Rp4 Promotion of Transfer of a Large Agrobacterium Plasmid Which Confers Virulence

Introduction of RP4 plasmid into Agrobacterium tumefaciens promotes the transfer on solid medium of large virulence-associated plasmids from virulent donor strains to a plasmidless avirulent recipient. Exconjugants were selected for the ability to utilize octopine or nopaline as the sole source of arginine, traits which are coded for by virulence-associated plasmids in the strains employed here. All exconjugants retained the arginine auxotrophy of the recipient strain, and were resistant to ampicillin and kanamycin, drugs to which RP4 confers resistance. Five exconjugant clones from one cross were shown by alkaline sucrose gradient analysis to contain both RP4 plasmid and the large virulence-associated plasmid of the donor strain. All five exconjugants exhibited virulence on carrot, sunflower and kalanchoe plants. These results indicate that virulence and the ability to degrade octopine are plasmid-borne traits in A. tumefaciens strains 15955 and A6, and extend the evidence that large plasmids in A. tumefaciens are vectors of virulence genes.     

Octopine and nopaline synthesis and breakdown genetically controlled by a plasmid of Agrobacterium tumefaciens

Summary Several nopaline degrading strains and one octopine degrading strain are shown to loose oncogenicity as well as the ability to utilize these guanidine compounds when they are cured of their TI plasmid. To investigate whether the specific genes involved in the utilization of one or the other compound are located on the plasmid, plasmid-transfer experiments have been performed.The plasmid from a nopaline degrading strain has been transferred to a naturally non oncogenic Agrobacterium namely A. radiobacter. Furthermore, the plasmid from an octopine degrading strain has been transferred to a plasmid-cured strain which originally had the capacity to utilize nopaline. Both kinds of experiments prove that the TI plasmid determines the strain specificity with regard to the utilization of either octopine or nopaline.They also demonstrate that the synthesis of either octopine or nopaline in crown gall cells is also determined by genes located on the TI plasmid harboured by the transforming A. tumefaciens strains.     

Characterization of Plasmid Deoxyribonucleic Acid in Streptococcus lactis subsp. diacetylactis: Evidence for Plasmid-Linked Citrate Utilization

The use of Streptococcus diacetylactis as a flavor producer in dairy fermentations is dependent upon its ability to produce diacetyl from citrate. Treatment of S. diacetylactis strains 18-16 and DRC1 with acridine orange resulted in the conversion of approximately 2% of the DRC1 population and 20% of the 18-16 population to citrate negative, which is indicative of the involvement of plasmid deoxyribonucleic acid (DNA). Growth in the presence of acridine orange also resulted in the appearance of 2% lactose-negative derivatives in S. diacetylactis 18-16 and 99% lactose-defective, proteinase-negative derivatives in S. diacetylactis DRC1. Cesium chloride-ethidium bromide equilibrium density gradients of cleared lysate material from each strain revealed the presence of covalently closed circular DNA. Samples of this covalently closed circular DNA were subjected to agarose gel electrophoresis to determine the plasmid composition of each strain. S. diacetylactis 18-16 was found to possess six plasmids, of approximately 41, 28, 6.4, 5.5, 3.4, and 3.0 megadaltons (Mdal). S. diacetylactis DRC1 contained six plasmids, of approximately 41, 31, 18, 5.5, 4.5, and 3.7 Mdal. Variants of S. diacetylactis 18-16 which failed to produce acetoin plus diacetyl from citrate (citrate negative) were missing a 5.5-Mdal plasmid. Lactose-negative mutants of the same strain were devoid of a 41-Mdal plasmid. Lactose-defective, proteinase-negative mutants of S. diacetylactis DRC1 were missing a 31-Mdal plasmid. The citrate-negative mutants of S. diacetylactis DRC1 isolated in this study did not possess a 5.5-Mdal plasmid. Thus, we have evidence that there is a correlation between the ability to utilize citrate and the presence of a 5.5-Mdal plasmid. A relationship was also noted between lactose fermentation and proteinase activity and plasmid DNA in S. diacetylactis.     

Plasmid Involvement in Acyclic Isoprenoid Metabolism by Pseudomonas putida

An organism identified as Pseudomonas putida was found to utilize citronellol or geraniol as the sole carbon and energy source. The ability to degrade these acyclic isoprenols was associated with pSRQ50, a 50-megadalton transmissible plasmid.     

