Genomics-based identification of a cold adapted clade in Deinococcus.

BACKGROUND: Microbes in the cold polar and alpine environments play a critical role in feedbacks that amplify the effects of climate change. Defining the cold adapted ecotype is one of the prerequisites for understanding the response of polar and alpine microbes to climate change. RESULTS: Here, we analysed 85 high-quality, de-duplicated genomes of Deinococcus, which can survive in a variety of harsh environments. By leveraging genomic and phenotypic traits with reverse ecology, we defined a cold adapted clade from eight Deinococcus strains isolated from Arctic, Antarctic and high alpine environments. Genome-wide optimization in amino acid composition and regulation and signalling enable the cold adapted clade to produce CO2 from organic matter and boost the bioavailability of mineral nitrogen. CONCLUSIONS: Based primarily on in silico genomic analysis, we defined a potential cold adapted clade in Deinococcus and provided an updated view of the genomic traits and metabolic potential of Deinococcus. Our study would facilitate the understanding of microbial processes in the cold polar and alpine environments.

Halocin H4 is activated through cleavage by halolysin HlyR4.

Halocins are antimicrobial peptides secreted by haloarchaea capable of inhibiting the growth of other haloarchaea or bacteria. Halocin H4 (HalH4) is secreted by the model halophilic archaeon Haloferax mediterranei ATCC 33500. Despite attempts to express halH4 heterologously in Escherichia coli and subsequent careful renaturation procedures commonly employed for haloarchaeal proteins, no active halocin was obtained. However, it was discovered that the antihaloarchaeal activity of this halocin could be activated through cleavage by halolysin R4 (HlyR4), a serine protease also secreted by Hfx. mediterranei ATCC 33500. Replacement of the cysteine at the number 115 amino acid with glycine and deletion of the internal trans-membrane region (15 aa) markedly abolished HalH4's antihaloarchaeal activity. Compared to the N-terminus, the C-terminal amino acid sequence was found to be more crucial for HalH4 to exert its antihaloarchaeal activity. Mass spectrometry analysis revealed that the biologically active antihaloarchaeal peptide produced after hydrolytic cleavage by HlyR4 was the C-terminus of HalH4, suggesting a potential mechanism of action involving pore formation within competitor species' cell membranes. Taken together, this study offers novel insights into the interplay between halocins and secreted proteases, as well as their contribution to antagonistic interaction within haloarchaea. IMPORTANCE: The antihaloarchaeal function of halocin H4 (HalH4) can be activated by extracellular proteases from haloarchaea, as demonstrated in this study. Notably, we report the first instance of halocin activation through proteolytic cleavage, highlighting its significance in the field. The C-terminus of HalH4 (CTH4) has been identified as the antihaloarchaeal peptide present in hydrolysates generated by HlyR4. The CTH4 exhibited inhibitory activity against a range of haloarchaeal species (Haloarchaeobius spp., Haloarcula spp., Haloferax spp., Halorubellus spp., and Halorubrum spp.), as well as selected bacterial species (Aliifodinibius spp. and Salicola spp.), indicating its broad-spectrum inhibitory potential across domains. The encoding gene of halocin HalH4, halH4, from the model halophilic archaeon Haloferax mediterranei ATCC 33500 can be expressed in Escherichia coli without codon optimization.

Genomic, Physiological, Biochemical, and Phenotypic Evidences Reveal a New Species, Halomicroarcula salaria sp. nov.

