Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N2.

Nitrogen (N2) gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown that Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed "Dirammox" (direct ammonia oxidation). However, the unique biochemistry of this process remains unknown. Here, we report an enzyme involved in Dirammox that catalyzes the conversion of hydroxylamine to N2. We tested previously annotated proteins involved in redox reactions, DnfA, DnfB, and DnfC, to determine their ability to catalyze the oxidation of ammonia or hydroxylamine. Our results showed that none of these proteins bound to ammonia or catalyzed its oxidation; however, we did find DnfA bound to hydroxylamine. Further experiments demonstrated that, in the presence of NADH and FAD, DnfA catalyzed the conversion of 15N-labeled hydroxylamine to 15N2. This conversion did not happen under oxygen (O2)-free conditions. Thus, we concluded that DnfA encodes a hydroxylamine oxidase. We demonstrate that DnfA is not homologous to any known hydroxylamine oxidoreductases and contains a diiron center, which was shown to be involved in catalysis via electron paramagnetic resonance experiments. Furthermore, enzyme kinetics of DnfA were assayed, revealing a Km of 92.9 ± 3.0 µM for hydroxylamine and a kcat of 0.028 ± 0.001 s-1. Finally, we show that DnfA was localized in the cytoplasm and periplasm as well as in tubular membrane invaginations in HO-1 cells. To the best of our knowledge, we conclude that DnfA is the first enzyme discovered that catalyzes oxidation of hydroxylamine to N2.

The sulfonamide-resistance dihydropteroate synthase gene is crucial for efficient biodegradation of sulfamethoxazole by Paenarthrobacter species.

Sulfonamide antibiotics (SAs) are serious pollutants to ecosystems and environments. Previous studies showed that microbial degradation of SAs such as sulfamethoxazole (SMX) proceeds via a sad-encoded oxidative pathway, while the sulfonamide-resistant dihydropteroate synthase gene, sul, is responsible for SA resistance. However, the co-occurrence of sad and sul genes, as well as how the sul gene affects SMX degradation, was not explored. In this study, two SMX-degrading bacterial strains, SD-1 and SD-2, were cultivated from an SMX-degrading enrichment. Both strains were Paenarthrobacter species and were phylogenetically identical; however, they showed different SMX degradation activities. Specifically, strain SD-1 utilized SMX as the sole carbon and energy source for growth and was a highly efficient SMX degrader, while SD-2 did could not use SMX as a sole carbon or energy source and showed limited SMX degradation when an additional carbon source was supplied. Genome annotation, growth, enzymatic activity tests, and metabolite detection revealed that strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation and a pathway of protocatechuate degradation. A new sulfonamide-resistant dihydropteroate synthase gene, sul918, was identified in strain SD-1, but not in SD-2. Moreover, the lack of sul918 resulted in low SMX degradation activity in strain SD-2. Genome data mining revealed the co-occurrence of sad and sul genes in efficient SMX-degrading Paenarthrobacter strains. We propose that the co-occurrence of sulfonamide-resistant dihydropteroate synthase and sad genes is crucial for efficient SMX biodegradation. KEY POINTS: • Two sulfamethoxazole-degrading strains with distinct degrading activity, Paenarthrobacter sp. SD-1 and Paenarthrobacter sp. SD-2, were isolated and identified. • Strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation. • A new plasmid-borne SMX resistance gene (sul918) of strain SD-1 plays a crucial role in SMX degradation efficiency.

Genetic Foundations of Direct Ammonia Oxidation (Dirammox) to N2 and MocR-Like Transcriptional Regulator DnfR in Alcaligenes faecalis Strain JQ135.

