Transfer of transformation and conjugation plasmids from Escherichia coli to Bacillus subtilis and Azospirillum brasilense]

The conjugative transfer of RP4 plasmid from Escherichia coli to Azospirillum brasilense was detected after introduction and subsequent incubation of these microorganisms in soil. The plasmid transfer via transformation from Escherichia coli to Bacillus subtilis was observed in case both bacteria were growing together in sand containing sucrose solution. The possible reason for low frequency interspecies plasmid transformation under conditions close to natural habitats is poor survival of "domesticated" rather than wild type Bacillus subtilis strains and lack of competence state in this case.     

Electroporation of Azospirillum brasilense with plasmid DNA

Abstract The feasibility of electric field mediated transformation of the nitrogen fixing bacterium Azospirillum was studied. The broad host range plasmid pRK290 was used throughout this study. Transformants were obtained with all A. brasilense strains tested, although with strain dependent efficiency. No transformants were obtained with an A. lipoferum strain. Transfer of the pRK290 plasmid DNA in the A. brasilense strains was confirmed by DNA extraction of the transformants and gel electrophoresis. The effects of the physiological status of the cells and the electric field strength during electroporation were studied in detail for one particular A. brasilense strain.     

Transfer of plasmid pRD1 from Escherichia coli to Azospirillum brasilense

Summary Data are presented which indicate that plasmid pRD1 can be transferred from Escherichia coli to strains of Azospirillum brasilense with a frequency of about 10^-7. The reverse was also possible; in this case the frequency of transfer appeared to be much higher, about 5×10^-1. Transfer of the plasmid was also obtained between strains of A. brasilense; in this cross the transfer frequency was very high (about 10^-1). Moreover the pRD1 plasmid seems very stable in A. brasilense cells.Abbreviations ade, his, and trp are requirements for adenine, histidine, and tryptophan, respectively - carb, kan, rif, spc, and tet are resistance to carbenicillin, kanamycin, spectinomycin, and tetracycline, respectively - recA56 recombination deficiency - nif genes for nitrogen fixation     

Characterization of two Azospirillum brasilense Sp7 plasmid genes homologous to Rhizobium meliloti nodPQ

Bacteria belonging to the Azospirillum genus are nitrogen fixers that colonize the roots of grasses, but do not cause the formation of differentiated structures. Sequences from total DNA of several Azospirillum strains are homologous to restriction fragments containing Rhizobium meliloti nodulation genes. A 10-kilobase (kb) EcoRI fragment from A. brasilense Sp7, sharing homology with a 6.8-kb EcoRI fragment carrying nodGEFH and part of nodP of R. meliloti 41, was cloned in pUC18 to yield pAB502. The nucleotide sequence of a 3.5-kb EcoRI-SmaI fragment of the pAB502 insert revealed 60% homology with R. meliloti nodP and nodQ genes. The nodP gene product shares no homology to any known protein sequence. The Azospirillum nodQ gene product shares homology with a family of initiation and elongation factors as does the R. meliloti nodQ gene product. Since the nodQ gene overlaps the nodP gene, the two genes might be cotranscribed. Azospirillum contains large plasmids, and the nodPQ genes were found on the 90-MDa plasmid (p90). A translational nodP-lacZ fusion was constructed in the broad host range plasmid pGD926. No beta-galactosidase activity was detected in Escherichia coli, but the fusion was functional in Azospirillum and constitutively expressed. Deletions and mutations of nodPQ did not modify growth, nitrogen fixation, or interaction with wheat seedlings.     

Transfer of plasmid pTO1 from Escherichia coli to various representatives of the order Actinomycetales by intergeneric conjugation

Plasmid pTO1 containing the oriT fragment from RK2, the Escherichia coli replication function from pBR322, and a DNA fragment of actinophage φC31 with the attachment site was transferred from E. coli S17-1 to strains of the genera Actinomadura, Arthrobacter, Micromonospora, Nocardia, Rhodococcus, and to 16 strains of the genus Streptomyces. The frequency of conjugant formation was 1×10⁻³–1×10⁻⁵ depending on the strain. Hybridization experiments demonstrated that plasmid pTO1 integrates into chromosomes of a number of the recipient strains examined.     

Plasmids of Azospirillum brasilense

The cells from natural isolates of A. Brasilense were found to harbour 1 to 4 plasmids with the molecular masses within the 27-300 Md range. 100 Md plasmids are specific for this bacterial species. Strains isolated from the roots of cereals (wheat, maize, barley) have more heterogeneous plasmid composition as compared to the strains isolated from soil.     