Characterization of a phenanthrene degradation plasmid from Alcaligenes faecalis AFK2

A plasmid pHK2 involved in phenanthrene degradation was isolated from Alcaligenes faecalis AFK2. Some mitomycin C treated cells lost the pHK2 plasmid. The plasmid could be transformed into Pseudomonas putida and A. faecalis. They gained the ability to utilize phenanthrene as well as o-phthalate, an intermediate of phenanthrene degradation. Phenanthrene dioxygenase could be detected from the transformants after induction with phenanthrene. The molecular size of pHK2 DNA was determined to be 42.5 kilobase (kb), and its physical map was constructed.     

Molecular Characterization of Endophytic Bacteria from Metal Hyperaccumulator Aquatic Plant (Eichhornia crassipes) and Its Role in Heavy Metal Removal

In this study, among a collection of heavy metals resistant endophytic bacterial strains isolated from aquatic hyperaccumulator plant (Eichhornia crassipes), one plant growth promoting endophytic bacteria (PGPE), SVUB4 was selected for its ability to utilize 1-aminocyclopropane-1-carboxylic acid (ACC) as the sole N source and accumulate different heavy metals. The SVUB4 strain was characterized as Enterobacter sp. on the basis of its 16S rDNA sequences. Assessment of the parameters of plant growth promotion revealed the intrinsic ability of the strain for the production of IAA, siderophore and solubilization of insoluble phosphate. Furthermore, plasmid DNA analysis of Enterobacter sp. strain SVUB4 indicated the presence of a single large plasmid element. The results of plasmid curing experiments demonstrated that the ability of this strain to grow in presence of Cd and Zn was encoded by the 98 kb plasmid, whereas the ability to grow in the presence of Pb appeared to be encoded by the chromosome. The Cd and Zn removal capacity of the respective metal sensitive strain (plasmidless) were about 36 and 45 μg/g-1 DW, respectively, while the removal capacity of the both metal by metal resistant strain (p SVUB4) showed a significantly higher Cd and Zn removal capacity of 153 and 228 μg/g^?1 DW, respectively. However, both strains exhibited a similar pattern of Pb accumulation. The present observation also showed that for wild-type strain SVUB4 (pSVUB4), the overall level of IAA production in the absence and in the presence of Cd²⁺ or Zn²⁺was approximately the same. Nevertheless, strain SVUB4M in this respect appeared to be more sensitive to heavy metals: a noticeable decrease in IAA production was observed under the effect of both metals, especially with Cd²⁺.     

Detection of Plasmid Transfer from Pseudomonas fluorescens to Indigenous Bacteria in Soil by Using Bacteriophage φR2f for Donor Counterselection

The transfer of a genetically marked derivative of plasmid RP4, RP4p, from Pseudomonas fluorescens to members of the indigenous microflora of the wheat rhizosphere was studied by using a bacteriophage that specifically lyses the donor strain and a specific eukaryotic marker on the plasmid. Transfer of RP4p to the wheat rhizosphere microflora was observed, and the number of transconjugants detected was approximately 10³ transconjugants per g of soil when 10⁷ donor cells per g of soil were added; transfer in the corresponding bulk soil was slightly above the limit of detection. All of the indigenous transconjugants which we analyzed contained a 60-kb plasmid and were able to transfer this plasmid to a Nx^r Rp^rP. fluorescens recipient strain. The indigenous transconjugants were identified as belonging to Pseudomonas spp., Enterobacter spp., Comamonas spp., and Alcaligenes spp.     

Improved Degradation of Monochlorophenols by a Constructed Strain

Pseudomonas sp. strain B13, a strain able to degrade 3-chlorobenzoate and, after prolonged adaptation (40 days), 4-chlorophenol, could transfer the ability to degrade chlorocatechols to a recipient, Alcaligenes sp. strain A7, which is able to grow with benzoate and phenol. Representative transconjugants, such as Alcaligenes sp. strain A7-2, were able to utilize all three isomeric chlorophenols; this property was not possessed by the donor or the recipient. The ability to grow readily with 4-chlorophenol may be attributable to a more rapid induction of phenol hydroxylase by Alcaligenes sp. strain A7-2 than by Pseudomonas sp. strain B13, a property which correlates with the greater level of resistance to chlorophenols shown by the transconjugant.     

