A Conjugative Transfer System for the Rumen Bacterium, Butyrivibrio fibrisolvens, Based on Tn916-Mediated Transfer of the Staphylococcus aureus Plasmid pUB110

A limitation of genetic studies of the rumen bacterium, Butyrivibrio fibrisolvens, has been the availability of suitable vectors and transfer systems. Using the conjugative tetracycline resistant transposon, Tn916, the Staphylococcus aureus plasmid, pUB110, and the pUB110-based shuttle vector, pUBLRS, a conjugative transfer system was developed for B. fibrisolvens. B. fibrisolvens donor strains H17c2 and H17c12, containing Tn916 and pUB110 or pUBLRS, respectively, were used in mating experiments with selected B. fibrisolvens strains. Kanamycin resistant transconjugants, containing pUB110, of strains 193, 194, and 195 were detected at a combined average frequency of 7.78 × 10^-7 per donor and 1.11 × 10^-5 per recipient. Transconjugants of strains 193 and 194, containing pUBLRS, were detected at an average frequency of 1.22 × 10^-6 per donor and 4.70 × 10^-8 per recipient. Southern hybridization analysis confirmed the presence of pUB110 and pUBLRS in transconjugants. Results indicated that Tn916 was necessary for mobilization of pUB110 as transconjugants were not detected when the transposon was absent from the donor strains. The ability to mobilize pUB110 and pUBLRS between B. fibrisolvens strains provides a conjugative transfer system that circumvents problems encountered with electroporation.     

Functional Comparison of Conjugative Transposons Tn916and Tn925

During interspecies matings betweenBacillus subtilisandBacillus thuringiensissubsp.israelensis,transfer of conjugative transposon Tn916was detected at a frequency of 1.1 × 10⁻⁴transconjugants per donor. Tn916-dependent transfer of plasmids pC194 and pE194 was detected at frequencies of 1.4 × 10⁻⁵and 3.2 × 10⁻⁷transconjugants per donor, respectively. Similar frequencies were obtained during parallel matings with otherwise isogenic strains that contain Tn925instead of Tn916. Tn916- or Tn925-dependent transfer of plasmids pC194 or pUB110 from the recipient to the donor (retrotransfer) was not observed during inter- or intraspecies matings. Transposon-mediated plasmid transfer by Tn916and Tn925is a Rec independent event. Thus, the data from studies in which otherwise isogenic donor and recipient strains were used indicated that Tn916and Tn925are, from a functional point of view, much more similar than previously suggested.     

In vivo transfer of genes from Escherichia coli to Bacillus subtilis

Summary Escherichia coli cells, carrying plasmid pRD1 with (a) drug resistance markers from Pseudomonas (km^r, carb^r, tc^r) and (b) the nif-gene group from Klebsiella, were incubated together with Bacillus subtilis cells (str^r), whose cell wall had been disintegrated with lysozyme. Upon plating the cell mixtures onto appropriately supplemented selective medium, multiple drug resistant Bacillus subtilis cells were obtained. Their nature was verified by suitable biochemical tests and checking for the presence of additional genetic markers. The majority of the isolates was unstable. Some however retained multiple drug resistance for longer periods of time, and several produced nitrogenase activity. The data are interpreted as evidence not only for the transfer of the respective genes but also for their expression in the gram-positive recipient cells.Abbreviations pRD1 a hybrid plasmid, renamed by Ray Dixon - pRP4 plasmid from Pseudomonas, originally described by Datta et al., J. Bacteriol 108, 1244 (1971) - km ^r, carb ^r, tc ^r, str ^r resistance against kanamycin, carbenicillin, tetracyclin and streptomycin, respectively - r restriction negative. For other bacterial markers refer to Bachmann, B.J. et al., Bacteriological Reviews 40, 116 (1976)     

