Phylogenetic position of Mesorhizobium huakuii subsp. rengei, a symbiont of Astragalus sinicus cv. Japan

The phylogenetic position of Rhizobium huakuii bv. renge, a symbiont of Astragalus sinicus cv. Japan (renge-sou), was studied. The following phylogenetic approaches were used: restriction fragment length polymorphism (RFLP)-polymerase chain reaction (PCR) analysis of a full-length 16S rDNA fragment, 16S rDNA analysis of the first 300-bp sequence, bacteriophage typing, and amplification of the genomic region by random primer. All the data suggest that strains of R. huakuii bv. renge should be classified into subspecies of the new genus Mesorhizobium (Jarvis et al., Inter. J. System. Bacteriol., 47, 895-898, 1997) and renamed M. huakuii subsp. rengei. All the strains fell into a tight cluster which included M. loti and M. huakuii. The strains isolated from root nodules on A. sinicus were divided into three groups as follows: group I, M. huakuii subsp. rengei B3, M. huakuii subsp. rengei My6, M. huakuii subsp. rengei My7, M. huakuii subsp. rengei My3, and M. huakuii subsp. rengei OUT30020; group II, M. huakuii subsp. huakuii CCBAU103(T), M. huakuii subsp. huakuii ACCC13005, M. huakuii subsp. huakuii 7653R, and Mesorhizobium sp. N-1; group III, Mesorhizobium sp. OUT30019. All the strains isolated in Japan except strains N-1 and OUT30019 were classified into group I. Strains in group I were sensitive to bacteriophage H1 which was isolated from rice-paddy soil in Japan. Strains in groups II and III except for M. huakuii subsp. huakuii 7653R were resistant to phage H1. Rhizobium sp. ACMP18, a native symbiont of Astragalus cicer that forms nodules on A. sinicus, showed close similarity to M. huakuii subsp. huakuii CCBAU103(T), and should thus be classified as a Mesorhizobium sp. Taken together, the results of the analyses indicate that M. huakuii subsp. rengei forms a subgroup which is distinct from M. huakuii subsp. huakuii strains isolated in China and that strain B3 is the type strain.     

Transformation of Candida albicans by electroporation

In contrast to a variety of other yeasts, Candida albicans has proved difficult to transform with high efficiency. Lithium acetate transformation is fast and simple but provides a very low efficiency of DNA transfer (50-100 transformants/microg DNA), while spheroplast transformation, although more efficient ( approximately 300 transformants/microg integrative DNA and 10(3)-10(4) transformants/microg replicative DNA), is complicated and time-consuming. In this study we applied various yeast transformation techniques to C. albicans and selected an electroporation procedure for further optimization. Transformation efficiencies of up to 300 transformants/microg were obtained for an integrative plasmid and up to 4500 transformants/microg for a CARS-carrying plasmid. This reasonably high transformation efficiency, combined with the ease and speed of electroporation in comparison to alternative techniques, make it the preferred method for transformation of C. albicans.     

多黏类芽孢杆菌JSa-9电转化方法的优化

为建立多黏类芽孢杆菌JSa-9菌株的高效电击转化体系,本实验研究菌体培养时间、电场强度、电击缓冲液、质粒用量以及电击后复苏培养时间等因素对JSa-9菌株电转化效率的影响。结果表明：Paenibacillus polymyxaJSa-9培养至OD595 nm为0.3时,电转化效率最高,为0.12×103 CFU/μg DNA;用0.5 mol/L甘露醇、0.5 mol/L山梨醇以及10%甘油的电击缓冲液洗涤细胞,在电场强度17.5 kV/cm、电阻200 Ω,电容25 μF的电击条件下,加入1.0 μg/100 mL质粒DNA,复苏培养时间18 h,电转化效率达到约为0.36×103 CFU/μg DNA;制备P. polymyxa JSa-9原生质体,在电场强度5.0 kV/cm的电击条件下,加入0.8 μg/100 mL质粒DNA,电转化效率较感受态电转提高到约1.0×103 CFU/μg DNA。     

Convenient transformation of anamorphic basidiomycetous yeasts belonging to genus pseudozyma induced by electroporation

A convenient procedure for carrying out transformation by electroporation was optimized for the genus Pseudozyma. Successful transformation was achieved using a plasmid, pUXV1, that confers resistance to hygromycin B; the maximum transformation efficiency was 48 transformants/mug of plasmid DNA. Transformants of Pseudozyma antarctica T-34 expressing a green fluorescent protein were obtained by the procedure.     