An extremely halophilic archaeon strain named FL173T was isolated from a salt mine (Anhui Province, China). Colonies on agar plate are orange-red, moist, and opaque. Cells are motile, Gram-stain-negative, polymorphic, and lyse in distilled water. Cells are able to grow at temperatures, NaCl concentrations, and pH ranging from 20 to 50 °C (optimum 42 °C), 2.6 to 5.1 M NaCl concentration (optimum 3.4 M), and 5.5 to 9.5 pH (optimum 7.0), respectively. Mg2+ is not necessary for growth. The major polar lipids of strain FL173T were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulfonate (PGS), sulfonated mannosyl glycolipid (S-DGD-1). It has two copies of the 16S rRNA gene, which share the highest sequence similarity (93.04-99.02% sequence similarity) to the 16S rRNA genes of Halomicroarcula salinisoli F24AT, respectively. The rpoB' gene of strain FL173T showed the highest sequence similarity (93.76%) to that of H. salinisoli F24AT. The genome-based analysis showed that the average amino-acid identity (AAI), orthologous average nucleotide identity (ANI) and in silico DNA-DNA hybridization values between strains FL173T and H. salinisoli F24AT were 84.80%, 85.29%, and 29.70%, respectively, which are far below the threshold for the delineation of a prokaryotic new species. The DNA G+C content of strain FL173T is 64.9%. Genomic, physiological, biochemical, and phenotypic evidences showed that strain FL173T (CGMCC 1.18851=NBRC 114260) represents a new species of the genus Halomicroarcula, for which the name Halomicroarcula salaria sp. nov. is proposed.

Genomic analyses reveal a low-temperature adapted clade in Halorubrum, a widespread haloarchaeon across global hypersaline environments.

BACKGROUND: Cold-adapted archaea have diverse ecological roles in a wide range of low-temperature environments. Improving our knowledge of the genomic features that enable psychrophiles to grow in cold environments helps us to understand their adaptive responses. However, samples from typical cold regions such as the remote Arctic and Antarctic are rare, and the limited number of high-quality genomes available leaves us with little data on genomic traits that are statistically associated with cold environmental conditions. RESULTS: In this study, we examined the haloarchaeal genus Halorubrum and defined a new clade that represents six isolates from polar and deep earth environments ('PD group' hereafter). The genomic G + C content and amino acid composition of this group distinguishes it from other Halorubrum and the trends are consistent with the established genomic optimization of psychrophiles. The cold adaptation of the PD group was further supported by observations of increased flexibility of proteins encoded across the genome and the findings of a growth test. CONCLUSIONS: The PD group Halorubrum exhibited denser genome packing, which confers higher metabolic potential with constant genome size, relative to the reference group, resulting in significant differences in carbon, nitrogen and sulfur metabolic patterns. The most marked feature was the enrichment of genes involved in sulfur cycling, especially the production of sulfite from organic sulfur-containing compounds. Our study provides an updated view of the genomic traits and metabolic potential of Halorubrum and expands the range of sources of cold-adapted haloarchaea.

Hly176B, a low-salt tolerant halolysin from the haloarchaeon Haloarchaeobius sp. FL176.

Extracellular proteases of haloarchaea can adapt to high concentrations of NaCl and can find useful applications in industrial or biotechnology processes where hypersaline conditions are desired. The diversity of extracellular proteases produced by haloarchaea is largely unknown though the genomes of many species have been sequenced and are publicly available. In this study, a gene encoding the extracellular protease Hly176B from the haloarchaeon Haloarchaeobius sp. FL176 was cloned and expressed in Escherichia coli. A related gene homolog to hly176B, hly176A, from the same strain was also expressed in E.coli, but did not show any proteinase activity after the same renaturation process. Therefore, we focus on the enzymatic properties of the Hly176B. The catalytic triad Asp-His-Ser was confirmed via site-directed mutagenesis, indicating that Hly176B belongs to the class of serine proteases (halolysin). Unlike previously reported extracellular proteases from haloarchaea, the Hly176B remained active for a relatively long time in an almost salt-free solution. In addition, the Hly176B displayed prominent tolerance to some metal ions, surfactants and organic solvents, and exerts its highest enzyme activity at 40 °C, pH 8.0 and 0.5 M NaCl. Therefore, this study enriches our knowledge of extracellular proteases and expands their applications for various industrial uses.

Halorhabdus amylolytica sp. nov. and Halorhabdus salina sp. nov., isolated from hypersaline environments.

