Thiopseudomonas acetoxidans sp. nov., an aerobic acetic and butyric acids oxidizer isolated from anaerobic fermentation liquid of food waste.

A Gram-stain-negative, oxidase-negative, rod-shaped, motile, facultatively anaerobic bacterial strain, designated as CY1220T, was isolated from an anaerobic fermentation liquid of food waste treatment plant. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain CY1220T belongs to the genus Thiopseudomonas, with the highest sequence similarity to Thiopseudomonas alkaliphila B4199T (95.91%), followed by Thiopseudomonas denitrificans X2T (95.56%). The genomic DNA G + C content of strain CY1220T was 48.6 mol%. The average nucleotide identity values and digital DNA-DNA hybridization values between strain CY1220T and the type species of T. alkaliphila and T. denitrificans were in the range of 70.8-71.6% and 19.2-20.0%, respectively, below the thresholds for species delineation. The strain was able to grow utilizing acetic acid and butyric acid (AABA) as the sole carbon source in aerobic conditions. Genomic analysis predicted that the strain could synthesize vitamin B12 and ectoine. The predominant cellular fatty acids were C18:1 ω7c and/or C18:1 ω6c, C16:0, C16:1 ω7c and/or C16:1 ω6c and C12:0. The polar lipids comprised diphosphatidylglycerol, unknown polar lipid, phosphatidylethanolamine, phosphatidylglycerol, and phospholipid. Q-8 (2.1%) and Q-9 (97.9%) were detected as the respiratory quinones. Based on its phenotypic, genotypic and genomic characteristics, strain CY1220T represents a novel species in the genus Thiopseudomonas, for which the name Thiopseudomonas acetoxidans sp. nov. is proposed. The type strain is CY1220T (= GDMCC 1.3503 T = JCM 35747 T).

Jeotgalibacillus haloalkalitolerans sp. nov., a novel alkalitolerant and halotolerant bacterium, isolated from the confluence of the Fenhe River and the Yellow River.

A Gram-stain positive, aerobic, alkalitolerant and halotolerant bacterium, designated HH7-29 T, was isolated from the confluence of the Fenhe River and the Yellow River in Shanxi Province, PR China. Growth occurred at pH 6.0-12.0 (optimum, pH 8.0-8.5) and 15-40℃ (optimum, 32℃) with 0.5-24% NaCl (optimum, 2-9%). The predominant fatty acids (> 10.0%) were iso-C15:0 and anteiso-C15:0. The major menaquinones were MK-7 and MK-8. The polar lipids were phosphatidylglycerol, diphosphatidylglycerol and two unidentified phospholipids. Phylogenetic analyses based on the 16S rRNA gene sequence revealed that strain HH7-29 T was a member of the genus Jeotgalibacillus, exhibiting high sequence similarity to the 16S rRNA gene sequences of Jeotgalibacillus alkaliphilus JC303T (98.4%), Jeotgalibacillus salarius ASL-1 T (98.1%) and Jeotgalibacillus alimentarius YKJ-13 T (98.1%). The genomic DNA G + C content was 43.0%. Gene annotation showed that strain HH7-29 T had lower protein isoelectric points (pIs) and possessed genes related to ion transport and organic osmoprotectant uptake, implying its potential tolerance to salt and alkali. The average nucleotide identity, digital DNA-DNA hybridization values, amino acid identity values, and percentage of conserved proteins values between strain HH7-29 T and its related species were 71.1-83.8%, 19.5-27.4%, 66.5-88.4% and 59.8-76.6%, respectively. Based on the analyses of phenotypic, chemotaxonomic, phylogenetic and genomic features, strain HH7-29 T represents a novel species of the genus Jeotgalibacillus, for which the name Jeotgalibacillus haloalkalitolerans sp. nov. is proposed. The type strain is HH7-29 T (= KCTC 43417 T = MCCC 1K07541T).

Heterologous expression, purification, and characterization of thermo- and alkali-tolerant laccase-like multicopper oxidase from Bacillus mojavensis TH309 and determination of its antibiotic removal potential.

