Isolation,transposon mutagenesis,and characterization of the novel nicotine-degrading strain Shinella sp. HZN7

Nicotine is a significant toxic waste generated in tobacco manufacturing. Biological methods for the degradation of nicotine waste are in high demand. In this study, we report the identification and characterization of the novel nicotine-degrading strain Shinella sp. HZN7. This strain can degrade 500 mg/L nicotine completely within 3 h at 30 °C and pH values of 6.5?～?8.0. The biodegradation of nicotine by Shinella sp. HZN7 involves five intermediate metabolites: 6-hydroxy-nicotine (6HN), 6-hydroxy-N-methylmyosmine, 6-hydroxypseudooxynicotine (6HPON), 6-hydroxy-3-succinoyl-pyridine (HSP), and 2,5-dihydroxypyridine, as detected by ultraviolet spectrophotometry, HPLC, and LC-MS. We generated three mutants, N7-W18, N7-X5, and N7-M17, by transposon mutagenesis, in which the nicotine-degrading pathway terminated at 6HN, 6HPON, and HSP, respectively. The production of the five intermediate metabolites and their order in the degradation pathway were confirmed in the three mutants, indicating that strain HZN7 degrades nicotine via a variant of the pyridine and pyrrolidine pathways. The mutant gene from strain N7-X5, orf2, was cloned by self-formed adaptor PCR, but the nucleotide and amino acid sequence showed no similarity to any gene or gene product with defined function. However, orf2 disruption and complementation suggested that the orf2 gene is essential for the conversion of 6HPON to HSP in strain HZN7. This is the first study to provide genetic evidence for this variant nicotine degradation pathway.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation: an efficient tool for targeted gene disruption in <Emphasis Type="Italic">Talaromyces marneffei</Emphasis>

Talaromyces marneffei causes life-threatening infections in immunocompromised hosts. An efficient tool for genetic manipulation of T. marneffei will allow for increased understanding of this thermally dimorphic fungus. Agrobacterium tumefaciens-mediated transformation (ATMT) was optimized for targeted gene disruption in T. marneffei using the plasmid pDHt/acuD::pyrG. Molecular analyses of transformants were performed by PCR, Southern blot and semi-quantitative RT-PCR. A. tumefaciens strain EHA105 was more efficient at transformation than strain AGL-1 in ATMT via solid co-cultivation. An A. tumefaciens:T. marneffei ratio of 1000:1 in an ATMT liquid co-cultivation led to a relatively high transformation efficiency of 90 transformants per 10⁶ yeast cells. Using ATMT-mediated knockout mutagenesis, we successfully deleted the acuD gene in T. marneffei. PCR and Southern blot analysis confirmed that acuD was disrupted and that the foreign pyrG gene was integrated into T. marneffei. Semi-quantitative RT-PCR analysis further confirmed that pyrG was expressed normally. These results suggest that ATMT can be a potential platform for targeted gene disruption in T. marneffei and that liquid co-cultivation may provide new opportunities to develop clinical treatments.     

Constitutive expression of the <Emphasis Type="Italic">tzs</Emphasis> gene from <Emphasis Type="Italic">Agrobacterium tumefaciens virG</Emphasis> mutant strains is responsible for improved transgenic plant regeneration in cotton meristem transformation

Key message

virG mutant strains of a nopaline type of Agrobacterium tumefaciens increase the transformation frequency in cotton meristem transformation. Constitutive cytokinin expression from the tzs gene in the virG mutant strains is responsible for the improvement.

Abstract

Strains of Agrobacterium tumefaciens were tested for their ability to improve cotton meristem transformation frequency. Two disarmed A. tumefaciens nopaline strains with either a virGN54D constitutively active mutation or virGI77V hypersensitive induction mutation significantly increased the transformation frequency in a cotton meristem transformation system. The virG mutant strains resulted in greener explants after three days of co-culture in the presence of light, which could be attributed to a cytokinin effect of the mutants. A tzs knockout strain of virGI77V mutant showed more elongated, less green explants and decreased cotton transformation frequency, as compared to a wild type parental strain, suggesting that expression of the tzs gene is required for transformation frequency improvement in cotton meristem transformation. In vitro cytokinin levels in culture media were tenfold higher in the virGN54D strain, and approximately 30-fold higher in the virGI77V strain, in the absence of acetosyringone induction, compared to the wild type strain. The cytokinin level in the virGN54D strain is further increased upon acetosyringone induction, while the cytokinin level in the virGI77V mutant is decreased by induction, suggesting that different tzs gene expression regulation mechanisms are present in the two virG mutant strains. Based on these data, we suggest that the increased cytokinin levels play a major role in increasing Agrobacterium attachment and stimulating localized division of the attached plant cells.

