Plasmids Controlling Synthesis of Hemolysin in Escherichia coli: Molecular Properties

Covalently closed extrachromosomal deoxyribonucleic acid (DNA) was isolated from alpha-hemolytic wild-type strains of Escherichia coli. Most strains examined were able to transfer the hemolytic property with varying frequencies to nonhemolytic recipient strains. Out of eight naturally isolated alphahemolytic E. coli strains, four contained a set of three different supercoiled DNAs with sedimentation coefficients of 76S (plasmid A), 63S (plasmid B), and 55S (plasmid C). The sedimentation coefficients and the contour lengths of the isolated molecules correspond to molecular weights of 65 x 10(6), 41 x 10(6), and 32 x 10(6). Three alpha-hemolytic wild-type strains carried only one plasmid with a molecular weight of 41 x 10(6), and one strain harbored two plasmids with molecular weights of 41 x 10(6) and 32 x 10(6). Alpha-hemolytic transconjugants were obtained by conjugation of E. coli K-12 with the hemolytic wild-type strains. A detailed examination revealed that plasmids with the same sizes as plasmids B and C of the wild-type strains can be transferred separately or together to the recipients. Both plasmids possess the hemolytic determinant and transfer properties. Plasmid A appears to be, at least in one wild-type strain, an additional transfer factor without a hemolytic determinant. In one case a hemolytic factor was isolated, after conjugation, that is larger in size than plasmid A and appears to be a recombinant of both plasmids B and C.     

Translocation of antibiotic resistance markers of a plasmid-free Streptococcus pyogenes (group A) strain into different streptococcal hemolysin plasmids

Summary Wild-type strain A454 (Streptococcus pyogenes) transferred en bloc its erythromycin (Em) and tetracycline (Tc) resistance markers into several plasmid-free streptococcal recipients. No plasmid DNA was detected in either the wild-type or the transconjugant strains. Crosses were performed between A454 and S. faecalis Rec⁺ or Rec^- recipients carrying hemolysin-bacteriocin plasmids, pIP964 or pAD1. The Em Tc-resistant transconjugants obtained harbored either the parental plasmid or an Em Tc resistance plasmid derived from pIP964 or pAD1. The restriction endonuclease analysis of 12 derivative plasmids showed insertions of various sizes into different fragments of pIP964 or pAD1. A454 and the Em Tc-resistant plasmid-free transconjugants were found to contain two EcoRI DNA fragments, that shared homology with ³²P-labeled pIP1077, one of the Em Tc resistance derivative plasmids, but not with ³²P-labeled pIP964. No homology was detected between pIP1077 and the cellular DNA of the antibiotic-susceptible recipients.Previously Thea Horodniceanu     

The plasmids of Acetobacter xylinum and their interaction with the host chromosome

Summary Acetobacter xylinum contains a complex system of plasmid DNA molecules. Plasmids of molecular weights or copy numbers different from the original wild-type, are found in different types of mutants. Restriction endonuclease digestion and DNA/DNA hybridization analysis, showed that the plasmids often contained partly, but not completely the same DNA sequences. Two of these plasmid classes were analysed in more detail, and could be shown to differ in size by about 5 kb. Hybridization analysis using cloned DNA fragments as probes, showed that sequences lacking in the smallest plasmid were still present in a DNA fraction co-migrating with linearized chromosomal DNA. In addition, at least part of the DNA in the smallest plasmid was present both in the plasmid and chromosomal DNA fraction. Analysis of a particular strain containing an insertion of transposon Tn1, also indicated the existence of complex interactions between plasmids and chromosomal DNA. Together with experiments on conjugative transfer and curing of the plasmids, the results indicate that at least part of the genetic system of A. xylinum is unusual when compared to that of other genetically characterized bacteria.     

Plasmids in avirulent strains of Agrobacterium.