Laccases or laccase-like multicopper oxidases have great potential in bioremediation to oxidase phenolic or non-phenolic substrates. However, their inability to maintain stability in harsh environmental conditions and against non-substrate compounds is one of the main reasons for their limited use. The gene (mco) encoding multicopper oxidase from Bacillus mojavensis TH309 were cloned into pET14b( +), expressed in Escherichia coli, and purified as histidine tagged enzyme (BmLMCO). The molecular weight of the enzyme was about 60 kDa. The enzyme exhibited laccase-like activity toward 2,6-dimethoxyphenol (2,6-DMP), syringaldazine (SGZ), and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). The highest enzyme activity was recorded at 80 °C and pH 8. BmLMCO showed a half-life of ~ 305, 99, 50, 46, 36, and 20 min at 40, 50, 60, 70, 80, and 90 °C, respectively. It retained more than 60% of its activity after pre-incubation in the range of pH 5-12 for 60 min. The enzyme activity significantly increased in the presence of 1 mM of Cu2+. Moreover, BmLMCO tolerated various chemicals and showed excellent compatibility with organic solvents. The Michaelis constant (Km) and the maximum velocity (Vmax) values of BmLMCO were 0.98 mM and 93.45 µmol/min, respectively, with 2,6-DMP as the substrate. BmLMCO reduced the antibacterial activity of cefprozil, gentamycin, and erythromycin by 72.3 ± 1.5%, 79.6 ± 6.4%, and 19.7 ± 4.1%, respectively. This is the first revealing shows the recombinant production of laccase-like multicopper oxidase from any B. mojavensis strains, its biochemical properties, and potential for use in bioremediation.

Two novel alkalitolerant species Pseudalkalibacillus spartinae sp. nov. and Pseudalkalibacillus sedimenti sp. nov.

In this study, two novel alkalitolerant strains (FJAT-53046T and FJAT-53715T) were isolated from sediment samples collected in Zhangzhou, PR China. Phylogeny based on 16S rRNA gene sequences suggested that strains FJAT-53046T and FJAT-53715T were new members of the genus Pseudalkalibacillus. The two novel strains showed the highest 16S rRNA gene sequence similarity to Pseudalkalibacillus hwajinpoensis DSM 16206T, with values of 97.4 and 97.6 %, respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the two strains and the reference strain were 77.2 and 79.6 %, 20.9 and 30.2 %, respectively, which were below the prokaryotic species delineation thresholds. The ANI and dDDH values between strains FJAT-53046T and FJAT-53715T were 86.0 and 30.2 %, respectively, suggesting that they belonged to different species in the genus Pseudalkalibacillus. The major respiratory quinone in both strains was MK-7 and the major cellular fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0. Diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine were the major polar lipids in both novel strains. Combined with results stemming from the determination of physical and biochemical characteristics, chemical properties, and genome analysis, strains FJAT-53046T and FJAT-53715T are proposed to represent two novel species of the genus Pseudalkalibacillus, for which the names Pseudalkalibacillus spartinae sp. nov. and Pseudalkalibacillus sedimenti sp. nov. are proposed. The type strains are FJAT-53046T (=GDMCC 1.3077T=JCM 35611T) and FJAT-53715T (=GDMCC 1.3076T=JCM 35610T), respectively.

Oleiharenicola alkalitolerans gen. nov., sp. nov., a new member of the phylum Verrucomicrobia isolated from an oilsands tailings pond.

