Extrachromosomal deoxyribonucleic acid in wild-type and photosynthetically incompetent strains of Rhodopseudomonas spheroides.

Three covalently closed circular species of extrachromosomal deoxyribonucleic acid have been identified by electron microscopic analysis in strains of Rhodopseudomonas spheroides. The weights of these plasmids, as determined from contour length, are about 75 X 10(6), 66 X 10(6), and 28 X 10(6) daltons for both aerobically grown and photosynthetically grown R. spheroides strain 2.4.1 (NRS) and for the photosynthetically incompetent strain V-2 (obtained by N-methyl-N-nitro-N'nitrosoguanidine mutagenesis) and 74 X 10(6), 66 X 10(6) and 34 X 10(6) daltons for a second photosynthetically incompetent strain, SLS I (obtained by incubating strain 2.4.1 [NRS] in medium containing sodium lauryl sulfate). Buoyant densities uere found to be 1.717 g/cm3 (58% guanine plus cytosine) for the plasmids of 66 X 10(6), 28 X 10(6), and 34 X 10(6) daltons in weight and 1.724 g/cm3 (65% guanine plus cytosine) for those weighing about 75 X 10(6) daltons. Possible functions of these plasmids are discussed.     

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.     

Mobilization of the gonococcal 5.2 kb beta-lactamase plasmid pSJ5.2 into Escherichia coli by cointegration with several gram-conjugative plasmids

We report the mobilization by cointegration of the gonococcal 5.2 kb beta-lactamase plasmid pSJ5.2 in an Escherichia coli background. Transfer of pSJ5.2 was measured by filter mating assays with five different conjugative plasmids from Enterobacteriaceae and the gonococcal 41 kb tet(M). Plasmid pSJ5.2 was mobilized to E. coli at frequencies of 1.7x10(-6), 9.3x10(-8) and 2.7x10(-5) by the tet(M), R64 drd-33 and N3 conjugative plasmids, respectively. Mobilization of pSJ5.2 by the 41 kb tet(M) conjugative plasmid resulted in stable Amp(R) E. coli transconjugants consisting of pSJ5.2 plasmid with an insertion located in the 2.4 kb BamHI-BamHI fragment. Mobilization of pSJ5.2 by R64drd-33 and N3 conjugative plasmids involved stable cointegrates as detected by Southern Blot with a DIG-labelled PstI-digested pSJ5.2 probe. Restriction analysis of the R64::pSJ5.2 and N3::pSJ5.2 cointegrates and Southern Blot with the pSJ5.2 probe showed that cointegrates formed by deletion of DNA regions within the 1.8 kb BamHI-HindIII fragment of pSJ5.2. The plasmid thus appears to use multiple recombination mechanisms for cointegration with different conjugative plasmids. The complete nucleotide sequence of pSJ5.2 was determined, and will be a useful tool to further investigate the molecular mechanisms leading to its cointegrative transfer.     

Isolation and characterization of antibiotic resistance plasmids from thermophilic bacilli and construction of deletion plasmids

Ten plasmids were isolated as covalently closed circular deoxyribonucleic acid from antibiotic-resistant thermophilic bacteria. Of the 10 plasmids tested, 2 could transform Bacillus subtilis, yielding resistance to specific antibiotics. Plasmid pTB20 (2.8 X 10(6) daltons, approximately 24 copies per chromosome) specifies resistance to tetracycline (Tcr), whereas pTB19 (17.2 X 10(6) daltons, approximately 1 copy per chromosome) renders the host resistant to both kanamycin and tetracycline (KMrTcr). Three plasmids were not self-transmissible. The restriction endonuclease cleavage maps of the two plasmids, pTB19 and pTB20, were constructed. pTB19 and pTB20, both of which were originally isolated from thermophilic bacilli, were tested for stability in B. subtilis. Digestion of pTB19 followed by ligation yielded deletion plasmids pTB512 (Kmr), pTB52 (Tcr), and pTB53 (KmrTcr). Determinants of Kmr, Tcr, and DNA replication were associated with EcoRI fragments R1b (4.2 X 10(6) daltons), R3 (2.8 X 10(6) daltons), and R1a (4.2 X 10(6) daltons), respectively. Restriction endonuclease cleavage maps of pTB51, pTB52, and pTB53 were constructed. Tetracycline resistance of pTB20 was confirmed to be in the EcoRI fragment (1.85 X 10(6) daltons).     

