Expression and Secretion of Bifidobacterium adolescentis Amylase by Bifidobacterium Longum

Bifidobacterium adolescentis Int-57 (INT57), isolated from human feces, secretes an amylase. We have shot-gun cloned, sequence analyzed and expressed the gene encoding this amylase in B. longum. The sequenced 2477 bp fragment was homologous to other extracellular amylases. The encoded protein was predicted to be composed of 595 amino acids with a molecular weight of 64 kDa, and was designated AmyB. Highly conserved amylase domains were found in AmyB. The signal sequence and cleavage site was predicted by sequence analysis. AmyB was subcloned into pBES2, a novel E. coli–Bifidobacterium shuttle vector, to construct pYBamy59. Subsequently, B. longum, with no apparent amylase activity, was transformed with pYBamy59. More than 90% of the amylase activity was detected in the culture broth. This approach may open the way for the development of more efficient expression and secretion systems for Bifidobacterium. Both authors contributed equally Received 17 June 2005; Revisions requested 13 July 2005 and 26 September 2005; Revisions received 12 September 2005 and 8 November 2005; Accepted 11 November 2005     

A novel binary expression vector for production of human IL-10 in Escherichia coli and Bifidobacterium longum

A novel expression vector (pLR) driven by hup promoter and Bifidobacterium β-galactosidase signal peptide was constructed. The pLR vector was used for the expression of the optimized human IL-10 synthetic gene in Escherichia coli and Bifidobacterium longum. In both microorganisms, rhIL-10 was in a soluble form in total extract cells. The recombinant hIL-10 was partially processed in E. coli, whereas in Bifidobacterium all rhIL-10 was found in the mature form.     

Phylogenetic Studies of <Emphasis Type="Italic">Gordonia</Emphasis> Species Based on <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">secA1</Emphasis> Gene Analyses

Phylogenetic relations within the genus Gordonia were analyzed using partial gyrB and secA1 gene sequences of 23 type species in comparison with those of 16S rRNA gene. The gyrB and secA1 phylogenies showed agreement with that constructed using 16S rRNA gene sequences. The degrees of divergence of the gyrB and secA1 genes were approximately 3.4 and 1.7 times greater, respectively, than that of 16S rRNA gene. The gyrB gene showed more discriminatory power than either the secA1 or 16S rRNA gene, facilitating clear differentiation of any two Gordonia species using gyrB gene analysis. Our data indicate that gyrB and secA1 gene sequences are useful as markers for phylogenetic study and identification at the species level of the genus Gordonia.     

The use of <Emphasis Type="Italic">gyrB</Emphasis> sequence analysis in the phylogeny of the genus <Emphasis Type="Italic">Amycolatopsis</Emphasis>

Partial gyrB sequences (>1 kb) were obtained from 34 type strains of the genus Amycolatopsis. Phylogenetic trees were constructed to determine the effectiveness of using this gene to predict taxonomic relationships within the genus. The use of gyrB sequence analysis as an alternative to DNA–DNA hybridization was also assessed for distinguishing closely related species. The gyrB based phylogeny mostly confirmed the conventional 16S rRNA gene-based phylogeny and thus provides additional support for certain of these 16S rRNA gene-based phylogenetic groupings. Although pairwise gyrB sequence similarity cannot be used to predict the DNA relatedness between type strains, the gyrB genetic distance can be used as a means to assess quickly whether an isolate is likely to represent a new species in the genus Amycolatopsis. In particular a genetic distance of >0.02 between two Amycolatopsis strains (based on a 315 bp variable region of the gyrB gene) is proposed to provide a good indication that they belong to different species (and that polyphasic taxonomic characterization of the unknown strain is worth undertaking). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The GenBank accession numbers for the gyrB gene sequences obtained in this study are shown in Table 1.     