     

Co-expression of Beta-Glucosidase and Laccase in <Emphasis Type="Italic">Trichoderma reesei</Emphasis> by Random Insertion with Enhanced Filter Paper Activity

Trichoderma reesei strain Rut-C30 was modified with enhanced beta-glycosidase (BGL) activity to balance the cellulase system and generated laccase (LAC) protein for lignin degradation. Initially, the binary plasmid p1300-w1 was constructed to express T. reesei bgl2 under the control of promoter P _pki and T-nos terminator. Random insertion was performed via Agrobacterium tumefaciens-mediated transformation. A total of 353 mutants were obtained, and 34PTrb2 was exceptionally stable with increased FPA and BGL activity after screening for extracellular enzyme activity. Subsequently, 34PTrb2 was used as parent strain via the same method to insert the lac gene from Fomes lignosus, with promoter P _gpd , followed by cbh1 signal peptide trss and T-nos as terminator. Several mutants successfully expressed enzyme LAC with stable activity of approximately 0.13 U/mL. The mutant 15Gsslac increased activity by 40.4% FPA compared with that of the host Rut-C30.     

<Emphasis Type="Italic">glyA</Emphasis> gene knock-out in <Emphasis Type="Italic">Escherichia coli</Emphasis> enhances L-serine production without glycine addition

In E. coli, glyA encodes for serine hydroxymethyltransferase (SHMT), which converts L-serine to glycine. When engineering L-serine-producing strains, it is therefore favorable to inactivate glyA to prevent L-serine degradation. However, most glyA knockout strains exhibit slow cell growth because of the resulting lack of glycine and C₁ units. To overcome this problem, we overexpressed the gcvTHP genes of the glycine cleavage system (GCV), to increase the C₁ supply before glyA was knocked out. Subsequently, the kbl and tdh genes were overexpressed to provide additional glycine via the L-threonine degradation pathway, thus restoring normal cell growth independent of glycine addition. Finally, the plasmid pPK10 was introduced to overexpress pgk, serA ^Δ197 , serC and serB, and the resulting strain E4G2 (pPK10) accumulated 266.3 mg/L of L-serine in a semi-defined medium without adding glycine, which was 3.18-fold higher than the production achieved by the control strain E3 (pPK10). This strategy can accordingly be applied to disrupt the L-serine degradation pathway in industrial production strains without causing negative side-effects, ultimately making L-serine production more efficient.     

<Emphasis Type="Italic">Spirosoma luteolum</Emphasis> sp. nov. isolated from water

A novel Gram-negative and rod-shaped bacterial strain, designated as 16F6E^T, was isolated from a water sample. Cells were yellowish in color and catalase- and oxidase-positive. The strain grew at 10–37°C (optimum at 25°C) but not at 4 and 42°C, and pH 5–7 (optimum at pH 7). It showed moderate resistance to gamma-ray irradiation. Comparative phylogenetic analysis showed that strain 16F6E^T belonged to the family Cytophagaceae of the class Cytophagia. Furthermore, this isolate showed relatively low 16S rRNA gene sequence similarities (90.7–93.1%) to the members of the genus Spirosoma. The major fatty acids were summed feature 3 (C_16:1 ω7c/C_16:1 ω6c), C_16:1 ω5c, C_16:0 N alcohol, and C_16:0. The polar lipid profile indicated presence of phosphatidylethanolamine, unknown aminophospholipids, an unknown amino lipid, unknown phospholipids, and unknown polar lipids. The predominant isoprenoid quinone was MK-7. The genomic DNA G+C content of strain 16F6E^T was 56.5 mol%. Phenotypic, phylogenetic, and chemotaxonomic properties indicated that isolate 16F6E^T represents a novel species within the genus Spirosoma, for which the name Spirosoma luteolum sp. nov. is proposed. The type strain is 16F6E^T (=KCTC 52199^T =JCM 31411^T).     