Twelve strains of Agrobacterium radiobacter isolated from naturally occurring crown galls or soil were found to be avirulent on sunflower, tomato, Kalanchoe, and carrot. Eleven strains contained plasmids of molecular weights 77 X 10(6) to 182 X 10(6) as determined by electron microscopy. One strain contained only a smaller plasmid (50 X 10(6) daltons). Several strains had both large and small (ca. 11 X 10(6) daltons) plasmids; one strain contained two large plasmids (112 X 10(6) and 136 X 10(6) daltons). Hybridization reactions of virulence plasmids from Agrobacterium tumefaciens strains C58 and A6 with plasmids from each of the A. radiobacter strains revealed that some A. radiobacter plasmids had less than 10% homology to either the C58 or A6 plasmids. Plasmids from some strains had approximately 50% homology with the C58 plasmid, but only one A. radiobacter plasmid contained more than 10% homology to the A6 plasmid. The presence of large plasmids in A. radiobacter strains did not correlate with sensitivity to agrocin 84; however, the utilization of the amino acid derivatives octopine and nopaline was generally correlated to partial base sequence homology to the C58 plasmid. We conclude that all large plasmids found in Agrobacterium strains are not virulence associated, although they may share base sequence homology with a virulence-associated plasmid. Further, plasmids from tumorigenic strains may be more closely related by base sequence homology to plasmids from nonpathogenic strains than to plasmids from other pathogenic strains.     

Isolation and characterization ofMicrococcus plasmids

Plasmids were detected in a small to moderate percentage of strains in the speciesMicrococcus kristinae (7%)M. agilis (20%),M. luteus (20%),M. varians (23%),M. nishinomiyaensis (41%), andM. roseus (55%). Plasmids were not detected inM. lylae (0/16) orM. sedentarius (0/20). Plasmid molecular sizes ranged from 1 to ca. 90 MDa. Most of the strains carrying plasmids exhibited only one or two types. Plasmid patterns appeared to be slightly more complex inM. nishinomiyaensis, which usually carried 2–3 different plasmids. The various plasmids detected remained cryptic, with the possible exception of a lincomycin resistance plasmid pWE2205 present inM. roseus strain KH6. DNA hybridization with Southern blots indicated that extensive deoxyribonucleotide sequence homologies were restricted to certain plasmids within a given species, e.g., withinM. nishinomiyaensis orM. roseus. Several plasmids ofM. nishinomiyaensis sharing extensive homology could be distinguished on the basis of restriction endonuclease analysis.Paper no. 9243 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina.     

Genetic isolation and physical characterization of pAgK84, the plasmid responsible for agrocin 84 production

The plasmid responsible for agrocin 84 biosynthesis by Agrobacterium radiobacter strain K84 has been genetically isolated free from any opine-catabolic plasmids. This was accomplished by mobilizing the agrocin plasmid, pAgK84, into a Ti plasmid-free A. tumefaciens strain, A136. The mobilizing element, pAt84a, was then cured from such a transconjugant by cultivation at 37 °C. Derivatives of strain A136 harboring both plasmids or pAgK84 only produce agrocin 84. The agrocin plasmid isolated from these strains is indistinguishable by restriction endonuclease analysis from that in strain K84. A physical map of pAgK84 has been constructed with respect to six restriction endonucleases. The plasmid is cut only once by XbaI and twice by HpaI. Hybridization analysis shows that pAgK84 is closely related to pAtBo542a, a 25-Mdal plasmid from a virulent, agrocinogenic A. tumefaciens strain of European origin. Similar analyses indicate, however, that pAgK84 shows no detectable homology to octopine or nopaline-type Agrobacterium plasmids.     

Homology of plasmids in strains of unicellular Cyanobacteria.

Six strains of unicellular cyanobacteria were examined for the presence of plasmids. Analysis of lysates of these strains by CsCl-ethidium bromide density centrifugation yielded a major chromosomal DNA band and a minor band containing covalently closed circular plasmid DNA, as shown by electron microscopy and agarose gel electrophoresis. The sizes of the various plasmid species were determined; in each of the Synechococcus strains 6301, 6707, and 6908 two plasmid species were found with molecular weights of 5.3 × 10⁶ and 32.7 × 10⁶. Synechococcus strain 7425 had two plasmids of molecular weight 5.4 × 10⁶ and 24 × 10⁶. Synechococcus strain 6312 and Synechocystis strain 7005 each contained one plasmid species with molecular weight of 15.9 × 10⁶ and 2.0 × 10⁶, respectively. Restriction enzyme analysis revealed identical cleavage patterns for the plasmids of identical molecular weight.     