Ammonia oxidation is an important process in both the natural nitrogen cycle and nitrogen removal from engineered ecosystems. Recently, a new ammonia oxidation pathway termed Dirammox (direct ammonia oxidation, NH3→NH2OH→N2) has been identified in Alcaligenes ammonioxydans. However, whether Dirammox is present in other microbes, as well as its genetic regulation, remains unknown. In this study, it was found that the metabolically versatile bacterium Alcaligenes faecalis strain JQ135 could efficiently convert ammonia into N2 via NH2OH under aerobic conditions. Genetic deletion and complementation results suggest that dnfABC is responsible for the ammonia oxidation to N2 in this strain. Strain JQ135 also employs aerobic denitrification, mainly producing N2O and trace amounts of N2, with nitrite as the sole nitrogen source. Deletion of the nirK and nosZ genes, which are essential for denitrification, did not impair the capability of JQ135 to oxidize ammonia to N2 (i.e., Dirammox is independent of denitrification). Furthermore, it was also demonstrated that pod (which encodes pyruvic oxime dioxygenase) was not involved in Dirammox and that AFA_16745 (which was previously annotated as ammonia monooxygenase and is widespread in heterotrophic bacteria) was not an ammonia monooxygenase. The MocR-family transcriptional regulator DnfR was characterized as an activator of the dnfABC operon with the binding motif 5'-TGGTCTGT-3' in the promoter region. A bioinformatic survey showed that homologs of dnf genes are widely distributed in heterotrophic bacteria. In conclusion, this work demonstrates that, besides A. ammonioxydans, Dirammox occurs in other bacteria and is regulated by the MocR-family transcriptional regulator DnfR. IMPORTANCE Microbial ammonia oxidation is a key and rate-limiting step of the nitrogen cycle. Three previously known ammonia oxidation pathways (i.e., nitrification, anaerobic ammonia oxidation [Anammox], and complete ammonia oxidation [Comammox]) are mediated by autotrophic microbes. However, the genetic foundations of ammonia oxidation by heterotrophic microorganisms have not been investigated in depth. Recently, a previously unknown pathway, termed direct ammonia oxidation to N2 (Dirammox), has been identified in the heterotrophic bacterium Alcaligenes ammonioxydans HO-1. This paper shows that, in the metabolically versatile bacterium Alcaligenes faecalis JQ135, the Dirammox pathway is mediated by dnf genes, which are independent of the denitrification pathway. A bioinformatic survey suggests that homologs of dnf genes are widely distributed in bacteria. These findings enhance the understanding of the molecular mechanisms of heterotrophic ammonia oxidation to N2.

Novel Alcaligenes ammonioxydans sp. nov. from wastewater treatment sludge oxidizes ammonia to N2 with a previously unknown pathway.

Heterotrophic nitrifiers are able to oxidize and remove ammonia from nitrogen-rich wastewaters but the genetic elements of heterotrophic ammonia oxidation are poorly understood. Here, we isolated and identified a novel heterotrophic nitrifier, Alcaligenes ammonioxydans sp. nov. strain HO-1, oxidizing ammonia to hydroxylamine and ending in the production of N2 gas. Genome analysis revealed that strain HO-1 encoded a complete denitrification pathway but lacks any genes coding for homologous to known ammonia monooxygenases or hydroxylamine oxidoreductases. Our results demonstrated strain HO-1 denitrified nitrite (not nitrate) to N2 and N2 O at anaerobic and aerobic conditions respectively. Further experiments demonstrated that inhibition of aerobic denitrification did not stop ammonia oxidation and N2 production. A gene cluster (dnfT1RT2ABCD) was cloned from strain HO-1 and enabled E. coli accumulated hydroxylamine. Sub-cloning showed that genetic cluster dnfAB or dnfABC already enabled E. coli cells to produce hydroxylamine and further to 15 N2 from (15 NH4 )2 SO4 . Transcriptome analysis revealed these three genes dnfA, dnfB and dnfC were significantly upregulated in response to ammonia stimulation. Taken together, we concluded that strain HO-1 has a novel dnf genetic cluster for ammonia oxidation and this dnf genetic cluster encoded a previously unknown pathway of direct ammonia oxidation (Dirammox) to N2 .

Candidatus Kaistella beijingensis sp. nov., Isolated from a Municipal Wastewater Treatment Plant, Is Involved in Sludge Foaming.

