-1 ATCC 700922, Hbt. salinarum R1 and Haloferax volcanii DSM 3757T. Most species synthesized PHAs when growing in synthetic as well as in complex medium. The polyesters were generally composed of PHB and poly-ß-hydroxybutyrate-co-3-hydroxyvalerate (PHBV). Available genomic data suggest the absence of PHA synthesis in some haloarchaea and in all other Euryarchaeota and Crenarchaeota. Homologies between haloarchaeal and bacterial PHA synthesizing enzymes had indicated to some authors probable horizontal gene transfer, which, considering the data obtained in this study, may have occurred already before Permian times.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-010-2611-6) contains supplementary material, which is available to authorized users.
PMCID: PMC2895300  PMID: 20437233
Polyhydroxybutyrate; Haloarchaea; Halococcus; Halobacterium; Haloalkaliphile
2.  Properties of Halococcus salifodinae, an Isolate from Permian Rock Salt Deposits, Compared with Halococci from Surface Waters 
Life : Open Access Journal  2013;3(1):244-259.
Halococcus salifodinae BIpT DSM 8989T, an extremely halophilic archaeal isolate from an Austrian salt deposit (Bad Ischl), whose origin was dated to the Permian period, was described in 1994. Subsequently, several strains of the species have been isolated, some from similar but geographically separated salt deposits. Hcc. salifodinae may be regarded as one of the most ancient culturable species which existed already about 250 million years ago. Since its habitat probably did not change during this long period, its properties were presumably not subjected to the needs of mutational adaptation. Hcc. salifodinae and other isolates from ancient deposits would be suitable candidates for testing hypotheses on prokaryotic evolution, such as the molecular clock concept, or the net-like history of genome evolution. A comparison of available taxonomic characteristics from strains of Hcc. salifodinae and other Halococcus species, most of them originating from surface waters, is presented. The cell wall polymer of Hcc. salifodinae was examined and found to be a heteropolysaccharide, similar to that of Hcc. morrhuae. Polyhydroxyalkanoate granules were present in Hcc. salifodinae, suggesting a possible lateral gene transfer before Permian times.
PMCID: PMC4187196  PMID: 25371342
Halococcus species; Halococcus salifodinae; haloarchaea; Permian salt deposit; cell wall polymer; polyhydroxyalkanoate; prokaryotic evolution
3.  Investigating the Effects of Simulated Martian Ultraviolet Radiation on Halococcus dombrowskii and Other Extremely Halophilic Archaebacteria 
Astrobiology  2009;9(1):104-112.
The isolation of viable extremely halophilic archaea from 250-million-year-old rock salt suggests the possibility of their long-term survival under desiccation. Since halite has been found on Mars and in meteorites, haloarchaeal survival of martian surface conditions is being explored. Halococcus dombrowskii H4 DSM 14522T was exposed to UV doses over a wavelength range of 200–400 nm to simulate martian UV flux. Cells embedded in a thin layer of laboratory-grown halite were found to accumulate preferentially within fluid inclusions. Survival was assessed by staining with the LIVE/DEAD kit dyes, determining colony-forming units, and using growth tests. Halite-embedded cells showed no loss of viability after exposure to about 21 kJ/m2, and they resumed growth in liquid medium with lag phases of 12 days or more after exposure up to 148 kJ/m2. The estimated D37 (dose of 37 % survival) for Hcc. dombrowskii was ≥ 400 kJ/m2. However, exposure of cells to UV flux while in liquid culture reduced D37 by 2 orders of magnitude (to about 1 kJ/m2); similar results were obtained with Halobacterium salinarum NRC-1 and Haloarcula japonica. The absorption of incoming light of shorter wavelength by color centers resulting from defects in the halite crystal structure likely contributed to these results. Under natural conditions, haloarchaeal cells become embedded in salt upon evaporation; therefore, dispersal of potential microscopic life within small crystals, perhaps in dust, on the surface of Mars could resist damage by UV radiation.
PMCID: PMC3182532  PMID: 19215203
Halococcus dombrowskii; Simulated martian UV radiation; LIVE/DEAD staining; Halite fluid inclusions; UV transmittance and reflectance; Desiccation
4.  Culturable halophilic archaea at the initial and crystallization stages of salt production in a natural solar saltern of Goa, India 
Aquatic Biosystems  2012;8:15.