Two novel extremely halophilic archaeal strains, designated H27T and FL145T, were isolated from a salt mine and a kelp salt sample, respectively. Cells of both strains were Gram-stain-negative, motile and pleomorphic. The 16S rRNA and rpoB' gene sequence similarities between strains H27T and FL145T were 96.60 and 88.77%. Strains H27T and FL145T were both closely related to Halorhabdus rudnickae WSM-64T, Halorhabdus tiamatea SARL4BT and Halorhabdus utahensis AX-2T, with a 16S rRNA gene sequence similarities of 98.14, 96.34 and 96.27% for strain H27T and 96.42, 95.82 and 96.17% for strain FL145T. The genome-based average nucleotide identity (ANI) values between strains H27T and FL145T, and these three species were 83.93, 79.79 and 79.09% (for strain H27T), and 78.32, 77.95 and 77.05% (for strain FL145T), respectively. The ANI value between strains H27T and FL145T was 78.65 %. The digital DNA-DNA hybridization values between strains H27T and FL145T, and these three species were less than 27.40%, which were below the recommended threshold for membership of the same species. The major polar lipids of both strains were found to consist of sulfated diglycosyl diether, triglycosyl diether, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol. The DNA G+C content was determined from genome to be 62.10 mol% for strain H27T and 61.51 mol% for strain FL145T. Based on phylogenetic, phenotypic, chemotaxonomic and genomic analyses, these two new isolates should be classified as representing two novel species in the genus Halorhabdus, with strain H27T (=CGMCC 1.16342T=NBRC 113589T) as the type strain of a new species for which we propose the name Halorhabdus amylolytica sp. nov., and strain FL145T (=CGMCC 1.13888T=NBRC 114260T) as the type strain of another new species for which we propose the name Halorhabdus salina sp. nov.

Structural asymmetry does not indicate hemiphosphorylation in the bacterial histidine kinase CpxA.

Histidine protein kinases (HKs) are prevalent prokaryotic sensor kinases that are central to phosphotransfer in two-component signal transduction systems, regulating phosphorylation of response regulator proteins that determine the output responses. HKs typically exist as dimers and can potentially autophosphorylate at each conserved histidine residue in the individual protomers, leading to diphosphorylation. However, analyses of HK phosphorylation in biochemical assays in vitro suggest negative cooperativity, whereby phosphorylation in one protomer of the dimer inhibits phosphorylation in the second protomer, leading to ∼50% phosphorylation of the available sites in dimers. This negative cooperativity is often correlated with an asymmetric domain arrangement, a common structural characteristic of autophosphorylation states in many HK structures. In this study, we engineered covalent dimers of the cytoplasmic domains of Escherichia coli CpxA, enabling us to quantify individual species: unphosphorylated, monophosphorylated, and diphosphorylated dimers. Together with mathematical modeling, we unambiguously demonstrate no cooperativity in autophosphorylation of CpxA despite its asymmetric structures, indicating that these asymmetric domain arrangements are not linked to negative cooperativity and hemiphosphorylation. Furthermore, the modeling indicated that many parameters, most notably minor amounts of ADP generated during autophosphorylation reactions or present in ATP preparations, can produce ∼50% total phosphorylation that may be mistakenly attributed to negative cooperativity. This study also establishes that the engineered covalent heterodimer provides a robust experimental system for investigating cooperativity in HK autophosphorylation and offers a useful tool for testing how symmetric or asymmetric structural features influence HK functions.

VOLN27B: A New Head-Tailed Halovirus Isolated from an Underground Salt Crystal and Infecting Halorubrum.

A novel halovirus, VOLN27B, was isolated from a drill core sample taken at a depth of approximately 430 m, from a layer formed during the Cretaceous period (Anhui, China). VOLN27B infects the halophilic archaeon Halorubrum sp. LN27 and has a head-tailed morphotype with a contractile tail, typical of myoviruses. The average head diameter is 64 ± 2.0 nm, and uncontracted tails are 15 ± 1.0 × 65 ± 2.0 nm. The latent period is about 10 h. The maturing time of VOLN27B in cells of Halorubrum sp. LN27 was nearly 8 h. The adsorption time of VOLN27B on cells of Halorubrum sp. LN27 was less than 1 min. Virus particles are unstable at pH values less than 5 or when the NaCl concentration is below 12% (w/v). VOLN27B and Halorubrum sp. LN27 were recovered from the same hypersaline environment and provide a new virus-host system in haloarchaea.

