Endogenous Plasmid Research Articles

Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC 6803.

Cyanobacteria are photosynthetic cell factories that use solar energy to convert CO2 into useful products. Despite this attractive feature, the development of tools for engineering cyanobacterial chassis has lagged behind that for heterotrophs such as Escherichia coli or Saccharomyces cerevisiae. Heterologous genes in cyanobacteria are often integrated at presumptively "neutral" chromosomal sites, with unknown effects. We used transcriptome sequencing (RNA-seq) data for the model cyanobacterium Synechocystis sp. strain PCC 6803 to identify neutral sites from which no transcripts are expressed. We characterized the two largest such sites on the chromosome, a site on an endogenous plasmid, and a shuttle vector by integrating an enhanced yellow fluorescent protein (EYFP) expression cassette expressed from either the Pcpc560 or the Ptrc1O promoter into each locus. Expression from the endogenous plasmid was as much as 14-fold higher than that from the chromosome, with intermediate expression from the shuttle vector. The expression characteristics of each locus correlated predictably with the promoters used. These findings provide novel, characterized tools for synthetic biology and metabolic engineering in cyanobacteria.

Curing a large endogenous plasmid by single substitution of a partitioning gene

To investigate whether plasmid-free cells of pathogenic Escherichia coli can be isolated by disrupting a single gene in an endogenous plasmid without further treatment, the effect of the disruption of partitioning genes on the inheritance of the endogenous plasmid pUTI89 of the uropathogenic E. coli strain UTI89 was studied. We found that mutation of parB, which encodes a type Ib partitioning protein, could cause loss of the endogenous plasmid at a ratio of about 1%. Clones derived from parB mutants, identified by antibiotic sensitivity, were all plasmid free. Plasmid instability caused by the parB mutation was found to correlate with a negative effect on host cell growth. Thus, in this pathogenic E. coli, an endogenous plasmid as large as 114kbp could be cured effectively by targeting a single type Ib partitioning gene followed by passaging, which may facilitate further investigations on the function of endogenous plasmids in their natural hosts.

Disruption of bbe02 by Insertion of a Luciferase Gene Increases Transformation Efficiency of Borrelia burgdorferi and Allows Live Imaging in Lyme Disease Susceptible C3H Mice.

Lyme disease is the most prevalent tick-borne disease in North America and Europe. The causative agent, Borrelia burgdorferi persists in the white-footed mouse. Infection with B. burgdorferi can cause acute to persistent multisystemic Lyme disease in humans. Some disease manifestations are also exhibited in the mouse model of Lyme disease. Genetic manipulation of B. burgdorferi remains difficult. First, B. burgdorferi contains a large number of endogenous plasmids with unique sequences encoding unknown functions. The presence of these plasmids needs to be confirmed after each genetic manipulation. Second, the restriction modification defense systems, including that encoded by bbe02 gene lead to low transformation efficiency in B. burgdorferi. Therefore, studying the molecular basis of Lyme pathogenesis is a challenge. Furthermore, investigation of the role of a specific B. burgdorferi protein throughout infection requires a large number of mice, making it labor intensive and expensive. To overcome the problems associated with low transformation efficiency and to reduce the number of mice needed for experiments, we disrupted the bbe02 gene of a highly infectious and pathogenic B. burgdorferi strain, N40 D10/E9 through insertion of a firefly luciferase gene. The bbe02 mutant shows higher transformation efficiency and maintains luciferase activity throughout infection as detected by live imaging of mice. Infectivity and pathogenesis of this mutant were comparable to the wild-type N40 strain. This mutant will serve as an ideal parental strain to examine the roles of various B. burgdorferi proteins in Lyme pathogenesis in the mouse model in the future.

Strategies and approaches in plasmidome studies-uncovering plasmid diversity disregarding of linear elements?

The term plasmid was originally coined for circular, extrachromosomal genetic elements. Today, plasmids are widely recognized not only as important factors facilitating genome restructuring but also as vehicles for the dissemination of beneficial characters within bacterial communities. Plasmid diversity has been uncovered by means of culture-dependent or -independent approaches, such as endogenous or exogenous plasmid isolation as well as PCR-based detection or transposon-aided capture, respectively. High-throughput-sequencing made possible to cover total plasmid populations in a given environment, i.e., the plasmidome, and allowed to address the quality and significance of self-replicating genetic elements. Since such efforts were and still are rather restricted to circular molecules, here we put equal emphasis on the linear plasmids which—despite their frequent occurrence in a large number of bacteria—are largely neglected in prevalent plasmidome conceptions.