Plasmid Content and Tumor Initiation Complementation by Agrobacterium tumefaciens IIBNV6

Avirulent strains IIBNV6 and NT1, derived from virulent strains of Agrobacterium tumefaciens, were tested for their ability to enhance tumor initiation (complement) on coinoculation with tumorigenic strains. Strain NT1, cured of the Agrobacterium virulence plasmid, failed to complement when inoculated with its virulent parental strain or with other virulent strains. Strain IIBNV6, however, complemented with all virulent strains tested. Attachment to host wound sites by both strain IIBNV6 and the virulent strain was essential for this effect. Inoculation of the tumorigenic strain at different times on leaves previously inoculated with IIBNV6 showed that the capacity to complement is lost during the period between 4 and 8 h after IIBNV6 inoculation. The rate of tumor appearance obtained with an inoculum containing IIBNV6 and a virulent auxotrophic strain was characteristic of the appearance rate obtained with prototrophic bacteria. Evidence is summarized which suggests that strain IIBNV6 can induce tumors when supplied with a substance produced or induced by a virulent bacterium at a separate site. A deoxyribonucleic acid plasmid about 40% the size of the Agrobacterium virulence plasmid was obtained from strain IIBNV6. We propose that this plasmid accounts for the ability of strain IIBNV6 to complement and that it contains part of the genetic information necessary for tumor initiation.     

A new way of stabilizing recombinant plasmids

A new method to stabilize recombinant plasmids extremely well was exploited using Escherichia coli Tna (trpAEI trpR tnaA) and pSC101trpI15-14 (tetracycline resistance, whole trp operon) as a model system. We mutagenized the Tna strain carrying pSC101trpI15-14 and isolated a mutant 6F484 that stably maintained the recombinant plasmid for 100 generations. From 6F484, plasmid-free cells (tetracycline sensitive) were screened for on selective agar plates containing fusaric acid. The host strain FA14 was found to have lost the ability for active transport of tryptophan, in addition to the phenotype of Trp⁻. Therefore, strain FA14 could not grow normally even in a complete medium. However, when the strain was transformed with the trp operon recombinant plasmid, its growth rate was almost restored to the original level. These results suggest that the recombinant plasmid is indispensable for the normal growth of host cells like FA14. Even if plasmid-free segregants appear during the cultivation, they cannot grow so rapidly and are diluted as a minority in total population. Consequently, owing to the deficiency of both the biosynthesis and uptake of tryptophan in host strain, the trp operun recombinant plasmid can be stably maintained.     

Method for the transfer of large cryptic, non-self-transmissible plasmids: ex planta transfer of the virulence plasmid of Agrobacterium rhizogenes

A method is presented for the (ex planta) transfer of large, cryptic plasmids that are (phenotypically) non-self-transmissible. The procedure consists of introducing a mobilizing R plasmid into the strain carrying the cryptic plasmid followed by random transposon mutagenesis (e.g., by conjugation with a bacterium carrying a suicide plasmid). Tn-carrying derivatives with a copy of the Tn in the cryptic plasmid are subsequently identified by their ability to transfer the Tn-encoded markers via R plasmid mobilization. The method was applied for the labeling of a large cryptic plasmid present in Agrobacterium rhizogenes 1855 and for its subsequent ex planta transfer to a Ti plasmidless A. tumefaciens strain. The plasmid gave the new host the capacity to induce the hairy root disease in plants, and thus turned out to be an Ri plasmid. From the labeled Ri plasmid derepressed mutants were isolated that were transmissible both in planta and ex planta.     

Enhancement of the Potential To Utilize Octopine in the Nonfluorescent Pseudomonas sp. Strain 92

The nonfluorescent Pseudomonas sp. strain 92 requires the presence of a supplementary carbon source for growth on octopine, whereas the spontaneous mutant RB100 has acquired the capacity to utilize this opine as the sole carbon and nitrogen source. Insertional mutagenesis of RB100 with transposon Tn5 generated mutants which were unable to grow on octopine and others which grew slowly on this substrate. Both types of mutants yielded revertants that had regained the ability to utilize octopine. Some of the revertants had lost the transposon, whereas in others the transposon was retained but with rearrangements of the insertion site. Genes of octopine catabolism from strain 92 were cloned on a cosmid vector to generate pK3. The clone pK3 conferred the ability to utilize octopine as the sole carbon and nitrogen source on the host Pseudomonas putida KT2440. Although they conferred an equivalent growth phenotype, the mutant genes carried by RB100 and the cloned genes on pK3 differed in their regulation. Utilization of [¹⁴C]octopine was inducible by octopine in RB100 and was constitutive in KT2440(pK3).     