Halolysin R4 of Haloferax mediterranei confers its host antagonistic and defensive activities.

Halolysins, which are subtilisin-like serine proteases of haloarchaea, are usually secreted into the extracellular matrix via the twin-arginine translocation pathway. A small number of activated molecules can greatly affect cell growth owing to their proteolytic activity. It is, however, unclear as to whether this proteolysis-based growth inhibition by halolysins conveys antagonistic or defensive effects against other resident abd potentially competitive microorganisms. Here, we report that halolysin R4 (HlyR4), encoded by the hlyR4 gene, is the key enzyme in the initial steps of extracellular protein utilization in Haloferax mediterranei HlyR4 shows significant antagonistic activity against other haloarchaeal strains. Deletion of hlyR4 completely halts the inhibition activity of Hfx. mediterranei towards other haloarchaea, while correspondingly, complementation of hlyR4 almost completely restores the inhibition activity. Furthermore, Hfx. mediterranei strains containing hlyR4 showed a certain amount of resistance to halocins and halolysins in milieu, and this function of hlyR4 is reproducible in Haloarcula hispanica The versatility of HlyR4 enables its host to outcompete other haloarchaea living in the same hypersaline environment. Intriguingly, unlike the growth phase-dependent halolysins SptA and Nep, it is likely that HlyR4 may be secreted independent of growth phase. This study provides a new peptide antibiotics candidate in haloarchaea, as well as new insight towards a better understanding of the ecological roles of halolysins.Importance: This study shows that halolysin R4 from Haloferax mediterranei provides its host antagonistic and defensive activities against other haloarchaea, which expands our knowledge on the traditional function of haloarchaeal extracellular proteases. Haloarchaeal extracellular serine proteases have been previously discussed as growth-phase-dependent proteins, whereas our study reports constitutive expression of halolysin R4. This work also clearly reveals a hidden diversity of extracellular proteases from haloarchaea. Studies on multifunctional halolysins reveal that they play an important ecological role in shaping microbial community composition and provide a new perspective towards understanding the intricate interactions between haloarchaeal cells in hypersaline environments. HlyR4 can lyse competing cells living in the same environment, and the cell debris may probably be utilized as nutrients, which may constitute an important part of nutrient cycling in extremely hypersaline environments.

Halomicroarcula amylolytica sp. nov., a novel halophilic archaeon isolated from a salt mine.

A halophilic archaeon named strain LR21T was isolated from a salt mine in Yunnan Province, PR China. Cells were spherical, Gram-stain-negative and motile. Strain LR21T grew at 20-50 °C (optimum, 42 °C), with 8-30 % (w/v) NaCl (optimum, 23 %) and at pH 5.5-9.5 (optimum, pH 7.5-8.5). Mg2+ was not required for growth. The major polar lipid profile comprised phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate. Strain LR21T had two dissimilar 16S rRNA genes (rrnA and rrnB) and they were closely related to Halomicroarcula limicola YGHS32T, Hma. pellucida BNERC31T and Hma. salina YGHS18T with sequence similarities of 95.3-99.0, 93.0-96.2 and 93.2-95.9 %, respectively, and much lower values to other members. The rpoB' gene sequence similarities between strain LR21T and Hma. limicola YGHS32T, Hma. pellucida BNERC31T and Hma. salina YGHS18T were 95.2, 91.2 and 91.2 % respectively. The values of average nucleotide identity (ANI) and average amino-acid identity (AAI) between strain LR21T and Hma. limicola YGHS32T, were 89.0 and 90.1 %, respectively. DNA relatedness between strains LR21T and Hma. limicola YGHS32T determined by in silico DNA-DNA hybridization was 36.8 %. Values of ANI and AAI between strain LR21T and other members in the genus Halomicroarcula were far below 95 % and the DNA-DNA relatedness values between strain LR21T and its close relatives were much lower than 70 %, which is far below the boundary for delineation of a new species prokaryote. The DNA G+C content of strain LR21T was 62.0 mol% (genome). The results suggested that strain LR21T represents a novel species of the genus Halomicroarcula, for which the name Halomicroarcula amylolytica sp. nov. is proposed. The type strain is LR21T (=CGMCC 1.13611T=NBRC 113588T).

