Instability of Cryptic Plasmids in Strain Sinorhizobium meliloti P108 in the Course of Symbiosis with Alfalfa Medicago sativa

Instability of cryptic plasmids in Sinorhizobium meliloti laboratory strains SXM1, DM7-R, and P108 as well as in their clones isolated from nodules of alfalfa grown during a long-term microvegetation experiment (120 days) was studied. The isolated clones of strains SXM1 and DM7-R manifested stable inheritance of plasmids, whereas 12.7–14.0% of clones with changed plasmid profile were detected in a population of clones from strain P108. These segregants were designated as P108c. Segregants P108c exhibited significantly decreased symbiotic effectiveness, nitrogenase activity, and the competitiveness with respect to alfalfa, compared to the original strain P108. It was established that a 80-kb deletion occurred in a larger of two cryptic plasmids (240 and 230 kb) of segregants P108c. It was concluded that genetic rearrangements are possible in rhizobial clones that did not undergo structural transformation and retained viability in the nodule during the natural vegetation period of alfalfa.     

Derivatives of Rhizobium meliloti strains carrying a plasmid of Rhizobium leguminosarum specifying hydrogen uptake and pea-specific symbiotic functions

pIJ1008, a Rhizobium leguminosarum plasmid which determines hydrogen uptake ability and symbiotic functions in pea was transferable to three of seven natural isolates of R. meliloti tested. In these three strains, pIJ1008 was maintained stably with the respective sym megaplasmid indigenous to each R. meliloti strain. These strains carrying both plasmids nodulated alfalfa but not pea. By reisolation and examination of the strains from alfalfa nodule tissue, it was shown that pIJ1008 continued to be maintained but that pea-nodulation ability was suppressed.In one strain of R. meliloti which carries a 200 kb cryptic plasmid (in addition to a megaplasmid), the transfer and selection for pIJ1008 resulted in the loss of the cryptic plasmid.In three separate plant growth experiments, alfalfa nodules induced by each of the R. meliloti strain carrying both sym plasmids were assayed for hydrogen uptake activity. The average activity was 40-, 3.5-and 2-fold higher than with the respective pIJ1008-free strains. However, this higher activity was not accompanied by an increase in plant biomass or nitrogen content of shoots.C.B.R.I. Contribution Number: 1478     

Genetic Diversity of a Natural Population of Sinorhizobium melilotiRevealed in Analysis of Cryptic Plasmids and ISRm2011-2 Fingerprints

Fifty-six natural strains of alfalfa nodule bacteria were isolated from samples of the soil under wild legume and alfalfa in two different field sites of Irkutsk oblast. Based on the results of analysis of plasmid profile, 11 different types of strains were detected, and 43 types were identified based on the results of hybridization with the insertion sequence element ISRm2011-2. Significant differences were found in the plasmid profile and IS fingerprints between strains isolated from the soil under alfalfa and the soil under legume. In contrast, strains growing at some distance from each other differed only in the IS fingerprints. From a comparison of results obtained in the assessment of plasmid profile and in analysis of IS fingerprints with results of RFLP analysis in strains, the conclusion about the transference of cryptic plasmids between strains and genetic rearrangements in strains of this population was drawn.     

Characterization and molecular cloning of cryptic plasmids isolated from Lactobacillus casei

Four small cryptic plasmids were isolated from Lactobacillus casei strains, and restriction endonuclease maps of these plasmids were constructed. Three of the small plasmids (pLZ18C, pLZ19E, and pLZ19F1; 6.4, 4.9, and 4.8 kilobase pairs, respectively) were cloned into Escherichia coli K-12 by using pBR322, pACYC184, and pUC8 as vectors. Two of the plasmids, pLZ18C and pLZ19E, were also cloned into Streptococcus sanguis by using pVA1 as the vector. Hybridization by using nick-translated cloned 32P-labeled L. casei plasmid DNA as the probe revealed that none of the cryptic plasmids had appreciable DNA-DNA homology with the large lactose plasmids found in the L. casei strains, with chromosomal DNAs isolated from these strains. Partial homology was detected among several plasmids isolated from different strains, but not among cryptic plasmids isolated from the same strain.     

