Halomontanus rarus gen. nov., sp. nov., a novel halophilic archaeon of the family Natrialbaceae from salt lakes on the Qinghai-Xizang Plateau.

Two halophilic archaeal strains TS33T and KZCA124 were isolated from two distant salt lakes on the Qinghai-Xizang Plateau, respectively. Culture-independent analysis indicated that these two strains were original inhabitants but low abundant taxa in respective salt lakes. Strains TS33T and KZCA124 were able to grow at 20-60 °C (optimum were 42 and 35 °C, respectively), with 0.9-4.8 M NaCl (optimum were 3.0 and 2.6 M, respectively), with 0-0.7 M MgCl2 (optimum, 0.3 M) and at pH 5.0-9.5 (optimum were pH 7.5 and pH 7, respectively). The 16S rRNA and rpoB' gene similarities between these two strains were 99.7% and 99.4%, and these two similarities among strains TS33T, KZCA124, and existing species of the family Natrialbaceae were 90.6-95.5% and 84.4-89.3%, respectively. Phylogenetic and phylogenomic analyses indicated that strains TS33T and KZCA124 formed an independent branch separated from neighboring genera, Saliphagus, Natronosalvus, and Natronobiforma. The averagenucleotideidentity (ANI), digital DNA-DNAhybridization (dDDH), and average amino acid identity (AAI) values between strains TS33T and KZCA124 were 96.4%, 73.1%, and 96.7%, respectively, higher than the thresholds for species demarcation. The overall genome-related indexes between these two strains and existing species of family Natrialbaceae were 73-77%, 21-25%, and 63-70%, respectively, significantly lower than the species boundary thresholds. Strains TS33T and KZCA124 may represent a novel species of a new genus within the family Natrialbaceae judged by the cutoff value of AAI (≤76%) proposed to differentiate genera within the family Natrialbaceae. The major polar lipids of strains TS33T and KZCA124 were phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, sulfated mannosyl glucosyl diether, and sulfated galactosyl mannosyl glucosyl diether. These two strains could be distinguished from the related genera according to differential phenotypic characteristics. These phenotypic, phylogenetic, and genomic analyses revealed that strains TS33T (=KCTC 4310T = MCCC 4K00132T) and KZCA124 (=CGMCC 1.17432 = JCM 34316) represent a novel species of a new genus of the family Natrialbaceae and were named Halomontanus rarus gen. nov., sp. nov.

Halobaculum halophilum sp. nov. and Halobaculum salinum sp. nov., isolated from salt lake and saline soil.

Two halophilic archaeal strains, Gai3-2T and NJ-3-1T, were isolated from salt lake and saline soil samples, respectively, collected in PR China. The 16S rRNA gene sequences of the two strains were 97.5% similar to each other. Strains Gai3-2T and NJ-3-1T had the highest sequence similarities to 'Halobonum tyrrellense' G22 (96.7 and 97.8%, respectively), and displayed similarities of 91.5-93.5% and 92.3-94.7%, respectively, to Halobaculum members. Phylogenetic analysis revealed that the two strains formed different branches and clustered tightly with 'H. tyrrellense' G22 and Halobaculum members. The average nucleotide identity (ANI), in silico DNA-DNA hybridization (isDDH) and amino acid identity (AAI) values between the two strains were 83.1, 26.9 and 77.9%, respectively, much lower than the threshold values proposed as a species boundary. These values between the two strains and 'H. tyrrellense' G22 (ANI 77.9-78.2%, isDDH 22.5-22.6% and AAI 68.8-69.3%) and Halobaculum members (ANI 77.53-77.63%, isDDH 21.8-22.3% and AAI 68.4-69.4%) were almost identical, and much lower than the recommended threshold values for species delimitation. These results suggested that strains Gai3-2T and NJ-3-1T represent two novel species of Halobaculum. Based on phenotypic, chemotaxonomic and phylogenetic properties, strains Gai3-2T (=CGMCC 1.16080T=JCM 33550T) and NJ-3-1T (=CGMCC 1.16040T=JCM 33552T) represent two novel species of the genus Halobaculum, for which the name Halobaculum halophilum sp. nov. and Halobaculum salinum sp. nov. are proposed.