<Emphasis Type="Italic">Spirosoma daeguensis</Emphasis> sp. nov., isolated from beach soil

A Gram-stain-negative, non-motile, non-spore-forming, rod-shaped, aerobic bacterium, designated 15J9-6^T, was isolated from beach soil on Jeju Island, South Korea. Strain 15J9-6^T, grew at 10–30°C (optimum growth at 25°C) and pH 7–8 (optimum growth at pH 7) on R2A, NA, and TSA agar. Phylogenetically, the strain was closely related to members of the genus Spirosoma (92.3–90.1% 16S rRNA gene sequence similarities) and showed highest sequence similarity to Spirosoma panaciterrae DSM 21099^T (92.3%). The G+C content of the genomic DNA of strain 15J9-6^T was 45.7 mol%. The strain contained phosphatidylethanolamine, two unidentified aminophospholipids, an unidentified phospholipid, and an unidentified lipid as the major polar lipids; menaquinone MK-7 as the predominant respiratory quinone and summed feature 3 (C_16:1 ω6c/C_16:1 ω7c; 30.1%), C_16:1 ω5c (23.1%), iso C_15:0 (13.3%), and C_16:0 (8.4%) as the major fatty acids which supported the affiliation of strain 15J9-6^T to the genus Spirosoma. The results of physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain 15J9-6^T from recognized Spirosoma species. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain 15J9-6^T represents a novel species of the genus Spirosoma, for which the name Spirosoma daeguensis sp. nov. is proposed. The type strain is 15J9-6^T (=KCTC 52036^T =JCM 31995T)     

Taxonomic description and draft genome of <Emphasis Type="Italic">Pseudomonas sediminis</Emphasis> sp. nov., isolated from the rhizospheric sediment of <Emphasis Type="Italic">Phragmites karka</Emphasis>

The taxonomic position of a Gram-stain-negative, rod-shaped bacterial strain, designated PI11^T, isolated from the rhizospheric sediment of Phragmites karka was characterized using a polyphasic approach. Strain PI11^T could grow optimally at 1.0% NaCl concentration with pH 7.0 at 30°C and was positive for oxidase and catalase but negative for hydrolysis of starch, casein, and esculin ferric citrate. Phylogenetic analysis of 16S rRNA gene sequences indicated that the strain PI11^T belonged to the genus Pseudomonas sharing the highest sequence similarities with Pseudomonas indoloxydans JCM 14246^T (99.72%), followed by, Pseudomonas oleovorans subsp. oleovorans DSM 1045^T (99.29%), Pseudomonas toyotomiensis JCM 15604^T (99.15%), Pseudomonas chengduensis DSM 26382^T (99.08%), Pseudomonas oleovorans subsp. lubricantis DSM 21016^T (99.08%), and Pseudomonas alcaliphila JCM 10630^T (99.01%). Experimental DNA-DNA relatedness between strain PI11^T and P. indoloxydans JCM 14246^T was 49.4%. The draft genome of strain PI11^T consisted of 4,884,839 bp. Average nucleotide identity between the genome of strain PI11^T and other closely related type strains ranged between 77.25–90.74%. The polar lipid pattern comprised of phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylcholine. The major (> 10%) cellular fatty acids were C_18:1ω6c/ω7c, C_16:1ω6c/ω7c, and C_16:0. The DNA G + C content of strain PI11^T was 62.4 mol%. Based on the results of polyphasic analysis, strain PI11^T was delineated from other closely related type strains. It is proposed that strain PI11^T represents represents a novel species of the genus Pseudomonas, for which the name Pseudomonas sediminis sp. nov. is proposed. The type strain is PI11^T (= KCTC 42576^T = DSMZ 100245^T).     

Characterization and Proteomic Analysis of the <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. HK-6 <Emphasis Type="Italic">xenB</Emphasis> Knockout Mutant Under RDX (Hexahydro-1,3,5-trinitro-1,3,5-triazine) Stress

