Extremophiles

Zhu, Yueming; Wei, Dongsheng; Zhang, Jun; Wang, Yufan; Xu, Hengyi; Xing, Laijun; Li, Mingchun

doi: 10.1007/s00792-009-0281-zpmid: 19779856

A thermostable trehalose synthase (TreS) gene from Meiothermus ruber CBS-01 was cloned and overexpressed in Escherichia coli. The purified recombinant TreS could utilize maltose to produce trehalose, and showed an optimum pH and temperature of 6.5 and 50°C, respectively. Kinetic analysis showed that the enzyme had a twofold higher catalytic efficiency (k cat/K m) for maltose than for trehalose, indicating maltose as the preferred substrate. The TreS also had a weak hydrolytic property with glucose as the byproduct, and glucose was a strong competitive inhibitor of the enzyme. The maximum production of trehalose by the enzyme reached 65% at 20°C. The most importantly the enzyme could maintain very high activity (above 90%) at pH 4.0–8.0 and 60°C 5 h. These results provided that the stable TreS was suitable for the industrial production of trehalose from maltose in a one-step reaction.

Hallberg, Kevin; González-Toril, Elena; Johnson, D.

doi: 10.1007/s00792-009-0282-ypmid: 19787416

Phenotypic and genotypic analysis was carried out on four iron- and sulfur-oxidizing acidophilic bacteria (the “NO-37 group”) isolated from different parts of the world. 16S rRNA phylogeny showed that they are highly related to each other, but are less related to the type strain of Acidithiobacillus ferrooxidans. The NO-37 group isolates are obligate chemolithoautotrophs, facultative anaerobes, diazotrophic, and psychrotolerant. They are less tolerant of extremely low pH, and in contrast to At. ferrooxidans T, all of the NO-37 group isolates are motile. The GC contents of genomic DNA of the NO-37 group isolates were around 56 mol% and the DNA–DNA hybridization value between genomic DNA of isolate NO-37 and At. ferrooxidans T was 37%. It also appears that the bacteria of the NO-37 group have a different biochemical mechanism for oxidizing ferrous iron than At. ferrooxidans T; the gene coding for the archetypal rusticyanin (RusA) was not detected in any of the NO-37 group isolates, rather a gene coding for a homologous protein (RusB) was amplified from three of the four novel isolates. Isolates of the NO-37 group clearly belong to a species that is different to those already recognized in the genus Acidithiobacillus, for which the name Acidithiobacillus ferrivorans is proposed.

Matsunaga, Fujihiko; Takemura, Kie; Akita, Masaki; Adachi, Akinori; Yamagami, Takeshi; Ishino, Yoshizumi

doi: 10.1007/s00792-009-0284-9pmid: 19787415

The initiation step is a key process to regulate the frequency of DNA replication. Although recent studies in Archaea defined the origin of DNA replication (oriC) and the Cdc6/Orc1 homolog as an origin recognition protein, the location and mechanism of duplex opening have remained unclear. We have found that Cdc6/Orc1 binds to oriC and unwinds duplex DNA in the hyperthermophilic archaeon Pyrococcus furiosus, by means of a P1 endonuclease assay. A primer extension analysis further revealed that this localized unwinding occurs in the oriC region at a specific site, which is 12-bp long and rich in adenine and thymine. This site is different from the predicted duplex unwinding element (DUE) that we reported previously. We also discovered that Cdc6/Orc1 induces topological changes in supercoiled oriC DNA, and that this process is dependent on the AAA+ domain. These results indicate that topological alterations of oriC DNA by Cdc6/Orc1 introduce a single-stranded region at the 12-mer site, that could possibly serve as an entry point for Mcm helicase.

