Genotypic alteration and competitive nodulation of Mesorhizobium muleiense against exotic chickpea rhizobia in alkaline soils.

Mesorhizobium muleiense, Mesorhizobium mediterraneum and Mesorhizobium ciceri are chickpea (Cicer arietinum L.) rhizobia that share a high similarity of the symbiotic genes nodC and nifH, but they have different geographic distributions. M. muleiense has been isolated and found only in alkaline soils of Xinjiang, China, whereas the other two strains have been found in the Mediterranean and India. To investigate the species stability of M. muleiense during natural evolution and its capability of competitive nodulation against the other two exotic species, re-sampling of nodules in the field and competition experiments between the three species were conducted. The results showed that the predominant microsymbiont associated with chickpea grown in Xinjiang was still M. muleiense, but the predominant genotypes of M. muleiense had changed significantly during the four years since a previous survey. The data also showed that M. mediterraneum and M. ciceri were more competitive than the residential strain of M. muleiense CCBAU 83963(T) in sterilized vermiculite or soils from Xinjiang. However, in non-sterilized soils, M. muleiense was the predominant nodule occupier. These results indicated that natural or adapting evolution of M. muleiense was occurring in fields subjected to changing environmental factors. In addition, the biogeography and symbiotic associations of rhizobia with their host legumes were also influenced by biological factors in the soil, such as indigenous rhizobia and other organisms.

A visual method for direct selection of high-producing Pichia pastoris clones.

BACKGROUND: The methylotrophic yeast, Pichia pastoris, offers the possibility to generate a high amount of recombinant proteins in a fast and easy way to use expression system. Being a single-celled microorganism, P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. A simple and direct method for the selection of high-producing clones can dramatically enhance the whole production process along with significant decrease in production costs. RESULTS: A visual method for rapid selection of high-producing clones based on mannanase reporter system was developed. The study explained that it was possible to use mannanase activity as a measure of the expression level of the protein of interest. High-producing target protein clones were directly selected based on the size of hydrolysis holes in the selected plate. As an example, the target gene (9elp-hal18) was expressed and purified in Pichia pastoris using this technology. CONCLUSIONS: A novel methodology is proposed for obtaining the high-producing clones of proteins of interest, based on the mannanase reporter system. This system may be adapted to other microorganisms, such as Saccharomyces cerevisiae for the selection of clones.

Myceligenerans halotolerans sp. nov., an actinomycete isolated from a salt lake, and emended description of the genus Myceligenerans.

A halotolerant actinomycete strain, designated XJEEM 11063(T), was isolated from a salt lake in Xinjiang province, north-western China. Strain XJEEM 11063(T) grew at pH 6.0-8.0 (optimal growth at pH 7.0), between 10 and 40 °C (optimal growth at 28-37 °C) and at salinities of 0-10 % (w/v) NaCl (optimal growth at 0-5 %, w/v). The peptidoglycan type was A4α, and the whole-cell hydrolysates contained glucose, mannose and arabinose. The major fatty acids were anteiso-C(15 : 0), iso-C(15 : 0) and anteiso-C(17 : 0). MK-9(H(4)) was the predominant menaquinone and the polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides, two unknown phospholipids and three unknown glycolipids. The genomic DNA G+C content was 71.8 mol%. The chemotaxonomic properties supported the affiliation of strain XJEEM 11063(T) to the genus Myceligenerans. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the organism was most closely related to Myceligenerans xiligouense XLG9A10.2(T) (98.3 %) and Myceligenerans crystallogenes DSM 17134(T) (97.0 %). However, it had relatively low values for DNA-DNA relatedness with the above strains (56.0 % and 47.5 %, respectively). Thus, on the basis of the results from this study, a novel species, Myceligenerans halotolerans sp. nov., is proposed. The type strain is XJEEM 11063(T) ( = DSM 21949(T) = CCTCC AA 208063(T)).

Expression and purification of an antimicrobial peptide by fusion with elastin-like polypeptides in Escherichia coli.

Different carrier molecules have been fused to antimicrobial polypeptides (AMPs) to facilitate recombinant protein expression and purification. Some of them have improved the stability of AMPs and reduced the toxicity to host cells, but most current strategies still have some problems to be solved such as poor yield, low purity, high expense, time-consumption, and difficulty in scaling-up. Here, we introduced the elastin-like polypeptides (ELPs) as a fusion partner to express an antimicrobial polypeptide halocidin18 (Hal18). By the reversible soluble-insoluble phase transition, 69 mg of the fusion protein were purified from 1 l of culture medium with the purity of nearly 95%. After cleavage with hydroxylamine, the ELP's tag was easily separated from Hal18 in the next round of inverse transition cycle and Hal18 (1.7 mg, approximately 1.9 kDa) was mainly found in the supernatant with a recovery of about 47% and purity of 60%. Antimicrobial activity showed that Hal18 had strong antimicrobial activity against Escherichia coli and Micrococcus luteus but weak activity against Pichia pastoris.