Biological foaming (or biofoaming) is a frequently occurring problem in wastewater treatment plants (WWTPs) and is attributed to the overwhelming growth of filamentous bulking and foaming bacteria (BFB). Biological foaming has been intensively investigated, with BFB like Microthrix and Skermania having been identified from WWTPs and implicated in foaming. Nevertheless, studies are still needed to improve our understanding of the microbial diversity of WWTP biofoams and how microbial activities contribute to foaming. In this study, sludge foaming at the Qinghe WWTP of China was monitored, and sludge foams were investigated using culture-dependent and culture-independent microbiological methods. The foam microbiomes exhibited high abundances of Skermania, Mycobacterium, Flavobacteriales, and Kaistella. A previously unknown bacterium, Candidatus Kaistella beijingensis, was cultivated from foams, its genome was sequenced, and it was phenotypically characterized. Ca. K. beijingensis exhibits hydrophobic cell surfaces, produces extracellular polymeric substances (EPS), and metabolizes lipids. Ca. K. beijingensis abundances were proportional to EPS levels in foams. Several proteins encoded by the Ca. K. beijingensis genome were identified from EPS that was extracted from sludge foams. Ca. K. beijingensis populations accounted for 4 to 6% of the total bacterial populations in sludge foam samples within the Qinghe WWTP, although their abundances were higher in spring than in other seasons. Cooccurrence analysis indicated that Ca. K. beijingensis was not a core node among the WWTP community network, but its abundances were negatively correlated with those of the well-studied BFB Skermania piniformis among cross-season Qinghe WWTP communities. IMPORTANCE Biological foaming, also known as scumming, is a sludge separation problem that has become the subject of major concern for long-term stable activated sludge operation in decades. Biological foaming was considered induced by foaming bacteria. However, the occurrence and deterioration of foaming in many WWTPs are still not completely understood. Cultivation and characterization of the enriched bacteria in foaming are critical to understand their genetic, physiological, phylogenetic, and ecological traits, as well as to improve the understanding of their relationships with foaming and performance of WWTPs.

The damage and tolerance mechanisms of Phaffia rhodozyma mutant strain MK19 grown at 28 °C.

BACKGROUND: Phaffia rhodozyma has many desirable properties for astaxanthin production, including rapid heterotrophic metabolism and high cell densities in fermenter culture. The low optimal temperature range (17-21 °C) for cell growth and astaxanthin synthesis in this species presents an obstacle to efficient industrial-scale astaxanthin production. The inhibition mechanism of cell growth at > 21 °C in P. rhodozyma have not been investigated. RESULTS: MK19, a mutant P. rhodozyma strain grows well at moderate temperatures, its cell growth was also inhibited at 28 °C, but such inhibition was mitigated, and low biomass 6 g/L was obtained after 100 h culture. Transcriptome analysis indicated that low biomass at 28 °C resulted from strong suppression of DNA and RNA synthesis in MK19. Growth inhibition at 28 °C was due to cell membrane damage with a characteristic of low mRNA content of fatty acid (f.a.) pathway transcripts (acc, fas1, fas2), and consequent low f.a. CONTENT: Thinning of cell wall and low mannose content (leading to loss of cell wall integrity) also contributed to reduced cell growth at 28 °C in MK19. Levels of astaxanthin and ergosterol, two end-products of isoprenoid biosynthesis (a shunt pathway of f.a. biosynthesis), reached 2000 µg/g and 7500 µg/g respectively; ~2-fold higher than levels at 21 or 25 °C. Abundance of ergosterol, an important cell membrane component, compensated for lack of f.a., making possible the biomass production of 6 g/L for MK19 at 28 °C. CONCLUSIONS: Inhibition of growth of P. rhodozyma at 28 °C results from blocking of DNA, RNA, f.a., and cell wall biosynthesis. In MK19, abundant ergosterol made possible biomass production 6 g/L at 28 °C. Significant accumulation of astaxanthin and ergosterol indicated an active MVA pathway in MK19 at 28 °C. Strengthening of the MVA pathway can be a feasible metabolic engineering approach for enhancement of astaxanthin synthesis in P. rhodozyma. The present findings provide useful mechanistic insights regarding adaptation of P. rhodozyma to 28 °C, and improved understanding of feasible metabolic engineering techniques for industrial scale astaxanthin production by this economically important yeast species.

Eleutheroside B, a selective late sodium current inhibitor, suppresses atrial fibrillation induced by sea anemone toxin II in rabbit hearts.