A novel member of the phylum Verrucomicrobia was isolated from an oilsands tailings pond in Alberta, Canada. Cells of isolate NVTT are Gram-negative, strictly aerobic, non-pigmented, non-motile cocci to diplococci 0.5-1.0 µm in diameter. The bacterium is neutrophilic (optimum pH 6.0-8.0) but alkalitolerant, capable of growth between pH 5.5 and 11.0. The temperature range for growth is 15-40 °C (optimum 25-37 °C). Carbon and energy sources include sugars and organic acids. Nitrogen sources include nitrate, urea, l-glycine, l-alanine, l-proline and l-serine. Does not fix atmospheric nitrogen. Does not require NaCl and is inhibited at NaCl concentrations above 3.0 % (w/v). The DNA G+C content of strain NVTT, based on a draft genome sequence, is 66.1 mol%. MK-6 and MK-7 are the major respiratory quinones. Major cellular fatty acids are anteiso-C15 : 0 and iso-C15 : 0. Phylogenetic analysis of 16S rRNA gene sequences revealed that the strain belongs to the family Opitutaceae of the phylum Verrucomicrobia. The most closely related validated species is Opitutus terrae (93.7 % 16S rRNA gene sequence identity to its type strain PB90-1T). Based on genotypic, phenotypic and chemotaxonomic characteristics, it was concluded that this strain represents a novel genus and species, for which the name Oleiharenicola alkalitolerans gen. nov., sp. nov. is proposed. The type strain of this novel species is NVTT (=ATCC BAA-2697T;=DSM 29249T).

Rhodanobacter hydrolyticus sp. nov., a novel DNA- and tyrosine-hydrolysing gammaproteobacterium isolated from forest soil.

A bacterial isolate, designated G-5-5T, was isolated from forest soil at Kyonggi University. Strain G-5-5T was acid-tolerant and alkali-tolerant. Cells were strictly aerobic, Gram-stain-negative, catalase- and oxidase-positive, non-motile, non-spore-forming, rod-shaped, and yellow-coloured. Strain G-5-5T hydrolysed DNA and tyrosine; assimilated d-glucose, maltose, N-acetyl-glucosamine and l-fucose; and tolerated only 0.5 % NaCl (w/v). Phylogenetic analysis based on its 16S rRNA gene sequence revealed that strain G-5-5T formed a lineage within the family Rhodanobacteraceae and that it grouped with but was distinct from various members of the genus Rhodanobacter. The closest member was Rhodanobacter umsongensis GR24-2T (97.8 % sequence similarity). The sole respiratory quinone was Q-8. The major polar lipids of strain G-5-5T were phosphatidylethanolamine, phosphatidyl-N-methylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The major cellular fatty acids were summed feature 9 (iso-C17 : 1ω9c and/or C16 : 0 10-methyl), iso-C15 : 0, iso-C17 : 0, iso-C16 : 0 and anteiso-C15 : 0. The DNA G+C content of strain G-5-5T was 64.1 mol%. DNA-DNA hybridization relatedness between strain G-5-5T and other close members of the genus Rhodanobacter ranged from 19 % to 45 %. On the basis of the polyphasic characterization and phylogenetic analyses, strain G-5-5T represents a novel species of the genus Rhodanobacter, for which the name Rhodanobacter hydrolyticus sp. nov. is proposed. The type strain is G-5-5T (=KEMB 9005-533T=KACC 19113T=NBRC 112685T).

Altererythrobacter fulvus sp. nov., a novel alkalitolerant alphaproteobacterium isolated from forest soil.

A novel α-proteobacterium, designated strain S-54T, was isolated from forest soil sampled at Kyonggi University and subjected to polyphasic study. Cells were aerobic, Gram-stain-negative, catalase- and oxidase-positive, non-motile, non-spore-forming, rod-shaped and yellow-pigmented. Flexirubin-type pigments were absent. Strain S-54T assimilated lactic acid, d-glucose and 4-hydroxybenzoic acid. Strain S-54T tolerated 4 % NaCl (w/v), and grew optimally at 45 °C and pH 10.5. Phylogenetic analysis based on 16S rRNA gene sequence data revealed that strain S-54T formed a lineage within the class Alphaproteobacteria of the phylum Proteobacteria that was distinct from various members of the genus Altererythrobacter, including Altererythrobacter troitsensis JCM 17037T (96.8 % sequence similarity), Altererythrobacterxinjiangensis S3-63T (96.6 %), Altererythrobacter dongtanensis KCTC 22672T (96.5 %) and Altererythrobacter mangrovi C9-11T (96.5 %). Q-10 was the sole isoprenoid quinone. The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and sphingoglycolipid. The major cellular fatty acids were C18 : 1ω7c, C16 : 0 and C18 : 1ω7c 11-methyl. The DNA G+C content of strain S-54T was 64.2 mol%. On the basis of the results of phenotypic, genotypic, chemotaxonomic and phylogenetic analysis, strain S-54T represents a novel species in the genus Altererythrobacter, for which the name Altererythrobacter fulvus sp. nov. is proposed. The type strain of Altererythrobacter fulvus is S-54T (=KEMB 9005-542T=KACC 19119T=NBRC 112676T).