Transgenic hairy roots of Tetrastigma hemsleyanum: induction, propagation, genetic characteristics and medicinal components

Tetrastigma hemsleyanum Diels & Gilg ex Diels (T. hemsleyanum) is an important medicinal plant in China. Its bulbous root is the most valuable part for medicine. There is an increasing demand for T. hemsleyanum in the medicinal market. However, the natural slow growth rate of T. hemsleyanum hardly meets the demand of the medicinal market. There is the need of alternative way for rapid and large-scale production of T. hemsleyanum. In this study, we investigated the induction of hairy roots by using Agrobacterium rhizogenes (A. rhizogenes) as an alternative method of increasing production of the secondary metabolites in hairy roots of T. hemsleyanum. Herein, we reported our successful induction of hairy roots of T. hemsleyanum by cucumopine type A. rhizogenes K599 and their rapid propagation. Firstly, we selected the optimal mediums for induction of hairy roots and for subculture of induced hairy roots. We found that when A. rhizogenes K599 carrying both an endogenous plasmid pRi2659 and an exogenous plasmid pRI101-AN-GFP was used to infect the leaves of T. hemsleyanum seedlings, the optimal medium for inducting hairy roots was the medium of MS + 0.4 mg/L 1-naphthaleneacetic acid. The optimal medium for subculture of hairy roots was the combination of MS + 1.0 mg/L indole-3-butyric acid + 0.5 or 1.0 mg/L kinetin. During suspension culture of hairy roots, the rapid growth phase occurred in the period of 15–28 days. Secondly, we employed polymerase chain reaction (PCR) assays to examine the integration of T-DNA of A. rhizogenes. The PCR results revealed the existence of a phenomenon of lacking of integration of one or more of 11 genes within T-DNA of plasmid pRi2659 into the genomic DNA of hairy roots. Thirdly, we conducted histocytological analysis to examine the structural features of the induced hairy roots and observed that xylem structures of the hairy roots were simplified as compared to those of the bulbous roots and ordinary fine roots of non-transgenic plant. Finally, we applied high performance liquid chromatography to analyze the medicinal components in several organs of the T. hemsleyanum plants. The results showed that the contents of kaempferol in all hairy root lines were significantly higher than those in bulbous roots, fine roots, stems and leaves. These results suggest that insertion and integration of T-DNA from A. rhizogenes can significantly increase the kaempferol content in hairy roots of T. hemsleyanum.

Extensive amplification of GI-VII-6, a multidrug resistance genomic island of Salmonella enterica serovar Typhimurium, increases resistance to extended-spectrum cephalosporins.

GI-VII-6 is a chromosomally integrated multidrug resistance genomic island harbored by a specific clone of Salmonella enterica serovar Typhimurium (S.Typhimurium). It contains a gene encoding CMY-2 β-lactamase (blaCMY−2), and therefore contributes to extended-spectrum cephalosporin resistance. To elucidate the significance of GI-VII-6 on adaptive evolution, spontaneous mutants of S. Typhimurium strain L-3553 were selected on plates containing cefotaxime (CTX). The concentrations of CTX were higher than its minimum inhibition concentration to the parent strain. The mutants appeared on the plates containing 12.5 and 25 mg/L CTX at a frequency of 10−6 and 10−8, respectively. No colonies were observed at higher CTX concentrations. The copy number of blaCMY−2 increased up to 85 per genome in the mutants, while the parent strain contains one copy of that in the chromosome. This elevation was accompanied by increased amount of transcription. The blaCMY−2 copy number in the mutants drastically decreased in the absence of antimicrobial selection pressure. Southern hybridization analysis and short-read mapping indicated that the entire 125 kb GI-VII-6 or parts of it were tandemly amplified. GI-VII-6 amplification occurred at its original position, although it also transposed to other locations in the genome in some mutants, including an endogenous plasmid in some of the mutants, leading to the amplification of GI-VII-6 at different loci. Insertion sequences were observed at the junction of the amplified regions in the mutants, suggesting their significant roles in the transposition and amplification. Plasmid copy number in the selected mutants was 1.4 to 4.4 times higher than that of the parent strain. These data suggest that transposition and amplification of the blaCMY−2-containing region, along with the copy number variation of the plasmid, contributed to the extensive amplification of blaCMY−2 and increased resistance to CTX.