Electroporation of Bradyrhizobium japonicum

Summary Electroporation offers a fast, efficient and reproducible way to introduce DNA into bacteria. We have successfully used this technique to transform two commercially important strains of Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont. Initially, electroporation conditions were optimized using plasmid DNA which had been prepared from the same B. japonicum strain into which the{imDNA was to b}e transformed. Efficiencies of 10⁵-10⁶ transformants/g DNA were obtained for strains USDA 110 and 61A152 with ready-to-use frozen cells. Successful electroporation of B. japonicum with plasmid DNA prepared from Escherichia coli varied with the E. coli strain from which the plasmid was purified. The highest transformation efficiencies (10⁴ transformants/g DNA) were obtained using DNA prepared from a dcm ^– dam ^– strain of E. coli. This suggests that routine isolation of DNA from an E. coli strain incapable of DNA modification should help in increasing transformation efficiencies for other strains of bacteria where DNA restriction appears to be a significant obstacle to successful transformation. We have also monitored the rate of spontaneous mutation in electroporated cells and saw no significant difference in the frequency of streptomycin resistance for electroporated cells compared to control cells.     

Characterization of the ntrBC genes of Azospirillam brasilense Sp7: Their involvement in the regulation of nitrogenase synthesis and activity

A 7.1 kb EcoRI fragment from Azospirillum brasilense, that hybridized with a probe carrying the ntrBC genes from Bradyrhizobium japonicum, was cloned. The nucleotide sequence of a 3.8 kb subfragment was established. This led to the identification of two open reading frames, encoding polypeptides of 401 and 481 amino acids, that were similar to NtrB and NtrC, respectively. A broad host range plasmid containing the putative Azospirillum ntrC gene was shown to restore nitrogen fixation under free-living conditions to a ntrC-Tn5 mutant of Azorhizobium caulinodans. Several Tn5 insertion mutants were isolated in the ntrBC coding region in A. brasilense. These mutants were prototrophic and Nif⁺. However, their nitrogenase activity was slightly lower than in the wild type and they were unable to grow on nitrate as sole nitrogen source. Under microaerobiosis and in the absence of ammonia, a nifA-lacZ fusion was expressed in the mutants at about 60% of the level in the wild type. In the presence of ammonia, the fusion was similarly expressed (60% of the maximum) both in the wild type and mutants. Addition of ammonia to a nitrogen-fixing culture of ntrBC mutants did not abolish nitrogenase activity, in contrast with the wild type. It thus appears that in Azospirillum the ntrBC genes are not essential for nitrogen fixation, although NtrC controls nifA expression to some extent. They are, however, required for the switch-off of nitrogenase activity.     

Evidence of abortive recombination in ruv mutants of Escherichia coli K12

Summary Genetic recombination in Escherichia coli was investigated by measuring the effect of mutations in ruv and rec genes on F-prime transfer and mobilization of nonconjugative plasmids. Mutation of ruv was found to reduce the recovery of F-prime transconjugants in crosses with recB recC sbcA strains by about 30-fold and with recB recC sbcB sbcC strains by more than 300-fold. Conjugative plasmids lacking any significant homology with the chromosome were transferred normally to these ruv mutants. Mobilization of the plasmid cloning vectors pHSG415, pBR322, pACYC184 and pUC18 were reduced by 20- to 100-fold in crosses with ruv rec ⁺ sbc ⁺ strains, depending on the plasmid used. Recombinant plasmids carrying ruv ⁺ were transferred efficiently. With both F-prime transfer and F-prime cointegrate mobilization, the effect of ruv was suppressed by inactivating recA. It is proposed that the failure to recover transconjugants in ruv recA ⁺strains is due to abortive recombination and that the ruv genes define activities which function late in recombination to help convert recombination intermediates into viable products.     