Eleutheroside B (EB) is the main active constituent derived from the Chinese herb Acanthopanax senticosus (AS) that has been reported to possess cardioprotective effects. In this study we investigated the effects of EB on cardiac electrophysiology and its suppression on atrial fibrillation (AF). Whole-cell recording was conducted in isolated rabbit atrial myocytes. The intracellular calcium ([Ca2+]i) concentration was measured using calcium indicator Fura-2/AM fluorescence. Monophasic action potential (MAP) and electrocardiogram (ECG) synchronous recordings were conducted in Langendorff-perfused rabbit hearts using ECG signal sampling and analysis system. We showed that EB dose-dependently inhibited late sodium current (INaL), transient sodium current (INaT), and sea anemone toxin II (ATX II)-increased INaL with IC50 values of 167, 1582, and 181 µM, respectively. On the other hand, EB (800 µM) did not affect L-type calcium current (ICaL), inward rectifier potassium channel current (IK), and action potential duration (APD). Furthermore, EB (300 µM) markedly decreased ATX II-prolonged the APD at 90% repolarization (APD90) and eliminated ATX II-induced early afterdepolarizations (EADs), delayed afterdepolarizations (DADs), and triggered activities (TAs). Moreover, EB (200 µM) significantly suppressed ATX II-induced Na+-dependent [Ca2+]i overload in atrial myocytes. In the Langendorff-perfused rabbit hearts, application of EB (200 µM) or TTX (2 µM) substantially decreased ATX II-induced incidences of atrial fibrillation (AF), ventricular fibrillation (VF), and heart death. These results suggest that augmented INaL alone is sufficient to induce AF, and EB exerts anti-AF actions mainly via blocking INaL, which put forward the basis of pharmacology for new clinical application of EB.

Casimicrobium huifangae gen. nov., sp. nov., a Ubiquitous "Most-Wanted" Core Bacterial Taxon from Municipal Wastewater Treatment Plants.

Microorganisms in wastewater treatment plants (WWTPs) play a key role in the removal of pollutants from municipal and industrial wastewaters. A recent study estimated that activated sludge from global municipal WWTPs harbors 1 × 109 to 2 × 109 microbial species, the majority of which have not yet been cultivated, and 28 core taxa were identified as "most-wanted" ones (L. Wu, D. Ning, B. Zhang, Y. Li, et al., Nat Microbiol 4:1183-1195, 2019, https://doi.org/10.1038/s41564-019-0426-5). Cultivation and characterization of the "most-wanted" core bacteria are critical to understand their genetic, physiological, phylogenetic, and ecological traits, as well as to improve the performance of WWTPs. In this study, we isolated a bacterial strain, designated SJ-1, that represents a novel cluster within Betaproteobacteria and corresponds to OTU_16 within the 28 core taxa in the "most-wanted" list. Strain SJ-1 was identified and nominated as Casimicrobium huifangae gen. nov., sp. nov., of a novel family, Casimicrobiaceae. C. huifangae is ubiquitously distributed and is metabolically versatile. In addition to mineralizing various carbon sources (including carbohydrates, aromatic compounds, and short-chain fatty acids), C. huifangae is capable of nitrate reduction and phosphorus accumulation. The population of C. huifangae accounted for more than 1% of the bacterial population of the activated sludge microbiome from the Qinghe WWTP, which showed seasonal dynamic changes. Cooccurrence analysis suggested that C. huifangae was an important module hub in the bacterial network of Qinghe WWTP.IMPORTANCE The activated sludge process is the most widely applied biotechnology and is one of the best ecosystems to address microbial ecological principles. Yet, the cultivation of core bacteria and the exploration of their physiology and ecology are limited. In this study, the core and novel bacterial taxon C. huifangae was cultivated and characterized. This study revealed that C. huifangae functioned as an important module hub in the activated sludge microbiome, and it potentially plays an important role in municipal wastewater treatment plants.

Pseudomonas sp. AOB-7 utilizes PHA granules as a sustained-release carbon source and biofilm carrier for aerobic denitrification of aquaculture water.