Transfer of the Ti plasmid from Agrobacterium tumefaciens into Escherichia coli cells

We have screened strains of Agrobacterium tumefaciens for spontaneous mutants showing constitutive transfer of the nopaline Ti plasmid pTiC58 during conjugation. The Ti plasmid derivatives obtained could be transferred not only to A. tumefaciens but also to E. coli cells. The Ti plasmid cannot survive as a freely replicating plasmid in E. coli, but it can occasionally integrate into the E. coli chromosome. However, insertion in tandem of plasmids carrying fd replication origins (pfd plasmids) into the T-DNA provides an indicator for all transfer events into E. coli cells, providing fd gene 2 protein is present in these cells. This viral protein causes the excision of one copy of the pfd plasmid and allows its propagation in the host cell. By using this specially designed Ti plasmid, which was also made constitutive in transfer functions, we found plasmid exchange among A. tumefaciens strains and between A. tumefaciens and E. coli cells to be equally efficient. A Ti plasmid with repressed transfer functions was transferred to E. coli with a rate similar to the low frequency at which it was transferred to A. tumefaciens. The expression of transfer functions of plasmid RP4 either in A. tumefaciens or in E. coli did not increase the transfer of the Ti plasmid into E. coli cells, nor did the addition of acetosyringone, an inducer of T-DNA transfer to plant cells. The results show that A. tumefaciens can transfer the Ti plasmid to E. coli with the same efficiency as within its own species. Conjugational transmission of extrachromosomal DNA like the narrow-host-range Ti plasmid may often not only occur among partners allowing propagation of the plasmid, but also on a 'try-all' basis including hosts which do not replicate the transferred DNA.     

Cloning and expression in Escherichia coli of the Azospirillum brasilense Sp7 gene encoding ampicillin resistance

The Azospirillum brasilense ATCC 29145 gene coding for beta-lactamase was cloned in Escherichia coli. The gene was expressed in E. coli from its own promoter as a 30-kilodalton protein, conferring resistance to high levels of beta-lactam antibiotics. The DNA sequence containing the beta-lactamase gene was found to be highly amplified in the Azospirillum genome, scattered in the chromosomal as well as in the plasmidic DNA.     

Insights into the 1.59-Mbp largest plasmid of Azospirillum brasilense CBG497

The plant growth-promoting proteobacterium Azospirillum brasilense enhances growth of many economically important crops, such as wheat, maize, and rice. The sequencing and annotation of the 1.59-Mbp replicon of A. brasilense CBG497, a strain isolated from a maize rhizosphere grown on an alkaline soil in the northeast of Mexico, revealed a GC content of 68.7?% and the presence of 1,430 potential protein-encoding genes, 1,147 of them classified into clusters of orthologous groups categories, and 16 tRNA genes representing 11 tRNA species. The presence of sixty-two genes representatives of the minimal gene set and chromid core genes suggests its importance in bacterial survival. The phaAB?→?G operon, reported as involved in the bacterial adaptation to alkaline pH in the presence of K(+), was also found on this replicon and detected in several Azospirillum strains. Phylogenetic analysis suggests that it was laterally acquired. We were not able to show its inference on the adaptation to basic pH, giving a hint about the presence of an alternative system for adaptation to alkaline pH.     

Mutants of Azospirillum brasilense resistant to methylammonium

One hundred and twenty-nine mutants of Azospirillum brasilense strain Sp6, resistant to methylammonium, were isolated. Three of the mutants were found to be able to reduce acetylene in the presence of 4 mM ammonium or 120mM methylammonium, concentrations which strongly reduced the nitrogenase activity of the parental strain. Under N₂-fixing conditions, two mutants failed to switch off nitrogenase when NH₄Cl was added. Moreover, the three mutants showed a reduced capacity to incorporate [¹⁴C]methylammonium. The level of glutamine synthetase activity found in the mutants was not reduced as compared to that of the parental strain. All of the data indicate an impairement in the mechanism of ammonium uptake by the bacterial cell.Abbreviations MEA Methylammonium - MSP minimal medium (ammonium free) - PY complete medium - GS glutamine synthetase     

Cloning and expression in Escherichia coli of the Azospirillum brasilense Sp7 gene encoding ampicillin resistance.

The Azospirillum brasilense ATCC 29145 gene coding for beta-lactamase was cloned in Escherichia coli. The gene was expressed in E. coli from its own promoter as a 30-kilodalton protein, conferring resistance to high levels of beta-lactam antibiotics. The DNA sequence containing the beta-lactamase gene was found to be highly amplified in the Azospirillum genome, scattered in the chromosomal as well as in the plasmidic DNA.     

Hexamerization of RepA from the Escherichia coli plasmid pKL1

The Escherichia coli plasmid pKL1 is one of the smallest bacterial plasmids. It encodes a single, autoregulating structural gene, repA, responsible for replication and copy number control. The oligomerization of RepA was previously proposed as the basis of a strategy for pKL1 copy number control. To elucidate the oligomerization properties of RepA in solution, RepA was expressed in E. coli; purified by ion exchange and hydrophobic chromatography; and examined in solution by spectrapolarimetry, light scattering, sedimentation velocity, and equilibrium ultracentrifugation. RepA behaved as a concentration-dependent equilibrium of dimers and hexamers. Conformational parameters of the RepA hexameric complex were determined. These results support the proposed autogenous regulatory model whereby RepA hexamers negatively regulate repA expression thereby affecting the copy number control of pKL1. RepA of pKL1 is the first plasmid replication initiation protein documented to be in dimeric-hexameric forms.     