Pseudomonas sp. HK-6 is able to utilize RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) as its sole nitrogen source. The role of the xenB gene, encoding xenobiotic reductase B, was investigated using HK-6 xenB knockout mutants. The xenB mutant degraded RDX to a level that was 10-fold less than that obtained with the wild-type HK-6 strain. After 60 days of culture with 25 or 50 μM RDX, no residual RDX was detected in the supernatants of the wild-type aerobically grown cultures, whereas approximately 90 % of the RDX remained in the xenB mutant cultures. The xenB mutant bacteria exhibited a 10²–10⁴-fold decrease in survival rate compared to the wild-type. The expression of DnaK and GroEL proteins, two typical stress shock proteins (SSPs), in the xenB mutant increased after immediate exposure to RDX, yet dramatically decreased after 4 h of exposure. In addition, DnaK and GroEL were more highly expressed in the cultures with 25 μM RDX in the medium but showed low expression in the cultures with 50 or 75 μM RDX. The expression levels of the dnaK and groEL genes measured by RT-qPCR were also much lower in the xenB genetic background. Analyses of the proteomes of the HK-6 and xenB mutant cells grown under conditions of RDX stress showed increased induction of several proteins, such as Alg8, alginate biosynthesis sensor histidine kinase, and OprH in the xenB mutants when compared to wild-type. However, many proteins, including two SSPs (DnaK and GroEL) and proteins involved in metabolism, exhibited lower expression levels in the xenB mutant than in the wild-type HK-6 strain. The xenB knockout mutation leads to reduced RDX degradation ability, which renders the mutant more sensitive to RDX stress and results in a lower survival rate and an altered proteomic profile under RDX stress.     

Expression of gene for <Emphasis Type="Italic">N</Emphasis>-acyl-homoserine lactonase <Emphasis Type="Italic">AiiA</Emphasis> affects properties of rhizospheric strain <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> 449

The introduction into strain Pseudomonas chlororaphis 449 of plasmid pME6863 that contains the cloned gene for N-acyl-homoserine lactonase, AiiA, leads to the degradation of all three types of N-acylhomoserine lactones produced by this strain (N-butanoyl-homoserine lactone, N-hexanoyl-homoserine lactone, and N-3-oxo-hexanoyl-homoserine lactone). This causes a drastic reduction in the synthesis of phenazine pigment and decreases the ability of cells to migrate on the surface of nutrient medium. However, the antagonistic activity of P. chlororaphis 449 toward phytopathogenic fungi Sclerotinia sclerotiorum and Rhizoctonia solani is not only decreased, but is even slightly increased; no essential changes in the exoprotease activity were observed. It is assumed that one of the QS systems of P. chlororaphis 449 may exert the repression effect on the expression of genes, which determine the two latter cell activities.     

<Emphasis Type="Italic">Tardibacter chloracetimidivorans</Emphasis> gen. nov., sp. nov., a novel member of the family <Emphasis Type="Italic">Sphingomonadaceae</Emphasis> isolated from an agricultural soil from Jeju Island in Republic of Korea

A pale yellow bacterial strain, designated JJ-A5^T, was isolated form an agricultural soil from Jeju Island in Republic of Korea. Cells of the strain were Gram-stain-negative, motile, flagellated and rod-shaped. The strain grew at 15–30°C, pH 6.0–9.0, and in the presence of 0–1.5% (w/v) NaCl. Growth occurred on R2A, but not on Luria-Bertani agar, nutrient agar, trypticase soy agar and MacConkey agar. The strain utilized alachlor as a sole carbon source for growth. The strain JJ-A5^T showed 16S rRNA gene sequence similarities lower than 95.4% with members of the family Sphingomonadaceae. Phylogenetic analysis showed that the strain belongs to the family Sphingomonadaceae and strain JJ-A5^T was distinctly separated from established genera of this family. The strain contained Q-10 as dominant ubiquinone and spermidine as major polyamine. The predominant cellular fatty acids were summed feature 8 (C_18:1ω7c and/or C_18:1ω6c), summed feature 3 (C_16:1ω7c and/or C_16:1ω6c), 11-methyl C_18:1ω7c, C_16:0 and C_14:0 2-OH. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, sphingoglycolipid, and phosphatidylcholine. The DNA G + C content of the strain was 62.7 mol%. On the basis of the phenotypic, genomic and chemotaxonomic characteristics, strain JJ-A5^T is considered to represent a novel genus and species within the family Sphingomonadaceae, for which the name Tardibacter chloracetimidivorans gen. nov., sp. nov. is proposed. The type strain of Tardibacter chloracetimidivorans is JJ-A5^T (= KACC 19450^T = NBRC 113160^T).     

An efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for aflatoxin generation fungus <Emphasis Type="Italic">Aspergillus flavus</Emphasis>

Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ～ 60 positive transformants per 10⁶ conidia using our protocol. A small-scale insertional mutant library (～ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.     

