Plasmid Screening Research Articles

The Influence of the Synthetic Organoselenium Preparation 974zh on the Immunogenic Activity of Yersinia pestis EV Vaccine Strain NIIEG.

In the present study, a stimulating effect of a new synthetic organoselenium compound 2,6-dipyridinium-9-selenabicyclo[3.3.1]nonandibromide (974zh) on the immunogenic activity of the vaccine strain Yersinia pestis EV NIIEG was revealed. After infection with the virulent plague strain, the survival rate of laboratory mice immunized with the vaccine strain grown on Hottinger's agar in the presence of 974zh (300 μg/ml) increased in comparison with control animals immunized with the Y. pestis EV NIIEG culture grown on agar without the studied compound. Plasmid screening of cultures grown on medium with and without 974zh showed that plasmid DNA of Y. pestis EV culture grown in the presence of 974zh had broader bands in the control grown without 974zh. This phenomenon can indicate activation of replication of plasmid DNA of Y. pestis EV under the influence of the experimental compound.

Sustainable Methodologies for Efficient Gel Electrophoresis and Streamlined Screening of Difficult Plasmids

We describe a workflow for efficient, environmentally attentive, and sustainable practices related to routine agarose gel electrophoresis. The methods reduce plastic waste and improve efficiency, especially for the exhaustive screening of difficult-to-obtain plasmids. Sustainability is increased when agarose is used ten times over by virtue of a thorough recycling regimen. The workflow optimizes workspaces and standardizes lab practices for handling potentially hazardous waste, minimizing environmental harm. Safety, efficiency, and sustainability improve laboratory productivity, help minimize environmental contamination, and increase cost-effectiveness.

Efficient De Novo Biosynthesis of Heme by Membrane Engineering in Escherichia coli

Heme is of great significance in food nutrition and food coloring, and the successful launch of artificial meat has greatly improved the application of heme in meat products. The precursor of heme, 5-aminolevulinic acid (ALA), has a wide range of applications in the agricultural and medical fields, including in the treatment of corona virus disease 2019 (COVID-19). In this study, E. coli recombinants capable of heme production were developed by metabolic engineering and membrane engineering. Firstly, by optimizing the key genes of the heme synthesis pathway and the screening of hosts and plasmids, the recombinant strain EJM-pCD-AL produced 4.34 ± 0.02 mg/L heme. Then, the transport genes of heme precursors CysG, hemX and CyoE were knocked out, and the extracellular transport pathways of heme Dpp and Ccm were strengthened, obtaining the strain EJM-ΔCyoE-pCD-AL that produced 9.43 ± 0.03 mg/L heme. Finally, fed-batch fermentation was performed in a 3-L fermenter and reached 28.20 ± 0.77 mg/L heme and 303 ± 1.21 mg/L ALA. This study indicates that E. coli recombinant strains show a promising future in the field of heme and ALA production.

Analysis of the Features of the Genomic Organization of Plague Microbe Strains Suitable for the Creation of a New Attenuated Vaccine

Relevance. To date, the specific prevention of plague remains the most effective measure to prevent to prevent the deterioration of the epidemiological situation for this especially dangerous infection in the territory of the Russian Federation. The development of new vaccine preparations created on the basis of avirulent strains of Yersinia pestis with specific genetic defects, can play a crucial importance in the issues of plague immunoprophylaxis.Aim. Determination and comparative analysis of the genomic organization of avirulent strains Y. pestis I-1, Y. pestis I-3536 and vaccine strain Y. pestis EV line NIIEG.Materials and methods. The features of the genetic structure of three Y. pestis strains were studied using plasmid screening and whole genome sequencing.Results and discussion. It was found that the Y. pestis I-1 and Y. pestis I-3536 strains, like Y. pestis EV, lack the chromosomal pgm region. Analysis the genome sequence of Y. pestis I-1 showed that this strain is devoid of the plasmid pMT1, while its individual genes were found in the chromosome structure and the unique 340 kb plasmid. It was revealed that the Y. pestis strain I-3536 lost the plasmid pCD1, however, fragments homologous to this plasmid were found in the structure of its genome.Conclusion. The data obtained allow us to recommend Y. pestis I-1 and Y. pestis I-3536 as the basis for the development of a modern live attenuated plague vaccine.