Industrial production of α-amylase by genetically engineered Bacillus

A genetically engineered Bacillus subtilis strain (ALKO 84) has been introduced for industrial production of α-amylase. This strain carries the α-amylase gene from a traditionally developed production strain B. amyloliquefaciens (ALKO 89) on the multicopy plasmid pUB110.⁸At laboratory scale the recombinant strain ALKO 84 produced in industrial medium about twice as much α-amylase as the traditional strain ALKO 89. The process for production of the enzyme was scaled-up to 60m³. At this scale B. subtilis ALKO 84 retained its relative superiority to B. amyloliquefaciens ALKO 89, producing about 85% of the activity obtained at laboratory scale. Stability of the recombinant plasmid was found acceptable during the large-scale cultivations with over 90% of cells retaining plasmid-encoded characteristics throughout.     

Expression of the Escherichia Coli TdcB gene encoding threonine dehydratase in L-isoleucine-overproducing Corynebacterium Glutamicum Yilw

Threonine dehydratase (EC 4.3.1.19, TDH) catalyzing the degradation of Thr to α-ketobutyrate, is a rate-limiting enzyme in L-Ile pathway. The tdcB gene encoding TDH was obtained from Escherichia coli K12 by PCR and expressed at E. coli BL21 (DE3). Then the tdcB gene was inserted into the shuttle expression vector pXMJ19 and the recombinant plasmid was electroporated into the L-isoleucine-producing strain of Corynebacterium glutamicum YILW. Crude extracts of the microbial strain containing the plasmid pXMJ19tdcB retained 60% of the original TDH activity even in the presence of 300 mM L-Ile. The recombinant strain of bacteria showed 7.5% higher enzyme activity and 11.3% higher L-Ile production compared to the original strain.     

Plasmid pCS1966, a New Selection/Counterselection Tool for Lactic Acid Bacterium Strain Construction Based on the oroP Gene, Encoding an Orotate Transporter from Lactococcus lactis

In this paper we describe the new selection/counterselection vector pCS1966, which is suitable for both sequence-specific integration based on homologous recombination and integration in a bacteriophage attachment site. This plasmid harbors oroP, which encodes a dedicated orotate transporter, and can replicate only in Escherichia coli. Selection for integration is performed primarily by resistance to erythromycin; alternatively, the ability to utilize orotate as a pyrimidine source in a pyrimidine auxotrophic mutant could be utilized. Besides allowing the cell to utilize orotate, the transporter renders the cell sensitive to 5-fluoroorotate. This sensitivity is used to select for loss of the plasmid. When expressed from its own promoter, oroP was toxic to E. coli, whereas in Lactococcus lactis the level of expression of oroP from a chromosomal copy was too low to confer 5-fluoroorotate sensitivity. In order to obtain a plasmid that confers 5-fluoroorotate sensitivity when it is integrated into the chromosome of L. lactis and at the same time can be stably maintained in E. coli, the expression of the oroP gene was controlled from a synthetic promoter conferring these traits. To demonstrate its use, a number of L. lactis strains expressing triosephosphate isomerase (tpiA) at different levels were constructed.     

Glutamate Dehydrogenase Activity Can Be Transmitted Naturally to Lactococcus lactis Strains To Stimulate Amino Acid Conversion to Aroma Compounds

Amino acid conversion to aroma compounds by Lactococcus lactis is limited by the low production of α-ketoglutarate that is necessary for the first step of conversion. Recently, glutamate dehydrogenase (GDH) activity that catalyzes the reversible glutamate deamination to α-ketoglutarate was detected in L. lactis strains isolated from a vegetal source, and the gene responsible for the activity in L. lactis NCDO1867 was identified and characterized. The gene is located on a 70-kb plasmid also encoding cadmium resistance. In this study, gdh gene inactivation and overexpression confirmed the direct impact of GDH activity of L. lactis on amino acid catabolism in a reaction medium at pH 5.5, the pH of cheese. By using cadmium resistance as a selectable marker, the plasmid carrying gdh was naturally transmitted to another L. lactis strain by a mating procedure. The transfer conferred to the host strain GDH activity and the ability to catabolize amino acids in the presence of glutamate in the reaction medium. However, the plasmid appeared unstable in a strain also containing the protease lactose plasmid pLP712, indicating an incompatibility between these two plasmids.     