Malpighian tubules are important determinants of Pseudomonas transstadial transmission and longtime persistence in Anopheles stephensi.

BackgroundPseudomonas is a genus of bacteria commonly found in investigations of gut microbes in malaria mosquitoes. Among those mosquitoes is the dominating malaria vector in Asia, Anopheles stephensi, where Pseudomonas is a prevailing bacterium and natural inhabitant of its breeding places. In order to explore the reason for finding Pseudomonas so frequently, an investigation of its localization and transstadial properties was undertaken.MethodsA Pseudomonas isolate from An. stephensi was transformed successfully with an endogenous plasmid modified to express green fluorescent protein (GFP). Subsequently, the Pseudomonas-GFP was added to the laboratory larval breeding place of An. stephensi and taken up by the larvae. After 24 hours, the larvae were cleaned and moved to a bath with double-distilled water. Also, female adults were fed sugar solution containing Pseudomonas-GFP. The Pseudomonas-GFP was traced in the alimentary canal of larvae, pupae and adults.ResultsFluorescent microscopy and PCR assays showed that the Pseudomonas bacteria underwent transstadial transmission from larvae to pupae and then to adults. In blood-fed female mosquitoes, the bacteria increased in numbers and remained in the mosquito body for at least three weeks after eclosion. In addition to the midgut, the Malpighian tubules of both larvae and adult mosquitoes were colonized by the bacteria. Also Pseudomonas-GFP that was distributed through sugar solution was able to colonize the Malpighian tubules of adult females.ConclusionsColonization of the Malpighian tubules by Pseudomonas bacteria seems to be important for the transstadial passage from larvae to adult and presumably for the longevity of the bacteria in the adult mosquito. The existence of an entry point in the larval stage, and the long duration in the female gut, opens up for a possible use of Pseudomonas in mosquito paratransgenesis.

Zinc biosorption by the purple non-sulfur bacterium Rhodobacter capsulatus.

This paper presents the first report providing information on the zinc (Zn) biosorption potentialities of the purple non-sulfur bacterium Rhodobacter capsulatus. The effects of various biological, physical, and chemical parameters on Zn biosorption were studied in both the wild-type strain B10 and a strain, RC220, lacking the endogenous plasmid. At an initial Zn concentration of 10 mg·L(-1), the Zn biosorption capacity at pH 7 for bacterial biomass grown in synthetic medium containing lactate as carbon source was 17 and 16 mg Zn·(g dry mass)(-1) for strains B10 and RC220, respectively. Equilibrium was achieved in a contact time of 30-120 min, depending on the initial Zn concentration. Zn sorption by live biomass was modelled, at equilibrium, according to the Redlich-Peterson and Langmuir isotherms, in the range of 1-600 mg Zn·L(-1). The wild-type strain showed a maximal Zn uptake capacity (Qm) of 164 ± 8 mg·(g dry mass)(-1) and an equilibrium constant (Kads) of 0.017 ± 0.00085 L·(mg Zn)(-1), compared with values of 73.9 mg·(g dry mass)(-1) and 0.361 L·mg(-1) for the strain lacking the endogenous plasmid. The Qm value observed for R. capsulatus B10 is one of the highest reported in the literature, suggesting that this strain may be useful for Zn bioremediation. The lower Qm value and higher equilibrium constant observed for strain RC220 suggest that the endogenous plasmid confers an enhanced biosorption capacity in this bacterium, although no genetic determinants for Zn resistance appear to be located on the plasmid, and possible explanations for this are discussed.

Use of an endogenous plasmid locus for stable in trans complementation in Borrelia burgdorferi.

