Crystal structure of an inulosucrase from Halalkalicoccus jeotgali B3T, a halophilic archaeal strain.

Several archaea harbor genes that code for fructosyltransferase (FTF) enzymes. These enzymes have not been characterized yet at structure-function level, but are of extreme interest in view of their potential role in the synthesis of novel compounds for food, nutrition, and pharmaceutical applications. In this study, 3D structure of an inulin-type fructan producing enzyme, inulosucrase (InuHj), from the archaeon Halalkalicoccus jeotgali was resolved in its apo form and with bound substrate (sucrose) molecule and first transglycosylation product (1-kestose). This is the first crystal structure of an FTF from halophilic archaea. Its overall five-bladed ß-propeller fold is conserved with previously reported FTFs, but also shows some unique features. The InuHj structure is closer to those of Gram-negative bacteria, with exceptions such as residue E266, which is conserved in FTFs of Gram-positive bacteria and has possible role in fructan polymer synthesis in these bacteria as compared to fructooligosaccharide (FOS) production by FTFs of Gram-negative bacteria. Highly negative electrostatic surface potential of InuHj, due to a large amount of acidic residues, likely contributes to its halophilicity. The complex of InuHj with 1-kestose indicates that the residues D287 in the 4B-4C loop, Y330 in 4D-5A, and D361 in the unique α2 helix may interact with longer FOSs and facilitate the binding of longer FOS chains during synthesis. The outcome of this work will provide targets for future structure-function studies of FTF enzymes, particularly those from archaea.

Physiological response to silver toxicity in the extremely halophilic archaeon Halomicrobium mukohataei.

Adaptive strategies responsible for heavy metal tolerance were explored in the extremely halophilic archaeon Halomicrobium mukohataei DSM 12286. The tested strain was seemingly able to overcome silver-induced oxidative stress (assessed by malondialdehyde quantification, catalase assay and total antioxidant capacity measurement) mainly through non-enzymatic antioxidants. Energy dispersive spectrometry analysis illustrated the presence of colloidal silver in Hmc. mukohataei cultures exposed to AgNO3. Bright-field and transmission electron microscopy images, as well as dynamic light scattering analysis, demonstrated the presence of intracellular nanoparticles, mostly spherical, within a size range of 20-100 nm. As determined by the zeta potential measurement, the biosynthesized nanoparticles were highly stable, with a negative surface charge. Our research is a first attempt in the systematic study of the oxidative stress and intracellular silver nanoparticle accumulation, generated by exposure to silver ions, in members of Halobacteria class, thus broadening our knowledge on mechanisms supporting heavy metal tolerance of microbial cells living under saline conditions.

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 3757T 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 3754T and Hfx. volcanii DSM 3757T , cells adhered within 1 day of culture and remained viable for at least 2 months in mature biofilms. Adherent cells of Hbt. salinarum DSM 3754T 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.

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 (1)H-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 1307(T), Halococcus saccharolyticus DSM 5350(T), Halococcus salifodinae DSM 8989(T), Halococcus dombrowskii DSM 14522(T), Halococcus hamelinensis JCM 12892(T), Halococcus qingdaonensis JCM 13587(T)), Halorubrum sp. (Hrr. coriense DSM 10284(T), Halorubrum chaoviator DSM 19316(T), Hrr. chaoviator strains NaxosII and AUS-1), haloalkaliphiles (Natronobacterium gregoryi NCMB 2189(T), Natronococcus occultus DSM 3396(T)) and Halobacterium noricense DSM 9758(T). No PHB was detected in Halobacterium salinarum NRC-1 ATCC 700922, Hbt. salinarum R1 and Haloferax volcanii DSM 3757(T). 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.

Two immunologically distinct types of protofilaments can be identified in Natrialba magadii flagella.

We examine distribution of flagellins along multicomponent flagellar filaments (FF) and protofilaments (PF) of the haloalkaliphilic archaeon Natrialba magadii using immunogold electron microscopy. A high specific polyclonal antibody raised to one of the flagellin types bound homogeneously to the undissociated FF along the full length. At the same time both uniformly labelled and completely unlabelled PF, outwardly indistinguishable one from another, were observed.

Natrialba hulunbeirensis sp. nov. and Natrialba chahannaoensis sp. nov., novel haloalkaliphilic archaea from soda lakes in Inner Mongolia Autonomous Region, China.

Two haloalkaliphilic archaeal strains, X21T and C112T, were isolated from soda lakes in Inner Mongolia Autonomous Region, China. Their morphology, physiology, biochemical features, polar lipid composition and 16S rRNA genes were characterized in order to elucidate their taxonomy. According to these data, strains X21T and C112T belong to the genus Natrialba, although there are clear differences with respect to their physiology and polar lipid composition between the two strains and the type species, Natrialba asiatica. On the basis of low DNA-DNA hybridizations, these two strains should be considered as new species of genus Natrialba. The names Natrialba hulunbeirensis sp. nov. (type strain X21T = AS 1.1986T = JCM 10989T) and Natrialba chahannaoensis sp. nov. (type strain C112T = AS 1.1977T = JCM 10990T) are proposed.