Characterization and molecular cloning of cryptic plasmids isolated from Lactobacillus casei.

Four small cryptic plasmids were isolated from Lactobacillus casei strains, and restriction endonuclease maps of these plasmids were constructed. Three of the small plasmids (pLZ18C, pLZ19E, and pLZ19F1; 6.4, 4.9, and 4.8 kilobase pairs, respectively) were cloned into Escherichia coli K-12 by using pBR322, pACYC184, and pUC8 as vectors. Two of the plasmids, pLZ18C and pLZ19E, were also cloned into Streptococcus sanguis by using pVA1 as the vector. Hybridization by using nick-translated cloned 32P-labeled L. casei plasmid DNA as the probe revealed that none of the cryptic plasmids had appreciable DNA-DNA homology with the large lactose plasmids found in the L. casei strains, with chromosomal DNAs isolated from these strains. Partial homology was detected among several plasmids isolated from different strains, but not among cryptic plasmids isolated from the same strain.     

Genetic diversity of a natural population of Sinorhizobium meliloti, detected during analysis of a cryptic plasmid and ISRm2011-2 fingerprints]

Fifty-six natural strains of alfalfa nodule bacteria were isolated from samples of the soil under wild legume and alfalfa in two different field sites of Irkutsk oblast. Based on the results of analysis of plasmid profile, 11 different types of strains were detected, and 43 types were identified based on the results of hybridization with the insertion sequence element ISRm2011-2. Significant differences were found in the plasmid profile and IS fingerprints between strains isolated from the soil under alfalfa and the soil under legume. In contrast, strains growing at some distance from each other differed only in the IS fingerprints. From a comparison of results obtained in the assessment of plasmid profile and in analysis of IS fingerprints with results of RFLP analysis in strains, the conclusion about the transference of cryptic plasmids between strains and genetic rearrangements in strains of this population was drawn.     

Conjugal transfer and characterization of bacteriocin plasmids in group N (lactic acid) streptococci.

Thirteen bacteriocin-producing strains of group N (lactic acid) streptococci were screened for their potential to transfer this property by conjugation to Streptococcus lactis subsp. diacetylactis Bu2-60. Bacteriocin production in three strains was plasmid encoded as shown by conjugal transfer and by analysis of cured, bacteriocin-negative derivatives of the donor strains and the transconjugants. With Streptococcus cremoris strains 9B4 and 4G6 and S. lactis subsp. diacetylactis 6F7 as donors, bacteriocin-producing transconjugants were isolated with frequencies ranging from ca. 2 X 10(-2) to 2 X 10(-1) per recipient cell. Bacteriocin-producing transconjugants had acquired a 39.6-megadalton plasmid from the donor strains 9B4 and 4G6, and a 75-megadalton plasmid from the donor strain 6F7. As shown by restriction endonuclease analysis, the plasmids from strains 9B4 and 4G6 were almost identical. The plasmid from strain 6F7 yielded some additional fragments not present in the two other plasmids. In hybridization experiments any of the three plasmids strongly hybridized with each other and with some other bacteriocin but nontransmissible plasmids from other S. cremoris strains. Homology was also detected to a variety of cryptic plasmids in lactic acid streptococci.     

Characteristics of Ti plasmids from broad-host-range and ecologically specific biotype 2 and 3 strains of Agrobacterium tumefaciens.

