Transfer of RP4::Mu plasmids to Agrobacterium tumefaciens

Transfers of RP4::Mu plasmids from Escherichia coli to Agrobacterium tumefaciens are very inefficient in contrast to the very efficient transfer of RP4. Apparently, one or more Mu functions prevent RPR::Mu plasmids from establishing in some Gram-negatives other than E. coli. This problem was eliminated by the use of a mutant Mu prophage, Mu cts62r23, in RP4. Moreover, the transfer of RP4::Mu cts62r23 to the Agrobacterium strain C58 was found to be affected by a restriction modification system. The target for this restriction was located on Mu DNA and not on RP4 DNA. The plaque-forming phage production of Mu cts62r23 in Agrobacterium was found to be 10⁶ times lower than in E. coli.     

IncP plasmids are most effective in mediating conjugation between Escherichia coli and streptomycetes

Mobilizable shuttle plasmids containing the origin of transfer (oriT) region of plasmid F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPα) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Streptomyces. The conjugative system of the IncPα plasmids was shown to be most effective in conjugative transfer, giving peak values of (2.7 ± 0.2) × 10⁻² S. lividans TK24 exconjugants per recipient cell. To assess whether the mating-pair formation system or the DNA-processing apparatus of the IncPα plasmids is crucial in conjugative transfer, an assay with an IncQ-based mobilizable plasmid (RSF1010) specifying its own DNA-processing system was developed. Only the IncPα plasmid mobilized the construct to S. lividans indicating that the mating-pair formation system is primarly responsible for the promiscuous transfer of the plasmids between E. coli and Streptomyces. Dynamic of conjugative transfer from E. coli to S. lividans was investigated and exconjugants starting from the first hour of mating were obtained. The text was submitted by the authors in English.     

Electroporation and conjugal plasmid transfer to members of the genus Aquaspirillum

Electroporation methods and conjugal matings were used to transfer several plasmid vectors to Aquaspirillum dispar and Aquaspirillum itersonii. The incompatibility P class plasmid RP4 was conjugally transferred from Escherichia coli HB101 to these spirilla, and the transconjugants subsequently donated the molecule to plasmid-free E. coli and A. dispar strains via conjugal matings. High-voltage electrotransformation was used to transfer plasmids pUCD2, pSa151 and RP4 to A. dispar and A. itersonii, at efficiencies as high as 3×10⁴ transformants per μg plasmid DNA. RP4 DNA isolated from spirillum hosts, but not RP4 from E. coli cells was successfully transferred to A. dispar and A. itersonii by electrotransformation, suggesting that modification and/or restriction activity may be present in these Aquaspirillum species.     

Expression of Escherichia coli tryptophan operon in Rhizobium leguminosarum.

Summary RP4-trp hybrid plasmid containing Escherichia coli whole tryptophan operon was conjugatively transferred from E. coli to Rhizobium leguminosarum strains carrying mutations in different trp genes, converting their Trp^– phenotype to Trp⁺. That the phenotype change of the R. leguminosarum cells was due to the presence of the E. coli tryptophan operon was verified by the isolation of RP4-trp hybrid plasmid from the R. leguminosarum conjugant cells, and by re-transfer of RP4-trp plasmid by conjugation back to E. coli trp and Pseudomonas putida trp strains. Enzymatic activities of anthranilate synthetase and subunit of tryptophan synthetase in crude extracts of R. leguminosarum cells containing RP4-trp plasmid were much higher than that of the wild-type cells and were not repressed by the presence of tryptophan in the culture medium.     

Lack of expression of RP4-specified β-lactamase in Azospirillum brasilense

Plasmid RP4, which normally confers resistance to ampicillin (Ap^r), tetracycline (Tc^r), and kanamycin (Km^r) to its hosts, failed to express enhanced Ap^r when transferred from Escherichia coli to Azospirillum brasilense which has its own intrinsic β-lactamase. Even in a β-lactamase-deficient mutant, A. brasilense RG-D16, no increase in β-lactamase or significant Ap^r appeared following transfer of RP4. However, A. brasilense RG (RP4) and A. brasilense RG-D16 (RP4) did exhibit Tc^r Km^r. When RP4 was transferred back from A. brasilense to E. coli all three drug resistances and β-lactamase activity were fully expressed.     

Isolation and Chemical Characterization of a Mating Pheromone Produced by Saccharomyces kluyveri

The pullulanase gene (pul) of Klebsiella aerogenes was transferred in vivo to Escherichia coli by using RP4:: Mu cts. The pul gene was expressed in E. coli, although the level of pullulanase activity in E. coli was lower than that in K. aerogenes, and the Pul⁺ transconjugants were relatively unstable in an unselective medium. Production of pullulanase, which is used to make maltose from starch, was induced in E. coli by pullulan, waxy maize amylopectin, soluble starch and maltose. When the transconjugant cells of E. coli were grown with pullulan or maltose, most pullulanase was produced intracellularly, whereas K. aerogenes produced pullulanase extracellularly. Retransfer of the pul_k gene from E. coli to K. aerogenes by conjugation resulted in an increase of the production of extracellular pullulanase.     