Paracoccus saliphilus sp. nov., a halophilic bacterium isolated from a saline soil.

A Gram-negative, coccoid to short rod-shaped, non-spore-forming, moderately halophilic bacterium, designated strain YIM 90738T, was isolated from a salt lake in Xinjiang province, north-west China, and was subjected to a polyphasic taxonomic study. The strain was non-motile and grew at pH 6.0-8.0 (optimal growth at pH 7.0), 10-55 degrees C (optimal growth at 37 degrees C) and salinities of 1-15% NaCl (w/v, optimal growth at 8% NaCl). Ubiquinone 10 was detected as the major respiratory quinone. The cellular fatty acid profile had C18:1omega7c (80.4% of the total) as the major component, similar to those of members of the genus Paracoccus. The nearest phylogenetic neighbour of strain YIM 90738T was the type strain of Paracoccus homiensis, as determined by 16S rRNA gene sequence analysis (97.5% similarity). The level of DNA-DNA relatedness between P. homiensis DSM 17862T and strain YIM 90738T was 51.5%. The G+C content of the genomic DNA of strain YIM 90738T was 60.3 mol%. On the basis of phenotypic and phylogenetic data and its genotypic distinctiveness, strain YIM 90738T (=CCTCC AB 206074T=KCTC 22163T) is proposed as the type strain of a novel species of the genus Paracoccus, Paracoccus saliphilus sp. nov.

Marinococcus luteus sp. nov., a halotolerant bacterium isolated from a salt lake, and emended description of the genus Marinococcus.

A Gram-positive, aerobic, motile, coccoid, orange-pigmented bacterium, designated strain YIM 91094(T), was isolated from a salt lake sample collected from Barkol Lake in Xinjiang Province, north-west China. The strain was able to grow at pH 6.0-8.0 (optimal growth at pH 7.0), at 10-37 degrees C (optimal growth at 28 degrees C) and in the presence of 0-25 % (w/v) NaCl [optimal growth in the presence of 10-15 % (w/v) NaCl]. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain YIM 91094(T) was affiliated with the genus Marinococcus and exhibited levels of sequence similarity of 99.2 % to Marinococcus halotolerans YIM 70157(T) and 99.7 % to Marinococcus halophilus DSM 20408(T). However, it showed moderately low levels of DNA-DNA relatedness with the above type strains (56.0 and 57.5 %, respectively). The peptidoglycan type of strain YIM 91094(T) was A1gamma, with meso-diaminopimelic acid as the diagnostic diamino acid. MK-7 was the predominant menaquinone and anteiso-C(15 : 0) (49.9 % of the total) and anteiso-C(17 : 0) (29.6 %) were the major cellular fatty acids. The DNA G+C content was 48.7 mol%. Strain YIM 91094(T) possessed chemotaxonomic markers that were consistent with its classification in the genus Marinococcus. On the basis of the data presented, strain YIM 91094(T) is considered to represent a novel species of the genus Marinococcus, for which the name Marinococcus luteus sp. nov. is proposed. The type strain is YIM 91094(T) (=KCTC 13214(T)=CCTCC AA 208014(T)). An emended description of the genus Marinococcus is provided.

Kocuria halotolerans sp. nov., an actinobacterium isolated from a saline soil in China.

A Gram-positive actinobacterium, designated strain YIM 90716(T), was isolated from a saline soil sample collected from Ganjiahu Suosuo Forest National Nature Reserve in Xinjiang Province, north-west China. The new isolate contained lysine, glutamic acid and alanine with peptidoglycan type Lys-Ala(3) (variation A3alpha). The major phospholipids were phosphatidylglycerol and diphosphatidylglycerol. The predominant menaqinone was MK-7(H(2)). The major fatty acids were anteiso-C(15 : 0), iso-C(16 : 0) and anteiso-C(17 : 0). The DNA G+C content of strain YIM 90716(T) was 68.0 mol%. Chemotaxonomic properties supported the affiliation of strain YIM 90716(T) to the genus Kocuria. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the organism was related most closely to Kocuria kristinae DSM 20032(T) (96.8 % similarity) and showed lower levels of 16S rRNA gene similarity (<96.5 %) with the type strains of other species of the genus Kocuria. The results of fatty acid analysis and physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strain YIM 90716(T) from its closest relatives. On the basis of data from the present polyphasic study, strain YIM 90716(T) is considered to represent a novel species of the genus Kocuria, for which the name Kocuria halotolerans sp. nov. is proposed. The type strain is YIM 90716(T) (=DSM 18442(T)=KCTC 19172(T)=CCTCC AB 206069(T)).

Gracilibacillus saliphilus sp. nov., a moderately halophilic bacterium isolated from a salt lake.