<Emphasis Type="Italic">Hymenobacter agri</Emphasis> sp. nov., a novel bacterium isolated from soil

A bacterial isolate was recovered from a soil sample collected in Jeollabuk-do Province, South Korea, and subjected to polyphasic taxonomic assessment. Cells of the isolate, designated strain S1-2-1-2-1^T, were observed to be rod-shaped, pink in color, and Gram-stain negative. The strain was able to grow at temperature range from 10 to 30 °C, with an optimum of 25 °C, and growth occurred at pH 6–8. Comparative 16S rRNA gene sequence analysis showed that strain S1-2-1-2-1^T belongs to the genus Hymenobacter, with closely related type strains being Hymenobacter daeguensis 16F3Y-2^T (95.8% similarity), Hymenobacter rubidus DG7B^T (95.8%), Hymenobacter soli PB^T (95.7%), Hymenobacter terrenus MIMtkLc17^T (95.6%), Hymenobacter terrae DG7A^T (95.3%), and Hymenobacter saemangeumensis GSR0100^T (95.2%). The genomic DNA G+C content of strain S1-2-1-2-1^T was 63.0 mol%. The main polar lipid of this strain was phosphatidylethanolamine, the predominant respiratory quinone was menaquinone-7, and the major fatty acids were C_15:0 iso (27.3%), summed feature 3 (C_16:1 ω7c/C_16:1 ω6c) (16.5%), C_15:0 anteiso (15.3%), and C_16:0 (14.7%), supporting the affiliation of this strain with the genus Hymenobacter. The results of this polyphasic analysis allowed for the genotypic and phenotypic differentiation of strain S1-2-1-2-1^T from recognized Hymenobacter species. On the basis of its phenotypic properties, genotypic distinctiveness, and chemotaxonomic features, strain S1-2-1-2-1^T is considered to represent a novel species of the genus Hymenobacter, for which the name Hymenobacter agri sp. nov. is proposed. The type strain is S1-2-1-2-1^T (=KCTC 52739^T?=?JCM 32194^T).     

Polymorphic variants of glutamate receptor (<Emphasis Type="Italic">GRIK5</Emphasis>, <Emphasis Type="Italic">GRIN2B</Emphasis>) and serotonin receptor (<Emphasis Type="Italic">HTR2A</Emphasis>) genes are associated with chronic

Chronic obstructive pulmonary disease (COPD) is a complex chronic inflammatory disease of the respiratory system that affects primarily distal respiratory pathways and lung parenchyma. Smoking tobacco is a major risk factor for COPD. The relationship of HTR4 (rs3995090), HTR2A (rs6313), GRIK5 (rs8099939), GRIN2B (rs2268132), and CHRNB4 (rs1948) gene polymorphisms and COPD, as well as the contribution of these polymorphisms to the variations in quantitative characteristics that describe respiratory function, smoking behavior, and nicotine dependence was assessed in an ethnically homogeneous Tatar population. The polymorphisms of HTR2A (rs6313) (P = 0.026, OR = 1.42 for the CC genotype) and GRIN2B (rs2268132) (P = 0.0001, OR = 2.39 for the TT genotype) were significantly associated with increased risk of COPD. The AA genotype of GRIK5 (rs8099939) had a protective effect (P = 0.02, OR = 0.61). Importantly, the HTR2A (rs6313), GRIN2B (rs2268132), and GRIK5 (rs8099939) polymorphisms were only associated with COPD in smokers. Smoking index (pack-years) was significantly higher in carriers of the GRIK5 genotype AC (rs8099939) (P = 0.0027). The TT genotype of GRIN2B (rs2268132) was associated with COPD in subjects with high nicotine dependence according to the Fagerström test (P = 0.002, OR = 2.98). The TT genotype of HTR2A (rs6313) was associated with a reduced risk of the disease in the group with moderate nicotine dependence (P = 0.02, OR = 0.22). The CC genotype of HTR2A (rs6313) and the TT genotype of GRIN2B (rs2268132) were associated with higher levels of nicotine dependence according to the Fagerström test (P = 0.0011 and P = 0.037). Our results may provide insight into potential molecular mechanisms that involve the glutamate (GRIK5, GRIN2B) and serotonin (HTR2A) receptor genes in the pathogenesis of COPD.     