Self-transfer and mobilisation capabilities of the pXO2-like plasmid pBT9727 from Bacillus thuringiensis subsp. konkukian 97-27

Recent characterisations of plasmids related to the anthrax virulence plasmids pXO1 and pXO2 in clinical isolates of Bacillus cereus and Bacillus thuringiensis have contributed to the emerging picture of a virulence-associated plasmid pool in the B. cereus sensu lato group. The family of pXO2-like plasmids includes the conjugative plasmid pAW63 from the biopesticide strain B. thuringiensis subsp. kurstaki HD73 and the heretofore cryptic plasmid pBT9727 from the clinical strain B. thuringiensis subsp. konkukian 97-27. Comparative sequence analysis of these three plasmids suggested that they were derived from an ancestral conjugative plasmid, with pAW63 retaining its self-transfer capabilities, and pXO2 having lost them through genetic drift. Such properties had not been investigated in pBT9727, but sequence homologies led us to predict that it may possess self-transfer capabilities. Here, we report that pBT9727 is indeed conjugative, and is able to promote its own transfer as well as that of small mobilisable plasmids.     

Isolation of a DNA sequence related to several plasmids from Bacillus thuringiensis after a mating involving the Streptococcus faecalis plasmid pAM beta 1

Summary The transmissible plasmid pAM1, which codes for resistance to erythromycin and lincomycin, was transferred from Streptococcus faecalis to several strains of Bacillus thuringiensis by a filter-mating process. Introduction of pAM1 into the Em^r transconjugant strains of B. thuringiensis was confirmed by Southern hybridisation using the ³²P-labelled pAM1 as a probe. In the B. thuringiensis transconjugant strains, used as donors, the plasmid conserved its ability to be transferred during intraspecific mating, with a frequency of 10^-4 per recipient cell. In addition, the transconjugant clones acted as donors of the erythromycin resistance marker and permitted the transfer of cryptic plasmids present in the B. thuringiensis () strains used as donors. From a transconjugant clone of B. thuringiensis a hybrid plasmid resulting from an in vivo insertion into pAM1 of a 3 Md DNA sequence was isolated. This 3 Md DNA molecule originated from a 54 Md plasmid of a kurstaki strain and is related to several plasmids found in different serotypes of B. thuringiensis.Abbreviations cry^– acrystalliferous mutant - CCC covalently closed circular DNA - Md megadalton - EMS ethyl methanesulphonate     

Horizontal transfer of catabolic plasmids in the process of naphthalene biodegradation in model soil systems

The process of naphthalene degradation by indigenous, introduced, and transconjugant strains was studied in laboratory soil microcosms. Conjugation transfer of catabolic plasmids was demonstrated in naphthalene-contaminated soil. Both indigenous microorganisms and an introduced laboratory strain BS394 (pNF142::TnMod-OTc) served as donors of these plasmids. The indigenous bacterial degraders of naphthalene isolated from soil were identified as Pseudomonas putida and Pseudomonas fluorescens. The frequency of plasmid transfer in soil was 10^?5–10^?4 per donor cell. The activity of the key enzymes of naphthalene biodegradation in indigenous and transconjugant strains was studied. Transconjugant strains harboring indigenous catabolic plasmids possessed high salicylate hydroxylase and low catechol-2,3-dioxygenase activities, in contrast to indigenous degraders, which had a high level of catechol-2,3-dioxygenase activity and a low level of salicylate hydroxylase. Naphthalene degradation in batch culture in liquid mineral medium was shown to accelerate due to cooperation of the indigenous naphthalene degrader P. fluorescens AP1 and the transconjugant strain P. putida KT2442 harboring the indigenous catabolic plasmid pAP35. The role of conjugative transfer of naphthalene biodegradation plasmids in acceleration of naphthalene degradation was demonstrated in laboratory soil microcosms.     

Growth and reactivation of single stranded DNA phage phiX174 in E. coli undergoing "thymineless death".

Summary Flac maintenance was aberrant at permissive temperature in a temperature-sensitive dnaC mutant of Salmonella typhimurium when the normally resident pLT2 plasmid was present. Flac was, however, efficiently transferred into the dnaC pLT2⁺ strain and the resulting Flac derivative was almost as efficient in transferring Flac as were dnaC ⁺ pLT2⁺ Flac strains indicating that aberrant Flac maintenance was not associated with appreciable inhibition of transfer replication. A range of F-like plasmids behaved like pLT2 in causing aberrant Flac maintenance when present in the dnaC pLT2^- strain. Flac was, however, stably maintained in the dnaC strain in the absence of other plasmids. Although the F-like plasmids destabilized Flac, each was stably maintained when introduced into strain 11G dnaC pLT2⁺ and pLT2 was also apparently stable under these conditions. The destabilizing effect of pLT2 and other fi ⁺ plasmids was not consequent upon their inhibiting the formation of a repressible F transfer component needed for Flac replication in the dnaC strain. Incompatibility between Flac and the other plasmids induced by the dnaC lesion also appeared unlikely to be a cause of the aberrant Flac maintenance. The possibility is discussed that the initiation of Flac replication differs from that of pLT2 and the F-like plasmids with F competing less effectively than the others for the DnaC gene product.     