A novel Gram-positive-staining, aerobic, spore-forming, rod-shaped bacterium, designated strain YIM 91119(T), was isolated from Ebinur Lake in Xinjiang Province, north-west China. Cells were motile, produced terminal endospores and grew at pH 6.0-8.0 (optimally at pH 7.0), 4-45 degrees C (optimally at 28-37 degrees C) and 1-22 % (w/v) NaCl, (optimally at 10-15 %, w/v). Comparative 16S rRNA gene sequence analysis showed that strain YIM 91119(T) belongs to the genus Gracilibacillus, exhibiting the highest sequence similarity with respect to the type strain of Gracilibacillus orientalis (97.8 %); the next most similar 16S rRNA gene sequences were those of the type strains of Gracilibacillus boraciitolerans (96.8 %), Gracilibacillus dipsosauri (96.5 %) and Gracilibacillus halotolerans (95.8 %). DNA-DNA hybridization with G. orientalis AS 1.4250(T) showed a relatedness of 55 %. The major fatty acids of strain YIM 91119(T) were anteiso-C(15 : 0), iso-C(15 : 0) and anteiso-C(17 : 0). The peptidoglycan type was A1gamma (directly cross-linked meso-diaminopimelic acid). The genomic DNA G+C content was 40.1 mol% and the predominant respiratory quinone was MK-7. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. On the basis of the evidence from this polyphasic study, strain YIM 91119(T) represents a novel species of the genus Gracilibacillus, for which the name Gracilibacillus saliphilus sp. nov. is proposed, with YIM 91119(T) (=DSM 19802(T) =CCTCC AA 208015(T)) as the type strain.

Transfer of Ephedra genomic DNA to yeasts by ion implantation.

The genomic DNA from Ephedra glauca was randomly transferred to Saccharomyces cerevisiae and Hansenula anomala by argon and nitrogen ion implantation. Through repeated subculturing and using reversed phase high-performance liquid chromatography analysis to quantify the concentrations of the secondary metabolites, l-ephedrine and d-pseudoephedrine, 12 recombinant strains of genetically stable yeast were obtained, each using glucose as a carbon source, NaNO3 as a nitrogen source and producing l-ephedrine and/or d-pseudoephedrine. After culturing in liquid medium for 72 h, extracellular l-ephedrine and d-pseudoephedrine concentrations of 18.85 and 4.11 mg/L, respectively, were detected. Using l-ephedrine and d-pseudoephedrine as the target products, the transformation efficiencies of the genomic DNA from E. glauca transferred to S. cerevisiae and H. anomala were 1.15% (1/87) and 2.13% (8/376), respectively. The addition of the amino acid, L-Phe, to culture media substantially changed the amount of l-ephedrine and/or d-pseudoephedrine produced by the recombined yeasts. However, the change in metabolite production was not consistent among strains, rising in some, while dropping to nondetectable levels in others. After random amplification of polymorphic DNA (RAPD) analysis, four RAPD primers were obtained from the initial 100 RAPD primers, each amplifying different fragments with the recombined yeast Ar_Han0458 genome. Using one primer as polymerase chain reaction primer, the result showed that the recombined yeast Ar_Han0458 genome matched E. glauca genomic DNA at 150 bp, indicating a successful transfer of genetic information, facilitated by ion implantation.

Microbulbifer halophilus sp. nov., a moderately halophilic bacterium from north-west China.

A Gram-negative, rod-shaped, moderately halophilic bacterium, designated strain YIM 91118(T), motile with a single polar flagellum, was isolated from Xinjiang province in north-west China and subjected to a polyphasic taxonomic study. Strain YIM 91118(T) grew optimally at 37 degrees C, pH 7.0-8.0 and 10% NaCl (w/v). Its major cellular fatty acids were iso-C(15:0), iso-C(17:0), C(16:0) and iso-C(17:1)omega9c. The predominant lipoquinone was Q-8. The DNA G+C content was 63.2 mol%. All of these chemotaxonomic data supported the assignment of the new isolate to the genus Microbulbifer. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 91118(T) belongs to the genus Microbulbifer and it formed a distinct subclade with Microbulbifer maritimus KCCM 41774(T). The levels of 16S rRNA gene sequence similarity to the type strains of Microbulbifer species were in the range 93.5-95.0%. The mean level of DNA-DNA relatedness between strain YIM 91118(T) and M. maritimus KCCM 41774(T) was 45.8%. On the basis of phenotypic properties, phylogeny and genomic data, strain YIM 91118(T) represents a novel species of the genus Microbulbifer, for which the name Microbulbifer halophilus sp. nov. is proposed. The type strain is YIM 91118(T) (=CCTCC AB 206094(T) =KCTC 12848(T)). Furthermore, it is necessary to emend the description of the genus Microbulbifer.