Highly efficient and simple SSPER and rrPCR approaches for the accurate site-directed mutagenesis of large and small plasmids

Advances are needed in the site-directed mutagenesis of large plasmids for protein structure-function studies, as current methods are often inefficient, complicated and time-consuming. Here two new methods are reported that overcome these difficulties, namely the single primer extension reaction (SSPER) strategy that reaches 100% efficiency and the reduce recycle PCR (rrPCR) method that is advantageous in generating single and pairwise combinations of mutations. Both methods are distinguished from current technologies by the addition of a step that easily removes the oligonucleotide primer(s) after the first reaction, thus allowing for the addition of a second reaction in chronological sequence to generate and isolate the appropriate DNA product with the site-directed mutation(s). High efficiency of the methods is demonstrated by generating single and paired combinations of the 11 site-directed mutations targeted on 5 different plasmid DNA templates ranging from 10 to 12 kb and 57–60% GC-content at a rate of 50–100%. Overall, the methods are demonstrated to be (i) highly accurate, allowing for screening of plasmids by DNA sequencing, (ii) streamlined to generate the mutations within a single day, (iii) cost-effective in requiring only two primers and two enzymes (DpnI and a proofreading DNA polymerase), (iv) straightforward in primer design, (v) applicable for both large and small plasmids, and (vi) easily implemented by entry level researchers.

A 4-α-Glucanotransferase from Thermus thermophilus HB8: Secretory Expression and Characterization.

4-α-glucanotransferase (4GT, EC 2.4.1.25) catalyzes the breakdown of the α-1,4 glycosidic bonds of the starch main chain and forms new α-1,4 glycosidic bonds in the side chain, which is often used to optimize the physical and chemical properties of starch and to improve the quality of starch-based food. However, the low enzyme activity of 4GT limits its production and widespread application. Herein, the 4GT gene encoding 500 amino acids from Thermus thermophilus HB8 was cloned and expressed in Escherichia coli. The purified 4GT exhibited maximum activity at pH 7.0 and 60°C and had a good stability at pH 6.0-8.0 and 30-60°C. It was confirmed that 4GT possessed the catalytic function of extending the branch length of potato starch. Furthermore, the 4GT gene was successfully expressed extracellularly in Bacillus subtilis. Then, the enzyme yield of 4GT increased by 4.1 times through screening of different plasmids and hosts. Additionally, the fermentation conditions were optimized to enhance 4GT extracellular enzyme yield. Finally, a recombinant Bacillus subtilis with 299.9U/mL enzyme yield of 4GT was obtained under the optimized fermentation process. In conclusion, this study provides a valuable reference for characterization and expression of food-grade enzymes.

Характеристика CRISPR/CAS-системы штамма Pseudomonas aeruginosa DSM 50071 на основе биоинформационного анализа её структур

An algorithm for bioinformatic search and analysis of the structures of CRISPR/Cas systems of bacteria and screening of phages and plasmids through spacer sequences of CRISPR cassettes in the genome of the Pseudomonas aeruginosa strain DSM 50071 is presented.Using several search algorithms in the CRISPR/Cas system of the studied strain, the presence of three CRISPR loci and a group of Cas genes characteristic of Type-I Subtype-I-F was determined.Analysis of the spacer composition of CRISPR cassettes showed the presence of 31 to 43 spacers and a universal consensus repeat in all cassettes.Screening of the spacer sequences of the CRISPR cassettes of the studied strain showed their correspondence to the protospacers of phages and plasmids of bacteria of the families Pseudomonadaceae and Enterobacteriaceae. A complete characterization of bacteriophages to which this strain is resistant is given with their accession number in NCBI. A complete identification of spacers to protospacers of phages specific for bacteria of the Pseudomonadaceae family, most often isolated from the lungs of patients with bronchiectasis, pneumonia, as well as from hospitals and reservoirs, has been established.Full correspondence between spacers and protospacers of bacterial plasmids with pan-resistance and causing the development of respiratory failure and pneumonia was revealed. Correspondence of a segment of one spacer with protospacers of several bacterial phages of the same family was noted. This may indicate that the bacterium “expediently” acquires new spacers from DNA regions that are conserved for phages of bacteria of the same family.Genes that have phage protospacers in their structure have been identified.It has been established that these genes are responsible for the synthesis of enzymes that regulate the processes of virus reproduction.Therefore, activation of the CRISPR/Cas system in the genome of this strain will allow the restriction endonuclease to introduce breaks into unmethylated DNA, which will lead to disruption of the synthesis of this enzyme, and, consequently, disruption of bacteriophage replication.Correspondences of spacer sequences with protospacers of plasmids included in the structure of genes responsible for the synthesis of conjugative transfer enzymes were revealed.These results suggested that activation of the CRISPR/Cas system of this strain would disrupt the processes replication of bacteriophage and conjugation.The proposed algorithm made it possible to obtain information about the structure of the CRISPR/Cas system of the P. aeruginosa DSM 50071 strain, about its resistance to certain phages and plasmids. In the future, this will serve as the basis for creating approaches for targeted therapy of infectious diseases.