Acinetobacter halotolerans sp. nov., a novel halotolerant, alkalitolerant, and hydrocarbon degrading bacterium, isolated from soil.

A novel aerobic, non-motile, halotolerant, alkalitolerant, hydrocarbon degrading, and rod shaped bacterium, designated strain R160T, was isolated from soil in South Korea. Cells were Gram-staining-negative, catalase-positive, and oxidase-negative. This strain grew up to 7% of NaCl and in the pH range of 6-11 (optimum 7.0-10.0). The isolate degraded 51.7 ± 1.3% of hydrocarbon components (C-18, C-20, and C-22) and 45.8 ± 1.4% oil components (kerosene, diesel, and gasoline). Phylogenetic analysis based on 16 S rRNA gene sequences revealed that strain R160T formed a lineage within the genus Acinetobacter, and was closely related to 'Acinetobacter oleivorans' DR1T (97.47%, sequence similarity). Other closely related members have sequence similarity between 97.47 to 96.52%. The predominant respiratory lipoquinones of strain R160T were ubiquinone 9 (Q-9) and ubiquinone 8 (Q-8). The major polar lipids were phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), and phosphatidylcholine (PC). The major cellular fatty acids were 9-octadecenoic acid (C18:1 ω9c), hexadecanoic acid (C16:0), and summed feature (comprising C16:1 ω7c and/or C16:1 ω6c). The DNA G + C content of strain R160T was 44.9 mol%. On the basis of phenotypic, genotypic, chemotaxonomic, and phylogenetic characteristics, strain R160T represents a novel species of the genus Acinetobacter, for which the name Acinetobacter halotolerans sp. nov. is proposed. The type strain is R160T (= KEMB 9005-333T = KACC 18453T = JCM 31009T).

Heterologous expression in Pichia pastoris and characterization of a novel GH11 xylanase from saline-alkali soil with excellent tolerance to high pH, high salt concentrations and ethanol.

A GH11 xylanase gene (xyn11-1) cloned from saline-alkali soil was successfully expressed in Pichia pastoris GS115. The purified recombinant Xyn11-1 showed its maximal activity at pH 6.0, and retained more than 60.4% of activity at pH 10.0, with good pH stability. Its optimal temperature was 50 °C and it was stable after incubation for 1 h at 30 °C. Furthermore, Xyn11-1 was highly salt-tolerant, retaining more than 77.4% of activity in the presence of 0.25-4 M NaCl and displaying more than 47.2% relative activity after being incubated in the presence of 5 M NaCl at 37 °C for 10 min. In addition, 5 mM ß-Mercaptoethanol, Cu2+, Co2+, and Mn2+ increased the xylanase activity by 22.3%, 8.8%, 7.1%, and 4.4%, respectively. Significantly, 93.4% and 59.8% of the optimal activity was retained in the presence of 2% and 10% (v/v) ethanol, respectively. Under optimal conditions, the Km,Vmax, and Kcat value of Xyn11-1 for beechwood xylan were 3.7 mg ml-1, 101.0 µmol min-1 mg-1 and 42.1 s-1, respectively. Xyn11-1 is a strict endo-ß-1,4-xylanase, its main enzymatic products being xylotetraose and xylopentaose. Xyn11-1 is the first reported GH11 xylanase isolated from saline-alkali soil, and has excellent tolerance of high pH, high salt concentrations and ethanol, which indicates its great potential for basic research and industrial applications.

Ramlibacter alkalitolerans sp. nov., alkali-tolerant bacterium isolated from soil of ginseng.