Molecular nature of two beta-lactamase-specifying plasmids isolated from Haemophilus influenzae type b.

The molecular nature of two beta-lactamase-specifying plasmids isolated from two separate ampicillin-resistant Haemophilus influenzae type b strains was examined. A 30 X 10(6)-dalton (30-Mdal) plasmid (RSF007) had a copy number of approximately 3 per chromosomal equivalent and a mole fraction guanine plus cytosine content of 0.39. By heteroduplex analysis the 30-Mdal plasmid was found to contain the entire ampicillin translocation DNA segment (TnA) found on R factors of enteric origin. A 3.0-Mdal plasmid (RSF0885) was found as a multicopy pool of approximately 28 copies per chromosomal equivalent, had a mole fraction guanine plus cytosine content of 0.40, and contained only about one-third of the transposable TnA sequence. RSF007 and RSF0885 appeared to be unrelated plasmids in that they share base sequence homology only within the confines of the TnA segment. The 3.0-Mdal Haemophilus plasmid was used to transform E. coli to ampicillin resistance but was found to be unstable in this host in the absence of antibiotic. The possibility that R-plasmids arose in Haemophilus by the translocation of TnA from a donor R-factor onto an indigenous H. influenzae plasmid is discussed.     

Isolation, by tetracycline selection, of small plasmids derived from R-factor R12 in Escherichia coli K-12.

The examination, by agarose gel electrophoresis, of tetracycline-resistant colonies of Escherichia coli K-12 carrying R-factor R12 reveals the presence of smaller plasmid deoxyribonucleic acids (DNAs), incompatible with R12, in many of the clones. These plasmids are demonstrated to be homologous with R12 DNA by electron microscope heteroduplex experiments and by the production of consistent fragment patterns upon digestion with various restriction endonucleases. These autonomously replicating plasmids form a related series of covalently closed circular DNA molecules ranging in size from 3.6 X 10(6) to 61 X 10(6) daltons. Plasmids of molecular weight between 3.6 X 10(6) and 37 X 10(6) confer no antibiotic resistances, but when jointly present with R12 by nonetheless enhance the expression of the tetracycline resistance associated with this latter molecule.     

Isolation and characterization of four types of plasmids from Bacillus subtilis (natto).

Covalently closed circular deoxyribonucleic acids were found in 10 strains of Bacillus natto. The plasmids could be classified into four types on the basis of the molecular weights as well as the patterns in agarose gel electrophoresis after digestion with restriction endonucleases: (i) plasmids (seven were detected) with a molecular weight of 3.6 X 10(6); (ii) plasmids (two were detected) with a molecular weight of 4.0 X 10(6); (iii) plasmids (eight were detected) with a molecular weight of about 34 X 10(6); and (iv), a plasmid with an approximate molecular weight of 46 X 10(6). Out of the 10 plasmid-carrying strains, 6 (IFO3009, IFO3013, IFO3335, IFO13169, IAM1143, and IAM1207) harbored both type 1 and 3 plasmids; 2 (IAM1114 and IAM1168) harbored both type 2 and 3 plasmids, and IFO3936 and IAM1163 carried type 1 and 4 plasmids, respectively.     

Sequence analysis of the family of penicillinase-producing plasmids of Neisseria gonorrhoeae

The exact nature of the sequence differences between the medically important family of gonococcal penicillinase-producing plasmids has been ascertained. The entire DNA sequence of the Asia-type plasmid, pJD4, demonstrated that it is 7426 bp and contains two direct repeats (DR30) that are implicated in the formation of deletion variant plasmids, such as the Africa-type plasmid. We have identified putative DnaA and IHF binding sites, various open reading frames that are thought to specify functional proteins, and some important DNA sequences involved with conjugative transfer of gonococcal beta-lactamase plasmids. The deletion in the Africa-type plasmid is 1827 bp and one of the DR30 repeats is also missing. The deletion in the Rio-type plasmid and several Toronto-type plasmids was determined to be 2273 bp and the sequence spanning the deletion was identical irrespective of geographic or temporal origin. The &Ncirc;imes-type plasmid is an Africa-type plasmid and also contains an IS5 insertion sequence. Since IS5 has not been identified in gonococcal isolates, we suggest that this sequence may have been inserted after the original gonococcal plasmid was transformed into Escherichia coli. The New Zealand plasmid is an Asia-type plasmid that contains an endogenous tandem duplication of 1883 bp and the direct DR2 is implicated in this duplication. The nature of the defined truncation of Tn2 present in the various plasmids is also discussed.     