Halomicrococcus hydrotolerans gen. nov., sp. nov., an extremely halophilic archaeon isolated from a subterranean salt deposit.

A halophilic archaeon, strain H22T, was isolated from a subterranean salt deposit sampled at Yunnan salt mine, PR China. Colonies of strain H22T were light pink-pigmented. Cells were coccus, non-motile, Gram-stain-negative, and did not lyse in distilled water. The strain was aerobic and grew at 20-55 °C (optimum, 37 °C), in the presence of 10-30 % (w/v) NaCl (20 %) and at pH 6.5-9.0 (pH 7.0). Mg2+ was required for growth (optimum, 0.005 M). Major polar lipids were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and sulfated mannosyl-glucosyl-glycerol diether-1. Sequence similarity search based on the multiple 16S rRNA genes (rrnA, rrnB and rrnC) of strain H22T revealed that it was most closely related to species of the genera Haloarchaeobius, Haladaptatus, Halorussus and Halorubellus with relative low sequence similarities (91.9-93.7 %). The strain, however, shared highest rpoB' gene sequence identities with Halorussus rarus TBN4T (90.8 % rpoB' gene sequence similarity). Phylogenetic trees based on 16S rRNA and rpoB' gene sequences revealed a robust lineage of the strain H22T with members of related genera of the family Halobacteriaceae. The DNA G+C content of strain H22T was 62.9 mol%. Genome-based analysis of average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) between strains H22T and its closest relative were equal or lower than 77.7 and 22.4 %, respectively, which were far below the threshold for delineation of a new species. Based on ANI values, in silico DDH, and distinct morphological and physiological differences from the previously described taxa, we suggest that strain H22T represents a novel species of a new genus within the family Halobacteriaceae, for which the name Halomicrococcus hydrotolerans gen. nov., sp. nov. is proposed. The type strain is H22T (=CGMCC 1.16291T=NBRC 113231T).

Halobellus captivus sp. nov., an extremely halophilic archaeon isolated from a subterranean salt mine.

An extremely halophilic archaeon, strain ZY21T, was isolated from a subterranean rock salt sample in Yunnan, China. Colonies of strain ZY21T on nutrient-rich agar plates are orange, wet and transparent. Cells are pleomorphic, motile, Gram-stain negative and lyse in distilled water. Cells can grow at 20-55 °C (optimum 42 °C), in the presence of 15-30% (w/v) NaCl (optimum 18-20%) and at pH 6.0-9.5 (optimum 7.5). Mg2+ is required for growth (optimum 0.3 M). The major polar lipids of strain ZY21T are phosphatidylglycerol, phosphatidylglycerol sulfate and phosphatidylglycerol phosphate methyl ester, sulfated mannosyl-glucosyl-glycerol diether-1 and seven unidentified glycolipids. Sequence similarity searches with the 16S rRNA gene and rpoB' gene showed that strain ZY21T is closely related to Halobellus rufus CBA1103T (sequence similarities: 97.5% for 16S rRNA gene and 93.3% for rpoB' gene). The DNA G+C content of strain ZY21T was determined to be 63.0 mol% based on the draft genome sequence. Genome-based sequence similarity analysis showed that the values of the ANI, AAI, and DDH were far below the boundary for delineation of new species. Phenotypic, chemotaxonomic characteristics and phylogenetic properties suggest that strain ZY21T represents a novel species in the genus Halobellus, for which the name Halobellus captivus sp. nov. is proposed. The type strain is ZY21T (= CGMCC 1.16343T = NBRC 113439T).

Postoperative chemoradiotherapy is superior to postoperative chemotherapy alone in squamous cell lung cancer patients with limited N2 lymph node metastasis.