Molecular characterization of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis> biovar <Emphasis Type="Italic">longum</Emphasis> NAL8 plasmids and construction of a novel replicon screening system

In this study, we performed molecular characterization and sequence analysis of three plasmids from the human intestinal isolate Bifidobacterium longum biovar longum NAL8 and developed a novel vector screening system. Plasmids pNAL8H (10 kb) and pNAL8M (4.9 kb) show close sequence similarity to and the same gene organization as the already characterized B. longum plasmids. The B. longum plasmid pNAC1 was identified as being most closely related to pNAL8L (3.5 kb). However, DNA sequence analysis suggested that direct repeat-rich sites could have promoted several recombination events to diversify the two plasmid molecules. We verified the likely rolling circle replication of plasmid pNAL8L and studied the phylogenetic relationship in all the Bifidobacterium plasmids fully sequenced to date based on in silico comparative sequence analysis of their replication proteins and iteron regions. Our transformation experiments confirmed that the ColE1 replication origin from high-copy-number pUC vectors could interfere with the replication apparatus of Bifidobacterium plasmids and give rise to false positive clones. As a result, we developed a system suitable for avoiding possible interference by other functional replication modules on the vector and for screening functional replicons from wild-type plasmids. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users only.     

Plasmids and saprophytic growth of Rhizobium leguminosarum bv. trifolii W14-2 in soil

Abstract: The role of plasmids in the saprophytic growth of Rhizobium is mostly unknown. Plasmid-cured and complemented derivatives of R. leguminosarum bv. trifolii strain W14-2 were used to investigate the role of plasmids in the growth of this strain in sterile soil incubated under favorable moisture and temperature conditions. Strain W14-2 contains four plasmids ( a,b,c,d ). Absence of single plasmids in plasmid-cured derivatives generally did not reduce growth in soil when compared to the wild-type but absence of plasmid a delayed growth. Derivatives were unable to grow in soil when only plasmids a or d were present in cells. When only plasmids b or c were present, growth was delayed and the final population in 7 days was approximately 10% of the wild-type population. When the wild-type was co-inoculated at equal population into soil with derivatives lacking plasmids a , c , or d, the population of the wild-type at 7 days incubation was approximately 10 times larger than those of the derivatives. Elimination of only plasmid b did not reduce the ability of the strain to grow in soil when competing with the wild-type. Plasmids were involved in saprophytic growth of strain W14-2 in soil and may be important to the ecology of Rhizobium .     

Plasmids of Yersinia pseudotuberculosis

Plasmids with the sizes of 5.7; 51; 70-77; and 120-130 kb were found in six strains among the ten strains collection of Yersinia pseudotuberculosis. The restriction endonucleases analysis. Southern-blot hybridization and physical maps construction were performed for the plasmids. The 70-77 kb plasmids were found to be analogous to the Ca2(+)-dependence plasmid pYVO19 from Yersinia pestis EV76. The difference between the plasmids of this type is in the insertions or deletions located on the similar fragments of the restriction maps. The 51 kb plasmid has no common fragments with the Ca2(+)-dependence plasmids and does not code for virulence properties of the strain harbouring it. No homology is shared by the 5.7 kb plasmid and the 10 kb plasmid from Yersinia pestis EV76. Replicon of the 5.7 kb plasmid has been used to construct the pVS11 vector plasmid.     

Plasmids in clinical isolates ofStreptococcus pneumoniae

Forty clinical isolates ofStreptococcus pneumoniae with various antibiotic resistance profiles were screened for the presence of plasmids. Plasmids were demonstrated in five isolates. Three procedures for plasmid isolation were evaluated. A 10.8-kb plasmid was demonstrated by all three methods, but a further four plasmids were detected with one method only. The sizes of these plasmids were 11.5 kb, 10.8 kb, and 3.0 kb (two strains). Curing experiments were performed, but no plasmid/antibiotic correlation was observed. Tetracycline, erythromycin, and clindamycin resistance was lost in one strain, although the plasmid was still present.     