植物乳杆菌G63电转化方法的优化

为获得植物乳杆菌G63高效的电转化方法,本研究从细胞生长状态、细胞弱化剂质量浓度、洗涤液、质粒添加量和电击参数等方面对菌株G63的电转化效率进行优化。结果表明：取菌株G63对数生长中期的细胞制备感受态,以1 g/100 mL甘氨酸作为细胞弱化剂,分别用1 mmol/L MgCl2和30 g/100 mL聚乙二醇1000洗涤细胞,并用30 g/100 mL聚乙二醇1000作为电击液,加入20 μg穿梭质粒,在1.5 kV和400 Ω条件下进行电击,可以获得最高的电转化效率,转化效率达到1.18×103 CFU/μg DNA,满足后续遗传学实验要求。     

粪肠球菌内源性质粒的序列分析及其穿梭载体的构建

为了对粪肠球菌EXW27的内源性质粒pXW进行DNA序列的测定和分析,并基于其最小复制子构建大肠杆菌-粪肠球菌穿梭载体。本研究从具有优良益生特性的粪肠球菌EXW27中分离得到了内源性质粒pXW,对其进行了DNA序列的测定及分析,然后利用质粒pXW的复制子构建大肠杆菌-乳酸菌穿梭载体,并研究大肠杆菌-乳酸菌穿梭载体的宿主范围、转化效率和稳定性。结果表明该质粒大小为8617 bp,GC含量为33.29%,包含8个ORF,推定为θ型复制质粒。质粒pXW在粪肠球菌EXW27中拷贝数最高可达32.09±0.93,表明质粒pXW属于高拷贝数质粒。本研究确定了质粒pXW的最小复制子,并基于该复制子构建了大肠杆菌-粪肠球菌穿梭载体pXWM1。该穿梭载体具有较宽的宿主范围,可成功转化至不同类型乳酸菌,转化效率介于1.96×102~8.96×104 CFU/μg （质粒DNA）之间,质粒丢失率介于28.54%~54.17%之间。本研究成功构建了一个具有宽宿主范围、高转化效率以及高稳定性的大肠杆菌-粪肠球菌穿梭载体,为乳酸菌基因操作提供了新工具。     

Development of a genetic transformation system for benzene-tolerant Rhodococcus opacus strains

Rhodococcus opacus B-4 and B-9 are tolerant to various organic solvents including benzene, toluene, ethylbenzene, xylenes and styrene, and are suitable bacterial hosts for the production of chemical products from hydrophobic substrates. A 4.4-kb endogenous plasmid (pKNR 01) was isolated from R. opacus B-4 and sequenced completely. Plasmid pKNR 01 encodes proteins that share similarity to replication proteins from the enteric bacterial and actinomycete theta-replication plasmids. A 7.4-kb chimeric plasmid, designated pKNR 01.1, was constructed by fusing XhoI-digested pKNR 01 and Escherichia coli vector pSTV 28. Plasmid pKNR 01.1 had the ability to replicate in B-4 and B-9. A protocol for transformation of B-9 by electroporation was optimized employing pKNR 01.1. Frequencies of 4.1 x 10(5) transformants per mug of plasmid DNA were obtained for B-9 cells, whereas B-4 harboring naturally occurring pKNR 01 was transformed at lower frequencies (approximately 1 x 10(4) transformants per mug of plasmid DNA). Deletion analysis of pKNR 01.1 showed that the 1.9-kb SphI-XhoI region containing the repA and rep B genes and the 0.6-kb region upstream of repA was essential for plasmid maintenance in R. opacus strains.     