Agrobacterium tumefaciens strains isolated from crown gall tumors on grapevines in California were consistently of the biotype 3 group. All 11 of these strains were limited in their host range and harbored Ti plasmids with molecular masses between 119 and 142 megadaltons (Mdal) as well as a larger cryptic plasmid of greater than 200 Mdal; occasionally a smaller cryptic plasmid of 65 Mdal was also present. Ti plasmids o these strains have DNA sequences in common with Ti plasmids of octopine and nopaline strains belonging to the biotype 1 group and exhibited sequence homologies with the conserved region of the T-DNA. Ten of the 11 strains utilized octopine as a sole source of carbon and nitrogen and 3 strains catabolized both octopine and nopaline, whereas 1 strain catabolized only nopaline. All of these strains were resistant to the bacteriocin agrocin-84, except one grapevine strain that belonged to the biotype 1 group and was agrocin sensitive; it is also differed in its plasmid and virulence characteristics. Isolations from Rubus ursinus ollalieberry galls yielded exclusively biotype 2 strains. These strans were insensitive to agrocin-84, utilized nopaline as a sole carbon and nitrogen source, and were highly virulent on all host plants tested. They contained Ti plasmids ranging between 100 and 130 Mdal and occasionally a cryptic plasmid of 69 Mdal. Their Ti plasmids have DNA sequences in common with Ti plasmids of biotype 1 strains and with the conserved region of the T-DNA.     

Cryptic heterokaryons in Saccharomyces cerevisiae strains: pseudopleiotropic suppression in the multiple marked YPH857 strain

The yeast strain YPH857 carrying multiple genetic markers was shown to segregate clones that had the pleiotropic suppression phenotype. The phenotype was designated Ppsu+. This suppression involves deletion alleles of the TRP1 and HIS3 genes and an insertion in the URA3 gene. Unlike the original YPH857 culture that carries an unidentified mutation of resistance to cycloheximide, the Ppsu+ clones exhibited a decreased level of resistance to this inhibitor of protein synthesis. In addition, they have a lower mating ability and can produce asci on a standard medium for sporulation. A comparative analysis of total DNA from the YPH857 strain and Ppsu+ segregants by Southern blotting provided evidence for the presence of an extraneous nucleus in these segregants. Ppsu+ strains were shown to contain wild-type alleles, apart from deletion and insertional alleles typical for the YPH857 strain. Moreover, they contain the 2 microns DNA of the Scp3 type with deletion of one of the two EcoRI and HpaI recognition sites (whereas the 2 microns DNA of YPH857 belongs to the Scp1 type) and exhibit heterogeneity with respect to the presence of one EcoR1 recognition site in the gene of 5S ribosomal RNA. It was supposed that the "cryptic" nucleus belongs to a strain of low viability and can survive as an unexpressed DNA in a small fraction of cells. Nuclei from the cryptic and YPH857 strains can be fused at a low rate to yield Ppsu+ cells capable of sporulation. In certain cases, a Ppsu+ clone may be heterogeneous: some of its cells contain nuclei in an unfused heterokaryotic state. This assumption has been confirmed by selecting Cyhr colonies with the original YPH857 genome among Ppsu+ clones on the cycloheximide-containing medium.     

Factors influencing the copy number of F-like plasmids in Escherichia coli and Salmonella typhimurium.

The copy numbers of Flac, four F-like plasmids and pLT2 were estimated in two strains of Salmonella typhimurium and (for all except pLT2) one strain of Escherichia coli. For organisms grown in casamino acids minimal medium, the plasmids spanned a 7--8 fold range of copy number with ColB-K98 having the highest copy number in each strain and R124 the lowest. The copy number of ColB-K98 was substantially greater than 1 in each of the strains tested. There was no clear relation between the plasmid size and copy number, although the plasmids studied spanned only a narrow size range. The copy number of individual plasmids was slightly reduced or not affected at all by the presence of a second plasmid in the same strain. Derivatives harbouring each of the plasmids were grown in three different media to ascertain how plasmid copy number responds to changes in growth rate. For each plasmid, the copy number increased with decreasing growth rate. Extracts from each of the three strains harbouring ColB-K98 contained two distinct plasmid species. One appeared to be about twice as large as the other and both were absent from Col- segregants.     