IS8- and Tn2353-mediated cointegration of the plasmids R15 and RP4::Tn1

Summary The plasmids R15 and RP4:: Tn1 form fused structures (85 Md and 92 Md cointegrates). The cointegrates do not resolve practically in recA ^– Escherichia coli cells and have a mean life-time of more than 50 generations in a recA ⁺ background.The 85 Md cointegrates were generated at a frequency of 4×10^–4 per R15 transconjugant during a mating between E. coli [R15; RP4:: Tn1] and E. coli [FColVBtrp:: Tn1755]. These plasmids carry two directly repeated copies of the mobile element IS8 at the junctions between R15 and RP4:: Tn1. The transposition of IS8 from RP4:: Tn1 to the R15 plasmid and the formation of hybrid molecules promoted by this process appear to be induced by the IS8 element of the Tn1755 structure during or after conjugal transfer of FColVBtrp:: Tn1755 into E. coli [R15; RP4:: Tn1] cells.The formation of the 92 Md cointegrates occurs at a frequency of 2×10^–5. The fused molecules of R15 and RP4:: Tn1 carry two direct copies of an 8.65 Md R15 fragment at the junctions between these replicons. The fragment has specific features of a new transposon. This element designated Tn2353 determines resistance to Hg, Sm and Su and contains two sites for each BamHI, BglII and SalI and three sites for both EcoRI and PstI. The physical map and some other characteristics of Tn2353 are presented.Abbreviations Ap ampicillin - EtBr ethidium bromide - Km kanamycin - Md megadaltons - Sm streptomycin - Su sulfanilamide - Tc tetracycline - [] brackets indicate plasmid-carrier state     

Cloning of a Microbispora bispora cellobiohydrolase gene in Streptomyces lividans

The cellobiohydrolase II (CBHII) of Microbispora bispora, originally cloned in Escherichia coli, was subcloned into Streptomyces lividans using shuttle vectors pSKN 01 and pSKN 02. The enzyme was secreted from Streptomyces, whereas it was intracellular in E. coli. The yields of CBHII produced by S. lividans transformants were 15–20-fold higher than those produced by E. coli transformants. The optimal pH of M. bispora native cellobiohydrolase and the cloned enzyme from S. lividans is 6.5. The thermal and pH stability of CHBII produced in M. bispora, E. coli and S. lividans were compared. Enzyme produced in E. coli was inactivated more rapidly (k = 0.252 min^–1 at 90° C; 90% inactivation after 10 min vs. 0.119 min^–1 for the others). CBHII was monitored following electrophoretic separation by reaction with a monoclonal antibody. The apparent molecular mass of the protein produced from the S. lividans clone was 93 kDa, the same as that of the native enzyme, but that of the enzyme produced in E. coli was smaller (82 kDa). Correspondence to: P. Hu     

Response of RP1+ and RP1− strains ofEscherichia coli to antibacterial agents and transfer of resistance toPseudomonas aeruginosa

The sensitivity of strains ofEscherichia coli, with and without the RP1 R-factor, to antibiotics and other antibacterial agents has been studied. RP1⁺ strains ofE. coli were resistant to kanamycin, carbenicillin, and tetracycline, resistance to the first two antibiotics being produced by destruction of the drugs. This resistance could be transferred to two strains ofPseudomonas aeruginosa. The parent strain ofE. coli UB 1005, its two mutant strains (DC2 and DC3), and two of the strains with the RP1 R-factor showed a similar order of sensitivity to phenylmercuric nitrate, chlorhexidine, thiomersal, and mercuric chloride.E. coli strains DC2 and DC2 (RP1⁺) were the most sensitive to benzalkonium chloride and cetrimide. RP1⁺ strains were more resistant than RP1⁻ strains to lysozyme-ethylenediaminetetraacetic acid, but treatment of the former strains with acriflavine rendered the cells more sensitive to the lytic system. There was no evidence thatP. aeruginosa (RP1⁺) strains possessed increased resistance to polymyxin or to disinfectants, although they became somewhat less sensitive to lysozyme-ethylenediaminetetraacetic acid.     

Chromosomal location of gene governing the trehalose utilization in Escherichia coli K12.

Summary Several mutants unable to utilize trehalose were isolated from Escherichia coli. Their genetic analysis led to determine the following gene order on the chromosomal map: pur B-dad R-tre-hem A-trp. Furthermore, the tre gene belongs to the inversion of the trp chromosomal region between E. coli and S. typhimurium.     