Natrinema versiforme sp. nov., an extremely halophilic archaeon from Aibi salt lake, Xinjiang, China.

A novel extremely halophilic archaeon, strain XF10T, was isolated from a salt lake in China. This organism was neutrophilic, non-motile and pleomorphic, and was rod, coccus or irregularly shaped. It required at least 1.5 M NaCl for growth and grew in a wide range of MgCl2 concentrations (0.005-0.5 M). Lipid extract of whole cells contained two glycolipids with the same chromatographic properties as two unidentified glycolipids found in the two described Natrinema species, Natrinema pellirubrum and Natrinema pallidum. Phylogenetic analysis based on 16S rDNA sequence comparison revealed that strain XF10T clustered with the two described Natrinema species and several other strains (strains T5.7, GSL-11 and Haloterrigena turkmenica JCM 9743) with more than 98.1% sequence similarities, suggesting that strain XF1OT belongs to the genus Natrinema. Comparative analysis of phenotypic properties and DNA-DNA hybridization between strain XF10T and the Natrinema species supported the conclusion that strain XF10T is a novel species within the genus Natrinema. The name Natrinema versiforme sp. nov. is proposed for this strain. The type strain is XF10T (=JCM 10478T=AS 1.2365T=ANMR 0149T).

Halorhabdus utahensis gen. nov., sp. nov., an aerobic, extremely halophilic member of the Archaea from Great Salt Lake, Utah.

Strain AX-2T (T = type strain) was isolated from sediment of Great Salt Lake, Utah, USA. Optimal salinity for growth was 27% (w/v) NaCl and only a few carbohydrates supported growth of the strain. Strain AX-2T did not grow on complex substrates such as yeast extract or peptone. 16S rRNA analysis revealed that strain AX-2T was a member of the phyletic group defined by the family Halobacteriaceae, but there was a low degree of similarity to other members of this family. The polar lipid composition comprising phosphatidyl glycerol, the methylated derivative of diphosphatidyl glycerol, triglycosyl diethers and sulfated triglycosyl diethers, but not phosphatidyl glycerosulfate, was not identical to that of any other aerobic, halophilic species. On the basis of the data presented, it is proposed that strain AX-2T should be placed in a new taxon, for which the name Halorhabdus utahensis is appropriate. The type strain is strain AX-2T (= DSM 12940T).

Halogeometricum borinquense gen. nov., sp. nov., a novel halophilic archaeon from Puerto Rico.

A novel extremely halophilic archaeon was isolated from the solar salterns of Cabo Rojo, Puerto Rico. The organism is very pleomorphic, motile and requires at least 8% (w/v) NaCl to grow. Polar lipid composition revealed the presence of a novel non-sulfate-containing glycolipid and the absence of the glycerol diether analogue of phosphatidylglycerosulfate. The G + C content of the DNA is 59 mol%. On the basis of 16S rRNA sequence data, the new isolate cannot be classified in one of the recognized genera, but occupies a position that is distantly related to the genus Haloferax. All these features justify the creation of a new genus and a new species for the family Halobacteriaceae, order Halobacteriales. The name Halogeometricum borinquense gen. nov., sp. nov. is proposed. The type strain is ATCC 700274T.

Crystallography of ribosomes: attempts at decorating the ribosomal surface.

Crystals of various ribosomal particles, diffracting best to 2.9 A resolution were grown. Crystallographic data were collected from shock frozen crystals with intense synchrotron radiation at cryo temperature. For obtaining phase information, monofunctional reagents were prepared from an undecagold and a tetrairidium cluster, by attaching to them chemically reactive handles, specific for sulfhydryl moieties. Heavy-atom derivatives were prepared by a specific and quantitative binding of the undecagold cluster to an exposed sulfhydryl prior to the crystallization. To create potential binding sites on the halophilic and thermophilic ribosomal particles, which yield our best and most interesting crystals, exposed reactive moieties were inserted, using genetic and chemical procedures. In order to choose the appropriate locations for these insertions, the surfaces of the ribosomal particles were mapped by direct chemical determination of exposed amino and sulfhydryl groups.

[New species of Haloarcula].

Three strains of pleomorphic halobacteria (A5, B2 and B-B2) were isolated from salt lakes of Xinjiang, China. Based on the Bergey's Manual of Systematic Bacteriology, Volume 3 (1989) and the analytical results of polar lipids, the three strains could be included in the genus Haloarcula. But since they differ from normally accepted two species of this genus in cell shape and physiological and biochemical features, it is suggested that the three strains belong to a new species, called Haloarcu la aidinensis sp. nov.

Phototactic behaviour of the archaebacterial Natronobacterium pharaonis.

Natronobacterium pharaonis can react tactically to photo- and chemostimuli. It moves by rotation of a flagellar bundle which is monopolarly inserted. Under sufficient oxygen supply the photophobic response of N. pharaonis has been measured. The resulting action spectrum matches the absorption spectrum of the purified retinylidene protein psR-II. Retical synthesis could be inhibited by nicotine. Cells grown in the presence of nicotine show a strongly reduced photoresponse, which could be restored by addition of retinal. These data identify psR-II as the receptor for negative phototaxis.