A novel bacterial strain, designated CJ661T, was isolated from soil of ginseng in Anseong, South Korea. Cells of strain CJ661T were white-coloured, Gram-staining-negative, non-motile, aerobic and rod-shaped. Strain CJ661T grew optimally at 30 °C and pH 7.0. The analysis of 16S rRNA gene sequence of strain CJ661T showed that it belongs to the genus Ramlibacter within the family Comamonadaceae and was most closely related to Ramlibacter ginsenosidimutans KCTC 22276T (98.1 %), followed by Ramlibacter henchirensis DSM 14656T (97.1 %). DNA-DNA relatedness levels of strain CJ661T were 40.6 % to R. ginsenosidimutans KCTC 22276T and 25.0 % to R. henchirensis DSM 14656T. The major isoprenoid quinone was ubiquinone (Q-8). The predominant polar lipids were phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. The major cellular fatty acids of strain CJ661T were summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c), C16 : 0 and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The G+C content of the genomic DNA was 65.4 mol%. On the basis polyphasic taxonomic data, strain CJ661T represents a novel species in the genus Ramlibacter, for which name Ramlibacter alkalitolerans sp. nov. is proposed; the type strain is CJ661T (=KACC 19305T=JCM 32081T).

Optimal secretion of alkali-tolerant xylanase in Bacillus subtilis by signal peptide screening.

Xylanases are industrially important enzymes for xylan digestion. We experimentally screened over 114 Sec and 24 Tat pathway signal peptides, with two different promoters, for optimal production of an alkaline active xylanase (XynBYG) from Bacillus pumilus BYG in a Bacillus subtilis host. Though both promoters yielded highly consistent secretion levels (0.97 Pearson correlation coefficient), the Sec pathway was found to be more efficient than the Tat pathway for XynBYG secretion. Furthermore, the optimal signal peptide (phoB) for XynBYG secretion was found to be different from the optimal peptides for cutinase and esterase reported in previous studies. A partial least squares regression analysis further identified several statistically important variables: helical properties, amino acid composition bias, and the discrimination score in Signal P. These variables explain the observed 23 % variance in the secretion yield of XynBYG by the different signal peptides. The results also suggest that the helical propensity of a signal peptide plays a significant role in the beta-rich xylanase, but not in the helix-rich cutinase, suggesting a coupling of the conformations between the signal peptide and its cargo protein for optimal secretion.

Gene cloning and enzymatic characterization of alkali-tolerant type I pullulanase from Exiguobacterium acetylicum.

UNLABELLED: A pullulanase gene (Pul3YH5) of 2568 bp, which encodes a protein containing 855 amino acid residues, was cloned from the alkaliphilic bacterium Exiguobacterium acetylicum YH5. The pullulanase (Pul3YH5) contains the YNWGYDP motif of type I pullulanase as well as four conserved glycoside hydrolase sequences of the GH13 (α-amylase) family. When the pullulanase gene was cloned and expressed in Escherichia coli BL21 (DE3) plysS, the recombinant pullulanase had a molecular mass of ˜100·0 kDa. It was optimally active at 50°C and pH 6·0, alkali-tolerant and displayed excellent stability (>93·0%) over a broad pH range (4·0-10·0) when incubated for 30 min without substrate. The enzyme activity of Pul3YH5 was significantly enhanced in the presence of Co(2+) , Fe(2+) and Mn(2+) and was inhibited by Cu(2+) , SDS, ß-mercaptoethanol and EDTA. The enzyme displayed the highest specificity for pullulan (Km  = 0·12 ± 0·02 mg ml(-1) ), followed by soluble starch (Km  = 0·69 ± 0·04 mg ml(-1) ). Substrate hydrolysis demonstrated that pullulanase from E. acetylicum liberated maltotriose from pullulan, although hydrolytic activity was also detected with soluble starch, amylopectin, ß-limited dextrin and glycogen. These enzymatic properties indicate that Pul3YH5 is alkali-tolerant pullulanase and that Pul3YH5 could be useful in the detergent industry. SIGNIFICANCE AND IMPACT OF THE STUDY: Pullulanases have great potential in various industries, ranging from food (high fructose and glucose syrups) to washing detergent industries. In this study, the gene encoding the novel pullulanase from E. acetylicum YH5 was cloned and sequenced, then expressed in E. coli. The properties of the recombinant enzyme in E. coli were also determined. The pullulanase from E. acetylicum YH5 is alkaline tolerant and has a high optimum temperature, making it a candidate for applications in the detergent industry.