Incompatibility behavior of a symbiotic plasmid pMH7653Rb in <Emphasis Type="Italic">Mesorhizobium huakuii</Emphasis> 7653R

Mesorhizobium huakuii strain 7653R harbored two indigenous plasmids named pMH7653Ra and pMH7653Rb. The larger plasmid pMH7653Rb (symbiotic plasmid) was transferred to M. huakuii HN308SR harboring three plasmids: pMHHN308a, pMHHN308b and pMHHN308c, and HN3015SR harboring three plasmids: pMHHN3015a, pMHHN3015b and pMHHN3015c by tri-parent mating. Two stable indigenous plasmids, pMHHN308b and pMHHN308c of HN308SR, were co-eliminated due to the introduction of pMH7653Rb, and the transconjugant was named HN308SRN14. The results implied that pMH7653Rb and pMHHN308b, pMHHN308c were incompatible and might have been ascribed to the same incompatible group. The plasmid profiles of transconjugant HN3015SRN14 showed that the second largest plasmid pMHHN3015b of HN3015SR was cured due to the introduction of pMH7653Rb. The results also implied that pMH7653Rb and pMHHN3015b were incompatible. Results from plant nodulation tests showed that pMH7653Rb could only maintain the nodulation ability in transconjugant HN308SRN14 and its nodule number was more than that of wild strain HN308SR, but could not replace the nitrogen fixation effect of pMHHN308b and pMHHN308c. The plasmid cured mutant HN308SRN14D harboring only pMHHN308a formed null nodules that demonstrated pMHHN308a was relevant to nodulation ability. HN3015SRN14 harboring pMH7653Rb, pMHHN3015a and pMHHN3015c formed null nodules while HN3015SRN14D containing pMHHN3015a and pMHHN3015c lost the nodulation ability. The plasmid replication repC-like gene sequences were detected by a polymerase chain reaction from 7653R, HN308, HN3015, HN308SRN14 and HN3015SRN14. The repC gene sequence similarities of the strains tested attained 99%.     

An integrative plasmid and multiple-sized plasmids of Pseudomonas syringae pv. phaseolicola have extensive homology

Summary Plasmids isolated from five strains of the bean pathogen Pseudomonas syringae pv. phaseolicola were characterized by restriction endonuclease and filter hybridization analyses. BamHI and EcoRI restriction patterns revealed that total plasmid DNA from each strain had a high level of sequence homology with pMC7105, a 148 kbp integrative plasmid found in a sixth strain. Only six BamHI fragments from the eight plasmids in these strains failed to hybridize with pMC7105 probe. Four of these fragments, three from pPP6520 and one from pPP6525 of strain PP652, hybridized strongly to plasmid DNA from a closely-related pathovar, P. syringae pv. glycinea. BamHI fragment 8, which is involved in the integration of pMC7105 into the host chromosome, contains a repeat sequence that was present on all the plasmids except pPP6120 (6.8 kbp), pPP6310 (40 kbp) and pPP6520 (45 kbp). Every plasmid but pPP6520 had fragments that showed weak hybridization to the small plasmid, pPP6120. This homology suggests that a second repetitive sequence is common to these plasmids. The large plasmids (148 to 151 kbp) were essentially identical to pMC7105. The intermediate plasmids (122 to 128 kbp) appeared to be derived mainly from pMC7105 or a related plasmid, whereas the smaller plasmids (6.8 to 45 kbp) appear to have been derived in part from sequences not present in pMC7105.     

Strain polymorphism of the plasmid profiles in <Emphasis Type="Italic">Sulfobacillus</Emphasis> species

Plasmids were discovered for the first time in strains belonging to different species of the genus Sulfobacillus: S. thermosulfidooxidans, S. sibiricus, S. thermotolerans, “S. olympiadicus”, and S. acidophilus. The plasmids were detected in the cells of four out of eight strains grown on a medium with ferrous iron. Adaptation to elementary sulfur was accompanied by changes in the plasmid profiles in two out of seven strains. Plasmids were detected in all the studied strains of sulfobacilli after adaptation to the pyrite-arsenopyrite ore concentrate from the Nezhdaninskoe deposit containing gold, silver, zinc, copper, and lead. No plasmids were found in S. thermotolerans Kr1^T after four transfers on a medium containing iron and 0.018 mM Ag⁺. After adaptation of the same strain to 765 mM Zn²⁺, only one plasmid was found in the cells, the largest among those detected earlier in this culture adapted to the Nezhdaninskoe ore concentrate. The strain S. thermotolerans Kr1^T, after four transfers on media with either 78 mM Cu²⁺ or 2 mM Pb²⁺, did not contain plasmids. The presence of plasmids in the cells of sulfobacilli did not influence their resistance to the ions of the studied metals.