Kato, Tomohisa; Miyanaga, Asuka; Kanaya, Shigenori; Morikawa, Masaaki

doi: 10.1007/s00792-009-0285-8pmid: 19787414

Geobacillus thermoleovorans B23 is capable of degrading long-chain alkanes at 70°C. Bt-aldh, an aldehyde dehydrogenase gene in B23, was located in the upstream region of p21 whose expression level was dramatically increased when alkane degradation was started (Kato et al. 2009, BMC Microbiol 9:60). Like p21, transcription level of Bt-aldh was also increased upon alkane degradation. Bt-Aldh (497 aa, MW = 53,886) was overproduced in Escherichia coli, purified, and characterized biochemically. Bt-Aldh acted as an octamer, required NAD+ as a coenzyme, and showed high activity against aliphatic long-chain aldehydes such as tetradecanal. The optimum condition for activity was 50–55°C and pH 10.0. The activity was elevated to two- to threefold in the presence of 2 mM Ba2+, Ca2+, or Sr2+, while Mg2+ and Zn2+ inhibited the enzyme activity. Bt-Aldh represents thermophilic aldehyde dehydrogenases responsible for degradation of long-chain alkanes.

Sorokin, Dimitri; Muyzer, G.

doi: 10.1007/s00792-009-0283-xpmid: 19779762

A possibility of dissimilatory MnO2 reduction at extremely high salt and pH was studied in sediments from hypersaline alkaline lakes in Kulunda Steppe (Altai, Russia). Experiments with anaerobic sediment slurries demonstrated a relatively rapid reduction of colloidal MnO2 in the presence of acetate and formate as electron donor at in situ conditions (i.e., pH 10 and a salt content from 0.6 to 4 M total Na+). All reduced Mn at these conditions remained in the solid phase. A single, stable enrichment culture was obtained from the slurries consistently reducing MnO2 at pH 10 and 0.6 M total Na+ with formate. A pure culture of a haloalkaliphilic Mn-reducing bacterium obtained from the positive enrichment was phylogenetically closely related to the anaerobic haloalkaliphilic Bacillus arseniciselenatis isolated from Mono Lake (CA, USA). Bacillus sp. strain AMnr1 was obligately anaerobic, able to grow either by glucose fermentation, or respiring few nonfermentable substrates by using MnO2 as the electron acceptor. Optimal growth by dissimilatory MnO2 reduction was achieved with glycerol as electron donor at pH 9.5–10 and salt content between 0.4 and 0.8 M total Na+.

Thomas-Hall, Skye; Turchetti, Benedetta; Buzzini, Pietro; Branda, Eva; Boekhout, Teun; Theelen, Bart; Watson, Kenneth

doi: 10.1007/s00792-009-0286-7pmid: 19898737

Worldwide glaciers are annually retreating due to global overheating and this phenomenon determines the potential lost of microbial diversity represented by psychrophilic microbial population sharing these peculiar habitats. In this context, yeast strains, all unable to grow above 20°C, consisting of 42 strains from Antarctic soil and 14 strains isolated from Alpine Glacier, were isolated and grouped together based on similar morphological and physiological characteristics. Sequences of the D1/D2 and ITS regions of the ribosomal DNA confirmed the previous analyses and demonstrated that the strains belong to unknown species. Three new species are proposed: Mrakia robertii sp. nov. (type strain CBS 8912), Mrakia blollopis sp. nov. (type strain CBS 8921) and a related anamorphic species Mrakiella niccombsii sp. nov. (type strain CBS 8917). Phylogenetic analysis of the ITS region revealed that the new proposed species were closely related to each other within the Mrakia clade in the order Cystofilobasidiales, class Tremellomycetes. The Mrakia clade now contains 8 sub-clades. Teliospores were observed in all strains except CBS 8918 and for the Mrakiella niccombsii strains.