Targeted mutagenesis and complementation are important tools for studying genes of unknown function in the Lyme disease spirochete Borrelia burgdorferi. A standard method of complementation is reintroduction of a wild-type copy of the targeted gene on a shuttle vector. However, shuttle vectors are present at higher copy numbers than B. burgdorferi plasmids and are potentially unstable in the absence of selection, thereby complicating analyses in the mouse-tick infectious cycle. B. burgdorferi has over 20 plasmids, with some, such as linear plasmid 25 (lp25), carrying genes required by the spirochete in vivo but relatively unstable during in vitro cultivation. We propose that complementation on an endogenous plasmid such as lp25 would overcome the copy number and in vivo stability issues of shuttle vectors. In addition, insertion of a selectable marker on lp25 could ensure its stable maintenance by spirochetes in culture. Here, we describe the construction of a multipurpose allelic-exchange vector containing a multiple-cloning site and either of two selectable markers. This suicide vector directs insertion of the complementing gene into the bbe02 locus, a site on lp25 that was previously shown to be nonessential during both in vitro and in vivo growth. We demonstrate the functional utility of this strategy by restoring infectivity to an ospC mutant through complementation at this site on lp25 and stable maintenance of the ospC gene throughout mouse infection. We conclude that this represents a convenient and widely applicable method for stable gene complementation in B. burgdorferi.

The genetic basis of plasmid tropism betweenChlamydia trachomatisandChlamydia muridarum

The development of genetic transformation technology for Chlamydia trachomatis using its endogenous plasmid has recently been described. Chlamydia muridarum cannot be transformed by the C. trachomatis plasmid, indicating a barrier between chlamydial species. To determine which regions of the plasmid conferred the species specificity, we used the novel approach of transforming wild-type C. muridarum carrying the endogenous plasmid pNigg and forced recombination with the C. trachomatis vector pGFP::SW2 which carries the complete C. trachomatis plasmid (pSW2). Penicillin and chloramphenicol-resistant transformants expressing the green fluorescent protein were selected. Recovery of plasmids from these transformants showed they were recombinants. The differences between the pSW2 and pNigg allowed identification of the recombination breakpoints and showed that pGFP::SW2 had exchanged a ∼ 1 kbp region with pNigg covering CDS 2. The recombinant plasmid (pSW2NiggCDS2) is maintained under antibiotic selection when transformed into plasmid-cured C. muridarum. The ability to select for recombinants in C. muridarum shows that the barrier is not at transformation, but at the level of plasmid replication or maintenance. Our studies show that CDS 2, together with adjoining sequences, is the main determinant of plasmid tropism.

Rhizobial plasmid pLPU83a is able to switch between different transfer machineries depending on its genomic background

Plasmids have played a major role in bacterial evolution, mainly by their capacity to perform horizontal gene transfer (HGT). Their conjugative transfer (CT) properties are usually described in terms of the plasmid itself. In this work, we analyzed structural and functional aspects of the CT of pLPU83a, an accessory replicon from Rhizobium sp. LPU83, able to transfer from its parental strain, from Ensifer meliloti, or from Rhizobium etli. pLPU83a contains a complete set of transfer genes, featuring a particular organization, shared with only two other rhizobial plasmids. These plasmids contain a TraR quorum-sensing (QS) transcriptional regulator, but lack an acyl-homoserine lactone (AHL) synthase gene. We also determined that the ability of pLPU83a to transfer from R. etli CFN42 genomic background was mainly achieved through mobilization, employing the machinery of the endogenous plasmid pRetCFN42a, falling under control of the QS regulators from pRetCFN42a. In contrast, from its native or from the E. meliloti background, pLPU83a utilized its own machinery for conjugation, requiring the plasmid-encoded traR. Activation of TraR seemed to be AHL independent. The results obtained indicate that the CT phenotype of a plasmid is dictated not only by the genes it carries, but by their interaction with its genomic context.

Is the role of Chlamydia trachomatis underestimated in reactive arthritis patients in India