Nitrate accumulation causes long-time threat to aquatic animals in recirculating aquaculture system (RAS); thus, nitrate removal is also required in RASs. However, the lack of carbon sources makes denitrification difficult to function. Nitrate removal performance of an aerobic denitrifying and extracellular polyhydroxyalkanoate depolymerase-producing bacterium, Pseudomonas sp. AOB-7, using polyhydroxyalkanoate (PHA) granules as a solid sustained-release carbon source in RAS was evaluated. With the initial nitrate-N concentration of 140 mg/L, the high denitrification rates of 0.056 g NO3--N L-1 day-1 and 0.035 g NO3--N L-1 day-1 were achieved in denitrification medium containing poly-ß-hydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), respectively. Significant erosions and pits formed on the surface of the granules made them a good biofilm carrier for AOB-7, and 3-hydroxybutyrate (3-HB) monomer was the major product released to aquatic phase, which was benefit to animals. SEM photos showed that AOB-7 entered and attached on the inside of the PHA particle holes. A 4-week application trial was conducted to reveal the effects of PHB (AOB-7) denitrifying agent and 3-HB produced on growth of zebrafish (Brachydanio rerio) by adding 0.1% (w/v) PHB (AOB-7) denitrifying agent. Result indicated that PHB (AOB-7) denitrifying agent can significantly reduce nitrate-N content in RASs. Compared with the control group, feed coefficient ratio reduced by 18% and weight gain ratio increased by 29% in the PHB (AOB-7) denitrifying agent group. 3-HB monomer produced during the denitrification was speculated to function as a prebiotic and promote zebrafish growth. KEY POINTS: • AOB-7 showed a good aerobic denitrifying ability on PHA granules as sustained-release C source. • PHB (AOB-7) denitrifying agent can significantly reduce nitrate content in RAS. • R-3-HB monomer was the major product released to aquatic phase and function as a prebiotic.

Pseudomonas nitrititolerans sp. nov., a nitrite-tolerant denitrifying bacterium isolated from a nitrification/denitrification bioreactor.

A nitrite-tolerant denitrifying bacterium, strain GL14T, was isolated from the nitrification/denitrification bioreactor in our laboratory. Strain GL14T was Gram-stain-negative, rod-shaped, non-spore-forming, facultatively anaerobic and motile by means of a single polar flagellum. Phylogenetic analyses based on 16S rRNA gene sequences indicated that it was assigned to the genus Pseudomonas with highest 16S rRNA gene sequence similarity (98.77 %) to Pseudomonas xanthomarina DSM 18231T and Pseudomonassongnenensis NEAU-ST5-5T, followed by Pseudomonasstutzeri ATCC 17588T (98.42 %), Pseudomonaskunmingensis HL22-2T (98.29 %) and Pseudomonaszhaodongensis NEAU-ST5-21T (98.22 %). Phylogenetic analysis based on both concatenated sequences of the 16S rRNA gene and two housekeeping genes (gyrB and rpoD) and genome sequences further clarified the intrageneric phylogenetic position of strain GL14T. The DNA G+C content of GL14T was 63.1 mol%. The results of digital DNA-DNA hybridization (highest 24.2 % of DNA-DNA relatedness) based on the Genome-to-Genome Distance Calculator and average nucleotide identity analyses (highest 80.23 %) confirmed that the strain was distinctly delineated from known species of the genus Pseudomonas. The major fatty acids were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c), C16 : 0, summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), C17 : 0cyclo and C12 : 0. The respiratory quinone was ubiquinone Q-9. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Based on the phylogenetic, genomic, phenotypic and chemotaxonomic analyses, it was concluded that strain GL14T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas nitrititolerans sp. nov. is proposed. The type strain is GL14T (=CGMCC 1.13874T=NBRC 113853T).

Barbaloin inhibits ventricular arrhythmias in rabbits by modulating voltage-gated ion channels.

Barbaloin (10-ß-D-glucopyranosyl-1,8-dihydroxy-3-(hydroxymethyl)-9(10H)-anthracenone) is extracted from the aloe plant and has been reported to have anti-inflammatory, antitumor, antibacterial, and other biological activities. Here, we investigated the effects of barbaloin on cardiac electrophysiology, which has not been reported thus far. Cardiac action potentials (APs) and ionic currents were recorded in isolated rabbit ventricular myocytes using whole-cell patch-clamp technique. Additionally, the antiarrhythmic effect of barbaloin was examined in Langendorff-perfused rabbit hearts. In current-clamp recording, application of barbaloin (100 and 200 µmol/L) dose-dependently reduced the action potential duration (APD) and the maximum depolarization velocity (Vmax), and attenuated APD reverse-rate dependence (RRD) in ventricular myocytes. Furthermore, barbaloin (100 and 200 µmol/L) effectively eliminated ATX II-induced early afterdepolarizations (EADs) and Ca2+-induced delayed afterdepolarizations (DADs) in ventricular myocytes. In voltage-clamp recording, barbaloin (10-200 µmol/L) dose-dependently inhibited L-type calcium current (ICa.L) and peak sodium current (INa.P) with IC50 values of 137.06 and 559.80 µmol/L, respectively. Application of barbaloin (100, 200 µmol/L) decreased ATX II-enhanced late sodium current (INa.L) by 36.6%±3.3% and 71.8%±6.5%, respectively. However, barbaloin up to 800 µmol/L did not affect the inward rectifier potassium current (IK1) or the rapidly activated delayed rectifier potassium current (IKr) in ventricular myocytes. In Langendorff-perfused rabbit hearts, barbaloin (200 µmol/L) significantly inhibited aconitine-induced ventricular arrhythmias. These results demonstrate that barbaloin has potential as an antiarrhythmic drug.