A standardized genome architecture for bacterial synthetic biology (SEGA)

Chromosomal recombinant gene expression offers a number of advantages over plasmid-based synthetic biology. However, the methods applied for bacterial genome engineering are still challenging and far from being standardized. Here, in an attempt to realize the simplest recombinant genome technology imaginable and facilitate the transition from recombinant plasmids to genomes, we create a simplistic methodology and a comprehensive strain collection called the Standardized Genome Architecture (SEGA). In its simplest form, SEGA enables genome engineering by combining only two reagents: a DNA fragment that can be ordered from a commercial vendor and a stock solution of bacterial cells followed by incubation on agar plates. Recombinant genomes are identified by visual inspection using green-white colony screening akin to classical blue-white screening for recombinant plasmids. The modular nature of SEGA allows precise multi-level control of transcriptional, translational, and post-translational regulation. The SEGA architecture simultaneously supports increased standardization of genetic designs and a broad application range by utilizing well-characterized parts optimized for robust performance in the context of the bacterial genome. Ultimately, its adaption and expansion by the scientific community should improve predictability and comparability of experimental outcomes across different laboratories.

Pitfalls of Restriction Enzyme Mapping Following Generation of CRISPR Constructs

The PX330 and the related PX459 plasmids are widely used for Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-mediated genome editing. Screening for plasmids containing the correct sgRNA template insertion is one of the most important steps in this system. Different methods for screening the sgRNA inserts have been deployed. One such method is Restriction Enzyme (RE) mapping. Restriction enzyme mapping can be used to screen for numerous plasmid recombinants simultaneously. In this study, the sgRNA templates were initially cloned into the above PX459 plasmids. Subsequently, the accuracy of the constructs was determined by RE mapping. This method was established to screen for sgRNA-bearing PX459 plasmids. However, numerous anomalies were detected after ligation of sgRNA templates into RE digested PX459 plasmids. Our data suggest that RE mapping is only appropriate as an initial screen and that the identity of all plasmids with the correctly identified RE maps should be confirmed by Sanger sequencing.

Molecular Characterization of Staphylococcus aureus Plasmids Associated With Strains Isolated From Various Retail Meats.

Staphylococcus aureus is considered one of the most important foodborne bacterial pathogens causing food poisoning and related illnesses. S. aureus strains harbor plasmids encoding genes for virulence and antimicrobial resistance, but few studies have investigated S. aureus plasmids, especially megaplasmids, in isolates from retail meats. Furthermore, knowledge about the distribution of genes encoding replication (rep) initiation proteins in food isolates is lacking. In this study, the prevalence of plasmids in S. aureus strains isolated from retail meats purchased in Oklahoma was investigated; furthermore, we evaluated associations between rep families, selected virulence and antimicrobial resistance genes, and food source origin. Two hundred and twenty-two S. aureus isolates from chicken (n = 55), beef liver (n = 43), pork (n = 42), chicken liver (n = 29), beef (n = 24), turkey (n = 22), and chicken gizzards (n = 7) were subjected to plasmid screening with alkaline lysis and PFGE to detect small-to-medium sized and large plasmids, respectively. The S. aureus isolates contained variable sizes of plasmids, and PFGE was superior to alkaline lysis in detecting large megaplasmids. A total of 26 rep families were identified by PCR, and the most dominant rep families were rep10 and rep7 in 164 isolates (89%), rep21 in 124 isolates (56%), and rep12 in 99 isolates (45%). Relationships between selected rep genes, antimicrobial resistance and virulence genes, and meat sources were detected. In conclusion, S. aureus strains isolated from retail meats harbor plasmids with various sizes and there is an association between rep genes on these plasmids and the meat source or the antimicrobial resistance of the strains harboring them.