High efficiency transformation of Schizosaccharomyces pombe pretreated with thiol compounds by electroporation

A highly efficient method for transformation of the fission yeast Schizosaccharomyces pombe by electroporation has been developed. Significantly higher transformation efficiency was obtained when intact cells grown in SD medium (0.67% Bacto yeast nitrogen base without amino acids, 2% glucose) were pretreated with thiol compounds before an electric pulse was applied to the cells. Among the thiol compounds tested, dithiothreitol (DTT) was the most effective for pretreatment. A high transformation efficiency was obtained when the cells were pretreated with 25 mM DTT at 30 degrees C for 15 min in an osmotically adjusted buffer, since the cells were sensitive to osmotic pressure. It was important to exclude glucose from the DTT pretreatment buffer, as it caused a drastic decrease in efficiency. The optimal cell concentration and amount of DNA during the electric pulse were 1x10(9) cells/ml and 10 ng, respectively. The maximum transformation efficiency, 1.2x10(7) transformants/microg plasmid DNA, was obtained when an electric pulse of 11.0 kV/cm was applied for 5 ms. Furthermore, the high competency of cells pretreated with DTT was maintained by freezing them in a non-permeating cryoprotectant such as sorbitol.     

Efficient electropulse transformation of intact Candida maltosa cells by different homologous vector plasmids.

Conditions for efficient and quick transformation by electroporation were developed in Candida maltosa. To investigate the efficiency of transformation with integrative as well as with autonomously replicating plasmids, a series of vectors was constructed for homologous transformation of this species. Transformants were obtained with different plasmids as covalently closed circular molecules and as linearized DNA. The influence of recipient strain and plasmid type as well as of cell number and parameters of the supplied electrical pulse on the transformation efficiency have been investigated. A maximum of 7000 transformants per 100 ng of plasmid DNA was reached. The efficiency of transformation was compared with that of the LiCl method.     

Autofluorescence and green fluorescent protein-derived fluorescence in Listeria innocua

Fluorescent L. innocua M1 was generated by transformation with plasmid SB2019 carrying gfp3. The transformed organism exhibited 2 h longer lag phase than the parental strain. The transformation and gfp3 expression did not affect the growth rates (0.49 ± 0.02 and 0.47 ± 0.05 h⁻¹) and the maximum optical densities (1.02 ± 0.01 and 0.90 ± 0.06) of the parental or the transformed strains. The transformation of L. innocua M1 with pSB2019 resulted in cell-concentration related fluorescence which was detectable from washed cells but not in growth media. Statistical discrimination between GFP3-driven and background fluorescence signals of transformed and parental strains occurred at cell optical densities of 0.1 and above which was partially due to a relatively high endogenous autofluorescence. Although the gfp-transformed L. innocua M1 developed in this study has the potential to be a marker organism for monitoring Listeria spp. responses in mixed cultures, more work is needed to optimize the GFP-based fluorescent signal.     

Antimicrobial activity of a 14-residue synthetic peptide against foodborne microorganisms

A chemically synthesized short-chain peptide composed of six leucine and eight lysine (6K8L) residues was demonstrated to be biocidal against several foodborne organisms including Escherichia coli O157:H7, Listeria monocytogenes, Pseudomonas fluorescens, and Kluyveromyces marxianus suspended in phosphate buffer at concentrations of 5 to 50 microg/ml. All strains were reduced by 3 log10 CFU/ml within 10 min at peptide concentrations of <10 microg/ml. The peptide reduced by 3 log10 CFU/ml E. coli O157:H7 counts in apple juice and was active over the pH range of 3.5 to 7. Peptide concentrations of 100 microg/ml inhibited the aerobic and anaerobic microorganisms present in meat exudate liquid. However, the peptide was not effective against E. coli O157:H7 in skim milk at concentrations up to 100 microg/ml.     