Specific amino acids responsible for the cold adaptedness of Micrococcus antarcticus ß-glucosidase BglU.

Psychrophilic enzymes display efficient activity at moderate or low temperatures (4-25 °C) and are therefore of great interest in biotechnological industries. We previously examined the crystal structure of BglU, a psychrophilic ß-glucosidase from the bacterium Micrococcus antarcticus, at 2.2 Å resolution. In structural comparison and sequence alignment with mesophilic (BglB) and thermophilic (GlyTn) counterpart enzymes, BglU showed much lower contents of Pro residue and of charged amino acids (particularly positively charged) on the accessible surface area. In the present study, we investigated the roles of specific amino acid residues in the cold adaptedness of BglU. Mutagenesis assays showed that the mutations G261R and Q448P increased optimal temperature (from 25 to 40-45 °C) at the expense of low-temperature activity, but had no notable effects on maximal activity or heat lability. Mutations A368P, T383P, and A389E significantly increased optimal temperature (from 25 to 35-40 °C) and maximal activity (~1.5-fold relative to BglU). Thermostability of A368P and A389E increased slightly at 30 °C. Mutations K163P, N228P, and H301A greatly reduced enzymatic activity-almost completely in the case of H301A. Low contents of Pro, Arg, and Glu are important factors contributing to BglU's psychrophilic properties. Our findings will be useful in structure-based engineering of psychrophilic enzymes and in production of mutants suitable for a variety of industrial processes (e.g., food production, sewage treatment) at cold or moderate temperatures.

Characterization and mechanism of anti-Aeromonas salmonicida activity of a marine probiotic strain, Bacillus velezensis V4.

The bacterium Aeromonas salmonicida is the causative agent of furunculosis, a systemic, ubiquitous disease of fish in the salmon family, characterized by high mortality and morbidity. Probiotics are a promising approach for prevention of furunculosis in aquaculture. A bacterial strain with anti-A. salmonicida properties, Bacillus velezensis V4, was isolated and the mechanisms underlying these properties were investigated. Anti-A. salmonicida compounds present in cell-free supernatant of V4 were purified and structurally identified as members of the iturin, macrolactin, and difficidin groups. The compounds contributed jointly to inhibition of A. salmonicida, and the diversity of the compounds was related to the versatility of their mode of action. Addition of the compounds to A. salmonicida cell suspensions reduced cell density. Analyses by confocal microscopy and scanning electron microscopy revealed cell membrane disruption, deletion of cellular content, and cell lysis of A. salmonicida. The V4 genome was sequenced, and gene clusters involved in synthesis of anti-Aeromonas compounds were detected and identified. A possible probiotic effect on growth performance of Oncorhynchus mykiss (rainbow trout) was investigated by addition of 0, 1, and 3 % (v/w) V4. Relative to control, mortality was reduced 27.25 % in the 1 % addition group and 81.86 % in the 3 % addition group. Feed coefficient ratio was reduced 19.49 % and weight gain ratio was increased 71.22 % in the 1 % addition group. Our findings demonstrate that V4 is an effective probiotic strain in O. mykiss and has clear potential for both control of furunculosis and growth promotion of aquaculture animals.

Prokaryotic Community Structure Driven by Salinity and Ionic Concentrations in Plateau Lakes of the Tibetan Plateau.