Phylogenetic Analysis of the Genus Bifidobacterium and Related Genera Based on 16S rDNA Sequences

The 16S rRNA gene sequences were determined for type strains of 21 Bifidobacterium species. A phylogenetic tree was constructed using the determined sequences and sequences from DNA databases, which contain the sequences of 11 type strains of Bifidobacterium species and 11 strains of related genera. All species of the genus Bifidobacterium and Gardnerella vaginalis ATCC 14018 belonged to a cluster phylogenetically distinct from the other genera. The cluster was divided into two subclusters: subcluster 1 composed of most species of Bifidobacterium and G. vaginalis, and subcluster 2 consisting of two species, B. denticolens and B. inopinatum; both of which were isolated from human dental caries. In the genus Bifidobacterium, four groups of species are known to be moderately to highly related by DNA-DNA hybridization. The four groups of species exhibited more than 99% similarity among their 16S rDNA sequences within each group. These results indicated that species with around 99% or more similarity in their 16S rDNA sequences should be confirmed for species identities.     

Application of <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">parE</Emphasis> sequence similarity analyses for differentiation of species within the genus <Emphasis Type="Italic">Geobacillus</Emphasis>

The primary structures of the genes encoding the β-subunits of a type II topoisomerase (gyrase, gyrB) and a type IV topoisomerase (parE) were determined for 15 strains of thermophilic bacteria of the genus Geobacillus. The obtained sequences were used for analysis of the phylogenetic similarity between members of this genus. Comparison of the phylogenetic trees of geobacilli constructed on the basis of the 16S rRNA, gyrB, and parE gene sequences demonstrated that the level of genetic distance between the sequences of the genes encoding the β-subunits of type II topoisomerases significantly exceeded the values obtained by comparative analysis of the 16S rRNA gene sequences of Geobacillus strains. It was shown that, unlike the 16S rRNA gene analysis, comparative analysis of the gyrB and parE gene sequences provided a more precise determination of the phylogenetic position of bacteria at the species level. The data obtained suggest the possibility of using the genes encoding the β-subunits of type II topoisomerases as phylogenetic markers for determination of the species structure of geobacilli.     

Both the <Emphasis Type="Italic">AOX1</Emphasis> promoter and the <Emphasis Type="Italic">FLD1</Emphasis> promoter work together in a <Emphasis Type="Italic">Pichia pastoris</Emphasis> expression vector

We constructed a Pichia pastoris expression vector with two strongly inducible promoters (an alcohol oxidase 1 promoter and a formaldehyde dehydrogenase 1 promoter) based on pPIC9 k. To test the function of these promoters, the vector was used to co-express two genes that encode for green fluorescent protein (GFP) and a portion of a gelatin gene (an intra- and extracellular protein). The gelatin gene was placed under the control of PAOX1, while the GFP was under the control of PFLD1. The two proteins were simultaneously expressed upon induction with 0.5% (v/v) methanol. The two promoters functioned effectively and their coexistence on one vector did not affect their efficiency in protein expression. Thus, it was possible to simultaneously induce the expression of at least two proteins from one vector, using two different promoters.     

<Emphasis Type="Italic">Aliihoeflea aestuarii</Emphasis> gen. nov., sp. nov., a novel bacterium isolated from tidal flat sediment

A novel Gram-negative and rod-shaped bacterium, designated N8^T, was isolated from tidal flat sediment. Phylogenetic analysis based on 16S rRNA gene sequences showed that N8^T strain is associated with the family Phyllobacteriaceae: two uncultured clones (98.4 and 99.8% 16S rRNA gene sequence similarity) and the genus Mesorhizobium (≤97.0%). The novel strain formed a separate clade with uncultured clones in the phylogenetic tree based on 16S rRNA gene sequences. Cellular fatty acid profiles predominately comprised C_18:1 ω7c and C_19:0 cyclo ω8c. The major isoprenoid quinone is ubiquinone-10 and genomic DNA G+C content is 53.4 mol%. The polyphasic taxonomic study indicates that the novel strain N8^T represents a novel species of the new genus in the family Phyllobacteriaceae, named Aliihoeflea aestuarii. The type strain is N8^T (= KCTC 22052^T= JCM 15118^T= DSM 19536^T).     