Loveland-Curtze, Jennifer; Miteva, Vanya; Brenchley, Jean

doi: 10.1007/s00792-009-0287-6pmid: 19888547

Three novel orange, ultramicrobacterial isolates, UMB10, UMB14, and UMB34T were isolated from enrichment cultures inoculated with a melted 3,043 m deep Greenland ice core sample. Phylogenetic analysis of the 16S rRNA gene sequences indicated that the isolates belonged to a single species within the genus Chryseobacterium. They were most closely related to Chryseobacterium aquaticum (99.3%), Chryseobacterium soli (97.1%), and Chryseobacterium soldanellicola (96.9%). Genomic hybridization showed low levels of relatedness between UMB34T and C. aquaticum and C. soldanellicola (19–30%) and C. soli and Chryseobacterium jejuense (45–56%). Comparative genomic fingerprinting analysis using the enterobacterial repetitive intergenic consensus (ERIC) sequence showed nearly identical banding patterns for the three isolates and these patterns were distinct from those of C. aquaticum, C. soldanellicola, C. soli, and C. jejuense. The cells were short rods, lacked flagella, had cell volumes of <0.1 μm3, formed buds and smaller protrusions (blebs), produced copious extracellular material and a flexirubin type pigment. UMB34T produced acids from carbohydrates and utilized glucose and maltose although it did not assimilate mannose. The DNA G + C was 39.6–41.6 mol%. Based on the differences from validly named Chryseobacterium species, it was concluded that these isolates represent a new species for which the name, Chryseobacterium greenlandense is proposed. The type strain is UMB34T (=CIP 110007T = NRRL B-59357).

Sorokin, D.; Detkova, E.; Muyzer, G.

doi: 10.1007/s00792-009-0288-5pmid: 19888546

Evidence on the utilization of simple fatty acids by sulfate-reducing bacteria (SRB) at extremely haloalkaline conditions are practically absent, except for a single case of syntrophy by Desulfonatronum on acetate. Our experiments with sediments from soda lakes of Kulunda Steppe (Altai, Russia) showed sulfide production with sulfate as electron acceptor and propionate and butyrate (but not acetate) as an electron donor at a pH 10–10.5 and a salinity 70–180 g l−1. With propionate as substrate, a highly enriched sulfidogenic culture was obtained in which the main component was identified as a novel representative of the family Syntrophobacteraceae. With butyrate as substrate, a pure SRB culture was isolated which oxidized butyrate and some higher fatty acids incompletely to acetate. The strain represents the first haloalkaliphilic representative of the family Desulfobacteraceae and is described as Desulfobotulus alkaliphilus sp. nov.

Yamamoto, Masahiro; Ikeda, Takeshi; Arai, Hiroyuki; Ishii, Masaharu; Igarashi, Yasuo

doi: 10.1007/s00792-009-0289-4pmid: 19894084

Hydrogenobacter thermophilus TK-6 is a thermophilic, chemolithoautotrophic, hydrogen-oxidizing bacterium that fixes carbon dioxide via the reductive tricarboxylic acid (rTCA) cycle. 2-Oxoglutarate:ferredoxin oxidoreductase (OGOR) is the key enzyme in this cycle that fixes carbon dioxide. The genome of strain TK-6 encodes at least two distinct OGOR enzymes, termed For and Kor. We report here a method for measuring the carboxylation of succinyl-CoA catalyzed by OGORs. The method involves the in vitro coupling of OGOR with ferredoxin and pyruvate:ferredoxin oxidoreductase from strain TK-6, and glutamate dehydrogenase from Sulfolobus tokodaii. Using this method, we determined both the apparent maximum velocities and the K m values of For and Kor for the carboxylation of succinyl-CoA. This is the first reported kinetic analysis of carbon fixation catalyzed by OGOR enzymes from the rTCA cycle.

Ellen, Albert; Albers, Sonja-Verena; Driessen, Arnold

doi: 10.1007/s00792-009-0290-ypmid: 19957093

Although a large number of potentially secreted proteins can be predicted on the basis of genomic distribution of signal sequence-bearing proteins, protein secretion in Archaea has barely been studied. A proteomic inventory and comparison of the growth medium proteins in three hyperthermoacidophiles, i.e., Sulfolobus solfataricus, S. acidocaldarius and S. tokodaii, indicates that only few proteins are freely secreted into the growth medium and that the majority originates from cell envelope bound forms. In S. acidocaldarius both cell-associated and secreted α-amylase activities are detected. Inactivation of the amyA gene resulted in a complete loss of activity, suggesting that the same protein is responsible for the a-amylase activity at both locations. It is concluded that protein secretion in Sulfolobus is a limited process, and it is suggested that the S-layer may act as a barrier for the free diffusion of folded proteins into the medium.