The prokaryotic community composition and diversity and the distribution patterns at various taxonomic levels across gradients of salinity and physiochemical properties in the surface waters of seven plateau lakes in the Qaidam Basin, Tibetan Plateau, were evaluated using Illumina MiSeq sequencing. These lakes included Lakes Keluke (salinity, <1 g/liter), Qing (salinity, 5.5 to 6.6 g/liter), Tuosu (salinity, 24 to 35 g/liter), Dasugan (salinity, 30 to 33 g/liter), Gahai (salinity, 92 to 96 g/liter), Xiaochaidan (salinity, 94 to 99 g/liter), and Gasikule (salinity, 317 to 344 g/liter). The communities were dominated by Bacteria in lakes with salinities of <100 g/liter and by Archaea in Lake Gasikule. The clades At12OctB3 and Salinibacter, previously reported only in hypersaline environments, were found in a hyposaline lake (salinity, 5.5 to 6.6 g/liter) at an abundance of ∼1.0%, indicating their ecological plasticity. Salinity and the concentrations of the chemical ions whose concentrations covary with salinity (Mg(2+), K(+), Cl(-), Na(+), SO4 (2-), and Ca(2+)) were found to be the primary environmental factors that directly or indirectly determined the composition and diversity at the level of individual clades as well as entire prokaryotic communities. The distribution patterns of two phyla, five classes, five orders, five families, and three genera were well predicted by salinity. The variation of the prokaryotic community structure also significantly correlated with the dissolved oxygen concentration, pH, the total nitrogen concentration, and the PO4 (3-) concentration. Such correlations varied depending on the taxonomic level, demonstrating the importance of comprehensive correlation analyses at various taxonomic levels in evaluating the effects of environmental variable factors on prokaryotic community structures. Our findings clarify the distribution patterns of the prokaryotic community composition in plateau lakes at the levels of individual clades as well as whole communities along gradients of salinity and ionic concentrations.

Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic ß-Glucosidase BglU in Micrococcus antarcticus.

Psychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic ß-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (Tm). Mutation of His299 to Tyr increased the optimal temperature, the Tm, and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures.

Psychroflexus salis sp. nov. and Psychroflexus planctonicus sp. nov., isolated from a salt lake.

Two Gram-stain-negative, catalase- and oxidase-positive, strictly aerobic, non-motile, moderately halophilic bacteria (strains X15M-6T and X15M-8T) were isolated from Lake Xiaochaidan, a salt lake in Qaidam basin, Qinghai Province, China. Cells of X15M-6T were rod-like or coccoid, 0.5-0.9 µm wide and 0.9-1.5 µm long; cells of X15M-8T were rods, 0.3-0.6 µm wide and 1.2-2.2 µm long. Growth was observed in the presence of 0.5-14.0 % (w/v) NaCl (optimum, 3.0 %) and at pH 6.5-10.0 (optimum, pH 7.0-7.5) for both. X15M-6T and X15M-8T grew at 10-35 °C (optimum, 20-25 °C) and 4-35 °C (optimum, 25 °C), respectively. Both contained iso-C15 : 0, anteiso-C15 : 0 and iso-C17 : 0 3-OH as the major fatty acids, phosphatidylethanolamine and an unknown lipid as the major polar lipids, and menaquinone MK-6 as the major respiratory quinone. The DNA G+C contents were 32.8 and 35.0 mol% for X15M-6T and X15M-8T, respectively. Phylogenetic trees based on 16S rRNA gene sequences showed that both strains belonged to the genus Psychroflexus and formed a separate lineage. In addition, strains X15M-6T and X15M-8T shared 96.8 % 16S rRNA gene sequence similarity and showed highest similarities to members of the genus Psychroflexus (92.7-93.5 and 91.8-93.1 %, respectively). Based on the above data, it is concluded that strains X15M-6T and X15M-8T represent two novel species of the genus Psychroflexus, for which the names Psychroflexus salis sp. nov. (type strain X15M-6T = CGMCC 1.12925T = JCM 30615T) and Psychroflexus planctonicus sp. nov. (type strain X15M-8T = CGMCC 1.12931T = JCM 30616T) are proposed.

Aquisalinus flavus gen. nov., sp. nov., a member of the family Parvularculaceae isolated from a saline lake.