Growth inhibition of Escherichia coli by E colicin plasmids

Plasmids were isolated from E colicinogenic strains and transformed into prototrophic Escherichia coli K 12 strain DB364. Screening of E colicinogenic transformants for growth on defined medium revealed an apparent amino acid auxotrophy mediated by E4 and, to a lesser extent, E7 colicin plasmids. The auxotrophy was further investigated in E4 colicinogenic strains. From such auxotrophic transformants, denoted Pmi+ (plasmid-mediated inhibition of growth), Pmi- variants were obtained at a frequency of 3 X 10(-4) per bacterium. Plasmid loss was not detected among Pmi- clones. Isolation of E4 colicin plasmids from Pmi- clones and retransformation of strain DB364 with these plasmids showed that 40% of the plasmids were unable to inhibit growth of DB364 and were inferred to have alterations in an E4 colicin plasmid gene termed pmi. All such plasmids were indistinguishable from native E4 colicin plasmids, with respect to colicin immunity, colicin production and excretion, and sensitivity to lysis by mitomycin C. Experiments examining the nutritional basis of the plasmid-mediated auxotrophy indicated that at least seven amino acids, isoleucine, leucine, valine, arginine, methionine, serine and glycine, were involved in the auxotrophy. However, supplementation with only these seven amino acids did not completely restore growth. Assays of the activities of enzymes involved in amino acid biosynthesis in colicinogenic and non-colicinogenic strains under repressing and derepressing growth conditions suggested that E4 colicin plasmids did not repress synthesis of the implicated amino acids.     

Characterization and restriction analysis of the P sex factor and the cryptic plasmid of Vibrio cholerae strain V58

The P plasmid of Vibrio cholerae is a derepressed sex factor restricted to V. cholerae and has been shown to express surface exclusion. We have isolated the plasmids of strain V58 and have found that in addition to P, two further cryptic plasmids are also present. P has a size of 68 kb as determined by both electron microscopy and restriction endonuclease analysis. These other plasmids are 34 and 4.7 kb in size. Restriction maps of P and the larger cryptic plasmid have been determined. It has been demonstrated that P differs from the standard Inc group test plasmids and also expresses a surface exclusion system. The ability of the type Inc plasmids to be transferred to V. cholerae by either liquid or filter matings and the stability of these plasmids in V. cholerae have also been examined.     

Molecular cloning of the plasmids of Vibrio cholerae 01 and the incidence of related plasmids in clinical isolates and other Vibrio species

Abstract We describe the subcloning of plasmids present in Vibrio cholerae 01 strain V58: the P factor, the large cryptic plasmid (lcp) and small cryptic plasmid (scp). Strains harbouring fragments of the P sex factor and the lcp were examined for plasmid encoded proteins by Coomassie blue staining and analysis in Escherichia coli K-12 minicells.
The distribution of these three plasmids in a variety of Vibrio species has been examined using some of the cloned fragments as DNA probes. Most recent clinical isolates of V. cholerae were found to contain the scp. None of the strains contained the lcp. The P factor was only detected in one clinical isolate in addition to the scp. While plasmids appear to be generally uncommon among V. cholerae , and do not appear to differentiate biovars, the presence of plasmids may be a useful epidemiological adjunct.     

Functional characteristics of plasmids of Shigella sonnei 47 strains

The phenotypic characteristics of Shigella sonnei strain 47 containing 7 plasmids of low molecular weight and 2 plasmids 60-100 Md large have been studied. The strains of Escherichia coli containing the single plasmids or plasmid groups from Shigella sonnei have been obtained by transformation and conjugation. The comparison of phenotypes of the obtained strains has helped to find the plasmid location of the determinants for streptomycin resistance (P7), genes for colicinogenicity and colicin immunity (P5), the enzymes of host cell specificity system Sso47I (P6), Sso47II (P4), and the genes for the conjugative DNA transfer (P9). Escherichia coli strains producing individual restriction enzymes SsoI and SsoII have been isolated.     