Alkalistable xylanase production by alkalitolerant Paenibacillus montaniterrae RMV1 isolated from red mud.

The alkalitolerant and xylanolytic bacterial strain (RMV1) isolated from red mud pond was identified as Paenibacillus montaniterrae based on both biochemical and 16S rDNA sequence analysis. The RMV1 bacterial isolate produced alkalistable and thermostable endoxylanase active over a broad range of pH (4.0-11.0) and temperature (20-100 °C), with optima at 50 °C and pH 9.0 with a T1/2 of 6.7 hours at 50 °C. This is the first report on the isolation of P. montaniterrae from bauxite residue with quite a high xylanase producing ability.

Application of PHA surface binding proteins of alkali-tolerant Bacillus as surfactants.

Amphiphilic protein has lipophilic and hydrophilic domains, displaying the potential for development as a biosurfactant. The polyhydroxyalkanoate (PHA) surface binding protein derived from Bacillus is a type of protein that has not been studied for its emulsifying properties. In this study, PHA granule-associated protein (PhaP), PHA regulatory protein (PhaQ), and PHA synthase subunit (PhaR) derived from an alkali-tolerant PHA-producing Bacillus cereus HBL-AI were found and heterologously expressed in E. coli and purified to investigate their application as biosurfactants. It showed that the emulsification ability and stability of three amphiphilic proteins were higher than those of widely used chemical surfactants in diesel oil, vegetable oil, and lubricating oil. In particular, the PhaQ protein studied for the first time can form a stable emulsion layer in vegetable oil at a lower concentration (50 µg/mL), which greatly reduced the amount of protein used in emulsification. This clearly demonstrated that the PHA-binding protein of HBL-AI can be well applied as an environmentally friendly biosurfactants.

Fine Mapping of QTLs for Alkaline Tolerance in Crucian Carp (Carassius auratus) Using Genome-Wide SNP Markers.

Crucian carp (Carassius auratus) is widely distributed in the world and has become an economically freshwater fish. The population in Lake Dali Nur can tolerate the extreme alkaline environment with alkalinity over 50 mmol/L (pH 9.6), thus providing a special model for exploring alkali-tolerant molecular markers in an extremely alkaline environment. In this study, we constructed a high-density and high-resolution linkage map with 16,224 SNP markers based on genotyping-by-sequencing (GBS) consisting of 152 progenies and conducted QTL studies for alkali-tolerant traits. The total length of the linkage map was 3918.893 cM, with an average distance of 0.241 cM. Two QTLs for the ammonia-N-tolerant trait were detected on LG27 and LG45. A QTL for the urea-N-tolerant trait was detected on LG27. Interestingly, mapping the two QTLs on LG27 revealed that the mapped genes were both located in the intron of CDC42. GO functional annotation and KEGG enrichment analysis results indicated that the biological functions might be involved in the cell cycle, cellular senescence, MAPK, and Ras signaling pathways. These findings suggest that CDC42 may play an important role in the process of dealing with extremely alkaline environments.

Haloalkalitolerant Fungi from Sediments of the Big Tambukan Saline Lake (Northern Caucasus): Diversity and Antimicrobial Potential.

We have performed a characterization of cultivated haloalkalitolerant fungi from the sediments of Big Tambukan Lake in order to assess their biodiversity and antimicrobial activity. This saline, slightly alkaline lake is known as a source of therapeutic sulfide mud used in sanatoria of the Caucasian Mineral Waters, Russia. Though data on bacteria and algae observed in this lake are available in the literature, data on fungi adapted to the conditions of the lake are lacking. The diversity of haloalkalitolerant fungi was low and represented by ascomycetes of the genera Acremonium, Alternaria, Aspergillus, Chordomyces, Emericellopsis, Fusarium, Gibellulopsis, Myriodontium, Penicillium, and Pseudeurotium. Most of the fungi were characterized by moderate alkaline resistance, and they tolerated NaCl concentrations up to 10% w/v. The analysis of the antimicrobial activity of fungi showed that 87.5% of all strains were active against Bacillus subtilis, and 39.6% were also determined to be effective against Escherichia coli. The majority of the strains were also active against Aspergillus niger and Candida albicans, about 66.7% and 62.5%, respectively. These studies indicate, for the first time, the presence of polyextremotolerant fungi in the sediments of Big Tambukan Lake, which probably reflects their involvement in the formation of therapeutic muds.

