Plasmid content in Yersinia pestis strains of different origin

Plasmid content in 242 Yersinia pestis strains from various natural plague foci of the U.S.S.R. and other countries was studied. Of these strains, 172 (71%) were shown to carry three plasmids described previously of about 6, 45-50 and 60 MDa, respectively. Twenty strains (8%) from different foci harboured additional cryptic plasmids, most often of about 20 mDa in size. Plasmid pPst displayed considerable constancy of its molecular mass. On the contrary, size variations of pCad (45-49 MDa) and, especially, pFra (60-190 MDa) were found. Molecular mass of these plasmids correlated with the host strain origin.     

Structural and regulatory genes for coli surface associated antigen 4 (CS4) are encoded by separate plasmids in enterotoxigenic Escherichia coli strains of serotype 025.H42

Two enterotoxigenic Escherichia coli strains of serotype 0.25.H42 that produced coli surface associated antigens CS4 and CS6 hybridized with a probe containing the cfaD sequence that regulates expression of colonization factor antigen CFA/I. Transformation of a cloned cfaD gene into some derivatives of the strains that were negative for CS4 and CS6 resulted in expression of CS4 but not CS6. By hybridization the sequence that regulated CS4 production in the wild type 025 strains was located on a plasmid that also encoded the CS6 antigen. The structural genes for the CS4 antigen were on a separate plasmid. The 025 strains carried a third plasmid encoding enterotoxin production which was therefore unlinked to regulation sequences or genes encoding CS antigens.     

Mobilization ofEscherichia coli R1 silver-resistance plasmid pJT1 by Tn5-Mob intoEscherichia coli C600

Summary Escherichia coli Rl is an Ag⁺-resistant strain that, as we have shown recently, harbours at least two large plasmids, pJT1 (83 kb) and pJT2 (77 kb). Tn5-Mob was introduced into theE. coli Rl host replicon via conjugation on membrane filters. The transfer functions of plasmid RP4-4 were provided in this process and Tn5-Mob clones mated withE. coli C600 yielded Ag⁺-resistant transconjugants. This mobilization procedure allowed transfer and expression of pJT1 Ag⁺ resistance inE. coli C600. Prior to use of Tn5-Mob mobilization, it was not possible to transfer Ag⁺-resistant determinant(s) intoE. coli by conjugation or transformation including high-voltage electroporation.E. coli C600 containing PJTI and PJT2 displayed decreased accumulation of Ag⁺ similar toE. coli R1.E. coli C600 could not tolerate 0.1 and 0.5 mM Ag⁺, rapidly accumulated Ag⁺ and became non-viable. Tn5-Mob mobilization may be useful in the study of metal resistance in bacteria, especially in strains not studied for resistance mechanisms.     

Analysis of restriction profiles of Mitochondrial DNA from Sporothrix schenckii and related fungi

Restriction profiles by HaeIII of mitochondrial DNA were studied for classification and distinction of Sporothrix schenckii (100 strains), S. schenckii var. luriei (1), S. curviconia (1), S. inflata (7), Ceratocystis stenoceras (17) and C. minor (7). These 6 species showed unique restriction profiles which could be discriminated from each other. S. schenckii was further separable into 11 types, S. inflata into 4 types, C. stenoceras into 4 types and C. minor into 7 types based on restriction profile heterogeneity.     

Catabolic instability,plasmid gene deletion and recombination in Alcaligenes sp. BR60

An Alcaligenes sp. BR60, isolated from surface runoff waters of the Hyde Park industrial landfill, contained a novel 85 kb catabolic plasmid (pBR60) functional in 3-chlorobenzoate (3Cba) degradation. The plasmid exhibited a spontaneous 3.2% frequency of deletion of a 14 kb fragment specifying 3Cba degradation. The deletion mutant BR40 and mitomycin C cured strains were not able to grow on 3Cba and had reversion frequencies of less than 10^-10 cell^-1 generation^-1. Transformation or conjugation of pBR60 into cured strains restored catabolic activity. An EcoRI, BgIII, HindIII and SaII restriction map of the deletion region was constructed, and EcoRI and HindIII fragments spanning the deletion region of the plasmid were cloned in pUC18. Conjugation of resistance plasmid R 68.45 into Alcaligenes sp. BR60, with selection on antibiotics, resulted in the elimination of pBR60 and maintenance of unaltered R68.45. In 30% of the exconjugants, 3Cba degradative capacity was retained, although variation in the regulation of 3Cba degradation was observed in these strains. Hybridization of deletion region fragments to BgIII digested total DNA of BR60 and the R68.45 cured exconjugants revealed the presence of pBR60 deletion region sequences in the chromosome of exconjugants. Hybridization also revealed a repeated sequence flanking the deletion region of pBR60. Selection on 4-chlorobenzoate as a sole source of carbon and energy resulted in the isolation of 4Cba⁺ mutants of Alcaligenes sp. BR60.Abbreviations 3 and 4 Cba chlorobenzoic acid isomers and growth phenotypes - HPLC high pressure liquid chromatography - ATCC American Type Culture Collection     