<Emphasis Type="Italic">Anoxybacillus rupiensis</Emphasis> sp. Nov., a novel thermophilic bacterium isolated from Rupi basin (Bulgaria)

Three strains of a novel thermophilic, strictly aerobic, Gram-positive, spore-forming hemo-organotrophic bacterium were isolated from three hot springs in the region of Rupi basin, Bulgaria as producers of amylolytic enzymes. Their 16S rRNA gene sequences (first 500 nucleotides) were very similar (99.8%). Strains were able to ferment a wide spectrum of carbohydrates such as sugars, polyols, and polysaccharides like xylan, glycogen and starch. Optimal growth was observed at 55–58°C, and pH at 6.0–6.5. Phylogenetic analysis of the whole 16S rRNA gene sequence clustered the strain R270^T with the representatives of the genus Anoxybacillus and with Geobacillus tepidamans. The G + C content of the genomic DNA was 41.7%. DNA–DNA hybridization analysis revealed low homology with the closest relatives (32.0 mol% homology to Geobacillus tepidamans). Fatty acid profile (major fatty acids iso-C15:0 and iso-C17:0) confirmed the affiliation of the strain to the genus Anoxybacillus. On the basis of the data presented here, we propose that strain R270^T, represents a new species of the genus Anoxybacillus for which, we recommend the name Anoxybacillus rupiensis sp. nov. (=DSM 17127^T = NBIMCC 8387^T). The 16S rRNA gene sequence data of a strain R270^T have been deposited in the EMBL databases under the accession number AJ879076.     

Prevention of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 infection in gnotobiotic mice associated with <Emphasis Type="Italic">Bifidobacterium</Emphasis> strains

Previous reports have shown that Escherichia coli O157:H7 infection is strongly modified by intestinal microbes. In this paper, we examined whether bifidobacteria protect against E. coli O157:H7 infections using gnotobiotic mice di-associated with Bifidobacterium strains (6 species, 9 strains) and E. coli O157:H7. Seven days after oral administration of each Bifidobacterium strain, the mice were orally infected with E. coli O157:H7 and their mortality was examined. Bifidobacterium longum subsp. infantis 157F-4-1 (B. infantis 157F) and B. longum subsp. longum NCC2705 (B. longum NS) protected against the lethal infection, while mice associated with all other Bifidobacterium strains, including type strains of B. longum subsp. infantis and B. longum subsp. longum, died. There were no significant differences in the numbers of E. coli O157:H7 in the faeces among the Bifidobacterium-associated mouse groups. However, the Shiga toxin concentrations in the cecal contents and sera of the GB mice associated with B. infantis 157F and B. longum NS were significantly lower than those of the other groups. However, there were no significant differences in the volatile fatty acid concentrations and histopathological lesions between these two groups. These data suggest that some strains of B. longum subsp. longum/infantis can protect against the lethal infections of E. coli O157:H7 by preventing Shiga toxin production in the cecum and/or Shiga toxin transfer from the intestinal lumen to the bloodstream.     

Expression of Rice Glutamate Decarboxylase in <Emphasis Type="Italic">Bifidobacterium Longum</Emphasis> Enhances γ-Aminobutyric Acid Production

Bifidobacteria are important for the production of fermented dairy products and probiotic formulas but have a low capacity for γ-aminobutyric acid (GABA) production. To develop a Bifidobacterium strain with an enhanced GABA production, we transformed Bifidobacterium longum with a rice glutamate decarboxylase (OsGADC⁻) gene by electroporation. When the transformed strain was cultured in medium containing monosodium glutamate, the amount of GABA increased significantly compared with those of untransformed Bifidobacterium. Thus, by introducing a plant derived GAD gene, a Bifidobacterium strain has been genetically engineered to produce high levels of GABA from glutamate.     