Sequence and conservation of genes at the distal end of the transfer region on plasmids F and R6-5

The nucleotide sequence of the region downstream of transfer gene traI, including fertility inhibition gene finO, on the conjugative plasmids F and R6-5, has been determined. Analysis of the F sequence revealed two open reading frames (ORF's), ORF248 and ORF186; ORF186 (finO) is interrupted by the insertion of IS3. The R6-5 sequence also contained ORF248 and an intact ORF186, although an additional ORF (ORF286) was located between the two genes. ORF248, which we have designated traX, and ORF186 (finO) are highly conserved on both plasmids. The organisation of these genes indicates that traI and traX on F, and traI, traX and ORF286 on R6-5 are co-transcribed from their respective promoters upstream of traI. Sequences homologous to traX were detected on a range of conjugative F-like plasmids, whereas sequences homologous to ORF286 were only found on plasmids R6-5, R100 and R1. The conservation of traX sequences suggests a functional importance for that gene and/or its product.     

Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: identity of laboratory-constructed plasmids and clinical isolates.

The structural gene for ampicillin resistance resides upon a 3.2 X 10(6)-dalton sequence of deoxyribonucleic acid, TnA that can be transposed from replicon to replicon in laboratory experiments. TnA was transposed from a large conjugative plasmid to a small nonconjugative plasmid, RSF1010. Several RSF1010::TnA plasmids isolated in these laboratory experiments have been shown to be identical to plasmids found in clinical isolates. These data provide direct support to the theory that transposition of drug resistance genes play a key role in the evolution of R plasmids.     

Drug resistance and R plasmids in Bordetella bronchiseptica isolates from pigs

A total of 207 strains of Bordetella bronchiseptica isolated from pigs in 1978 and 1979 were tested for drug resistance and for the properties of their R plasmids. Apart from intrinsic resistance to spectinomycin, single (sulfadimethoxine), double (sulfadimethoxine and streptomycin), andt riple (sulfadimethoxine, streptomycin, and ampicillin) resistance were found in 54.1%, 1.0%, and 15.9% of the strains, respectively. All of the triple-resistance determinants were associated with mercury resistance and were conjugative. pBB1, one of these R plasmids, was identified as Fi- (F) and Spp- (no suppression of phage multiplication) type, and as a member of incompatability group IncP. The single- and double-resistance determinants were nonconjugative. pBB2, one of the double-resistance determinants, was mobilized by an R plasmid, RP4, with the high efficiency of 80% and at a frequency of 3.3% without cotransfer of RP4. The molecular weight of pBB1 and pBB2 was estimated at 36 X 10(6) and 13 X 10(6) daltons, respectively, by electron microscopy and agarose gel electrophoresis. pBB1 had five cleavage sites for EcoRI endonuclease, and four sites for HindIII. pBB2 had two EcoRI sites, one HindIII, and one BamHI site. Cells carrying pBB1 or pBB2 produced enzymic activity tha inactivated streptomycin in the presence of ATP.     

Isolation of extrachromosomal deoxyribonucleic acids from extremely thermophilic bacteria

Eight strains of thermophilic bacteria were examined for the presence of covalently closed circular deoxyribonucleic acid molecules by caesium chloride-ethidium bromide density gradient centrifugation. Four of the eight strains tested, Thermus flavus BS1, AT61, AT62 and Thermus thermophilus HB8 carried covalently closed circular DNA molecules. Thermus flavus BS1 haboured two species of plasmids with molecular weights of 6.1 X 10(6) and 17.0 X 10(6) as determined by electron microscopy. Thermus thermophilus HB8, T. flavus AT61 and T. flavus AT62 carried plasmids with molecular weights of 6.2 X 10(6), 6.6 X 10(6) and 6.6 X 10(6), respectively. Plasmids from T. flavus AT61 and AT62 were indistinguishable in their electrophoretic patterns in agarose or acrylamide gel after digestion with various restriction endonucleases. This is the first evidence for the presence of plasmids in extremely thermophilic bacteria, though their functions are unknown.     