Development of a Genetic System for Eikenella corrodens: Transfer of Plasmids pFM739 and pLES2

Eikenella corrodens is a Gram-negative microaerophilic rod which is emerging as an important human pathogen. Elucidation of the mechanisms by which it causes disease require efficient methods for the transfer of DNA to E. corrodens. Plasmids pFM739 and pLES2 have been transferred by conjugation from Escherichia coli S17-1 to E. corrodens ATCC 23834 at frequencies of 2.5 × 10^-7 and 2.42 × 10^-7, respectively. In addition, both plasmids could be transferred to four additional, clinical strains of E. corrodens at a similar frequency. The use of bacteriophage T4 as a counterselecting agent is also described.     

Transduction in Rhizobium meliloti

Summary Of 21Rhizobium meliloti temperate phages, 12 transduced streptomycin resistance (str-r) with a frequency of 10⁻⁵ to 10⁻⁷. Lysine dependence was transduced with frequency of 10⁻⁷. Five transducing phages were used in experiments on the transfer of effectiveness from effective donor strain to 11 ineffective strains. However, plant tests did not reveal changes in recipient effectiveness.     

Ars region in TL-DNA on octopine type Ti plasmids

Summary In the TL-DNA region of the octopine type Ti plasmids, an ars region was assigned as the DNA segment conferring the replicational ability to YIp5 in Saccharomyces cerevisiae. T-DNA:YIp5 hybrid plasmids containing a particular T-DNA region could transform yeast cells at a frequency of 10³–10⁴ transformants per g plasmid DNA and they were rescued in Escherichia coli, although the transformed phenotype was mitotically unstable. The instability was inferred to be caused by segregation of the plasmids due to their low efficiency of replication. The ars region was mapped on the noncoding region between the coding regions corresponding to no. 5 and no. 7 mRNA, and its minimal length determined in this experiment was about 150 bp.Abbreviations Ti plasmid tumor inducing plasmid - T-DNA transferred DNA or tumor DNA - TL-DNA left T-DNA - ars autonomously replicating sequences     

Nif-hybrids of Enterobacter cloacae: Selection for nif-gene integration with nif-plasmids containing the Mu transposon

Summary The nitrogen fixation (nif)-gene group of Klebsiella can be transferred onto Enterobacter cloacae by conjugation, using Escherichia coli donor cells carrying the composite self-transmissible nif-plasmid pRD1. To enforce integration and stabilisation, in the present study a derivative of pRD1, viz plasmid pCE1, containing the Mu transposon was used. pCE1:: Mu cts makes Enterobacter cloacae cells nif ⁺, and sensitive to temperature induction of Mu. Few cells survive treatment at 42°C. Seventy-two isolates thus obtained were screened for location of their nif-genes. At least four were found to contain the nif-genes integrated into the chromosome. This was documented by gel electrophoresis of their DNA, and by Southern hybridisation of their DNA with Klebsiella nif-KDH DNA as radioactive probe. The Mu transposon had also become part of their chromosome.     

The maintenance affinities of KLF-1 proximal F merogenotes inEscherichia coli

Summary In conjugation with donor strains carrying proximal F merogenotes of KLF-1 type about 100-fold lower frequency ofLeu ⁺ orLac ⁺ recombinants was found. The determination of the level of β-galactosidase synthesis during the initial period of mating indicated that the transfer process of plasmid DNA was not impaired. Among the recombinants selected a large fraction have not expressed the plasmic fertility functions. This phenomenon was found to be replicon specific and was observed only with proximal F merogenotes but not with classical F'lac and F'ORF-1 elements or RI-19 plasmid. The expression of KLF-1 plasmid functions in the cell seems to be affected by a chromosomal gene of the proximal F merogenote closely linked toleu marker.     