Goa is a coastal state in India and salt making is being practiced for many years. This investigation aimed in determining the culturable haloarchaeal diversity during two different phases of salt production in a natural solar saltern of Ribandar, Goa. Water and sediment samples were collected from the saltern during pre-salt harvesting phase and salt harvesting phase. Salinity and pH of the sampling site was determined. Isolates were obtained by plating of the samples on complex and synthetic haloarchaeal media. Morphology of the isolates was determined using Gram staining and electron microscopy. Response of cells to distilled water was studied spectrophotometrically at 600nm. Molecular identification of the isolates was performed by sequencing the 16S rRNA.
Salinity of salt pans varied from 3-4% (non-salt production phase) to 30% (salt production phase) and pH varied from 7.0-8.0. Seven haloarchaeal strains were isolated from water and sediment samples during non-salt production phase and seventeen haloarchaeal strains were isolated during the salt production phase. All the strains stained uniformly Gram negative. The orange-red acetone extract of the pigments showed similar spectrophotometric profile with absorption maxima at 393, 474, 501 and 535 nm. All isolates obtained from the salt dilute phase were grouped within the genus Halococcus. This was validated using both total lipid profiling and 16S rRNA data sequencing. The isolates obtained from pre-salt harvesting phase were resistant to lysis. 16S rRNA data showed that organisms belonging to Halorubrum, Haloarcula, Haloferax and Halococcus genera were obtained during the salt concentrated phase. The isolates obtained from salt harvesting phase showed varied lysis on suspension in distilled water and /or 3.5% NaCl.
Salterns in Goa are transiently operated during post monsoon season from January to May. During the pre-salt harvesting phase, all the isolates obtained belonged to Halococcus sp. During the salt harvesting phase, isolates belonging to Halorubrum, Haloarcula, Haloferax and Halococcus genera were obtained. This study clearly indicates that Halococcus sp. dominates during the low salinity conditions.
PMCID: PMC3444409  PMID: 22747590
Archaea; Haloarchaea; Hypersaline; Solar saltern
5.  Responses of Haloarchaea to Simulated Microgravity 
Astrobiology  2011;11(3):199-205.
Various effects of microgravity on prokaryotes have been recognized in recent years, with the focus on studies of pathogenic bacteria. No archaea have been investigated yet with respect to their responses to microgravity. For exposure experiments on spacecrafts or on the International Space Station, halophilic archaea (haloarchaea) are usually embedded in halite, where they accumulate in fluid inclusions. In a liquid environment, these cells will experience microgravity in space, which might influence their viability and survival. Two haloarchaeal strains, Haloferax mediterranei and Halococcus dombrowskii, were grown in simulated microgravity (SMG) with the rotary cell culture system (RCCS, Synthecon). Initially, salt precipitation and detachment of the porous aeration membranes in the RCCS were observed, but they were avoided in the remainder of the experiment by using disposable instead of reusable vessels. Several effects were detected, which were ascribed to growth in SMG: Hfx. mediterranei's resistance to the antibiotics bacitracin, erythromycin, and rifampicin increased markedly; differences in pigmentation and whole cell protein composition (proteome) of both strains were noted; cell aggregation of Hcc. dombrowskii was notably reduced. The results suggest profound effects of SMG on haloarchaeal physiology and cellular processes, some of which were easily observable and measurable. This is the first report of archaeal responses to SMG. The molecular mechanisms of the effects induced by SMG on prokaryotes are largely unknown; haloarchaea could be used as nonpathogenic model systems for their elucidation and in addition could provide information about survival during lithopanspermia (interplanetary transport of microbes inside meteorites). Key Words: Haloferax mediterranei—Halococcus dombrowskii—Simulated microgravity—Rotary cell culture system—Antibiotic resistance—Lithopanspermia. Astrobiology 11, 199–205.
PMCID: PMC3079168  PMID: 21417742
6.  Microbiology Study of a Hypersaline Lake in French Somaliland 
Applied Microbiology  1974;27(5):819-822.
In a study of a lake having a higher concentration of salts than the Dead Sea, all of the heterotrophic bacteria isolated were aerobes; no strictly anaerobic strains were found. Ninety percent of the strains were euryhalines and ten percent were strict halophiles. The extreme halophiles belonged to the species Halobacterium trapanicum and Halococcus morrhuae.