Conjugative plasmids in Neisseria gonorrhoeae.

A conjugation system initially discovered in beta-lactamase-producing gonococci mobilized small non-selftransmissible R plasmids encoding beta-lactamase (penicillinase) production into other gonococci, Neisseria, and Escherichia coli. This conjugation system was mediated by a separate selftransmissible plasmid of 23.9 X 10(6) daltons, pFA2. Conjugative plasmids capable of mobilizing R plasmids were also found in nearly 8% of the non-penicillinase-producing gonococci. These were similar to pFA2 in size, buoyant density, and restriction endonuclease digest patterns but were less efficient than pFA2 in mobilization of the penicillinase plasmid pFA3. The presence of conjugative plasmids in gonococci isolated before the appearance of penicillinase-producing strains indicates that a conjugation system for plasmid transfer predated the appearance of R plasmids in gonococci.     

Relationships among some R plasmids found in Haemophilus influenzae.

Tetracycline resistance in a strain of Haemophilus influenzae isolated in the United Kingdom was found to be determined by an apparently non-selftransmissible plasmid of 31 X 10(6) daltons (31 MDal), designated pUB701. Deoxyribonucleic acid hybridization studies indicated that pUB701 shares about 70% base sequence homology with the 30-MDal ampicillin resistance R plasmid RSF007 isolated in the United States from H. influenzae, and 64% sequence homology with the 38-MDal tetracycline and chloramphenicol resistance R plasmid pRI234, isolated in the Netherlands. Heteroduplex studies between RSF007 and pUB701 confirmed the fact that these plasmids were largely homologous, except that pUB701 contained the tetracycline resistance transposon TnD, whereas RSF007 contained the ampicillin resistance transposon TnA. A strain of H. parainfluenzae resistant to both chloramphenicol and tetracycline carried two species of plasmid deoxyribonucleic acid of 2.7 and 0.75 MDal. We were unable to prove that either resistance was plasmid-borne in this strain. Hybridization studies with a [3H]thymine-labeled tetracycline resistance enteric plasmid suggested that the tetracycline transposon was integrated into the chromosome of H. parainfluenzae UB2832. We conclude either that the strains we studied received R factors of the same incompatibility group bearing different resistance genes, or that different resistance genes were translocated to a commom resident plasmid of H. influenzae.     

Conjugative R plasmids in group C and G streptococci.

Two streptococcal isolates of groups C and G harbored conjugative R plasmids with molecular weights of 17 X 10(6) (pIP646) and 20 X 10(6) (pIP920). These plasmids carried genetic markers for resistance to macrolides and related drugs, as well as to chloramphenicol (pIP920), and have very similar HindIII restriction enzyme patterns.     

Incidence of plasmid DNA in Salmonella strains isolated from clinical sources in Ontario, Canada, during 1979 and 1980

Gel electrophoresis of DNA from 70 clinical strains of Salmonella revealed a heterogenous plasmid population. Plasmid DNA, ranging in molecular weight from 1.4 X 10(6) to 145 X 10(6), was demonstrated in 26 of 32 antibiotic-resistant strains. Several resistant strains carried up to six plasmids; however, of these, five strains which were multiply resistant contained a single plasmid of molecular weight 54 X 10(6) to 145 X 10(6). Only one incompatibility group H2 (IncH2) plasmid (pDT28) was detected in a strain of S. heidelberg; thus, this represents a reduction in the prevalence of these plasmids in Ontario Salmonella strains since 1974. The pDT28 plasmid resembled other IncH2 plasmids by its high molecular weight (145 X 10(6) ) and by virtue of its temperature-sensitive mode of transfer, resistance to tellurium, and inhibition of coliphage development. Of the 38 antibiotic-susceptible Salmonella strains, approximately half contained plasmids, ranging in molecular weight from 1.4 X 10(6) to 60 X 10(6). The plasmid-containing antibiotic-susceptible strains carried either a group of two to four small plasmids, with molecular weights less than 4.5 X 10(6), or a single large plasmid of molecular weight 23 X 10(6) or 60 X 10(6).     