BACKGROUND: The aim of the present study was to assess the efficacy of postoperative chemoradiotherapy (POCRT) following surgery in non-small-cell lung cancer patients with N2 lymph node metastasis (N2-NSCLC). METHODS: The clinical data of patients with N2-NSCLC treated with POCRT or postoperative chemotherapy (pCT) alone were retrospectively collected and reviewed. The overall survival (OS) rates were analyzed utilizing the Kaplan-Meier method and compared by the log-rank test. Cox regression analysis was used to determine factors significantly associated with survival. Propensity score matching (PSM) analysis was used to compensate for differences in baseline characteristics and OS was compared after matching. RESULTS: Between 2004 and 2014, a total of 175 patients fulfilled the inclusion criteria, 60 of whom were treated with POCRT, while 115 were administered pCT. The 1, 3 and 5-year OS rates in the POCRT and pCT groups were 98.3 vs. 86.1%, 71.7 vs. 53.0% and 45.7 vs. 39.0%, respectively (P = 0.019). Compared with pCT, POCRT improved OS in patients with squamous cell subtype (P = 0.010), no lymphovascular invasion (P = 0.006), pN2a (P = 0.006) or total number of metastatic lymph nodes ≤7 (P = 0.016). After PSM, these survival differences between POCRT and pCT remained significant in patients with squamous cell lung cancer (P = 0.010). CONCLUSIONS: POCRT following complete resection may be beneficial for patients with squamous cell lung cancer, particularly those with limited nodal involvement.

Halorubrum amylolyticum sp. nov., a novel halophilic archaeon isolated from a salt mine.

A pleomorphic and non-motile halophilic archaeon forming light-red pigmented colonies, strain ZC67T, was isolated from the Yuanyongjing Salt Mine, Yunnan, China. Based on similarity search and phylogenetic analysis of the 16S rRNA gene sequence, strain ZC67T belongs to the genus Halorubrum and is closely related to the species of Halorubrum (Hrr.) saccharovorum JCM 8865T, Hrr. persicum C49T, Hrr. halophilum B8T, Hrr. lipolyticum 9-3T, Hrr. salsamenti Y69T and Hrr. depositum Y78T with 16S rRNA gene sequence similarities of 99.0%, 98.7%, 98.5%, 98.4%, 98.1% and 97.7%, respectively. The values of average nucleotide identity (ANI) and average amino-acid identity (AAI) between strain ZC67T and its close relatives were less than 90.5% and 89.3%, respectively. In silico DNA-DNA hybridization (DDH) analysis showed that DNA-DNA relatedness between strain ZC67T and its relatives is less than 45%. Values of ANI, AAI and in silico DDH were clearly below the thresholds used for the delineation of a new species. The major polar lipids of strain ZC67T were similar to other neutrophilic members in the genus Halorubrum containing phosphatidylglycerol, phosphatidylglycerolphosphate methyl ester, phosphatidylglycerol sulfate and sulfated mannosyl-glucosyl-glycerol diether-1. The DNA G+C content was determined to be 66.3 mol% (based on the draft genome). Combined with other diagnostic characteristics, e.g. phenotypic and chemotaxonomic differences, strain ZC67T is concluded to represent a novel species in the genus Halorubrum, for which the name Halorubrum amylolyticum sp. nov. is proposed. The type strain is ZC67T (=CGMCC 1.15718T = JCM 31850T).

Halalkalicoccus subterraneus sp. nov., an extremely halophilic archaeon isolated from a subterranean halite deposit.

An extremely halophilic archaeon, designated strain GSM28T, was isolated from a subterranean halite deposit in a Yunnan salt mine, China. Cells of the strain were observed to be cocci, non-motile and Gram-variable, and to require at least 15% (w/v) NaCl for growth (optimum 20%). Growth was found to occur in the ranges of 20-45 °C (optimum 42 °C) and pH 6.0-8.5 (optimum 7.5). Cells did not lyse in distilled water. Phylogenetic analysis based on 16S rRNA gene sequences indicated that this strain belongs to the genus Halalkalicoccus and shows 99.1% similarities with its close phylogenetic relative Halalkalicoccus paucihalophilus DSM 24557T. Genomic ANI analysis showed that the DNA-DNA relatedness between strain GSM28T and the closely related species Hac. paucihalophilus DSM 24557T and Halalkalicoccus jeotgali B3T was 83.7% and 83.1%, respectively. The major polar lipids were determined to be phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, sulfated mannosyl-glucosyl-glycerol diether-1 and two unidentified glycolipids. The DNA G + C content was determined to be 61.8 mol %. On the basis of physiological, biochemical tests and phylogenetic differentiations, strain GSM28T is concluded to represent a novel species in the genus Halalkalicoccus, for which the name Halalkalicoccus subterraneus sp. nov. is proposed. The type strain is GSM28T (= CGMCC 1.16344T = NBRC 113432T).