PMCID: PMC380149  PMID: 4833284
7.  Kocuria marina BS-15 a biosurfactant producing halophilic bacteria isolated from solar salt works in India 
Biosurfactant screening was made among the eight halophilic bacterial genera isolated from Kovalam solar salt works in Kanyakumari of India. After initial screening, Kocuria sp. (Km), Kurthia sp. (Ku) and Halococcus sp. (Hc) were found to have positive biosurfactant activity. Biosurfactant derived from Kocuria sp. emulsified more than 50% of the crude oil, coconut oil, sunflower oil, olive oil and kerosene when compared to the other strains. Further, Kocuria marina BS-15 derived biosurfactant was purified and characterized by TLC, FTIR and GC–MS analysis. The TLC analysis revealed that, the purified biosurfactants belong to the lipopeptide group. The IR spectrum results revealed that functional groups are R2C 000000000000 000000000000 000000000000 111111111111 000000000000 111111111111 000000000000 000000000000 000000000000 NN, alkenes and N–H. The GC–MS analysis confirmed the compound as Nonanoic acid and Cyclopropane with the retention time of 12.78 and 24.65, respectively.
PMCID: PMC4250494  PMID: 25473358
Kocuria marina; Biosurfactant; Emulsification; Nonanoic acid
8.  Archaeosomes varying in lipid composition differ in receptor-mediated endocytosis and differentially adjuvant immune responses to entrapped antigen 
Archaea  2003;1(3):151-164.
Archaeosomes prepared from total polar lipids extracted from six archaeal species with divergent lipid compositions had the capacity to deliver antigen for presentation via both MHC class I and class II pathways. Lipid extracts from Halobacterium halobium and from Halococcus morrhuae strains 14039 and 16008 contained archaetidylglycerol methylphosphate and sulfated glycolipids rich in mannose residues, and lacked archaetidylserine, whereas the opposite was found in Methanobrevibacter smithii, Methanosarcina mazei and Methanococcus jannaschii. Annexin V labeling revealed a surface orientation of phosphoserine head groups in M. smithii, M. mazei and M. jannaschii archaeosomes. Uptake of rhodamine-labeled M. smithii or M. jannaschii archaeosomes by murine peritoneal macrophages was inhibited by unlabeled liposomes containing phosphatidylserine, by the sulfhydryl inhibitor N-ethylmaleimide, and by ATP depletion using azide plus fluoride, but not by H. halobium archaeosomes. In contrast, N-ethylmaleimide failed to inhibit uptake of the four other rhodamine-labeled archaeosome types, and azide plus fluoride did not inhibit uptake of H. halobium or H. morrhuae archaeosomes. These results suggest endocytosis ofarchaeosomes rich in surface-exposed phosphoserine head groups via a phosphatidylserine receptor, and energy-independent surface adsorption of certain other archaeosome composition classes. Lipid composition affected not only the endocytic mechanism, but also served to differentially modulate the activation of dendritic cells. The induction of IL-12 secretion from dendritic cells exposed to H. morrhuae 14039 archaeosomes was striking compared with cells exposed to archaeosomes from 16008. Thus, archaeosome types uniquely modulate antigen delivery and dendritic cell activation.
PMCID: PMC2685569  PMID: 15803661
antibody; archaea; cytotoxic T lymphocyte; liposomes; phagocytosis; phosphatidylserine
9.  Halorubrum chaoviator sp. nov., a haloarchaeon isolated from sea salt in Baja California, Mexico, Western Australia and Naxos, Greece 
Three halophilic isolates, strains Halo-G*T, AUS-1 and Naxos II, were compared. Halo-G* was isolated from an evaporitic salt crystal from Baja California, Mexico, whereas AUS-1 and Naxos II were isolated from salt pools in Western Australia and the Greek island of Naxos, respectively. Halo-G*T had been exposed previously to conditions of outer space and survived 2 weeks on the Biopan facility. Chemotaxonomic and molecular comparisons suggested high similarity between the three strains. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that the strains clustered with Halorubrum species, showing sequence similarities of 99.2–97.1 %. The DNA–DNA hybridization values of strain Halo-G*T and strains AUS-1 and Naxos II are 73 and 75 %, respectively, indicating that they constitute a single species. The DNA relatedness between strain Halo-G*T and the type strains of 13 closely related species of the genus Halorubrum ranged from 39 to 2 %, suggesting that the three isolates constitute a different genospecies. The G+C content of the DNA of the three strains was 65.5–66.5 mol%. All three strains contained C20C20 derivatives of diethers of phosphatidylglycerol, phosphatidylglyceromethylphosphate and phosphatidylglycerolsulfate, together with a sulfated glycolipid. On the basis of these results, a novel species that includes the three strains is proposed, with the name Halorubrum chaoviator sp. nov. The type strain is strain Halo-G*T (=DSM 19316T =NCIMB 14426T =ATCC BAA-1602T).