Halomonas lutea sp. nov., a moderately halophilic bacterium isolated from a salt lake.

A Gram-negative, moderately halophilic bacterium, designated YIM 91125(T), was isolated from a salt lake in Xinjiang province, north-west China. The isolate grew at salinities in the range 1-20% (w/v) and at 4-45 degrees C. Optimal growth occurred at 37 degrees C, pH 7.5 and 5-10% (w/v) NaCl. Cells were short rods motile by means of single polar flagella. The major fatty acids were C(18:1)omega7c, C(16:0), C(19:0) cyclo omega8c and C(12:0) 3-OH. The DNA G+C content was 60.8 mol%. The predominant respiratory quinone was Q-9. A comparison of 16S rRNA gene sequences revealed its relationship to Halomonas species, its closest neighbours being Halomonas pantelleriensis (95.9% similarity to the type strain) and Halomonas muralis (95.4 % similarity). On the basis of chemotaxonomic, phylogenetic and phenotypic evidence, strain YIM 91125(T) represents a novel member of the genus Halomonas, for which the name Halomonas lutea sp. nov. is proposed. The type strain is YIM 91125(T) (=KCTC 12847(T) =CCTCC AB 206093(T)).

[Production of l-ephedrine and d-pseudoephedrine in recombined yeasts obtained by argon ion implantation mediated Ephedra genome DNA transformation].

The genome DNA from Ephedra glauca was randomly transferred into Hansenula anomala, respectively, by argon ion bombardment. Then, after screening by the motheds of bromothymol blue indicator selection, slant cultivation, l-ephedrine and d-pseudoephedrine copper chromic salt qualitative test and RP-HPLC determination, 3 strains, producing recombined yeasts were obtained, which can use glucose as a carbon source, NaNO3 as nitrogen source and be genetically stable. After cultivated in liquid medium for 72 hours and analyzed by the RP-HPLC, the recombined strains can produce l-ephedrine 11.87 mg/L and d-pseudoephedrine 4.11 mg/L excellular, d-pseudoephedrine 294.86 mg/g dry cell incellular, but l-ephedrine not detected incellular. The transformation efficiency of Ephedra genome DNA transferred into yeasts via argon ion bombardment was 0.65%. The effects of Ephedra genome DNA macromolecule integrity on yeast transformation system were discussed. The results shown that DNA macromolecule with integrated structure used as exogenous donor can obtain higher transformation efficiency than DNA macromolecule random fragments by ion implantation mediated DNA transformation. It was inferred that biosynthesis of l-ephedrine and the d-pseudoephedrin were controlled by linked together genes or gene clusters.

Streptomonospora alba sp. nov., a novel halophilic actinomycete, and emended description of the genus Streptomonospora Cui et al. 2001.

A halophilic actinomycete, strain YIM 90003(T), was isolated from a soil sample collected from Xinjiang Province, China, by using starch-casein agar with a salt concentration of 20 % (w/v), pH 7.0. The strain grew well on most media tested. No diffusible pigment was produced. Aerial mycelium and substrate mycelium were well developed on most media. The aerial mycelium formed short spore chains, bearing non-motile, straight to flexuous spores with wrinkled surfaces. The cell walls of strain YIM 90003(T) contained meso-diaminopimelic acid as the diagnostic diamino acid. Cell-wall hydrolysates contained galactose and arabinose. Menaquinone composition varied with the medium used for cell cultivation; on glucose-yeast extract medium supplemented with 10 % NaCl, the major menaquinone was MK-9(H(4)), while, on vitamin-enriched ISP 2 medium, the major menaquinones were MK-10(H(2)), MK-9(H(8)) and MK-10(H(4)). Phospholipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, diphosphatidyl glycerol, methylphosphatidylethanolamine, phosphatidylserine, phosphatidylcholine and an unidentified phospholipid. 16S rRNA gene sequence analysis showed Streptomonospora salina as the closest phylogenetic neighbour. On the basis of these analyses, strain YIM 90003(T) is a member of the genus Streptomonospora, though its properties do not match the generic description fully with respect to the menaquinone composition and peptidoglycan amino acid. Analyses of mechanically disrupted cell walls of the type species, Streptomonospora salina DSM 44593(T), and strain YIM 90003(T), purified by tryptic digestion and subsequent SDS treatment, revealed the exclusive presence of meso-diaminopimelic acid as the diagnostic diamino acid of peptidoglycan. Thus, the genus description of Streptomonospora, indicating the presence of several amino acids usually not found in the peptidoglycan moiety, is therefore emended. DNA-DNA hybridization and comparison of physiological and chemotaxonomic characteristics demonstrated strain YIM 90003(T) to be different from Streptomonospora salina. The name Streptomonospora alba sp. nov. is proposed, with strain YIM 90003(T) (=CCTCC AA001013(T)=DSM 44588(T)) as the type strain.