The use of plasmid R1162 and derivatives for gene cloning in the methanol-utilizing Pseudomonas AM1

Summary A physical map for plasmid R1162 (Sm, Su, IncP4) was constructed. Neither EcoRI, PstI nor EcaI cut within a region essential for replication, mobilization or streptomycin resistence. Plasmid R1162 can replicate in E. coli as well as in Pseudomonas species and shows a strong dependence for DNA polymerase I in E. coli. By RP4 induced mobilization, R1162 can be transferred from E. coli to Pseudomonas AM1. A hybrid plasmid pFG7 (MW=8.4×10⁶, Sm, Su, Ap, Tc) was constructed between pBR322 and R1162, which allows the selection of hybrid plasmids by insertional inactivation with the restriction enzymes HindIII, BamHI, SalI, ClaI. Transformation of E. coli SK1592 with EcaI-cut and ligated R1162-DNA and Pseudomonas AM1-DNA and subsequent mobilization of the hybrid plasmids into Pseudomonas AM1/M15a (methanol dehydrogenase^-) led to the isolation of Pseudomonas AM1/M15a colonies, which could grow on methanol again. Back-conjugation into E. coli SK1592, subsequent mobilization studies and plasmid analysis suggests that the gene for Pseudomonas methanol dehydrogenase has been cloned in this vector.     

Development of a genetic system for a purple sulfur bacterium: Conjugative plasmid transfer inChromatium vinosum

Establishment of a system for manipulative genetics in phototrophic sulfur bacteria of the family Chromatiaceae has mainly been hampered by the lack of reliable methods for growth of these organisms on agar surfaces, techniques for streaking, growth on selective media, screening for antibiotic resistance markers, and most importantly by the lack of a system for DNA transfer. We, therefore, developed minimal and complex agar media for Chromatium vinosum strain D (DSM 180^T), a representative of the purple sulfur bacteria. Sensitivity of C. vinosum towards a broad range of antibiotics was tested in liquid cultures and solidified media, allowing us to select appropriate antibiotic resistance markers. Furthermore, a system for conjugative transfer of IncP-mobilizable plasmids from Escherichia coli to C. vinosum was established. Broad-host-range IncQ vectors were mobilized to C. vinosum with the aid of plasmid RP4 either present extrachromosomally or integrated in the chromosome of E. coli S17-1. Conjugation efficiencies of up to 1 were observed. Agarose gel electrophoretic analysis showed that transconjugants contained the transferred plasmids in addition to the two detectable plasmids of wild-type C. vinosum. All genetic markers tested (kanamycin, gentamicin, ampicillin, amikacin, tetracycline) were expressed in C. vinosum. Furthermore, high-frequency transfer of plasmid RP4 from C. vinosum to E. coli and to Rhodospirillum rubrum K100 was demonstrated. Received: 3 March 1995 / Accepted: 22 May 1995     

The restriction-modification system in Streptomyces flavopersicus

To clone bifunctional vectors in streptomycetes, it was necessary to define the restriction-modification system ofStreptomyces flavopersicus. Plasmid DNA from bifunctional vectors pIJ699 and pXED3-13, isolated fromE. coli strains with different methylation systems:E. coli DH5α (dam ⁺ dcm ⁺),E. coli MB5386(dam dcm), E. coli CB51 (dam dcm ⁺),E. coli NM544 (dam ⁺ dcm), was used for transformation of protoplasts from strainS. flavopersicus. Restriction ofdcm-methylated DNA fromS. flavopersicus was established. As a host in the intermediate cloning strainE. coli NM544 (dam ⁺ dcm) should be used, as thedcm-transmethylase-dependent strainS. flavopersicus does not process DNA from this strain.     

Conjugational transfer systems of Rhodopseudomonas capsulata mediated by R plasmids

Plasmids RP1, R68.45 and RP4::Mu cts 61 were transferred into Rhodopseudomonas capsulata from Escherichia coli. The frequency of intraspecies transfer of these plasmids in R. capsulata was 10^-4–10^-5 per donor. The plasmids also mobilized chromosomal genes at a low frequency. Phototrophic recombinants from matings between recipient strains defective in the photosynthetic-apparatus and wild type donors were obtained at a frequency of 10^-7–10^-8 per donor.     

Negative effects of agrocin 84-encoding Agrobacterium plasmids on symbiotic properties of Rhizobium meliloti

Two plasmids, pAgK84::Tn5-Mob from Agrobacterium radiobacter carrying genes for the production of agrocin 84, and RP4-4 from E. coli were inserted either separately or together into a strain of Rhizobium meliloti. Each of these plasmid-containing R. meliloti transconjugants was less effective than the wild type strain in their ability to fix nitrogen in Medicago tornata. The pAgK84::Tn5-Mob-containing transconjugant was significantly less effective than that containing RP4-4. The transconjugant strains were inferior to the wild type strain in their ability to nodulate seedlings and to compete for nodulation.     