Background: Although Chlamydia trachomatis-induced Reactive Arthritis (ReA) constitutes a significant disease burden in various countries, however it remains largely unrecognized and probably underestimated in India. Due to availability of multiple serovars, biphasic lifecycle and asymptomatic infection, diagnosis of this pathogen is difficult, thereby leading to chronic, persistent chlamydial infection in the synovium. The aim of the present prospective study was to find the presence of intra-articular C. trachomatis infection by nucleic acid amplification tests and secretory IgA determination in patients with ReA and in forme fruste of ReA, viz.: Undifferentiated Spondyloarthropathy (uSpA) attending a major tertiary Army hospital inNew Delhi (India). Methods & Materials: Synovial fluid was aspirated from the knee joints of age and sex-matched 30 arthritic patients, viz.: ReA/uSpA (n=15) and Osteoarthritis controls (OA) (n =15) following the European Spondyloarthropathy Study Group criteria for ReA while clinical and radiological diagnosis was done for OA patients. The clinical details were recorded in all patients. Both C. trachomatis-specific surface Major Outer Membrane Protein (MOMP) and endogenous plasmid genes were targeted using semi-nested Polymerase Chain Reaction (snPCR) and nested PCR (nPCR), while anti-C. trachomatis IgA antibodies were estimated by commercial enzyme-linked immunosorbent assay kit (Savyon Diagnostics, Israel). Statistical evaluation of data was done by using trial version of Graph Prism Pad software (version 5.0; GraphPad Software, Inc., San Diego, California, USA). Results: Overall, 33.3% (5/15) ReA/uSpA patients were found to be C. trachomatis-positive for either MOMP/plasmid gene in the joint fluid. 20% (3/15) were positive for plasmid gene while MOMPgenewas present in 13.3% (2/15) patients in the study group (ReA/uSpA). Intra-articular anti-C. trachomatis IgA antibodies were detected in 26.6% (4/15) patients in the synovial fluid. Significant correlation (‘p’ value <0.05) was found between PCR and serologic findings. None of OA controls was found positive for either chlamydial antibody/MOMP gene/plasmid gene. Conclusion: The observations in the present study indicate that intra-articular C. trachomatis infection in ReA/uSpA patients is both underestimated as well as underdiagnosed inIndia. It also suggests the usefulness of serology for better clinical management of such patients.

PZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis

BackgroundThe ethanol-producing bacterium Zymomonas mobilis has attracted considerable scientific and commercial interest due to its exceptional physiological properties. Shuttle vectors derived from native plasmids have previously been successfully used for heterologous gene expression in this bacterium for a variety of purposes, most notably for metabolic engineering applications.ResultsA quantitative PCR (qPCR) approach was used to determine the copy numbers of two endogenous double stranded DNA plasmids: pZMO1A (1,647 bp) and pZMO7 (pZA1003; 4,551 bp) within the NCIMB 11163 strain of Z. mobilis. Data indicated pZMO1A and pZMO7 were present at ca. 3-5 and ca. 1-2 copies per cell, respectively. A ca. 1,900 bp fragment from plasmid pZMO7 was used to construct two Escherichia coli – Z. mobilis shuttle vectors (pZ7C and pZ7-184). The intracellular stabilities and copy numbers of pZ7C and pZ7-184 were characterized within the NCIMB 11163, ATCC 29191 and (ATCC 10988-derived) CU1 Rif2 strains of Z. mobilis. Both shuttle vectors could be stably maintained within the ATCC 29191 strain (ca. 20-40 copies per cell), and the CU1 Rif2 strain (ca. 2-3 copies per cell), for more than 50 generations in the absence of an antibiotic selectable marker. A selectable marker was required for shuttle vector maintenance in the parental NCIMB 11163 strain; most probably due to competition for replication with the endogenous pZMO7 plasmid molecules. N-terminal glutathione S-transferase (GST)-fusions of four endogenous proteins, namely the acyl-carrier protein (AcpP); 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA); DNA polymerase III chi subunit (HolC); and the RNA chaperone protein Hfq; were successfully expressed from pZ7C-derived shuttle vectors, and their protein-protein binding interactions were analyzed in Z. mobilis ATCC 29191. Using this approach, proteins that co-purified with AcpP and KdsA were identified.ConclusionsWe show that a shuttle vector-based protein affinity ‘pull-down’ approach can be used to probe protein interaction networks in Z. mobilis cells. Our results demonstrate that protein expression plasmids derived from pZMO7 have significant potential for use in future biological or biotechnological applications within Z. mobilis.