Simultaneous detection of Escherichia coli O157:H7, Salmonella, and Shigella in apple cider and produce by a multiplex PCR

With three pairs of primers, a multiplex PCR assay was established for the simultaneous detection of Escherichia coli 0157:H7, Salmonella, and Shigella. Under the optimized conditions, the assay yielded a 252-bp product from E. coli O157:H7, a 429-bp product from Salmonella Typhimurium, and a 620-bp product from Shigella flexneri, respectively. When the DNA extraction of multiple target organisms was included in the same reaction, two or three corresponding amplicons of different sizes were observed. In the specificity test, 10 E. coli O157:H7 strains and one E. coli O157:NM strain showed the expected 252-bp amplicon. Seven other E. coli strains yielded no signal. Additionally, the 429-bp amplicon was produced from 20 Salmonella strains covering 16 serotypes, whereas the 620-bp amplicon was generated from 11 Shigella strains covering 4 species. No nonspecific amplification was observed with DNA from 48 other bacterial strains. Following a 24-h enrichment, the developed assay could concurrently detect the three pathogens at initial inoculation levels of approximately 8 x 10(-1) CFU/g (or CFU/ml) in apple cider, cantaloupe, lettuce, tomato, and watermelon and 8 x 10(1) CFU/g in alfalfa sprouts. The whole procedure can be easily completed within 30 h. The multiplex PCR assay can potentially be a simple, rapid, and efficient tool for presumptive and simultaneous screening of apple cider and produce for contamination by E. coli O157:H7, Salmonella, and/or Shigella.     

Development of a real-time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples

A highly sensitive real-time PCR (qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C gene (pc-plc), was developed for specific detection and quantification of strains belonging to Bacillus cereus group. The target region was selected based on the enterotoxigenic profiles of 75 Bacillus strains. The inclusivity and exclusivity of the RTi-PCR assay were assessed with 59 isolates of the B. cereus group, 16 other Bacillus spp., and 4 non-Bacillus strains. The assay was also used to construct calibration curves for different food matrices, and it had a wide quantification range of 6 log units using both serial dilutions of purified DNA and calibrated cell suspensions of B. cereus CECT 148^T. The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted infant formula was about 3 CFU per reaction or 60 CFU/ml of food, with a relative accuracy of 86.27% to 116.12% in artificially contaminated liquid egg. Naturally contaminated food samples were tested for the presence of B. cereus with the standard method, a conventional PCR and the new developed RTi-PCR assay. Results showed that the new developed RTi-PCR assay is very suitable for detection and quantification of strains of B. cereus group in food samples without an enrichment step.     

Characterization of the L41 gene in Cryptococcus neoformans: its application as a selectable transformation marker for cycloheximide resistance

A transformation system using resistance to the antibiotic cycloheximide as a dominant selectable marker was developed for the pathogenic yeast Cryptococcus neoformans. A 3.5 kb DNA fragment containing a gene encoding the ribosomal protein L41 was cloned from a wild-type strain of C. neoformans which is sensitive to cycloheximide. The open reading frame of the L41 gene contains five introns and encodes a protein of 107 amino acids, which is similar to those reported for other yeasts. The cycloheximide resistance gene to be used as a marker was constructed by replacing a DNA segment of the wild-type L41 gene, which contained the amino acid proline at its 56th position with a homologous DNA segment from a mutant strain resistant to cycloheximide that contained leucine in that position. Cycloheximide resistant transformants were obtained by electroporation on YEPD plates, supplemented with 10-20 microg/ml cycloheximide, at a maximum efficiency of 300 transformants/microg plasmid DNA. While with other genes, most transformants of serotype D in C. neoformans maintain the transforming DNA as episomes, the cycloheximide-resistant transformants were all the result of ectopic genomic integration events.     