Plasmid transformation of Azospirillum brasilense

Abstract Plasmid transformation of the nitrogen-fixing bacterium Azospirillum brasilense is described. A modification of the method of Hanahan [1] was used to transform this bacterium with the 20-kb plasmid pRK290. The efficiency of transformation ranged from 200–1000 transformants per μg of plasmid DNA according to DNA concentration. Ca²⁺, Mn²⁺ and K⁺ were essential for competence, while Rb⁺ and hexamine cobalt(III) chloride did not appear necessary. The length and the temperature of heat-pulse during transformation affected the efficiency of transformation. The response to different numbers of plasmid molecules was linear, in the range of 0.05–1.0 μg of DNA. No transformants were obtained with pRK290 plasmid DNA linearized with Eco RI. The transformability of different strains of Azospirillum has been compared.     

Conjugative transfer of plasmid pTO1 from Escherichia coli to Rhodococcus spp.

Summary We demonstrated the conjugative transfer of plasmid pTO1 from Escherichia coli S17-1 to different Rhodococcus spp. The plasmid contains the oriT fragment from RK2 and a fragment of Streptomyces C31 actinophage with the attachment site and the integration genes. Experiments on hybridization showed that plasmid pTO1 is chromosomally integrated into the Rhodococcus cells.     

Structural characterization of glutamine synthetase from Azospirillum brasilense

CD spectroscopic study of the secondary structure of partly adenylylated glutamine synthetase (GS) of the bacterium Azospirillum brasilense showed both the native and cation-free (EDTA-treated) enzyme to be highly structured (58 and 49% as alpha-helices, 10 and 20% as beta-structure, respectively). Mg(2+), Mn(2+), or Co(2+), when added to the native GS, had little effect on its CD spectrum, whereas their effects on the cation-free GS were more pronounced. Emission ((57)Co) M?ssbauer spectroscopic (EMS) study of (57)Co(2+)-doped cation-free GS in frozen solution and in the dried state gave similar spectra and M?ssbauer parameters for the corresponding spectral components, reflecting the ability of the Co(2+)-enzyme complex to retain its properties upon drying. The EMS data show that (a) A. brasilense GS has 2 cation-binding sites per active center and (b) one site has a higher affinity to Co(2+) than the other, in line with the data on other bacterial GSs.     

Physical map and properties of a 90-MDa plasmid of Azospirillum brasilense Sp7

Homology was previously detected between the DNA restriction fragments containing Rhizobium meliloti nodulation genes and the 90-MDa plasmid, p90, of Azospirillum brasilense Sp7. Two DNA loci from Sp7 genome that complement mutations in the exopolysaccharide synthesis genes, exoB and exoC, of R. meliloti were also shown to be present on the plasmid. A more detailed characterization of the plasmid was undertaken to establish its physical map and to localize the nod homologies and other specific regions. Six loci were mapped, the region homologous to the nodulation genes, nodPQ, of R. meliloti, the exoB and exoC mutation-correcting loci, a locus for Ap resistance, a bla homology region different from the Ap resistance locus, and a region necessary for the maintenance of p90 as an independent replicon. Mobilization into Agrobacterium tumefaciens of p90-Tn5-Mob was obtained at a frequency of 10(-4), with the plasmid helper pJB3JI. Self-transfer of p90 was not demonstrated. Fragments of p90 hybridized with a plasmid of 90 MDa present in most A. brasilense and some A. lipoferum strains, suggesting a plasmid family in Azospirillum.     

Factors inducing transition from growth to dormancy in rhizobacteria Azospirillum brasilense

The factors suppressing division of the cells of the rhizobacterium Azospirillum brasilense and inducing their transition to a dormant state were analyzed. These included the presence of hexylresorcinol or heavy metals (Cu and Co) in the medium, oxygen stress, and transfer of the cells into the physiological saline or phosphate buffer solution. The results were used to develop a protocol for obtaining of nonculturable cells of A. brasilense Sp245, a natural symbiont of wheat. The cells lost their ability to grow on synthetic agar medium, but could revert to growth when incubated in freshly prepared liquid medium. Needle-shaped crystals differing from struvite, which has been previously reported for this strain, were found in the dormant culture of A. brasilense Sp245.     

Transfer of plasmid RSF1010 by conjugation from Escherichia coli to Streptomyces lividans and Mycobacterium smegmatis.

The plasmid RSF1010 belongs to a class of plasmids (IncQ) that replicate in a range of bacterial hosts. Although non-self-transmissible, it can be mobilized at high frequency between different gram-negative bacterial species if transfer functions are supplied in trans. We report the transfer of RSF1010 by conjugation from Escherichia coli to the gram-positive actinomycetes Streptomyces lividans and Mycobacterium smegmatis. In its new hosts, the plasmid was stable with respect to structure and inheritance and conferred high-level resistance to streptomycin and sulfonamide. This is the first reported case of conjugative transfer of a naturally occurring plasmid between gram-negative and gram-positive bacteria.