Conditions affecting cell surface properties of human intestinal bifidobacteria

The cell surface properties of human intestinal bifidobacteria have been characterized for 30 strains isolated from a fecal sample. Strain identification to the species level was obtained by restriction analysis of the amplified 16S rRNA gene and confirmed by DNA/DNA reassociation experiments. The isolates were grouped in four genetically homogeneous clusters whose members belonged to Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium longum and Bifidobacterium pseudocatenulatum species. Cell surface properties of Bifidobacterium strains were evaluated by determining the level of hydrophobicity, adhesion to hydrocarbons and contact angle measurements, and their autoaggregation ability. The results showed high and homogeneous level of hydrophobicity in all tested strains when contact angle measurements values were considered. On the contrary, autoaggregation assays and bacterial adhesion to hydrocarbons detected interesting differences in cell surface properties among the tested Bifidobacterium strains. The highest levels of autoaggregation, detected in B. bifidum and B. adolescentis strains, were strictly dependent on the pH of the medium. Moreover, protease treatment experiments suggested that proteins had a key role in the autoaggregating ability of B. bifidum and B. adolescentis strains.     

Evaluation of using a short region of the recA gene for rapid and sensitive speciation of dominant bifidobacteria in the human large intestine

The feasibility of intragenerically characterizing bifidobacteria by a comparison of a short region within the recA gene was tested. An ∼300 bp fragment of the recA gene was PCR-amplified from six species from the genus Bifidobacterium using primers directed to two universally conserved regions of the recA gene. A phylogenetic analysis of the sequenced recA products compared favorably to classification based on the 16S rRNA sequences of the species tested. To apply this rapid methodology to unknown human intestinal bifidobacteria, 46 isolates were randomly chosen from the feces of four subjects and initially characterized by RFLP analysis of a PCR-amplified region of their 16S RNA genes. From a representative of the dominant RFLP family in each of the subjects, the recA segment was PCR-amplified, sequenced and phylogenetically analyzed. All four isolates were found to be related to one another and to B. longum and B. infantis. These results illustrate that the recA gene may be useful for intrageneric phylogenetic analysis as well as for the identification of unknown fecal bifidobacteria.     

Expression of hepatitis B surface antigen in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> utilizing glyceraldeyhyde-3-phosphate dehydrogenase promoter of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An expression vector constructed from genes of Pichia pastoris was applied for heterologous gene expression in Saccharomyces cerevisiae. Recombinant hepatitis B surface antigen was synthesized by cloning hepatitis B virus ‘S’ gene under the control of glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter of Pichia pastoris in Saccharomyces cerevisiae. Hepatitis B surface antigen was constitutively expressed, was stable and exhibited ∼20–22 nm particle formation. Stability and absence of toxicity to the host with the expression vector indicates the expression system can be applied for large-scale production.     

<Emphasis Type="Italic">Halorubrum cibi</Emphasis> sp. nov., an extremely halophilic archaeon from salt-fermented seafood

Strain B31^T is a Gram-staining-negative, motile, and extremely halophilic archaeon that was isolated from salt-fermented seafood. Its morphology, physiology, biochemical features, and 16S rRNA gene sequence were determined. Phylogenetic analysis of its 16S rRNA gene sequence and composition of its major polar lipids placed this archaeon in the genus Halorubrum of the family Halobacteriaceae. Strain B31^T showed 97.3, 97.2, and 96.9 % 16S rRNA similarity to the type strains of Halorubrum alkaliphilum, Hrr. tibetense, and Hrr. vacuolatum, respectively. Its major polar lipids were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and sulfated diglycosyl diether (S-DGD). Genomic DNA from strain B31^T has a 61.7 mol% G+C content. Analysis of 16S rRNA gene sequences, as well as physiological and biochemical tests, identified genotypic and phenotypic differences between strain B31^T and other Halorubrum species. The type strain of the novel species is B31^T (=JCM 15757^T =DSM 19504^T).     