Insights into the physiological and genomic characterization of three bacterial isolates from a highly alkaline, terrestrial serpentinizing system.

The terrestrial serpentinite-hosted ecosystem known as "The Cedars" is home to a diverse microbial community persisting under highly alkaline (pH ~ 12) and reducing (Eh < -550 mV) conditions. This extreme environment presents particular difficulties for microbial life, and efforts to isolate microorganisms from The Cedars over the past decade have remained challenging. Herein, we report the initial physiological assessment and/or full genomic characterization of three isolates: Paenibacillus sp. Cedars ('Paeni-Cedars'), Alishewanella sp. BS5-314 ('Ali-BS5-314'), and Anaerobacillus sp. CMMVII ('Anaero-CMMVII'). Paeni-Cedars is a Gram-positive, rod-shaped, mesophilic facultative anaerobe that grows between pH 7-10 (minimum pH tested was 7), temperatures 20-40°C, and 0-3% NaCl concentration. The addition of 10-20 mM CaCl2 enhanced growth, and iron reduction was observed in the following order, 2-line ferrihydrite > magnetite > serpentinite ~ chromite ~ hematite. Genome analysis identified genes for flavin-mediated iron reduction and synthesis of a bacillibactin-like, catechol-type siderophore. Ali-BS5-314 is a Gram-negative, rod-shaped, mesophilic, facultative anaerobic alkaliphile that grows between pH 10-12 and temperatures 10-40°C, with limited growth observed 1-5% NaCl. Nitrate is used as a terminal electron acceptor under anaerobic conditions, which was corroborated by genome analysis. The Ali-BS5-314 genome also includes genes for benzoate-like compound metabolism. Anaero-CMMVII remained difficult to cultivate for physiological studies; however, growth was observed between pH 9-12, with the addition of 0.01-1% yeast extract. Anaero-CMMVII is a probable oxygen-tolerant anaerobic alkaliphile with hydrogenotrophic respiration coupled with nitrate reduction, as determined by genome analysis. Based on single-copy genes, ANI, AAI and dDDH analyses, Paeni-Cedars and Ali-BS5-314 are related to other species (P. glucanolyticus and A. aestuarii, respectively), and Anaero-CMMVII represents a new species. The characterization of these three isolates demonstrate the range of ecophysiological adaptations and metabolisms present in serpentinite-hosted ecosystems, including mineral reduction, alkaliphily, and siderophore production.

Proline-Specific Fungal Peptidases: Genomic Analysis and Identification of Secreted DPP4 in Alkaliphilic and Alkalitolerant Fungi.

Proline-specific peptidases (PSP) play a crucial role in the processing of fungal toxins, pheromones, and intracellular signaling. They are of particular interest to biotechnology, as they are able to hydrolyze proline-rich oligopeptides that give a bitter taste to food and can also cause an autoimmune celiac disease. We performed in silico analysis of PSP homologs in the genomes of 42 species of higher fungi which showed the presence of PSP homologs characteristic of various kingdoms of living organisms and belonging to different families of peptidases, including homologs of dipeptidyl peptidase 4 (DPP4) and prolyl aminopeptidase 1 found in almost all the studied fungal species. Homologs of proliniminopeptidases from the S33 family absent in humans were also found. Several studied homologs are characteristic of certain taxonomic groups of fungi. Phylogenetic analysis suggests a duplication of ancestral DPP4 into transmembrane and secreted versions, which predate the split of ascomycete and basidiomycete lineages. Comparative biochemical analysis of DPP4 in alkaliphilic and alkali-tolerant strains of fungi showed that, notwithstanding some individual features of these enzymes, in both cases, the studied DPP4 are active and stable under alkaline conditions and at high salt concentrations, which makes them viable candidates for biotechnology and bioengineering.