Curing the Plasmid pMC1 from the Poly (γ-glutamic Acid) Producing Bacillus amyloliquefaciens LL3 Strain Using Plasmid Incompatibility

Bacillus amyloliquefaciens LL3 is a glutamate-independent poly-γ-glutamic acid (γ-PGA) producing strain which consists of a circular chromosome (3,995,227bp) and an endogenous plasmid pMC1 (6,758bp). The study of the function of native plasmid and the genome-size reduction of the B. amyloliquefaciens LL3 strain requires elimination of the endogenous plasmid. Traditional plasmid-curing procedures using sodium dodecyl sulfate (SDS) or acridine orange combined with heat treatment have been shown to be ineffective in this strain. Plasmid incompatibility is an effective method for curing which has been studied before. In our research, the hypothetical Rep protein gene and the origin of replication of the endogenous plasmid were cloned into the temperature-sensitive vector yielding the incompatible plasmid pKSV7-rep-ori. This plasmid was transformed into LL3 by electroporation. The analysis of the strain bearing incompatible plasmids after incubation at 30°C for 30 generations showed the production of plasmid cured strains. High frequency of elimination was achieved with more than 93% of detected strains showing to be plasmid-cured. This is the first report describing plasmid cured in a γ-PGA producing strain using this method. The plasmid-cured strains showed an increase of γ-PGA production by 6% and led to a yield of 4.159g/l, compared to 3.918g/l in control and cell growth increased during the early stages of the exponential phase. Gel permeation chromatography (GPC) characterization revealed that the γ-PGA produced by plasmid-cured strains and the wild strains were identical in terms of molecular weight. What is more, the further study of plasmid function showed that curing of the endogenous plasmid did not affect its sporulation efficiency.

Plasmid-Encoded ComI Inhibits Competence in the Ancestral 3610 Strain of Bacillus subtilis

Natural competence is a process by which bacteria construct a membrane-associated machine for the uptake and integration of exogenous DNA. Many bacteria harbor genes for the DNA uptake machinery and yet are recalcitrant to DNA uptake for unknown reasons. For example, domesticated laboratory strains of Bacillus subtilis are renowned for high-frequency natural transformation, but the ancestral B. subtilis strain NCIB3610 is poorly competent. Here we find that endogenous plasmid pBS32 encodes a small protein, ComI, that inhibits transformation in the 3610 strain. ComI is a single-pass trans-membrane protein that appears to functionally inhibit the competence DNA uptake machinery. Functional inhibition of transformation may be common, and abolishing such inhibitors could be the key to permitting convenient genetic manipulation of a variety of industrially and medically relevant bacteria.

Edwardsiella tarda mutant disrupted in type III secretion system and chorismic acid synthesis and cured of a plasmid as a live attenuated vaccine in turbot

Edwardsiella tarda is an intractable Gram-negative pathogen in many fish species to cause edwardsiellosis. Its infection leads to extensive losses in a diverse array of commercially important fish. The type III secretion system (T3SS) has been considered as one of the major virulence factors and plays important roles in its intracellular lifestyle. In this study, an E. tarda EIB202 mutant WED with deletions in the T3SS genes for EseB, EseC, EseD and EscA, along with the aroC gene for the biosynthesis of chorismic acid, as well as the curing of endogenous plasmid pEIB202 was constructed by allelic exchange strategy. Compared to the wild-type EIB202 which was highly virulent towards turbot (Scophthamus maximus) via intraperitoneal (i.p.), intramuscular (i.m.) injection or immersion and caused systemic infection in turbot as well as the unexpected red mouth symptom when immersion challenged, WED was highly attenuated when inoculated into turbot via i.m., i.p. and immersion routes, and exhibited significantly impaired capacity to survive in fish tissues. WED showed 5700-fold higher 50% lethal dose (LD50) than that of the wild type when i.m. or i.p. challenged. Inoculation with WED by i.p. or immersion injection routes elicited significant protection against the challenge of the wild-type E. tarda after 5 weeks of vaccination. The vaccinated fish produced low while significant level of specific antibody and showed increased expression of immune-related factors including IL-1β, IFN-γ, MHC II, MHC-I and CD8, indicating that WED possesses significant immunoprotective potential. Furthermore, our data indicated that a single dose of i.p. and immersion vaccination with WED could produce significant protection as long as 12 and 6 months, respectively. These results demonstrated the feasibility of WED as a live attenuated vaccine in turbot against edwardsiellosis by immersion or i.p. injection routes.