Transfer and maintenance of IncP and IncW group plasmids into and between extra-slow-growing Bradyrhizobium japonicum strains

Abstract IncP group plasmid pRL180 was conjugally transferred from Agrobacterium tumefaciens LBA928 into extra-slow-growing (ESG) Bradyrhizobium japonicum strains and between ESG strains, RJ17W and RJ12S. pRL180 was integrated into the chromosome of RJ12S, RJ17W and RJ19FY. ESG strains efficiently transferred pRL180 into Escherichia coli at about a 3 × 10⁻⁵ frequency. IncW group plasmid pTY97 was transferred in intergeneric matings from E. coli into ESG strains at a high frequency of 2.5 × 10⁻³; between RJ17W and RJ12S transfer was about 5.6 × 10⁻⁴. pTY97 was maintained as an R' plasmid in RJ12S. The R' plasmid was resolved upon transfer into E. coli C where only pTY97 was autonomously replicated.     

Site-specific integration of plasmid pSAM2 in Streptomyces lividans and S. ambofaciens

Summary Streptomyces ambofaciens strain ATCC23877 contains the 11.1 kb plasmid pSAM2 stably integrated into its chromosome. This plasmidic sequence is able to loop out and to be transferred at high frequency to S. lividans where it is found simultaneously as both free and integrated plasmid. When a UV derivative of strain ATCC23877 (strain ATCC15154) is used, the resident copy of pSAM2 can be transferred to S. lividans, but only the integrated form is found in this strain. In both cases, the integration occurs at a unique chromosomal region through the same plasmidic integration site as that in strain ATCC23877. The resident copy of strain ATCC15154 can also be transferred at low frequency to S. ambofaciens DSM40697 (devoid of any pSAM2 sequence). In this case, as several copies of pSAM2 are integrated, the integration pattern is complicated. Integration of a complete pSAM2 sequence in this strain occurs in a region that hybridizes with the integration zones of S. lividans and of S. ambofaciens strain ATCC23877. Comparison of the cloned integration zone of S. lividans before and after the integration event showed that the restriction pattern of the resident pSAM2 in strain ATCC15154 is similar to that of the free form of pSAM2 found naturally in another UV derivative of strain ATCC23877 (strain JI3212).     

Killing of Escherichia coli cells modulated by components of the stability system ParD of plasmid R1

Summary The proteins P10 and P12 have been shown to be gene products of a new stability system, ParD, of plasmid R1. It is now shown that an R1 miniplasmid, pAB112, carrying a trans-complementable amber mutation in the gene of the P10 protein, is lethal for the host in the absence of suppression. This lethal effect is suppressed in a supF background and also by deletions in pAB112 that affect the gene of the P12 protein. These data indicate that the P12 protein has a lethal effect on the host and that this effect is neutralized by the P10 protein. The possibility that the stabilization conferred by the ParD system could be due to a counterselection, mediated by P12, of cells that lose the plasmid at cell division, is discussed.     

Interplasmidic illegitimate recombination in Bacillus subtilis

Summary The illegitimate recombination between Staphylococcus aureus plasmids pE194 (or pGG20, the hybrid between pE194 and Escherichia coli plasmid pBR322) and pBD17 (plasmid pUB110 without HpaII C-fragment) was studied in Bacillus subtilis. Cointegrates were generated with the frequency of 1–3x10^-8. Among 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all three parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions revealed that in 8 cases recombination occurred between short homologous regions (9–15 bp). One recombinant was formed using nonhomologous sites. The similarity was demonstrated between nucleotide sequences of the recombination sites of two types of cointegrates and those used for pE194 integration into the B. subtilis chromosome. Possible mechanisms of illegitimate recombination are discussed.     

The profile distribution of total and extractable boron in cacao-growing soils in southwestern Nigeria

The profile distribution of total and extractable B was determined in 16 Nigerian cacao-growing soil profiles formed from different parent materials. Total B for all soils ranged from 8 to 54μgg⁻¹ with a mean of 24μgg⁻¹. The soils formed from sandstones in the rainforest zone contained higher amounts of total B than soils derived from basement complex. Boron extractable in hot water, in 0.1% CaCl₂, and in 1N NH₄OAc varied from 0.13 to 1.38, 0.44 to 1.20 0.03 to 0.56μgg⁻¹ respectively. The corresponding means were 0.66, 0.75 and 0.27μgg⁻¹ B. Soils on metamorphic rocks gave the highest values. All extractable B values were related to organic matter while only CaCl₂-extractable B correlated with total B. Generally total and extractable B values were higher in the top soils than in the subsoils.