Germ line transformation of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>, using the transposable element <Emphasis Type="Italic">Minos</Emphasis>

We investigated the use of Minos as a vector for transgenesis in the silkworm, Bombyx mori. We first constructed a vector plasmid with the green fluorescent protein (GFP) gene fused with the silkworm cytoplasmic actin gene (A3) promoter, and a helper plasmid with the Minos transposase gene controlled by the same A3 promoter. Injection of the vector and helper plasmid DNA into silkworm eggs produced transgenic animals in the following generation. The efficiency of transgenic silkworm production using this method was much lower than that obtained using piggyBac-mediated germ line transformation. However, >40-fold increase in the efficiency of producing transgenic silkworms was obtained using an in vitro synthesized source of Minos transposase mRNA. We conclude that the Minos transposon is a useful vector for construction of transgenic silkworms, particularly when in vitro synthesized mRNA is used. This is the first report showing that Minos can be used as a vector for germ-line transformation in lepidopteran insects.     

Functional expression of amine oxidase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> (AO-I) in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The aim of this work was to prepare recombinant amine oxidase from Aspergillus niger after overexpressing in yeast. The yeast expression vector pDR197 that includes a constitutive PMA1 promoter was used for the expression in Saccharomyces cerevisiae. Recombinant amine oxidase was extracted from the growth medium of the yeast, purified to homogeneity and identified by activity assay and MALDI-TOF peptide mass fingerprinting. Similarity search in the newly published A. niger genome identified six genes coding for copper amine oxidase, two of them corresponding to the previously described enzymes AO-I a methylamine oxidase and three other genes coding for FAD amine oxidases. Thus, A. niger possesses an enormous metabolic gear to grow on amine compounds and thus support its saprophytic lifestyle.     

Culturable Actinobacteria from the Marine Sponge Hymeniacidon perleve: Isolation and Phylogenetic Diversity by 16S rRNA gene-RFLP Analysis

A total of 106 actinobacteria associated with the marine sponge Hymeniacidon perleve collected from the Yellow Sea, China were isolated using eight different media. The number of species and genera of actinobacteria recovered from the different media varied significantly, underlining the importance of optimizing the isolation conditions. The phylogenetic diversity of the actinobacteria isolates was assessed using 16S rRNA gene amplification–restriction fragment length polymorphism (RFLP) analysis of the 106 strains with different morphologies. The RFLP fingerprinting of selected strains by HhaI-digestion of the 16S rRNA genes resulted in 11 different patterns. The HhaI-RFLP analysis gave good resolution for the identification of the actinobacteria isolates at the genus level. A phylogenetic analysis using 16S rRNA gene sequences revealed that the isolates belonged to seven genera of culturable actinobacteria including Actinoalloteichus, Micromonospora, Nocardia, Nocardiopsis, Pseudonocardia, Rhodococcus, and Streptomyces. The dominant genus was Streptomyces, which represented 74% of the isolates. Three of the strains identified are candidates for new species.     

16S rRNA及rpoC1基因用于螺旋藻、节旋藻系统发育的比较北大核心CSCD

【目的】利用16S rRNA和rpoC1基因分子标记研究螺旋藻、节旋藻的系统发育关系,并对其区分能力进行比较。【方法】以84株螺旋藻、节旋藻为研究对象,对其进行16S rRNA、rpoC1基因序列的扩增、测序及分析,并对构建的系统发育树进行对比。【结果】rpoC1基因序列保守位点所占比例49.7%、平均G+C百分含量47.7%和序列相似度76%–100%明显低于16S rRNA基因序列的79.4%、55.6%和91%–100%,其变异程度高于16S rRNA基因;基于16S rRNA、rpoC1基因构建的系统发育NJ树拓扑结构基本一致,84株实验藻株分为2个属3个类群,其中仅F-351、F-904-2、F-1070和TJBC14-1藻株为螺旋藻,其余均为节旋藻;虽然2个基因都不能区分形态种和地理种,但rpoC1基因NJ树的置信度(100%)高于16S rRNA基因(99%),属内分群效果也明显优于16S rRNA基因。【结论】支持了螺旋藻、节旋藻为两个不同属的结论,且在属内分类时rpoC1基因比16S rRNA基因具有更高的区分度。