Determination of the Structure of the Repeated Unit of the Azospirillum brasilense SR75 O-Specific Polysaccharide and Homology of the lps Loci in the Plasmids of Azospirillum brasilense strains SR75 and Sp245

The structural identity of the repeated unit in O-specific polysaccharides (OPSs) present in the outer membrane of strain SR75 of the bacterium Azospirillum brasilense, isolated from wheat rhizosphere in Saratov oblast, and the previously studied OPSs of A. brasilense strain Sp245, isolated from surfacesterilized wheat roots in Brazil, has been demonstrated. Plasmid profiles, DNA restriction, and hybridization assays suggested that A. brasilense strains SR75 and Sp245 have different genomic structures. It was shown that homologous lps loci of both strains were localized in their plasmid DNA. This fact allows us to state that, despite their different origin, the development of the strains studied was convergent. Presumably, the habitation of these bacteria in similar ecological niches influenced this process in many respects. __________ Translated from Mikrobiologiya, Vol. 74, No. 5, 2005, pp. 626–632. Original Russian Text Copyright ? 2005 by Fedonenko, Borisov, O. Konnova, Zdorovenko, Katsy, S. Konnova, Ignatov.     

Plasmid-mediated transfer of nitrogen-fixing capability to bacteria from the rhizosphere of grasses

Summary Cells of a non-nitrogen-fixing, drug-sensitive Enterobacter cloacae strain, isolated from the rhizosphere of Festuca heterophylla, were mated to Escherichia coli cells harboring plasmid pRD1. This plasmid carries the nitrogen-fixation (nif⁺) genes as well as three markers of drug resistance. After mating, triple-resistant Enterobacter transferants could be selected. These were screened for plasmids, acetylene reduction, and stability of the transferred markers.Transferants contained plasmid pRD1. Of 48, 43 were acetylene-reducing and therefore carried the nif⁺ genes. Triple-resistance was stable upon passage in liquid minimal medium, but the number of cells with nif⁺ genes decreased. Both the triple-resistant and the nif⁺ genotypes decreased in complete medium, although by different rates, depending on the particular line. The most stable line, M14, was chosen and checked further.Samples taken after 8–14 passages in minimal medium contained cells with different genotypes, plasmid sizes smaller than the original plasmid pRD1 and no free plasmids. Progeny of the latter cells, in addition to being triple-resistant, were the best acetylene reducers. It is concluded that in these cells the plasmid pRD1 with all its relevant genes had become integrated into the recipients' chromosome.Grass seedlings were inoculated with the bacteria containing integrated plasmid pRD1. They were then planted into pots with sterile ash and watered with a nutrient salt solution of limited nitrogen content. Sampling after 8 weeks showed that the inoculated bacteria were preserved, as demonstrated by their triple-resistance. They could also still fix nitrogen.     

Physical characterization of plasmids isolated fromStreptosporangium

Plasmids were isolated from two species ofStreptosporangium by CsCl-ethidium bromide equilibrium density gradient centrifugation. A plasmid isolated fromS. brasiliense, designated pSgB-1, was characterized by electron microscopy and agarose gel electrophoresis. The pSgB-1 plasmid is a closed circular DNA molecule of 9.4 × 10⁶ Da. A restriction endonuclease map was generated and unique cleavage sites were found forEcoRI, ClaI, XbaI, and MstII. Another plasmid, pSgV-1, isolated fromS. viriodogriseum, has an estimatedM_r of 54 × 10⁶. The pSgB-1 plasmid is phenotypically cryptic but an unusual phenotypic trait, resembling phage plaques, may be associated with theS. viridogriseum plasmid pSgV-1.     

Properties of transposon SCTn1 of Streptomyces coelicolor A3(2)

Summary Chloramphenicol resistance (Cml^r) of Streptomyces coelicolor A3(2) behaves like a transposon locus, not being localisable in any region of the map and yet being transferable in crosses at a rate comparable to that of chromosomal markers. It can, also be transposed onto a plasmid (SCP1) and back to the chromosome. Some traits, such as arginino-succinate synthase production (ArgG), aerial mycelium formation (AmyA), resistance to tetracycline and to rifamycin C appear to be joined to Cml in three processes: co-mutation, i.e. simultaneous loss, post-mutation, i.e. spontaneous loss at high, frequency in subclones from Cml^s strains, co-transfer, i.e. joint transfer with the cml locus in crosses or during infection by the aggregate SCP1::SCTn1 plasmid. All these processes have been consistently observed with special attention to the argG locus.     