Conjugative Transfer of a Novel Staphylococcal Plasmid Encoding the Biocide Resistance Gene, qacA.

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTI). Some S. aureus strains harbor plasmids that carry genes that affect resistance to biocides. Among these genes, qacA encodes the QacA Multidrug Efflux Pump that imparts decreased susceptibility to chlorhexidine, a biocide used ubiquitously in healthcare facilities. Furthermore, chlorhexidine has been considered as a S. aureus decolonization strategy in community settings. We previously conducted a chlorhexidine-based SSTI prevention trial among Ft. Benning Army trainees. Analysis of a clinical isolate (C02) from that trial identified a novel qacA-positive plasmid, pC02. Prior characterization of qacA-containing plasmids is limited and conjugative transfer of those plasmids has not been demonstrated. Given the implications of increased biocide resistance, herein we characterized pC02. In silico analysis identified genes typically associated with conjugative plasmids. Moreover, pC02 was efficiently transferred to numerous S. aureus strains and to Staphylococcus epidermidis. We screened additional qacA-positive S. aureus clinical isolates and pC02 was present in 27% of those strains; other unique qacA-harboring plasmids were also identified. Ten strains were subjected to whole genome sequencing. Sequence analysis combined with plasmid screening studies suggest that qacA-containing strains are transmitted among military personnel at Ft. Benning and that strains carrying qacA are associated with SSTIs within this population. The identification of a novel mechanism of qacA conjugative transfer among Staphylococcal strains suggests a possible future increase in the prevalence of antiseptic tolerant bacterial strains, and an increase in the rate of infections in settings where these agents are commonly used.

PCR-Based Analysis of ColE1 Plasmids in Clinical Isolates and Metagenomic Samples Reveals Their Importance as Gene Capture Platforms.

ColE1 plasmids are important vehicles for the spread of antibiotic resistance in the Enterobacteriaceae and Pasteurellaceae families of bacteria. Their monitoring is essential, as they harbor important resistant determinants in humans, animals and the environment. In this work, we have analyzed ColE1 replicons using bioinformatic and experimental approaches. First, we carried out a computational study examining the structure of different ColE1 plasmids deposited in databases. Bioinformatic analysis of these ColE1 replicons revealed a mosaic genetic structure consisting of a host-adapted conserved region responsible for the housekeeping functions of the plasmid, and a variable region encoding a wide variety of genes, including multiple antibiotic resistance determinants. From this exhaustive computational analysis we developed a new PCR-based technique, targeting a specific sequence in the conserved region, for the screening, capture and sequencing of these small plasmids, either specific for Enterobacteriaceae or specific for Pasteurellaceae. To validate this PCR-based system, we tested various collections of isolates from both bacterial families, finding that ColE1 replicons were not only highly prevalent in antibiotic-resistant isolates, but also present in susceptible bacteria. In Pasteurellaceae, ColE1 plasmids carried almost exclusively antibiotic resistance genes. In Enterobacteriaceae, these plasmids encoded a large range of traits, including not only antibiotic resistance determinants, but also a wide variety of genes, showing the huge genetic plasticity of these small replicons. Finally, we also used a metagenomic approach in order to validate this technique, performing this PCR system using total DNA extractions from fecal samples from poultry, turkeys, pigs and humans. Using Illumina sequencing of the PCR products we identified a great diversity of genes encoded by ColE1 replicons, including different antibiotic resistance determinants, supporting the previous results achieved with the collections of bacterial isolates. In addition, we detected cryptic ColE1 plasmids in both families with no known genes in their variable region, which we have named sentinel plasmids. In conclusion, in this work we present a useful genetic tool for the detection and analysis of ColE1 plasmids, and confirm their important role in the dissemination of antibiotic resistance, especially in the Pasteurellaceae family of bacteria.

Genotypic heterogeneity of emetic toxin producing Bacillus cereus isolates from China.