A Gram-stain-negative bacterium, strain D11M-2T, was isolated from a saline lake (Lake Dasugan) in Qaidam basin, Qinghai Province, China. Its taxonomic position was determined by using a polyphasic approach. Cells were non-spore-forming rods, 0.5-0.7 µm wide and 1.2-1.6 µm long, and motile by means of a single subpolar or lateral flagellum. Strain D11M-2T was strictly heterotrophic and aerobic, and catalase- and oxidase-positive. Growth was observed in the presence of 0-14.0% (w/v) NaCl (optimum, 2.0%), and at 10-35 °C (optimum, 30 °C) and pH 6.0-10.5 (optimum, pH 8.0). Strain D11M-2T contained Q-10 and Q-11 as the respiratory quinones and three unknown glycolipids as the major polar lipids. The major cellular fatty acids (>10.0%) were summed feature 8 (C18:1ω7c and/or C18:1ω6c) and C16:0. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain D11M-2T belonged to the family Parvularculaceae and formed a separate lineage that was independent of the two genera within the family Parvularculaceae. Strain D11M-2T exhibited 92.8-93.4% 16S rRNA gene sequence similarity to members of the genus Parvularcula (highest to Parvularcula bermudensis HTCC 2503T), and 90.2% to a member of the genus Amphiplicatus. The DNA G+C content was 59 mol% (Tm). Based on the phenotypic, chemotaxonomic and phylogenetic data, strain D11M-2T is considered to represent a novel species of a new genus in the family Parvularculaceae, for which the name Aquisalinus flavus gen. nov., sp. nov. is proposed. The type strain of Aquisalinus flavus is D11M-2T (=CGMCC 1.12921T=KCTC 42673T).

Planktosalinus lacus gen. nov., sp. nov., a member of the family Flavobacteriaceae isolated from a salt lake.

A Gram-staining-negative bacterium, strain X14M-14T, was isolated from a salt lake (Lake Xiaochaidan) in Qaidam basin, Qinghai Province, China. Its taxonomic position was determined by using a polyphasic approach. Cells of strain X14M-14T were non-spore-forming, non-motile rods. Strain X14M-14T was strictly heterotrophic and aerobic, catalase-positive and oxidase-negative. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain X14M-14T belonged to the family Flavobacteriaceae and formed a distinct lineage that was independent of the most closely related genera: Aequorivita (16S rRNA gene sequence similarities, 91.8-93.1 %) and Salinimicrobium (91.5-92.4 %). Strain X14M-14T contained MK-6 as the major respiratory quinone, and phosphatidylethanolamine and two unknown lipids as the major polar lipids. The major cellular fatty acids were iso-C15 : 0, iso-C15 : 1 G and iso-C17 : 0 3-OH. The presence of iso-C15 : 1 G as a predominant fatty acid could distinguish this strain clearly from the most closely related genera in the family Flavobacteriaceae. The DNA G+C content was 36.6 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain X14M-14T represents a novel genus and species of the family Flavobacteriaceae, for which the name Planktosalinus lacus gen. nov., sp. nov. is proposed. The type strain is X14M-14T ( = CGMCC 1.12924T = KCTC 42675T).

Membranicola marinus gen. nov., sp. nov., a new member of the family Saprospiraceae isolated from a biofilter in a recirculating aquaculture system.

A Gram-staining-negative bacterial strain (termed CZ-AZ5T) was isolated from a biological filter in a marine recirculating aquaculture system in Tianjin, China. Its taxonomic status was determined using a polyphasic approach. CZ-AZ5T cells were non-spore-forming, non-motile rods, 0.6-0.7 µm wide and 3.0-3.7 µm long. CZ-AZ5T was strictly heterotrophic, aerobic, oxidase-negative and catalase-positive. Growth occurred in the temperature range 20-40 °C (optimal: 30 °C), pH range 6.0-8.5 (optimal: pH 7.5) and salinity range 0-5 % (w/v) NaCl (optimal: 1 %). In phylogenetic analyses based on 16S rRNA gene sequences, CZ-AZ5T was assigned to the family Saprospiraceae (phylum Bacteroidetes) and was clustered with the genera Saprospira and Aureispira within this family. It showed highest sequence similarity to 'Candidatus Haliscomenobacter calcifugiens' (86.2 %), followed by Saprospira grandis ATCC 23119T (85.7 %) and Lewinella persica T-3T (85.6 %). DNA G+C content was 40.1 mol%, the major menaquinone was MK-7, and the major cellular fatty acids (>10 %) were C16 : 1ω7c and iso-C15 : 0. Our phenotypic, chemotaxonomic and phylogenetic observations, taken together, led us to conclude that strain CZ-AZ5T represents a novel species and genus of the family Saprospiraceae, for which the name Membranicola marinus gen. nov., sp. nov. is proposed. The type strain is CZ-AZ5T ( = CGMCC 1.13179T = JCM 18886T).