PMCID: PMC3182535  PMID: 19567575
10.  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.
PMCID: PMC241517  PMID: 16346609
11.  A traditional Japanese-style salt field is a niche for haloarchaeal strains that can survive in 0.5% salt solution 
Saline Systems  2007;3:2.
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.
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.
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.
PMCID: PMC1828056  PMID: 17346353
12.  Oceanobacillus aidingensis sp. nov., a moderately halophilic bacterium 
Antonie Van Leeuwenhoek  2014;105(5):801-808.
Two Gram-positive, rod-shaped moderately halophilic bacterial strains, designated AD7-25T and AB-11, were isolated from Aiding and Manasi salt lakes in Xinjiang of China, respectively. The strains were found to be able to grow at NaCl concentrations of 0–21 % (w/v), with optimum growth occurring at 6–8 % (w/v) NaCl. The optimal temperature and pH for growth were determined to be 33–37 °C and pH 7.0–7.5. Cells of the strains are motile by means of polar flagella. Both strains can produce ellipsoidal spores. The major cellular fatty acids were identified as anteiso-C15:0, iso-C15:0, iso-C14:0, anteiso-C17:0 and iso-C16:0. The diamino acid in the peptidoglycan and the major quinone system were determined to be meso-diaminopimelic acid (meso-DAP) and MK-7, respectively. The DNA G+C contents of stains AD7-25T and AB-11 were 39.8 and 40.0 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that these two novel strains are closely related to the genus Oceanobacillus showing 90–99.5 % similarity with respect to type strains. These two novel strains were most closely related to Oceanobacillus oncorhynchi subsp. incaldanensis DSM 16557T (99.1 and 99.5 %), followed by O. oncorhynchi subsp. oncorhynchi JCM 12661T (99.1 and 99.4 %), Oceanobacillus neutriphilus CGMCC 1.7693T (97.0 and 97.5 %), Oceanobacillus sojae JCM 15792T (97.6 and 98.0 %) and Oceanobacillus locisalsi KCTC 13253T (96.5 and 96.9 %). The DNA–DNA hybridization data indicated that DNA relatedness between strains AD7-25T and AB-11 was 91.0 %, and the genomic homology of representative strain AD7-25T with O. oncorhynchi subsp. incaldanensis DSM 16557T, O. oncorhynchi subsp. oncorhynchi JCM 12661T, O. neutriphilus CGMCC 1.7693T, O. sojae JCM 15792T and O. locisalsi KCTC 13253T were 41, 39, 20, 23 and 17 %, respectively. On the basis of phenotypic and phylogenetic distinctiveness, strains AD7-25T and AB-11 should be assigned to the genus Oceanobacillus as a new species, for which the name Oceanobacillus aidingensis sp. nov. was proposed. The type strain is AD7-25T (=CGMCC 1.9106 T = NBRC 105904T).
Electronic supplementary material
The online version of this article (doi:10.1007/s10482-014-0128-1) contains supplementary material, which is available to authorized users.
PMCID: PMC3982209  PMID: 24595860
Oceanobacillus aidingensis sp. nov; Moderate halophile; Gram-positive; Salt lake
13.  Lysis of Halobacteria in Bacto-Peptone by Bile Acids 
All tested strains of halophilic archaebacteria of the genera Halobacterium, Haloarcula, Haloferax, and Natronobacterium lysed in 1% Bacto-Peptone (Difco) containing 25% NaCl, whereas no lysis was observed with other strains belonging to archaebacteria of the genera Halococcus, Natronococcus, and Sulfolobus, methanogenic bacteria, and moderately halophilic eubacteria. Substances in Bacto-Peptone which caused lysis of halobacteria were purified and identified as taurocholic acid and glycocholic acid. High-performance liquid chromatography analyses of peptones revealed that Bacto-Peptone contained nine different bile acids, with a total content of 9.53 mg/g, whereas much lower amounts were found in Peptone Bacteriological Technical (Difco) and Oxoid Peptone. Different kinds of peptones can be used to distinguish halophilic eubacteria and archaebacteria in mixed cultures from hypersaline environments.