Emetic toxin-producing Bacillus cereus (emetic B. cereus) is the third member of B. cereus group whose toxins are encoded by megaplasmids, beside anthrax and insecticidal toxins of B. anthracis and B. thuringiensis, respectively. A total of 18 emetic isolates collected from food poisoning events, clinical and non-random food samples in Zhejiang province of China, were analyzed by plasmid screening, pulse field gel electrophoresis, multilocus sequence typing, and toxic gene identification to investigate their genotypic diversity. In this study, 13 plasmid profile types, 14 pulse types and 6 different STs from emetic isolates were detected, in which ST 1035,1038,1053,1054 and 1065 were first assigned and reported. The toxic gene ces existed on its own, or coexisted with other toxic genes bceT, cytk, entFM and nhe, but never with hbl in emetic isolates. The results demonstrated that the emetic B. cereus strains from China were heterologous at genotypic level.

Rapid Screening of Recombinant Plasmids by Direct Colony Quantitative Real-Time PCR

Quantitative real-time PCR (qPCR) was applied to rapid screening of positive plasmid clones. Insert-specific primer pairs were used in qPCR colony screening, and false positive colonies could easily be distinguished from true positive ones by comparing their Ct values. In addition, qPCR is particularly suitable when amplicon is small (<150 bp). This method is sensitive, simple and fast, obviates the need for gel electrophoresis, and is a cost-effective alternative to the traditional PCR approach.

A Case of Human Plague in Kosh-Agach Region of the Republic of Altai in 2015. Communication 2. Microbiological and Molecular-Genetic Characteristics of the Isolated Strains

Objective of the study was a complex pheno- and genotypic investigation of Yersinia pestis I-3595 and Y. pestis I-3596 strains, isolated from marmots that were the source of human infection in 2015. Materials and methods. A comprehensive microbiological, molecular-genetic and mass-spectrometric studying of Y. pestis ssp. pestis strains, isolated from two grey marmots that became a cause of the plague case in Gorno-Altai high-mountain focus in 2015, was performed using up-to-date methods. Results and conclusions. It was established that the studied Y. pestis strains belonged to the main subspecies. Results of plasmid screening, multilocus VNTR-and mass-spectrometric analyses showed that those strains were closely related to Y. pestis variant detected in the focus in 2012 and in 2014, and circulating in Northwest Mongolia and Tuva natural focus.

Isolation of a novel plasmid from Couchioplanes caeruleus and construction of two plasmid vectors for gene expression in Actinoplanes missouriensis

To date, no plasmid vector has been developed for the rare actinomycete Actinoplanes missouriensis. Moreover, no small circular plasmid has been reported to exist in the genus Actinoplanes. Here, a novel plasmid, designated pCAZ1, was isolated from Couchioplanes caeruleus subsp. azureus via screening for small circular plasmids in Actinoplanes (57 strains) and Couchioplanes (2 strains). Nucleotide sequencing revealed that pCAZ1 is a 5845-bp circular molecule with a G + C content of 67.5%. The pCAZ1 copy number was estimated at 30 per chromosome. pCAZ1 contains seven putative open reading frames, one of which encodes a protein containing three motifs conserved among plasmid-encoded replication proteins that are involved in the rolling-circle mechanism of replication. Detection of single-stranded DNA intermediates in C. caeruleus confirmed that pCAZ1 replicates by this mechanism. The ColE1 origin from pBluescript SK(+) and the oriT sequence with the apramycin resistance gene aac(3)IV from pIJ773 were inserted together into pCAZ1, to construct the Escherichia coli–A. missouriensis shuttle vectors, pCAM1 and pCAM2, in which the foreign DNA fragment was inserted into pCAZ1 in opposite directions. pCAM1 and pCAM2 were successfully transferred to A. missouriensis through the E. coli-mediated conjugative transfer system. The copy numbers of pCAM1 and pCAM2 in A. missouriensis were estimated to be one and four per chromosome, respectively. Thus, these vectors can be used as effective genetic tools for homologous and heterologous gene expression studies in A. missouriensis.

Identification of regulatory factors of the stationary phase sigma factor RpoS in Escherichia coli

The sigma factor RpoS is the master regulator of the general stress response that helps cells survive harsh environmental conditions by associating with the RNA polymerase and regulating gene expression. While numerous stresses are known to up‐regulate RpoS, many of the molecular steps leading from stresses to changes in RpoS levels remain unknown. To screen for the novel regulators, we used an RpoS’‐‘LacZ fusion. We transformed the reporter strains with an overexpression library, screening for plasmids that resulted in colonies with either more or less RpoS as observed on Xgal indicator plates. We verified that RpoS levels were altered in the presence of the plasmid with Western blots and quantified the effect on RpoS expression beta‐galactosidase assays in both RpoS transcription and translation lacZ fusions. We identified the genes on the plasmids as gadY, rbsD, yhhI, yhjR, ykgR and part of the dsdA gene in the screen. Our results suggested all of the factors, with the exception of rbsD, regulated RpoS in a post‐transcriptional manner either at the level of translation or protein stability. The small RNA gadY caused a strong inhibitory effect on RpoS expression and yhhI, ykgR and yhjR causing a mild increase expression of RpoS. In summary, we have found multiple new factors that affect RpoS at the post‐transcriptional level.