Development of an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for <Emphasis Type="Italic">Taxus</Emphasis> suspension cultures

The FDA-approved anti-cancer compound paclitaxel is currently produced commercially by Taxus plant cell suspension cultures. One major limitation to the use of plant cell culture as a production platform is the low and variable product yields. Therefore, methods to increase and stabilize paclitaxel production are necessary to ensure product security, especially as the demand for paclitaxel continues to rise. Although a stable transformation method for Taxus suspension cultures has been developed, stable transformant yields are low (around 1% of experiments) and the method does not translate to the Taxus cuspidata Siebold and Zucc. and Taxus canadensis Marshall cell lines utilized in this study. Therefore, a new method for Agrobacterium-mediated transformation of Taxus callus and suspension cultures was developed through identification of the optimal Agrobacterium strain, inclusion of an anti-necrotic cocktail (silver nitrate, cysteine, and ascorbic acid) and increased recovery time for cells after cocultivation, the time following infection with Agrobacterium tumefaciens. Application of the increased recovery time to transformation of T. cuspidata line PO93XC resulted in 200 calluses staining positive for GUS. Additionally, two transgenic lines have been maintained with stable transgene expression for over 5 yr. This method represents an improvement over existing transformation methods for Taxus cultures and can be applied for future metabolic engineering efforts.     

<Emphasis Type="Italic">Zunongwangia flava</Emphasis> sp. nov., belonging to the family <Emphasis Type="Italic">Flavobacteriaceae</Emphasis>, isolated from <Emphasis Type="Italic">Salicornia europaea</Emphasis>

A yellow pigmented bacterium designated strain MBLN094^T within the family Flavobacteriaceae was isolated from a halophyte Salicornia europaea on the coast of the Yellow Sea. This strain was a Gram-stain negative, aerobic, non-spore forming, rod-shaped bacterium. Phylogenetic analysis of the 16S rRNA gene sequence of strain MBLN094^T was found to be related to the genus Zunongwangia, exhibiting 16S rRNA gene sequence similarity values of 97.0, 96.8, 96.4, and 96.3% to Zunongwangia mangrovi P2E16^T, Z. profunda SM-A87^T, Z. atlantica 22II14-10F7^T, and Z. endophytica CPA58^T, respectively. Strain MBLN094^T grew at 20?37°C (optimum, 25?30°C), at pH 6.0?10.0 (optimum, 7.0?8.0), and with 0.5?15.0% (w/v) NaCl (optimum, 2.0?5.0%). Menaquinone MK-6 was the sole respiratory quinone. The polar lipids were phosphatidylethanolamine, two unidentified aminolipids, and four unidentified lipids. Major fatty acids were iso-C_17:0 3-OH, summed feature 3 (C_16:1ω6c and/or C16:1 ω7c), and iso-C_15:0. The genomic DNA G + C content was 37.4 mol%. Based on these polyphasic taxonomic data, strain MBLN094^T is considered to represent a novel species of the genus Zunongwangia, for which the name Zunongwangia flava sp. nov. is proposed. The type strain is MBLN094^T (= KCTC 62279^T = JCM 32262^T).     

Locating a modifier gene of <Emphasis Type="Italic">Ovum mutant</Emphasis> through crosses between DDK and C57BL/6J inbred strains in mice

A striking infertile phenotype has been discovered in the DDK strain of mouse. The DDK females are usually infertile when crossed with males of other inbred strains, whereas DDK males exhibit normal fertility in reciprocal crosses. This phenomenon is caused by mutation in the ovum (Om) locus on chromosome 11 and known as the DDK syndrome. Previously, some research groups reported that the embryonic mortality deviated from the semilethal rate in backcrosses between heterozygous (Om/ + ) females and males of other strains. This embryonic mortality exhibited an aggravated trend with increasing background genes of other strains. These results indicated that some modifier genes of Om were present in other strains. In the present study, a population of N₂ (Om/ + ) females from the backcrosses between C57BL/6J (B6) and F₁ (B6 ♀ × DDK ♂) was used to map potential modifier genes of Om. Quantitative trait locus showed that a major locus, namely Amom1 (aggravate modifier gene of Om 1), was located at the middle part of chromosome 9 in mice. The Amom1 could increase the expressivity of Om gene, thereby aggravating embryonic lethality when heterozygous (Om/ +) females mated with males of B6 strain. Further, the 1.5 LOD-drop analysis indicated that the confidence interval was between 37.54 and 44.46 cM, ～6.92 cM. Amom1 is the first modifier gene of Om in the B6 background.