Dispersion of a gene that codifies fosfomycin resistance among plasmids from enterobacteriaceae isolated from sewage

Abstract The presence of plasmid encoded resistance to fostomycin among enterobacteriaceae isolated from sewage was studied. These plasmids found were classified into 7 varieties according to their original host, size, resistance determinants, restriction pattern and incompatibility group. All of them were related to the Inc M group, indicating a probable common origin from an ancestral replicon. The bacteria carrying these plasmids modified fosfomycin as did those carrying fosfomycin resistant (Fo^r) hospital plasmids. The plasmids showed positive hybridization with a 1-kb DNA fragment which carried a Fo^r-determinant from a hospital plasmid.     

Large plasmids in an actinomycete

Abstract Using a modified procedure large indigenous plasmids were detected in cells of three strains belonging to a group of phenotypically similar actinomycetes isolated from the rhizoplane and root nodules of Alnus spp. M _r values of the plasmids were estimated to be about 80 · 10⁶ and 120 · 10⁶. The plasmid profiles of different strains of the group were found to be almost identical. This remarkable plasmid similarly is discussed in relation to the common source of isolation.     

Demonstration of tra+ plasmid activity in bacteria indigenous to the phyllosphere of sugar beet; gene transfer to a recombinant pseudomonad

Abstract The presence of transfer proficient plasmids in bacteria isolated from the leaves of sugar beet ( Beta vulgaris L.) was studied. Of 435 bacteria sampled 79 (18%) contained plasmids. Pseudomonads (30%), Erwinia (12%) and Klebsiella (9%) were the largest populations sampled of which 22%, 33% and 29%, respectively, contained plasmids. The ability of these plasmids to self-transfer or mediate the mobilization of the tra⁻ mob⁺ broad host range IncQ plasmid R300B was determined. R300B was maintained in 61/79 natural plasmid containing isolates, the Gram positive isolates could not support R300B. Pseudomonas aureofaciens SBW25, isolated from sugar beet leaves, was chromosomally marked with a tetracycline resistance gene and used as a recipient (SBW25ETc). Five isolates of Erwinia herbicola and one of Erwinia salicis containing natural plasmids were able to mobilize R300B into the recombinant, SBW25ETc. These mobolizing ( tra⁺ ) plasmids were not maintained in transconjugant SBW25 cells. Analysis of the fragment patterns of Pst I digested plasmid DNA demonstrated that four (pSB139, pSB140, pSB142, pSB146; 110 kb) were identical, one (pSB153; 65 kb) was common to a subset of fragments in these four and another (pSB169; 100 kb) was unique. Other natural isolates were able to transfer copper resistance ( Erwinia rhapontici , 2 strains) or mercury resistance ( Pseudomonas fluorescens SBW340) to a rifampicin resistant recipient Pseudomonas putida UWC1 but not to SBW25ETc. These self-transferable plasmids were not able to mobilize R300B. These data demonstrate that the phyllosphere supports indigenous microbial populations which have the capacity to transfer genetic material between bacteria of different genera.     

Plasmid-borne mercury resistance in aquatic bacteria

Abstract Bacteria isolated from the River Mersey were analysed for their tolerance to mercury (HgCl₂). About 40% of the population was tolerant to mercury and in 13 of 52 mercury-tolerant isolates tested the mercury resistance (Hg^®) was transferred to Escherichia coli in conjugal matings. These 13 isolates represented a range of gram-negative genera and in each case mercury resistance was coded by a conjugative plasmid. These plasmids (75 kb to > 250 kb in size) all expressed mercury resistance of the narrow spectrum variety, volatilised HgCl₂ to elemental Hg° vapour and showed some degree of temperature sensitivity of transfer. None expressed resistance to nine different antibiotics. These 13 Hg^R plasmids were classified by restriction mapping into three distinct groups typified by pMER11, pMER327 and pMER610. The eight pMER610 group plasmids are identical and belong to the IncHI-2 group. Two of the four pMER327 group plasmids are closely related while the other two contain some common restriction fragments. pMER11 is quite distinct from the other groups. These results imply that within this aquatic environment plasmids play an important role in the response of bacteria to contaminating mercury and that there is widespread plasmid transfer and considerable genetic rearrangement.     