Characterization of a new Agrobacterium tumefaciens strain from alfalfa (Medicago sativa L.)

Agrobacterium tumefaciens strain 1D1609 is reported here as the first field isolate from alfalfa (Medicago sativa L.). Unlike well-characterized A. tumefaciens strains such as C58 and Ach5, strain 1D1609 is highly virulent on alfalfa and has a distinctive host range. Interestingly, strain 1D1609 is naturally resistant to kanamycin and spectinomycin. The Ti plasmid in strain 1D1609 is an octopine-type; thus, tumors formed by strain 1D1609 synthesize octopine, which is utilized by the bacterium as a sole carbon source. Reciprocal exchange of Ti plasmids between strains 1D1609 and C58 showed that both chromosomal and Ti plasmid genes in strain 1D1609 contribute specifically to tumor formation on alfalfa. In addition, the nondormant CUF101 alfalfa cultivar from which strain 1D1609 was isolated was significantly more susceptible to all Agrobacterium strains tested than was the dormant Agate cultivar. Received: 17 October 1997 / Accepted: 8 December 1997     

Study of the transmissibility of multiple drug resistance and the capacity to ferment lactose in a clinical strain of Kl. pneumoniae]

The donor properties of K. pneumoniae PI 220 with multiple drug resistance were studied. It was shown that the above strain carried 2 plasmids, i.e. R-plasmid pPI 220 controling resistance to ampicillin, streptomycin, tetracycline, chloramphenicol, kanamycin and sulphanylamides and plasmid pPI 221 controlling lactose fermentation. Both plasmids can be transfered on conjugation to strain E. coli P678 at a temperture of 28 degrees C at a rate of 10(-5) for pPI 220 and 10(-4) for pPI 221. The drug resistance controlled by pPI 221 was transfered mainly in a "blocks" simultaneously to 6 drugs. Deletion of plasmid pPI 220 was observed rarely. The donor properties of the strain were defined by the conjugative plasmid pPI 220 controlling the self-transfer and mobilization of plasmid pPI 221 incapable of the self-transfer. E. coli P678 (pPI 220) (PPI 221) acquired the donor properties and transfered both plasmids to E. coli J62 on crossing simultaneously at a rate of 10(-2), as well as to S. typhimurium LT2 and P. rettgeri at a rate of 10(-5). In all the recipient strains studied the transfered plasmids were unstable and segregated also simultaneously at a rate being the highest for P. retgari PI 230. The clones with stable preservation of the plasmids could be obtained by selection.     

Efficient use of DNA molecular markers to construct industrial yeast strains

Saccharomyces cerevisiae yeast strains exhibit a huge genotypic and phenotypic diversity. Breeding strategies taking advantage of these characteristics would contribute greatly to improving industrial yeasts. Here we mapped and introgressed chromosomal regions controlling industrial yeast properties, such as hydrogen sulphide production, phenolic off-flavor and a kinetic trait (lag phase duration). Two parent strains derived from industrial isolates used in winemaking and which exhibited significant quantitative differences in these traits were crossed and their progeny (50-170 clones) was analyzed for the segregation of these traits. Forty-eight segregants were genotyped at 2212 marker positions using DNA microarrays and one significant locus was mapped for each trait. To exploit these loci, an introgression approach was supervised by molecular markers monitoring using PCR/RFLP. Five successive backcrosses between an elite strain and appropriate segregants were sufficient to improve three trait values. Microarray-based genotyping confirmed that over 95% of the elite strain genome was recovered by this methodology. Moreover, karyotype patterns, mtDNA and tetrad analysis showed some genomic rearrangements during the introgression procedure.     