Interactions between Escherichia coli and the plant‐parasitic nematode Meloidogyne javanica

Aims: To determine the potential of the plant‐parasitic nematode Meloidogyne javanica to serve as a temporary reservoir for Escherichia coli. Methods and Results: The adhesion to and persistence of E. coli on the surface of M. javanica were evaluated at different times and temperatures. A pure culture of green fluorescent protein (GFP) tagged E. coli was mixed with ca. 1000 J2 M. javanica for 2 h at 25°C. The nematodes were then washed and the rate of the adhesion of the bacteria to the nematodes was determined by counting the viable nematode‐associated E. coli, and by fluorescence microscopy. A dose‐dependent adhesion rate was observed only at a bacterium to nematode ratio of 10⁴–10⁶ : 1. The adhesion of E. coli to the nematodes was also tested over a 24 h‐period at 4°C, 25°C and 37°C. At 4°C and 37°C, maximal adhesion was observed at 5 h; whereas at 25°C, maximal adherence was observed at 8 h. Survival experiments showed that the bacteria could be detected on the nematodes for up to 2 weeks when incubated at 4°C and 25°C, but not at 37°C. Conclusions: Under laboratory conditions, at 4°C and 25°C, M. javanica could serve as a temporary vector for E. coli for up to 2 weeks. Significance and Impact of the Study: These findings support the hypothesis that, in the presence of high concentrations of E. coli, M. javanica might serve as a potential vehicle for the transmission of food‐borne pathogens.     

Production of serotonin by dual expression of tryptophan decarboxylase and tryptamine 5-hydroxylase in <Emphasis Type="BoldItalic">Escherichia coli</Emphasis>

A plant-specific biogenic amine, serotonin, was produced by heterologous expression of two key biosynthetic genes, tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H), in Escherichia coli. The native T5H, a cytochrome P450 enzyme, was unable to be functionally expressed in E. coli. Through a series of N-terminal deletions or additions of tagging proteins, we generated a functional T5H enzyme construct (GST∆37T5H) in which glutathione S transferase (GST) was translationally fused with the N-terminal 37 amino acid deleted T5H. Dual expression of GST∆37T5H and TDC using a pCOLADuet-1 E. coli vector produced serotonin at concentrations of approximately 24 mg l⁻¹ in the culture medium and 4 mg l⁻¹ in the cells. An optimum temperature of approximately 20°C was required to achieve peak serotonin production in E. coli because the low induction temperature gave rise to the highest soluble expression of GST∆37T5H.     

Cloning and expression in Escherichia coli of the homoserine kinase (thrB) gene from Brevibacterium lactofermentum

Summary Five DNA fragments carrying the thrB gene (homoserine kinase E.C. 2.7.1.39) of Brevibacterium lactofermentum were cloned by complementation of Escherichia coli thrB mutants using pBR322 as vector. All the cloned fragments contained a common 3.1 kb DNA sequence. The cloned fragments hybridized among themselves and with a 9 kb BamHI fragment of the chromosomal DNA of B. lactofermentum but not with the DNA of E. coli. None of the cloned fragments were able to complement thrA and thrC mutations of E. coli. Plasmids pULTH2, pULTH8 and pULTH11 had the cloned DNA fragments in the same orientation and were very stable. On the contrary, plasmid pULTH18 was very unstable and showed the DNA inserted in the opposite direction. E. coli minicells transformed with plasmids pULTH8 or pULTH11 (both carrying the common 3.1 kb fragment) synthesize a protein with an M _r of 30,000 that is similar in size to the homoserine kinase of E. coli.Abbreviations SSC 0.15 M NaCl, 0.015 M sodium citrate - SDS sodium dodecyl sulphate - TSB tripticase soy broth - m-DAP meso-diaminopimelic acid - Sm^r, Cp^r, Km^r, Am^r, Ap^r, Tc^r, MA15^r resistance to streptomycin, cephalotin, kanamycin, amykacin, ampicillin, tetracycline and microcin A 15, respectively     

Effects of carbon dioxide concentration on anaerobic fermentations of Escherichia coli

Summary The effect of dissolved carbon dioxide concentration in the anaerobic growth of Escherichia coli was investigated. E. coli was grown anaerobically with the dissolved CO₂ concentration controlled over the range from 8x10^-6 M to 3.7x10^-2 M in the liquid phase. The maximum specific growth rate was 0.75h^-1 at 1.3x10^-3 M CO₂ and the maximum yield of cells on glucose was 0.32 at 1.75x10^-4 M CO₂. The maximum specific growth rate occurs close to the concentration of CO₂ prevalent in the mammalian gut where E. coli naturally resides.Alberta Research Council contribution, paper 1364