Structural relationships among chloramphenicol-resistance plasmids of Staphylococcus aureus

Abstract Twelve different chloramphenicol-resistance (Cm^r) plasmids detected in Staphylococcus aureus strains isolated between 1952 and 1981 were characterized by restriction endonuclease, DNA hybridization and heteroduplex analyses. These studies revealed three families of Cm^r plasmids which were distinguished by their chloramphenicol acetyltransferase sequences; the prototype plasmids of the families were pC221, pC223 and pC194. The cat and replication ( rep ) genes of the plasmid pC221 were highly conserved in other pC221 family members and were related to their homologs in the pC223 family plasmids; however, the cat and rep genes of the pC194 family plasmids were distinct.     

Instability of the morpholine-degradative phenotype in mycobacteria isolated from activated sludge

The stability of the morpholine-degradative phenotype was studied in a group of nine distinct strains of mycobacteria, all isolated from an activated sludge plant treating morpholine.
Variants incapable of morpholine degradation arose from all strains at high frequency following growth under non-selective conditions. However, strains differed with respect to the frequency at which Mor⁻ variants arose. No spontaneous reversion to wild type could be demonstrated. Six of the nine mycobacterial strains contained plasmids, each had a different plasmid profile. The plasmid profiles of wild type and Mor⁻ variants were compared. In no case could loss of the ability to grow on morpholine be correlated with loss of a complete plasmid. However, evidence is presented which suggests that in two strains loss of the morpholine-degradative phenotype may be correlated with a decrease in the size of a very large plasmid implying deletion of the DNA encoding morpholine degradation. The techniques employed to screen for plasmids in mycobacteria are discussed.     

Instability of plasmid-encoded citrate permease in Leuconostoc

M. KIHAL, H. PRÉVOST, M.E. LHOTTE, D.Q. HUANG AND C. DIVIÈS. 1996. The conversion from citrate positive (Cit⁺) to citrate negative (Cit^-) phenotype of six strains of Leuconostoc mesetiteroides was followed during growth in milk and buffered or unbuffered MRS medium at 30 or 37°C. High rate of loss of Cit⁺ phenotype was observed. The Cit^- phenotype was found to be linked to the loss of 22 to 23 kb plasmids. All Cit^- mutants isolated from Leuc. mesenteroides subsp. cremoris 195 reverted spontaneously to the Cit⁺ phenotype. Hybridization experiments using a 0.8 kb fragment of the citP gene of Leuc. mesenteroides showed that all the plasmids which were lost in Cit^- mutants encoded for a citrate permease. However, neither plasmid nor genomic DNA from Leuc. mesenteroides subsp. cremoris 195 hybridized with the citP probe.     

Transformation of Aspergillus parasiticus using autonomously replicating plasmids from Aspergillus nidulans

Abstract A genetic transformation system for the aflatoxin-producing fungus Aspergillus parasiticus using two autonomously replicating plasmids from A. nidulans (ARp1 and pDHG25) is reported. Transformation frequencies using the plasmid pDHG25 were from 5 × 10² to 2.5 × 10⁴ transformants per 10⁶ viable protoplasts and μg DNA. The stability of the plasmids in the transformants was also studied. This transformation system offers a new opportunity to clone genes related to aflatoxin production using appropriate aflatoxin-defective mutants.     

Species variation and plasmid incidence among fluorescent Pseudomonas strains isolated from agricultural and industrial soils

Abstract: A total of 132 different fluorescent Pseudomonas strains were isolated from several agricultural and industrial soils. The bacteria from the two different soil environments were compared for species and biotype variation, antibiotic and heavy metal resistance profiles, ability to degrade polyaromatic hydrocarbons, and plasmid incidence. Irrespective of the soil type, the isolates belonged to Pseudomonas fluorescens biotypes I–VI and Pseudomonas putida biotype B. Except for a streptomycin resistant isolate from one of the industrial soils, all the strains had the same antibiotic resistance profile. However, there was a higher incidence of heavy metal resistance and polyaromatic hydrocarbon degradation phenotypes in the isolates from industrial soils than from the agricultural soils. Only 2 out of 68 strains from agricultural soil were found to carry plasmids, while 28 out of 64 strains from industrial soil had plasmids. A majority of the plasmids (56%) were estimated to be larger than 50 kb, indicating that they could encode transfer functions. However, transferability as indicated by the ability to mobilize an IncQ plasmid (tra⁻, mob⁺), was observed with only one plasmid. None of the plasmid(s) containing isolates hybridized to a ³²P-labelled repP probe suggesting that none of the indigenous plasmids in the soil fluorescent Pseudomonas strains was related to the IncP group of conjugative plasmids commonly associated with resistance and catabolic genes.     