PMCID: PMC202585  PMID: 16347619
14.  Comparative analysis of ribonuclease P RNA structure in Archaea. 
Nucleic Acids Research  1996;24(7):1252-1259.
Although the structure of the catalytic RNA component of ribonuclease P has been well characterized in Bacteria, it has been little studied in other organisms, such as the Archaea. We have determined the sequences encoding RNase P RNA in eight euryarchaeal species: Halococcus morrhuae, Natronobacterium gregoryi, Halobacterium cutirubrum, Halobacteriurn trapanicum, Methanobacterium thermoautotrophicum strains deltaH and Marburg, Methanothermus fervidus and Thermococcus celer strain AL-1. On the basis of these and previously available sequences from Sulfolobus acidocaldarius, Haloferax volcanii and Methanosarcina barkeri the secondary structure of RNase P RNA in Archaea has been analyzed by phylogenetic comparative analysis. The archaeal RNAs are similar in both primary and secondary structure to bacterial RNase P RNAs, but unlike their bacterial counterparts these archaeal RNase P RNAs are not by themselves catalytically proficient in vitro.
PMCID: PMC145784  PMID: 8614627
15.  Halophilic Microorganisms Are Responsible for the Rosy Discolouration of Saline Environments in Three Historical Buildings with Mural Paintings 
PLoS ONE  2014;9(8):e103844.
A number of mural paintings and building materials from monuments located in central and south Europe are characterized by the presence of an intriguing rosy discolouration phenomenon. Although some similarities were observed among the bacterial and archaeal microbiota detected in these monuments, their origin and nature is still unknown. In order to get a complete overview of this biodeterioration process, we investigated the microbial communities in saline environments causing the rosy discolouration of mural paintings in three Austrian historical buildings using a combination of culture-dependent and -independent techniques as well as microscopic techniques. The bacterial communities were dominated by halophilic members of Actinobacteria, mainly of the genus Rubrobacter. Representatives of the Archaea were also detected with the predominating genera Halobacterium, Halococcus and Halalkalicoccus. Furthermore, halophilic bacterial strains, mainly of the phylum Firmicutes, could be retrieved from two monuments using special culture media. Inoculation of building materials (limestone and gypsum plaster) with selected isolates reproduced the unaesthetic rosy effect and biodeterioration in the laboratory.
PMCID: PMC4118916  PMID: 25084531
16.  Characterization of the Deoxyribonucleic Acid of Various Strains of Halophilic Bacteria 
Journal of Bacteriology  1969;99(1):248-254.
Bacteria classified as extreme halophiles, in the genera Halobacterium and Halococcus, contain deoxyribonucleic acid (DNA) which displays two components in a CsCl equilibrium density gradient. The base composition of the major DNA component ranges from 66 to 68% guanine plus cytosine (GC), whereas that of the satellite DNA comprising some 11 to 36% of the total, is between 57 and 60% GC. Purification of the bacterial cells in a CsCl density gradient and other more conventional strain purification procedures both indicated that the presence of the satellite DNA component is not a result of mixed cultures.
PMCID: PMC249995  PMID: 4979441
17.  Enteractinococcus coprophilus gen. nov., sp. nov., of the family Micrococcaceae, isolated from Panthera tigris amoyensis faeces, and transfer of Yaniella fodinae Dhanjal et al. 2011 to the genus Enteractinococcus as Enteractinococcus fodinae comb. nov. 