Transfer and expression of Klebsiella nif genes in Alcaligenes faecalis, a nitrogen-fixing bacterium associated with rice root

Plasmid pRD1 carrying Klebsiella nif genes was found to be transferable by conjugation from Escherichia coli JC5466 (pRD1) to Alcaligenes faecalis A-15 at a frequency of 5 X 10(-4). Nitrogenase activity of four A-15 (pRD1) strains tested was found to be higher than that of their parent A-15, as determined by the acetylene reduction assay. A-15-1 was a Nif- mutant derived from A-15. After mating with JC5466 (pRD1), the nitrogenase activity was restored. PRD1 was stable in A. faecalis and could be transferred to E. coli JC5466-1 by conjugation. The fact that Klebsiella nif genes carried in pRD1 can be expressed in A. faecalis makes it possible to use pRD1 as a tool for genetic analysis and genetic engineering of the nitrogen fixation system in A. faecalis.     

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.     

The conjugative transposon Tn925: enhancement of conjugal transfer by tetracycline in Enterococcus faecalis and mobilization of chromosomal genes in Bacillus subtilis and E. faecalis

Summary The effects of tetracycline on transfer of the conjugative, tetracycline-resistance transposon, Tn925, as well as the ability of the transposon to promote the transfer of chromosomal genes was examined in Enterococcus faecalis and Bacillus subtilis. To test for chromosomal transfer, multiply-marked strains of each organism, each carrying a single chromosomal copy of Tn925, were mated on filters with suitable recipient strains, under conditions where transformation and transduction were precluded. In both cases, transfer of a variety of chromosomal genes, at frequencies comparable to the frequency of Tn925 transfer, was detected readily. The presence of Tn925 in one of the members of the mating pair was absolutely required for chromosomal transfer, but transfer of Tn925 did not accompany every chromosomal transfer event. The results were consistent with a mating event resembling a type of cell fusion, allowing for extensive recombination between the genomes of the mating partners. Growth of Tn925-containing donor cells in the presence of tetracycline increased the transfer frequency of Tn925 by about tenfold in E. faecalis, but not in B. subtilis.Deceased, 7/89. O. Torres and R. Korman contributed equally to this work     

Plasmids controlling synthesis of hemolysin inEscherichia coli

Summary Plasmids of three different sizes, designated as plasmid A (mw: 65×10⁶), plasmid B (mw: 41×10⁶) and plasmid C (mw: 32×10⁶) respectively, have been isolated from various hemolytic wild-type strains ofE. coli. DNA-DNA hybridization was performed to determine their relationship. The wild-type strain, PM167a, harbours plasmids of all three sizes. Hybridization studies indicate that all three plasmids share extented sequence homologies but that plasmid A is not composed of plasmids B and C. Hybridization between plasmids of the donor strain and those of appropriate transconjugants demonstrates that in some cases plasmids with identical size are not longer completely homologous in their nucleotide sequences. This indicates that despite their defined sizes these plasmids are not stable genetic entities, but rather they undergo frequently recombination and dissociation during conjugation. In one particular transconjugant strain, K12-PM152/1, a plasmid D was found which is a stable recombined molecule of plasmids B and C of the original strain. Plasmids of size B found as the only extrachromosomal elements in a hemolytic wild-type strain (P224) and two transconjugant strains (e.g. K12-CM20 and K12-PM167/1) share extended nucleotide sequence homologies but are not identical. Little sequence homology was observed between two different hemolytic plasmids and the F and the Col Ib plasmids suggesting that the former do not belong to either the F-like or the I-like group of plasmids. Another hemolytic plasmid is F-like based on its sequence homologies with the F factor.