The First Case of &lt;I&gt;Yersinia Pestis&lt;/I&gt; Subsp. &lt;I&gt;Pestis&lt;/I&gt; Isolation in the Territory of Altai Mountain Natural Plague Focus. Communication 1. Microbiological Characteristics, Molecular-Genetic and Mass-Spectrometric Identification of the Isolate

Performed is a complex microbiological, molecular-genetic and mass-spectrometric identification of Yersinia pestis main ssp. strain, which was isolated for the first time in the history of surveillance over the Altai mountain natural plague focus in June, 2012. Determined is its high universal virulence. Plasmid screening, multi-locus VNTR- and mass-spectrometric analyses have revealed the strain to be more closely related to the plague agent variant, circulating in the territory of the natural focus Khuukh-Serkh-Munkh-Khairkhan, Bayan-Ul’giisk aimak, Mongolia.

Construction of temperature sensitive bone marrow mesenchymal stem cells model

Objective Bone marrow mesenchymal stem cells of temperature sensitive simian virus 40 large T antigen gene (tsSV40LTag - BMSCs ) was constructed successfully.The proliferation of tsSV40LTag - BMSCs at different temperatures was observed.Method Isolation and cultivation of primary BMSCs from SD rats was performed.Antibodies of CD29,CD31,CD44,CD45 and CD90 marked by FITC and CD34 were added to the cell suspensions respectively.Identification of surface markers of BMSCs was performed by flow cytometry.pRNS - 1 plasmid transfection,screening and positive amplification were done.RT - PCR was used to explore the expression of tsSV40LTag in BMSCs.Growth curve of tsSV40LTag -BMSCswas determined at temperature of 33℃ and 37℃.Results TsSV40LTag was transfected to BMSCs of SD rats with pRNS - 1 plasmid.TsSV40LTag - BMSCs grew fast at 33℃,serial passage was available in vitro culture.But cells grew significantly slowly at 37℃,even stopped growing.Conclusions The model of tsSV40LTag- BMSCs is set up successfully,which provides experimental proof for tsSV40LTag - BMSCs transplantation treatment against TBI. Key words: Bone marrow; Mesenchymal stem cells; Transfection

Screening of degradative plasmids from Arthrobacter sp. HW08 and their ability to degrade swainsonine

Arthrobacter sp. HW08 is capable of swainsonine (SW) degradation with high efficiency. The aim of this study was to screen the degradative plasmids from Arthrobacter sp. HW08 and investigate their ability of degrading SW. A genomic library of Arthrobacter sp. HW08 was successfully constructed by BamHI restriction digestion of genomic DNA and pUC19 vector, ligation and transformation into DH5. Five plasmids of different sizes were screened from 100 genomic library strains, and were named as pUCSW-1, pUCSW-2, pUCSW-3, pUCSW-4 and pUCSW-5, and their insertion elements had sizes of 6 684, 5 093, 136, 1 452 and 2 082 bp, respectively. They contained 7 major open reading frames. SW (400 mg l -1 ), as only carbon source, cultivated with the mixture of the five plasmid-transformants was degraded within 6 h. The degrading capability was equivalent to that of strain HW08. SW degradative ability of minimum combinations was pUCSW-2, 3, 5 > pUCSW-1, 2, 4 > pUCSW-1, 2, 5 > pUCSW-1, 3, 5, which were all slower than that of strain HW08. Any two or single transformant had no degradative ability. The results of this study support the search of the key genes of SW degradation and the investigating mechanism of SW biodegradation or exploration of its metabolic pathway, and even construction of transgene engineering bacteria to protect animals from lesion when locoweed is consumed. Key words : Swainsonine, biodegradation, degradative plasmids, Arthrobacter sp. HW08.