Construction of isogenic gonococcal strains varying in the presence of a 4.2-kilobase cryptic plasmid.

A 4.2-kilobase (kb) cryptic plasmid is present in 96% of isolates of Neisseria gonorrhoeae. An inability to construct isogenic derivatives which vary in the presence of the 4.2-kb plasmid has prevented the study of its function. We report a method to deliver an intact 4.2-kb plasmid into plasmidless gonococcal strains. The method involved transformation with novel 15.7-kb hybrid penicillinase-producing (Pcr) plasmids, which were cointegrates containing two copies of the 4.2-kb plasmid arranged in tandem direct repeat plus one copy of the 7.2-kb Pcr plasmid pFA3. When the 15.7-kb hybrid Pcr plasmids were introduced into a gonococcal recipient lacking evident plasmids, they dissociated at a relatively high frequency into plasmids identical to their parents: the 4.2-kb cryptic plasmid and pFA10 (a stable 11.5-kb plasmid containing one copy of each of the 7.2-kb Pcr plasmid pFA3 and the 4.2-kb cryptic plasmid pFA1). Curing strains of their Pcr plasmids resulted in isogenic strains which varied only in the presence of the 4.2-kb plasmid. The presence of the autonomously replicating 4.2-kb plasmid did not affect a number of tested phenotypes, including auxotype, antibiotic sensitivity, and frequencies of variation of outer membrane protein II. The interpretation of the functional significance of the 4.2-kb plasmid was complicated, however, by the additional finding that each of three tested plasmid-free strains contained a chromosomal fragment of about 1.6 kb that hybridized under moderate stringency with a 1.65-kb HinfI fragment of the 4.2-kb plasmid.     

Detection and characterization of plasmids in Pseudomonas glycinea.

Pathogenic strains of Pseudomonas glycinea were shown to possess plasmid deoxyribonucleic acid by dye-buoyant density gradient centrifugation. The size and number of plasmids of four different isolates were determined by neutral sucrose gradient centrifugation. Two isolates were found to harbor a single plasmid; however, they differed in size, having molecular weights of 43 X 10(6) and 54 X 10(6). Two other isolates each contained two different plasmids. Plasmids with molecular weights of 43 X 10(6) and 73 X 10(6) were observed in one isolate, and the other carried plasmids with molecular weights of 25 X 10(6) and 87 X 10(6). An auxotrophic mutant derived from the latter strain was found to contain plasmids of identical size. The plasmids were found to be under stringent control of replication, having plasmid copies of 1.0 to 2.7 per chromosome equivalent. By the dye-cesium chloride technique, the mutant showed twice as much covalently closed circular deoxyribonucleic acid as did the parental strain.     

Cloned bacteriophage phi X174 DNA sequence interferes with synthesis of the complementary strand of infecting bacteriophage phi X174.

The insertion of a particular phi X DNA sequence in the plasmid pACYC177 strongly decreased the capacity of Escherichia coli cells containing such a plasmid to propagate bacteriophage phi X174. The smallest DNA sequence tested that showed the effect was the HindII fragment R4. This fragment does not code for a complete protein. It contains the sequence specifying the C-terminal part of the gene H protein and the N-terminal part of the gene A protein, as well as the noncoding region between these genes. Analysis of cells that contain plasmids with the "reduction sequence" showed that (i) the adsorption of the phages to the host cells is normal, (ii) in a single infection cycle much less phage is formed, (iii) only 10% of the infecting viral single-stranded DNA is converted to double-stranded replicative-form DNA, and (iv) less progeny replicative form DNA is synthesized. The reduction process is phi X174 specific, since the growth of the related G4 and St-1 phages was not affected in these cells. The effect of the recombinant plasmids on infecting phage DNA shows similarity to the process of superinfection exclusion.     

Isolation and characterization of Streptomyces erythreus plasmids

Streptomyces erythreus strains were found to carry several plasmids of molecular weights ranging from about 2 X 10(6) Mr to 40 X 10(6) Mr. Restriction enzyme maps for the streptomycete plasmids pPC7 and pPC8 were constructed for the enzymes Bg/II, EcoRI, XbaI, HindIII, BamHI and SalI. The smaller, pPC8, plasmid appears to be a naturally occurring deletion variant of pPC7. These plasmids belong to the group of conjugative streptomycete plasmids.