Cloning of heterologous genes specifying detrimental proteins on pUC-derived plasmids inEscherichia coli

A system is described that enables the cloning of genes specifying detrimental proteins inEscherichia coli. The system is based on pUC plasmids and was developed for the expression of theBacillus subtilis csaA gene, which is lethal when expressed at high levels. Suppressor strains that tolerate the presence of plasmids for high-level expression ofcsaA were isolated, which contained small cryptic deletion variants of the parental plasmid in high copy numbers. The cryptic plasmids consisted mainly of the pUC replication functions and lacked thecsaA region and selectable markers. The co-resident, incompatible, cryptic plasmids enabled the maintenance of thecsaA plasmids by reducing their copy number 20-fold, which resulted in a concomitant 3- to 7-fold reduction in the expression of plasmid-encoded genes. Strains carrying these cryptic endogenous plasmids proved to be useful for the construction of pUC-based recombinant plasmids carrying other genes, such as theskc gene ofStreptococcus equisimilis, which cannot be cloned in high copy numbers inE. coli. Several strategies to reduce production levels of heterologous proteins specified by plasmids are compared.     

Cloning of heterologous genes specifying detrimental proteins on pUC-derived plasmids inEscherichia coli

A system is described that enables the cloning of genes specifying detrimental proteins inEscherichia coli. The system is based on pUC plasmids and was developed for the expression of theBacillus subtilis csaA gene, which is lethal when expressed at high levels. Suppressor strains that tolerate the presence of plasmids for high-level expression ofcsaA were isolated, which contained small cryptic deletion variants of the parental plasmid in high copy numbers. The cryptic plasmids consisted mainly of the pUC replication functions and lacked thecsaA region and selectable markers. The co-resident, incompatible, cryptic plasmids enabled the maintenance of thecsaA plasmids by reducing their copy number 20-fold, which resulted in a concomitant 3- to 7-fold reduction in the expression of plasmid-encoded genes. Strains carrying these cryptic endogenous plasmids proved to be useful for the construction of pUC-based recombinant plasmids carrying other genes, such as theskc gene ofStreptococcus equisimilis, which cannot be cloned in high copy numbers inE. coli. Several strategies to reduce production levels of heterologous proteins specified by plasmids are compared.     

Involvement of plasmids in total degradation of chlorinated biphenyls.

Acinetobacter sp. strain P6 has previously been reported to utilize biphenyl (BP) and chlorinated BPs, with accumulation of corresponding chlorobenzoic acids. Arthrobacter sp. strain M5 was isolated as a contaminant in the culture of Acinetobacter sp. strain P6 growing on 4-chlorobiphenyl and showed properties similar to P6 in the degradation of chlorinated BPs. Both strains harbored an identical plasmid of 53.7 megadaltons. These strains spontaneously lost the ability to utilize BP and 4-chlorobiphenyl with high frequency (4 to 8%) after overnight growth in nutrient broth. The BP- derivatives could not regain the BP-assimilating ability (reversion frequency, less than 10(-9) per cell per generation) but retained the plasmid with small, detectable deletions. BP+ P6 cells grown on BP or benzoate oxidized BP and 2,3-dihydroxybiphenyl and produced meta cleavage compounds from the latter compound (lambda max, 434 nm) and also from catechol (lambda max, 375 nm) through the meta pathway. On the other hand, benzoate-grown BP- segregants totally lost the BP-metabolizing activities and oxidized catechol through the ortho pathway. A combined culture of the chlorinated BP-dissimilating P6 or M5 strain (harboring the putative 53.7-megadalton plasmid specifying conversion of chlorobiphenyls to chlorobenzoic acids) and genetically constructed mono- or dichlorobenzoate-utilizing pseudomonads (harboring plasmids encoding complete utilization of mono- or dichlorobenzoates) allowed greater than 98% utilization of mono- and dichlorobiphenyls, with the liberation of equivalent amounts of chloride ions.