A simple and efficient procedure for transformation of Schizosaccharomyces pombe

We describe a simple and efficient procedure for transformation of Schizosaccharomyces pombe. Sz. pombe colonies grown on minimal (SD) plates were directly removed and suspended in a 100 microl reaction mixture containing 70 microl PLATE solution (50% polyethylene glycol-4000, 100 mM lithium acetate, 10 mM Tris-HCl, pH 4.9, and 1 mM EDTA), 10 microl plasmid DNA (1 microg), 10 microl carrier DNA (100 microg) and 10 microl sterile distilled water. After incubation at 30 degrees C for 1 h followed by heat shock treatment at 42 degrees C for 15 min, the reaction mixture was spread on a selection plate. The transformation efficiency obtained using the procedure was approximately 8000 transformants/microg DNA. The method is simple and time-saving, making it especially useful for a large number of samples and when a high transformation efficiency is not required.     

Detection and quantification of spoilage and pathogenic Bacillus cereus, Bacillus subtilis and Bacillus licheniformis by real-time PCR

A new primer-probe set for the detection and quantification of Bacillus cereus, Bacillus licheniformis and Bacillus subtilis by real-time PCR (Rti-PCR) was developed. For it, forty-eight strains belonging to these species were considered. The DNA of these strains was isolated and a fragment of the 16S rRNA gene amplified. The amplicons were sequenced and the obtained sequences were aligned with reference sequences from the GenBank. For the development of the Real-Time PCR (RTi-PCR) methodology based on TaqMan probes, a primer pair and probe, specific for the studied Bacillus spp., were designed. To establish the quantification method, two RTi-PCR standard curves were constructed; one with DNA extracted from a serially-diluted B. cereus culture and a second curve with DNA extracted from a sterilised food product inoculated with serial dilutions of B. cereus. The curves exhibited R² values of 0.9969 and 0.9958 respectively. Linear correlations between the log₁₀ input DNA concentration and the threshold cycle (Ct) values were observed with a magnitude of linearity in the range of 1.65 × 10¹ CFU/mL to 1.65 × 10⁶ CFU/mL for both standard curves. The specificity of the designed primers and probe was tested with DNA extracted from B. cereus, B. licheniformis and B. subtilis strains, which gave Ct values between 14 and 15, whereas non-specific amplifications of the DNA from other microbial species of food interest exhibited a Ct value above 28.5. To our knowledge, this method represents the first study about the quantification of spoilage and/or pathogenic B. cereus, B. licheniformis and B. subtilis in food products, with the aim to prevent the presence of these undesirable species in the food chain.     

酵母细胞的高效转化方法

运用醋酸锂处理酵母细胞，建立并优化了酵母完整细胞的高效质粒转化体系，转化率达１０４μｇ－１．通过对影响转化的诸因子的研究，发现载运ＤＮＡ是影响酵母完整细胞转化效率的最主要因素，热休克处理及处理时间、聚乙二醇相对分子质量及浓度等对转化率也有显著影响。     

Monitoring of phytopathogenic Ralstonia solanacearum cells using green fluorescent protein-expressing plasmid derived from bacteriophage phiRSS1

A green fluorescent protein (GFP)-expressing plasmid was constructed from a filamentous bacteriophage phiRSS1 that infects the phytopathogen Ralstonia solanacearum. This plasmid designated as pRSS12 (4.7 kbp in size) consists of an approximately 2248 bp region of the phiRSS1 RF DNA, including ORF1-ORF3 and the intergenic region (IG), and a Km cassette in addition to the GFP gene. It was easily introduced by electroporation and stably maintained even without selective pressure in strains of R. solanacearum of different races and biovars. Strong green fluorescence emitted from pRSS12-transformed bacterial cells was easily monitored in tomato tissues (stem, petiole, and root) after infection as well as from soil samples. These results suggest that pRSS12 can serve as an easy-to-use GFP-tagging tool for any given strain of R. solanacearum in cytological as well as field studies.