Haloterrigena salifodinae sp. nov., an extremely halophilic archaeon isolated from a subterranean rock salt.

A novel extremely halophilic strain, designated ZY19T, was isolated from a rock salt sample from Yunnan salt mine, PR China. Strain ZY19T is neutrophilic, non-motile and requires at least 10% (w/v) NaCl for growth. Optimal growth is observed at 20-25% (w/v) NaCl, pH 7.5-8.0 and 42 °C. Mg2+ is not required for growth. The cells do not lyse in distilled water. On the basis of 16S rRNA gene sequence analysis, strain ZY19T belongs to the genus Haloterrigena (Htg.) and is closely related to Haloterrigena salina XH-65T (98.5% sequence similarity) and Haloterrigena turkmenica DSM 5511T (97.9%). Phylogenetic and phylogenomic analysis showed that strain ZY19T clusters with the species Htg. salina and Htg. turkmenica forming an independent clade separated from other members of the genus. The value of genomic average nucleotide identity (ANI) between strains ZY19T and its close relative, Htg. salina XH-65T was 94.2%. DNA-DNA relatedness between strains ZY19T and Htg. salina XH-65T revealed by in silico DNA-DNA hybridization (DDH) was 56.3%. Both the ANI value and the degree of in silico DDH are below the accepted threshold for members of the same species. The major polar lipids were found to consist of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, sulfated diglycosyl-diether-1 and mannose-2, 6-disulfate (1 → 2)-glucose glycerol diether. The genomic DNA G+C content was determined to be 64.5 mol%. Based on the results of the phenotypic, chemotaxonomic, genetic similarity and inferred phylogeny, strain ZY19T is distinct from other validly named species and thus represents a novel species within the genus Haloterrigena, for which the name Haloterrigena salifodinae sp. nov. is proposed. The type strain is ZY19T (=CGMCC 1.16114T=NBRC 112981T).

Halorubrum glutamatedens sp. nov., a Halophilic Archaeon Isolated from a Rock Salt.

An extremely halophilic archaeon, strain ZY8T, was isolated from a rock salt of Yunnan salt mine. It was able to grow at 12-30% (w/v) NaCl (optimum, 15-20%), pH 7.0-9.0 (optimum, pH 8.5), and 20-45 °C (optimum, 42 °C). Sequence similarity search of its 16S rRNA gene showed that strain ZY8T belonged to the genus Halorubrum, and it is closely related to species of H. aethiopicum SAH-A6T (98.6%), H. aquaticum EN-2T (98.6%), and H. halodurans Cb34T (98.5%), respectively. Strain ZY8T contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate as its major phospholipids, and a sulfated diglycosyl diether as its major glycolipid. The DNA G+C content was 66.7 mol%. DNA-DNA relatedness between strains ZY8T and closely related species were far below 70%. Based on the phenotypic and phylogenetic analyses, it is proposed that strain ZY8T represents a novel species of the genus Halorubrum, for which the name Halorubrum glutamatedens sp. nov. is proposed. The type strain is ZY8T (=CGMCC 1.16026T=NBRC 112866T).

Halococcus salsus sp. nov., a novel halophilic archaeon isolated from rock salt.