A novel actinobacterium, designated strain YIM 100590T, was isolated from Panthera tigris amoyensis faeces collected from Yunnan Wild Animal Park in Yunnan province, south-west China. Phylogenetic analysis based on 16S rRNA gene sequence data showed that strain YIM 100590T is a member of the family Micrococcaceae. Cells were coccoid to oval (0.7–1.5 µm in diameter) occurring singly or in clusters. Growth was observed at 10–37 °C (optimum 28 °C) and at pH 7.0–11.0 (optimum pH 8.0). The major fatty acids were iso-C15 : 0 (32.22 %), anteiso-C15 : 0 (31.64 %) and iso-C16 : 0 (17.38 %). The peptidoglycan was of A4α type (l-Lys–Gly–l-Glu). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides, dimannosyl diacylglycerol, an unknown glycolipid and two unknown phospholipids. The quinone system comprised menaquinones MK-7 (91.9 %) and MK-8 (8.3 %). The DNA G+C content of strain YIM 100590T was 56.2 mol%. Chemotaxonomic data indicated that the strain belongs to the family Micrococcaceae. On the basis of morphological and chemotaxonomic data and phylogenetic analysis, strain YIM 100590T is considered to represent a novel species of a new genus within the family Micrococcaceae, for which the name Enteractinococcus coprophilus gen. nov., sp. nov. is proposed. The type strain of Enteractinococcus coprophilus is YIM 100590T ( = DSM 24083T = JCM 17352T). Yaniella fodinae DSM 22966T was transferred to the new genus as Enteractinococcus fodinae comb. nov. (type strain G5T = DSM 22966T = JCM 17931T = MTCC 9846T).
PMCID: PMC4080730  PMID: 22228667
18.  Limnohabitans planktonicus sp. nov., and Limnohabitans parvus sp. nov., two novel planktonic Betaproteobacteria isolated from a freshwater reservoir 
Two strains II-B4T and II-D5T, isolated from the meso-eutrophic freshwater Římov Reservoir (Czech Republic) were phenotypic ally, phylogenetically and chemotaxonomically characterized. Both strains are chemoorganotrophic, facultatively anaerobic, rod-shaped, non-motiles with identical G+C contents of their DNA of 59.9 mol%. Their major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine, and their major fatty acids were C16:1ω7c/C16:1ω6c, C16:0, C18:1ω7c/C18:1ω6c and C12:0. Both strains contained Q-8 as the only respiratory quinone component. The 16S rRNA sequences of the two strains posses a similarity of 99.0%, however the DNA-DNA reassociation value was only 26.7%, and the strains can be discriminated from each other by several chemotaxonomic and biochemical traits. The phylogenetic analysis of the 16S rRNA gene sequences revealed the affiliation of both strains with the genus Limnohabitans within the family Comamonadaceae. The two investigated strains represent a narrow phylogenetic cluster (so-called R-BT065 cluster) formed by a large number of environmental sequences obtained from the pelagic zones of various freshwater habitats. We propose to place the two strains in separate new species within the genus Limnohabitans. The two proposed species are Limnohabitans planktonicus sp. nov. with the type strain II-D5T (= DSM 21594T = CIP 109844T), and Limnohabitans parvus sp. nov. with the type strain II-B4T (= DSM 21592T = CIP 109845T).
PMCID: PMC3091486  PMID: 20061501
19.  High quality draft genome sequence of the slightly halophilic bacterium Halomonas zhanjiangensis type strain JSM 078169T (DSM 21076T) from a sea urchin in southern China 
Standards in Genomic Sciences  2014;9(3):1020-1030.
Halomonas zhanjiangensis Chen et al. 2009 is a member of the genus Halomonas, family Halomonadaceae, class Gammaproteobacteria. Representatives of the genus Halomonas are a group of halophilic bacteria often isolated from salty environments. The type strain H. zhanjiangensis JSM 078169T was isolated from a sea urchin (Hemicentrotus pulcherrimus) collected from the South China Sea. The genome of strain JSM 078169T is the fourteenth sequenced genome in the genus Halomonas and the fifteenth in the family Halomonadaceae. The other thirteen genomes from the genus Halomonas are H. halocynthiae, H. venusta, H. alkaliphila, H. lutea, H. anticariensis, H. jeotgali, H. titanicae, H. desiderata, H. smyrnensis, H. salifodinae, H. boliviensis, H. elongata and H stevensii. Here, we describe the features of strain JSM 078169T, together with the complete genome sequence and annotation from a culture of DSM 21076T. The 4,060,520 bp long draft genome consists of 17 scaffolds with the 3,659 protein-coding and 80 RNA genes and is a part of Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes (KMG) project.