Halosimplex halophilum sp. nov. and Halosimplex salinum sp. nov., isolated from saline soil and a salt mine.

A polyphasic study was undertaken to determine the taxonomic position of two halophilic archaeal strains, TH32T and YPL4T, isolated from saline soil and a salt mine in PR China, respectively. Strains TH32T and YPL4T both have two dissimilar 16S rRNA genes. The two strains exhibited sequence similarities of 91.5-95.5 % for 16S rRNA genes and 90.9 % for the rpoB' gene. Sequence similarities of 16S rRNA genes and the rpoB' gene between the two strains and the current four members of Halosimplex were 90.6-97.4 % and 91.4-93.5 %, respectively. Phylogenetic analysis revealed that the two strains formed different branches separating them from the current Halosimplex members. Several phenotypic characteristics differentiate strains TH32T and YPL4T from current Halosimplex members. The polar lipids of the two strains are phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and four glycolipids. Two of the glycolipids are chromatographically identical to disulfated mannosyl glucosyl diether and sulfated mannosyl glucosyl diether, respectively, and the remaining two glycolipids are unidentified. The average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) values between the two strains and the current members of Halosimplex (ANI 80.4-89.2 % and in silico DDH 24.0-41.8 %) were much lower than the threshold values proposed as a species boundary, suggesting that the two strains represent novel species of Halosimplex. The values between the two strains (ANI 81.3 % and in silico DDH 24.9 %) were also much lower than the recommended threshold values, which revealed that the two strains represent two genomically different species of Halosimplex. These results showed that strains TH32T (=CGMCC 1.15190T=JCM 30840T) and YPL4T (=CGMCC 1.15329T=JCM 31108T) represent two novel species of Halosimplex, for which the names Halosimplex halophilum sp. nov. and Halosimplex salinum sp. nov. are proposed.

Biochemical characterization of a low salt-adapted extracellular protease from the extremely halophilic archaeon Halococcus salifodinae.

Extracellular proteases from haloarchaea can expand the application fields of proteases. Exploring novel robust proteases is of great importance. An extracellular protease HlyA from Halococcus salifodinae was obtained by heterologous expression, affinity chromatography, in vitro refolding and gel filtration chromatography. Its activity was optimal at 45 °C, pH 9.0 and 1.5-2 M NaCl. Interestingly, although HlyA was from an extremely halophilic archaeon, it retained >75% of maximal activity in a broad NaCl concentration of 0.5-4 M. It displayed relatively stable activities over a wide range of temperature, pH and salinity. Thus, HlyA exhibited good temperature, pH and especially, salinity tolerance. Ca2+, Mg2+ and Sr2+ significantly enhanced the protease activity. HlyA activity was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), suggesting it is a serine protease. HlyA showed good tolerance to some surfactants and organic solvents. The Km and Vmax values of HlyA for azocasein were calculated to be 0.72 mM and 21.98 U/µg, respectively. HlyA was able to effectively degrade several protein substrates, including bovine hemoglobin, casein and azocasein. Generally, HlyA from the extremely halophilic archaeon Hcc. salifodinae is an alkaliphilic and low salt-adapted halolysin with high activity, thus representing an attractive candidate for various industrial uses.

Natronomonas halophila sp. nov. and Natronomonas salina sp. nov., two novel halophilic archaea.