Two pink-pigmented halophilic archaea, designated strains ZJ1T and J81, were isolated from rock salt of Yunnan Salt Mine, China, and commercial salt imported from Bolivia, respectively. Cells were non-motile, coccoid, approximately 0.8-1.6 µm in diameter, stained Gram-negative and often occurred in pairs. Colonies were wet, opaque and smooth-edged. Strain ZJ1T grew optimally with 20 % (w/v) NaCl, at pH 7.5 and at 38-40 °C, which was the same as for strain J81. 16S rRNA gene sequence similarity between strains ZJ1T and J81 was 99.7 %. Sequence similarity searches based on the 16S rRNA gene and cell morphology suggested that strains ZJ1T and J81 belong to the genus Halococcus in the family Halococcaceae. The major polar lipids of the type strain, ZJ1T, were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and sulfated diglycosyl-diether-1. The profile of polar lipids, cell shape, motility and lack of lysis of cells in distilled water show that strains ZJ1T and J81 were similar to other members of the genus Halococcus. Strain ZJ1T shared the highest 16S rRNA gene and rpoB' gene sequence similarities of 99.0 and 95.3 % with Halococcus hamelinensis 100A6T, respectively, followed by less than 94.6 % with sequences of other species in the genus Halococcus. DNA-DNA relatedness between strains ZJ1T and J81 was 90.1±0.7 %, while 27±0.5 % was found between strain ZJ1T and H. hamelinensis JCM 12892T (=100A6T), and 29.0±0.5 % between strains J81 and H. hamelinensis JCM 12892T. The DNA G+C content of strain ZJ1T was 66.5 mol% (Tm). The stable phylogenetic position, differential physiological and biochemical properties and extensive sequence divergence suggest that strains ZJ1T and J81 represent a novel species, for which the name Halococcus salsus sp. nov. is proposed. The type strain is ZJ1T (=CGMCC 1.16025T=NBRC 112867T).

A Simple Scoring System for Quick, Accurate, and Reliable Early Diagnosis of Hand, Foot, and Mouth Disease.

BACKGROUND To construct an accurate, reliable, and simple scoring system of improving HFMD diagnosis. MATERIAL AND METHODS Based on the following 3 steps, a simple scoring diagnostic system was built: (1) we selected basic markers (age and sex), markers recommended in HFMD diagnosis guidelines, and significant biomarkers among severity groups found in a large dataset; (2) we used positive constituent ratio for determining scores of each marker; and (3) we applied receiving operating curve in an external dataset to determine the optimal cut-off score. RESULTS The selected markers were sex, age, fever, skin rashes, nervous system disorder, respiratory system disorder, digestive system disorder and cardiopulmonary complications, C-reactive-protein, White Blood Cell, Creatinine Kinase, Creatinine Kinase Isoenzyme, Gamma-Glutamyl Transpeptidase, Albumin, Globulin, Albumin/Globulin Ratio, Natrium, Chloride, Calcium, and Glucose. A simple scoring system with 3.9684 as the lower cut-off was constructed. The AUC was 0.918 (95% CI: 0.874-0.963, P<0.01). The sensitivity, specificity, and Youden Index, which were based on the validation dataset of 200 subjects (80 cases, 120 non-cases with skin rashes or fever), were 0.95, 0.90, and 0.85, respectively. CONCLUSIONS This simple scoring system is an effective method to diagnose HFMD.

Halorubrum depositum sp. nov., a Novel Halophilic Archaeon Isolated from a Salt Deposit.

A non-motile, pleomorphic rod-shaped or oval, red-pigmented (nearly scarlet), extremely halophilic archaeon, strain Y78T, was isolated from a salt deposit of Yunnan salt mine, China. Analysis of the 16S rRNA gene sequence showed that it was phylogenetically related to species of the genus Halorubrum, with a close relationship to Halorubrum rutilum YJ-18-S1T (98.6%), Halorubrum yunnanense Q85T (98.3%), and Halorubrum lipolyticum 9-3T (98.1%). The temperature, NaCl, and pH ranges for growth were 25-50 °C, 12-30% (w/v), and 6.5-9.0, respectively. Mg2+ was required for growth. The polar lipids of strain Y78T were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, and a sulfated diglycosyl diether. The DNA G+C content was 66.6 mol%. DNA-DNA hybridization values between strain Y78T and two closely related species of the genus Halorubrum were far below 70%. Based on the data presented in this study, strain Y78T represents a novel species for which the name Halorubrum depositum sp. nov. is proposed; the type strain is Y78T (= CGMCC 1.15456T = JCM 31272T).