PMCID: PMC4148996  PMID: 25197480
strictly aerobic; motile Gram-negative; chemoorganotrophic; slightly halophilic; Halomonadaceae
20.  Rhodoluna lacicola gen. nov., sp. nov., a planktonic freshwater bacterium with stream-lined genome 
A pure culture of an actinobacterium previously described as ‘Candidatus Rhodoluna lacicola’ strain MWH-Ta8 was established and deposited in two public culture collections. Strain MWH-Ta8T represents a free-living planktonic freshwater bacterium obtained from hypertrophic Meiliang Bay, Lake Taihu, PR China. The strain was characterized by phylogenetic and taxonomic investigations, as well as by determination of its complete genome sequence. Strain MWH-Ta8T is noticeable due to its unusually low values of cell size (0.05 µm3), genome size (1.43 Mbp), and DNA G+C content (51.5 mol%). Phylogenetic analyses based on 16S rRNA gene and RpoB sequences suggested that strain MWH-Ta8T is affiliated with the family Microbacteriaceae with Pontimonas salivibrio being its closest relative among the currently described species within this family. Strain MWH-Ta8T and the type strain of Pontimonas salivibrio shared a 16S rRNA gene sequence similarity of 94.3 %. The cell-wall peptidoglycan of strain MWH-Ta8T was of type B2β (B10), containing 2,4-diaminobutyric acid as the diamino acid. The predominant cellular fatty acids were anteiso-C15 : 0 (36.5 %), iso-C16 : 0 (16.5 %), iso-C15 : 0 (15.6 %) and iso-C14 : 0 (8.9 %), and the major (>10 %) menaquinones were MK-11 and MK-12. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and two unknown glycolipids. The combined phylogenetic, phenotypic and chemotaxonomic data clearly suggest that strain MWH-Ta8T represents a novel species of a new genus in the family Microbacteriaceae, for which the name Rhodoluna lacicola gen. nov., sp. nov. is proposed. The type strain of the type species is MWH-Ta8T ( = DSM 23834T = LMG 26932T).
PMCID: PMC4156110  PMID: 24984700
21.  Lipids of the ultra-thin square halophilic archaeon Haloquadratum walsbyi  
Archaea  2008;2(3):177-183.
The lipid composition of the extremely halophilic archaeon Haloquadratum walsbyi was investigated by thin-layer chromatography and electrospray ionization-mass spectrometry. The analysis of neutral lipids showed the presence of vitamin MK-8, squalene, carotene, bacterioruberin and several retinal isomers. The major polar lipids were phosphatidylglycerophosphate methyl ester, phosphatidylglycerosulfate, phosphatidylglycerol and sulfated diglycosyl diether lipid. Among cardiolipins, the tetra-phytanyl or dimeric phospholipids, only traces of bisphosphatidylglycerol were detected. When the cells were exposed to hypotonic medium, no changes in the membrane lipid composition occurred. Distinguishing it from other extreme halophiles of the Halobacteriaceae family, the osmotic stress did not induce the neo-synthesis of cardiolipins in H. walsbyi. The difference may depend on the three-laminar structure of the cell wall, which differs significantly from that of other Haloarchaea.
PMCID: PMC2685597  PMID: 19054744
Archaea; archaeal phospholipids; ether lipids; Halobacteriaceae
22.  Arthrobacter nitrophenolicus sp. nov. a new 2-chloro-4-nitrophenol degrading bacterium isolated from contaminated soil 
3 Biotech  2012;3(1):29-32.
Strain SJConT, a 2-chloro-4-nitrophenol (2C4NP) degrading bacterium, was isolated from soil collected from a pesticide-contaminated site in Punjab, India. The strain, which stained Gram positive, displayed a rod-coccus life cycle, and possessed a type A3α peptidoglycan (l-Lys–l-Ala3), MK-9(H2) as the major menaquinone, anteiso-C15 and iso-C15:0 as the major cellular fatty acids, and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and a glycolipid as the major polar lipids, showed morphological and chemotaxonomic properties consistent with those reported for members of the genus Arthrobacter. Phylogenetic analysis of the 16S rRNA gene sequence of strain SJConT confirmed that it was a member of this genus with Arthrobacter globiformis DSM 20124T being the closest relative (sequence similarity of 97 %). The DNA G + C content of strain SJConT was 69 ± 1 mol% and DNA homology with A. globiformis DSM 20124T was 45 %, suggesting that strain SJConT represented a novel species of the genus Arthrobacter, which we have named Arthrobacter nitrophenolicus sp. nov The type strain is SJConT (=MTCC 10104T =DSM 23165T).