Evaluation of bioremediation and detoxification potentiality for papermaking black liquor by a new isolated thermophilic and alkali-tolerant Serratia sp. AXJ-M.

There is a great need for efficiently treating papermaking black liquor because it can seriously pollute both soil and water ecosystems. In this study, the Plackett-Burman (PB) experimental design combined with response surface methodology (RSM) was used for improving the biodegradation efficiency of lignin by a new isolated thermophilic and alkali-tolerant strain Serratia sp. AXJ-M, and the results showed that a biodegradation efficiency of 70.5% was achieved under optimal culture conditions. The bacterium with ligninolytic activities significantly decreased target the parameters (color 80%, lignin 60%, phenol 95%, BOD 80% and COD 80%). The control and treated samples were analyzed by gas chromatography-mass spectrometer (GC-MS), which showed that the concentrations of a majority of low-molecular-weight compounds were decreased after biological treatment. Furthermore, toxicological, genotoxicity and phytotoxicity studies have supported the detoxification by the bacterium of black liquor. Finally, the genome sequence of the thermophilic, alkali-tolerant and lignin-degrading bacterium AXJ-M was completed, and the genetic basis of the thermophilic and alkali-resistant properties of AXJ-M was preliminarily revealed. The dyp-type peroxidase was first reported to have the potential to catalyze lignin degradation structurally. These findings suggest that Serratia sp. AXJ-M may be potentially useful for bioremediation applications for papermaking black liquor.

Salicibibacter halophilus sp. nov., a moderately halophilic bacterium isolated from kimchi.

A Gram-stain-positive, rod-shaped, alkalitolerant, and halophilic bacterium-designated as strain NKC3-5T-was isolated from kimchi that was collected from the Geumsan area in the Republic of Korea. Cells of isolated strain NKC3-5T were 0.5-0.7 µm wide and 1.4-2.8 µm long. The strain NKC3-5T could grow at up to 20.0% (w/v) NaCl (optimum 10%), pH 6.5-10.0 (optimum pH 9.0), and 25-40°C (optimum 35°C). The cells were able to reduce nitrate under aerobic conditions, which is the first report in the genus Salicibibacter. The genome size and genomic G + C content of strain NKC3-5T were 3,754,174 bp and 45.9 mol%, respectively; it contained 3,630 coding sequences, 16S rRNA genes (six 16S, five 5S, and five 23S), and 59 tRNA genes. Phylogenetic analysis based on 16S rRNA showed that strain NKC3-5T clustered with bacterium Salicibibacter kimchii NKC1-1T, with a similarity of 96.2-97.6%, but formed a distinct branch with other published species of the family Bacillaceae. In addition, OrthoANI value between strain NKC3-5T and Salicibibacter kimchii NKC1-1T was far lower than the species demarcation threshold. Using functional genome annotation, the result found that carbohydrate, amino acid, and vitamin metabolism related genes were highly distributed in the genome of strain NKC3-5T. Comparative genomic analysis revealed that strain NKC3-5T had 716 pan-genome orthologous groups (POGs), dominated with carbohydrate metabolism. Phylogenomic analysis based on the concatenated core POGs revealed that strain NKC3-5T was closely related to Salicibibacter kimchii. The predominant polar lipids were phosphatidylglycerol and two unidentified lipids. Anteiso-C15:0, iso-C17:0, anteiso-C17:0, and iso-C15:0 were the major cellular fatty acids, and menaquinone-7 was the major isoprenoid quinone present in strain NKC3-5T. Cell wall peptidoglycan analysis of strain NKC3-5T showed that meso-diaminopimelic acid was the diagnostic diamino acid. The phephenotypic, genomic, phylogenetic, and chemotaxonomic properties reveal that the strain represents a novel species of the genus Salicibibacter, for which the name Salicibibacter halophilus sp. nov. is proposed, with the type strain NKC3-5T (= KACC 21230T = JCM 33437T).