Development of a Propionibacterium-Escherichia coli Shuttle Vector for Metabolic Engineering of Propionibacterium jensenii, an Efficient Producer of Propionic Acid

Propionic acid (PA) is an important chemical building block and is widely applied for organic synthesis, food, feedstuff, and pharmaceuticals. To date, the strains that can efficiently produce PA have included Propionibacterium thoenii, P. freudenreichii, and P. acidipropionici. In this report, we show that P. jensenii ATCC 4868 is also able to produce PA in much higher yields than the previously reported strains. To further improve the production capacity, a P. jensenii-Escherichia coli shuttle vector was developed for the metabolic engineering of P. jensenii. Specifically, a 6.9-kb endogenous plasmid, pZGX01, was isolated from P. acidipropionici ATCC 4875 and sequenced. Since the sequencing analysis indicated that pZGX01 could encode 11 proteins, the transcriptional levels of the corresponding genes were also investigated. Then, a P. jensenii-Escherichia coli shuttle vector was constructed using the pZGX01 plasmid, the E. coli pUC18 plasmid, and a chloramphenicol resistance gene. Interestingly, not only could the developed shuttle vector be transformed into P. jensenii ATCC 4868 and 4870, but it also could be transformed into freudenreichii ATCC 6207 subspecies of P. freudenreichii. Finally, the glycerol dehydrogenase gene (gldA) from Klebsiella pneumoniae was expressed in P. jensenii ATCC 4868 with the constructed shuttle vector. In a 3-liter batch culture, the PA production by the engineered P. jensenii ATCC 4868 strain reached 28.23 ± 1.0 g/liter, which was 26.07% higher than that produced by the wild-type strain (22.06 ± 1.2 g/liter). This result indicated that the constructed vector can be used a useful tool for metabolic engineering of P. jensenii.

Membrane vesicles, nanopods and/or nanotubes produced by hyperthermophilic archaea of the genus Thermococcus

Thermococcus species produce MVs (membrane vesicles) into their culture medium. These MVs are formed by a budding process from the cell envelope, similar to ectosome formation in eukaryotic cells. The major protein present in MVs of Thermococci is a peptide-binding receptor of the OppA (oligopeptide-binding protein A) family. In addition, some of them contain a homologue of stomatin, a universal membrane protein involved in vesiculation. MVs produced by Thermococcus species can recruit endogenous or exogenous plasmids and plasmid transfer through MVs has been demonstrated in Thermococcus kodakaraensis. MVs are frequently secreted in clusters surrounded by S-layer, producing either big protuberances (nanosphere) or tubular structures (nanotubes). Thermococcus gammatolerans and T. kodakaraensis produce nanotubes containing strings of MVs, resembling the recently described nanopods in bacteria, whereas Thermococcus sp. 5-4 produces filaments whose internal membrane is continuous. These nanotubes can bridge neighbouring cells, forming cellular networks somehow resembling nanotubes recently observed in Firmicutes. As suggested for bacteria, archaeal nanopods and/or nanotubes could be used to expand the metabolic sphere around cells and/or to promote intercellular communication.

Isolation and Molecular Characterization of Pesticide (Fenitrothion) Resistant Bacteria from Agricultural Field

Elimination of pollutants from contaminated sites using microorganisms is a cheaper alternative to chemical technology. There is therefore a need to isolate, identify and characterize the microorganisms that exist and interact in contaminated environment and to isolate the genetic determinants of resistance, frequently located on plasmids and transposons. Investigations were carried out to isolate microbial strains from the fenitrothion polluted soil. Bacteria were isolated using enrichment culture technique containing fenitrothion as sole source of carbon and energy was isolated in pure culture and selected for their ability to degrade fenitrothion. Growth curve experiments showed that the bacterial consortium was able to grow in mineral medium containing fenitrothion as the only carbon source. The microorganisms were identified as Bascillus sp. The identity of the isolate was determined through biochemical and morphological characteristics. For more confirmation, 16s rRNA gene analysis was done using PCR with universal primers. Comparative analysis of the 16s rRNA gene sequences in the Gene Bank database revealed that this bacteria was related to the genus Basicllus substilis. The bacterial strains were found to harbor endogenous plasmids. KEYWORDSBacteria, Biochemical characters, Fenitrothion, Gene sequencing

Establishment of TaqMan Real-time Quantitative PCR Assay for Foreign Gene Copy Numbers in Transgenic Soybean

TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y=−3.422x+35.201, R2=0.998; the standard curve equation of exogenous gene was y =−3.495x+35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.