PMCID: PMC3563742
Biodegradation;  2-Chloro-4-nitrophenol; 4-Nitrophenol; 3-Methyl-4-nitrophenol
23.  Isolation of Extreme Halophiles from Seawater 
Extreme halophilic bacteria were isolated from the ocean off the coast of Spain. All were gram-negative cocci. One isolate was compared to Halococcus sp. NCMB 757 and was found to have similar characteristics.
PMCID: PMC243452  PMID: 16345409
24.  Marinobacter salarius sp. nov. and Marinobacter similis sp. nov., Isolated from Sea Water 
PLoS ONE  2014;9(9):e106514.
Two non-pigmented, motile, Gram-negative marine bacteria designated R9SW1T and A3d10T were isolated from sea water samples collected from Chazhma Bay, Gulf of Peter the Great, Sea of Japan, Pacific Ocean, Russia and St. Kilda Beach, Port Phillip Bay, the Tasman Sea, Pacific Ocean, respectively. Both organisms were found to grow between 4°C and 40°C, between pH 6 to 9, and are moderately halophilic, tolerating up to 20% (w/v) NaCl. Both strains were found to be able to degrade Tween 40 and 80, but only strain R9SW1T was found to be able to degrade starch. The major fatty acids were characteristic for the genus Marinobacter including C16:0, C16:1ω7c, C18:1ω9c and C18:1ω7c. The G+C content of the DNA for strains R9SW1T and A3d10T were determined to be 57.1 mol% and 57.6 mol%, respectively. The two new strains share 97.6% of their 16S rRNA gene sequences, with 82.3% similarity in the average nucleotide identity (ANI), 19.8% similarity in the in silico genome-to-genome distance (GGD), 68.1% similarity in the average amino acid identity (AAI) of all conserved protein-coding genes, and 31 of the Karlin's genomic signature dissimilarity. A phylogenetic analysis showed that R9SW1T clusters with M. algicola DG893T sharing 99.40%, and A3d10T clusters with M. sediminum R65T sharing 99.53% of 16S rRNA gene sequence similarities. The results of the genomic and polyphasic taxonomic study, including genomic, genetic, phenotypic, chemotaxonomic and phylogenetic analyses based on the 16S rRNA, gyrB and rpoD gene sequence similarities, the analysis of the protein profiles generated using MALDI-TOF mass spectrometry, and DNA-DNA relatedness data, indicated that strains R9SW1T and A3d10T represent two novel species of the genus Marinobacter. The names Marinobacter salarius sp. nov., with the type strain R9SW1T ( =  LMG 27497T  =  JCM 19399T  =  CIP 110588T  =  KMM 7502T) and Marinobacter similis sp. nov., with the type strain A3d10T ( =  JCM 19398T  =  CIP 110589T  =  KMM 7501T), are proposed.
PMCID: PMC4157798  PMID: 25198502
25.  Thermophilic and halophilic β-agarase from a halophilic archaeon Halococcus sp. 197A 
Extremophiles  2013;17(6):931-939.
An agar-degrading archaeon Halococcus sp. 197A was isolated from a solar salt sample. The agarase was purified by hydrophobic column chromatography using a column of TOYOPEARL Phenyl-650 M. The molecular mass of the purified enzyme, designated as Aga-HC, was ~55 kDa on both SDS-PAGE and gel-filtration chromatography. Aga-HC released degradation products in the order of neoagarohexose, neoagarotetraose and small quantity of neoagarobiose, indicating that Aga-HC was a β-type agarase. Aga-HC showed a salt requirement for both stability and activity, being active from 0.3 M NaCl, with maximal activity at 3.5 M NaCl. KCl supported similar activities as NaCl up to 3.5 M, and LiCl up to 2.5 M. These monovalent salts could not be substituted by 3.5 M divalent cations, CaCl2 or MgCl2. The optimal pH was 6.0. Aga-HC was thermophilic, with optimum temperature of 70 °C. Aga-HC retained approximately 90 % of the initial activity after incubation for 1 hour at 65–80 °C, and retained 50 % activity after 1 hour at 95 °C. In the presence of additional 10 mM CaCl2, approximately 17 % remaining activity was detected after 30 min at 100 °C. This is the first report on agarase purified from Archaea.
Electronic supplementary material
The online version of this article (doi:10.1007/s00792-013-0575-z) contains supplementary material, which is available to authorized users.
PMCID: PMC3824881  PMID: 23949137
Halococcus; Thermophilic; Halophilic; β-agarase

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