Isolation and characterization of crude-oil-degrading bacteria from oil-water mixture in Dagang oilfield,China

Isolating novel crude-oil-degrading bacteria from oil-water mixture of oil production well and evaluating their degradation capacities are vitally important in the remediation of oil-polluted environments and crude oil exploitation. According to the molecular screening with degenerate primers of alkane hydroxylase gene (alk B), a strain Acinetobacter sp. LS-1 with alk B gene was isolated. This strain exhibited a 99.9% similarity with genus Acinetobacter. This alk B gene which is one of the key enzymes of metabolic process was identified. This gene sequence showed 98% similarity of its nucleotide and related amino acids to the genus Marinobacter but exhibited below 70% similarity to the genus Acinetobacter. This phylogenetic analysis indicated that alk B may have been transferred horizontally between bacteria. The isolated strain could utilize crude oil as the sole carbon, achieving a high degradation (70.3%) in 7 days. Microcalorimetric analysis of the metabolic processes for hexadecane degradation also demonstrated the ability of this strain to utilize hydrocarbons. Thus, this strain enables to degrade hydrocarbons as the sole carbon source from the gene level, combined with material and energy metabolism. These findings will benefit this strain in the remediation of oil-polluted environments and oil exploitation.     

Plasmid-mediated Molluscicide Bayluscide Degradation by Bacterial Strain Isolated from Schistosome Vector Snail Bulinus truncates

Pseudomonas spp. strain Bal1 was isolated from Schistosome vector snails Bulinus Truncates. Strain Bal1 was identified as Pseudomonas putida using partial sequence of 16s rRNA. This strain was able to utilize a Bayluscide as a sole source of carbon and nitrogen. The degradation of Bayluscide by Bal1 strain is mediated by pBal1 (110 Kb) plasmid. The loss of the plasmid resulted irreversibly in a derivative strain that was unable to degrade Bayluscide. The transfer of these plasmids from wild-type strain Bal1 to Bal1M derivative restored completely its capability to degrade the molluscicide. It is proposed that pBal1 is a conjugative plasmid and is involved in the Bayluscide degradation. The effect of bacterial degradation upon toxicity was tested, and it was shown that the molluscicidal action of Bayluscide was significantly reduced by bacterial action.     

Variable Bacteriocin Production in the Commercial Starter Lactococcus lactis DPC4275 Is Linked to the Formation of the Cointegrate Plasmid pMRC02

Lactococcus lactis DPC4275 is a bacteriocin-producing transconjugant of the industrial starter strain DPC4268. Strain DPC4275 was generated through conjugal transfer by mating DPC4268 with L. lactis MG1363 containing the 60-kb plasmid pMRC01, which encodes the genetic determinants for the lantibiotic lacticin 3147 and for a phage resistance mechanism of the abortive infection type. The many significant applications of this strain prompted a genetic analysis of its apparently unstable bacteriocin-producing phenotype. Increased levels of lacticin 3147 produced by DPC4275 were associated with the appearance of an 80-kb plasmid, designated pMRC02, which was derived from DNA originating from pMRC01 (60 kb) and a resident DPC4268 proteinase plasmid, pMT60 (60 kb). Indeed, pMRC02 was shown to be derived from the insertion of a 17-kb fragment of pMRC01, encompassing the lacticin 3147 operon, into pMT60. The presence of pMRC02 at a high copy number was found to correlate with increased levels of lacticin 3147 in DPC4275 compared to the wild-type containing pMRC01. Subsequent transfer of pMRC02 into the plasmid-free strain MG1363 by electroporation allowed a direct phenotypic comparison with pMRC01, also studied in the MG1363 background. Plasmid pMRC02 displayed phage resistance similar to that by pMRC01, although it was less potent, as demonstrated by a larger plaque size for phage c2 infection of MG1363(pMRC02). While this locus is flanked by IS946 elements, the sequencing of pMT60-pMRC01 junction sites established that this event was unlikely to be insertion sequence mediated and most probably occurred by homologous recombination followed by deletion of most of pMRC01. This was not a random occurrence, as nine other transconjugants investigated were found to have the same junction sites. Such derivatives of commercial strains producing increased levels of bacteriocin could be exploited as protection cultures for food applications.     

High-level production of recombinant glutamic acid-specific protease from Bacilllus licheniformis in Bacillus subtilis expression system

To obtain large quantities of glutamic acid-specific protease isolated originally from Bacillus licheniformis (BLase), an expression plasmid was constructed by inserting the BLase gene into a plasmid vector (pUB110) for Bacillus subtilis. B. subtilis strain ISW1214 harboring the resultant recombinant plasmid containing the coding and 5′-promoter and 3′-terminator regions of BLase gene secreted approximately 0.25 g/l of BLase in a culture medium contained in a 90-l jar fermentor, corresponding to nearly 10 times the natural production level and resulting in a stable large-scale production. The amount of BLase in the culture medium accounted for roughly 60% of the total extracellular proteins secreted from the recombinant strain, simplifying enzyme purification.