Two halophilic archaeal strains, C90T and YPL13T, were isolated from a salt lake and a salt mine in PR China. The two strains were found to form two clusters (97.5 and 89.5 % similarity between them, respectively) separating them from the three current members of the genus Natronomonas (95.4-97.0 % and 86.6-89.3 % similarity, respectively) on the basis of the 16S rRNA and rpoB' gene sequence similarities and phylogenetic analysis. Diverse phenotypic characteristics differentiate strains C90T and YPL13T from current Natronomonas members. The polar lipids of strain C90T were phosphatidic acid, phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulphate, two unidentified glycolipids, a major glycolipid and a minor glycolipid, while those of strain YPL13T were PG, PGP-Me, two unidentified phospholipids and a glycolipid. The average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) values between the two strains were 79.8 and 27.1 %, respectively, which were much lower than the threshold values proposed as a species boundaries (ANI 95-96 % and isDDH 70 %), which revealed that the two strains represent two novel species; these values (ANI 76.6-80.0 % and isDDH 21.6-27.0 %) of the strains examined in this study and the current members of Natronomonas are much lower than the recommended threshold values, suggesting that strains C90T and YPL13T represent two genomically different species of Natronomonas. These results showed that strains C90T (=CGMCC 1.13738T=JCM 32961T) and YPL13T (=CGMCC 1.13884T=JCM 31111T) represent two novel species of Natronomonas, for which the names Natronomonas halophila sp. nov. and Natronomonas salina sp. nov. are proposed.

[Gene therapy for tongue carcinoma mediated by oral administration of attenuated Salmonella harboring tip30 and IFN-γ genes in nude mouse model].

PURPOSE: To observe the effect of gene therapy mediated by oral administration of attenuated Salmonella carrying tip30 and IFN-γ genes in human tongue carcinoma nude mouse model. METHODS: 25 four-week-old BALB/C male nude mice were divided randomly into 5 groups based on the differently harboring genes in the recombinant attenuated Salmonella typhimuriums SL7207, which included blank group (PBS), control group (SL7207), IFN treatments group (SL7207-pCI-IFN), tip30 treatment group (SL7207-pCI-tip30) and the combination of IFN and tip30 treatment group (SL7207-pCI-tip30/IFN). On 10d after submandibular subcutaneous injection of Tca8113 cells into the mice, the recombinant attenuated Salmonella typhimuriums were orally administrated 3 times at 1 week interval. The treatment effect indicators included the growth curve, the tumor inhabitation rate, the survival rate, the apoptosis typical DNA ladder and the protein expressions of tip30 and IFN-γ in tumor cells. Statistical analysis was performed with SPSS15.0 software package. RESULTS: The growth curve, the tumor inhabitation rate and the survival rate indicated that the tumor in the PBS group constantly grew up and induced the animal death in earlier time, while the SL7207 treatment had a slight inhibiting effect compared with PBS group. However, the SL7207-pCI-IFN group and the SL7207-pCI-tip30 had a moderate inhibiting effect compared with the SL7207 group, while the combination of IFN and tip30 had the strongest inhibiting effect. The protein expressions of tip30 and IFN-γ were detected in tumor cells while the typical DNA ladders of apoptosis were observed only in the tip30 gene transfer groups (SL7207-pCI-tip30 and SL7207-pCI-tip30/IFN). CONCLUSIONS: The recombinant attenuated Salmonella typhimurium possesses a capacity of gene transfer in vivo through oral administration. The combination of the harboring tip30 and IFN-γ genes have synergistic inhibiting tumor effect for tongue carcinoma.

[Induction of apoptosis and related genes by five kinds of dental materials on L929 cell].

OBJECTIVE: To evaluate a new type of diatomite-based machinable ceramic biocompatibility by studying its induced apoptosis on L929 cell in contrasted with other prosthodontics materials. METHODS: Cell line was treated with extracting liquid containing different concentrations of diatomite-based machinable ceramic and other materials. Flow cytometry tested cell cycle progression and induced cell apoptosis. Annexin V-FITC/PI apoptosis staining kit quantitative detected cell death patterns. The expression of Bcl-2 and Bax mRNA were determined by reverse transcription-polymerase chain reaction. RESULTS: The experimental groups had no special influence on cell cycle. Apoptosis rates of the new ceramic closed to the negative group (P > 0.05). The apoptosis rate of resin was the highest, and the cell necrosis level of resin was increased, which had significant difference to the new ceramic (P < 0.05). The Bcl-2 and Bax mRNA levels of the new ceramic and the negative group were closed to each other, which had no statistical significance (P > 0.05). CONCLUSION: The new diatomite-based machinable ceramic has no apparent cytotoxicity, which is consistent with the clinical application of the basic requirements of biocompatibility.