Distribution of plasmids in cyanobacteria of the LPP group

Abstract The distribution of plasmid DNA has been studied in 23 strains and variants of non-heterocystous filamentous cyanobacteria that are susceptible to infection by the LPP-1 cyanophage (archetype). 21 have identical plasmid profiles and contain 3 plasmids of M _rs 0.9, 10 and approx. 12 · 10⁶ respectively. In one strain, Plectonema FS180, the plasmids have been designated pMP1, pMP2, and pMP3, respectively. pMP1 shows sequence homology with pMP2 and pMP3 but not with DNA from an LPP-type cyanophage. Plectonema UTEX 598 lacks the small plasmid only, while Plectonema UTEX 1541 is distinct amongst all these strains with 3 plasmids of M _rs 3, 10, and > 30 · 10⁶, respectively. The findings support the view that the majority of these strains may be independent isolates of a single species.     

Structure and putative origin of a plasmid from Staphylococcus hyicus that mediates chloramphenicol and streptomycin resistance

The structure and functional organization of Staphylococcus hyicus plasmid pSCGp3EB that mediates chloramphenicol and streptomycin resistance (Cm^rSm^r) is described and compared with another Cm^rSm^r plasmid, pSCS12, from Staphylococcus sciuri. Both plasmids appeared to be formed by co-integrate formation between plasmids that very closely resemble the chloramphenicol resistance (Cm^r) plasmid pC221 and the streptomycin resistance (Sm^r) plasmid pS194. In addition to the established recombination site B (RS_B) in pC221 and pS194, another area suitable for recombination immediately downstream of the cat gene in pC221 and upstream of the str gene in pS194 has been identified. Co-integration at these sites would lead to the structures we have observed in the wild-type Cm^rSm^r plasmids pSCGp3EB and pSCS12.     

Electrotransformation of Lactobacillus manihotivorans LMG 18010T and LMG 18011

An electroporation procedure was developed for the genetic transformation of intact cells of Lactobacillus manihotivorans , a new Lactobacillus species isolated from cassava sour starch fermentation in Colombia. Transformation efficiency of Lact. manihotivorans strains LMG 18010^T and LMG 18011 was measured and compared with electroporability of Lact. plantarum strains NCIMB 8299 and LMG 9211, by investigating the effect of changes in various parameters. For Lact. manihotivorans strain LMG 18010^T, the composition of the culture medium, such as the type of peptone and the presence of Tween-80, was found to be the most critical parameter, as well as the aeration conditions of the culture; better electroporation was obtained without air. The presence of MgCl₂ in the recovery medium was favourable to regeneration of electroporated cells. Plasmid-curing of the cells did not improve their electroporability. Transformants were obtained with Lact. manihotivorans strain LMG 18010^T and the plasmids pLZ12 and pGK13, whereas Lact. manihotivorans strain LMG 18011 was transformable with plasmids pLP825 and pLZ12, with different electroporation conditions.     

Transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA

Abstract A method for efficient polyethylene glycol (PEG)-mediated transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA is described. The best conditions found for protoplast regeneration included using 0.45 M sucrose both during the cultivation of the cells and (as an osmotic stabilizer) during their treatment with lysozyme, whereas 0.25 M sodium-succinate was added to the regeneration plates. Under these conditions about 5–10% of input cells regenerated. The highest transformation frequency with plasmid DNA was obtained with a PEG 6000 concentration of 22.5% (w/v). Transforming B. amyloliquefaciens strains with the plasmid pUB110 isolated from B. amyloliquefaciens resulted in 2–4 · 10⁵ transformants/μg DNA, 100–1 000-times as high as with DNA from Bacillus subtilis , suggesting a restriction barrier between the two species. Transformation of B. amyloliquefaciens with plasmids pC194 or pE194 cop -6 gave poor yields and no restriction barrier could be demonstrated for these plasmids. However, by curing pC194 from one of the transformants, a mutant strain compatible to both the plasmids could be isolated, yielding 2–3·10⁴ transformants/μg DNA. Both laboratory and industrial B. amyloliquefaciens strains could be transformed with the procedure.     

Plasmid profiles of ten strains of Lactobacillus plantarum

Abstract The presence of extrachromosomal DNA elements has been investigated in 10 strains of Lactobacillus plantarum .
8 of the strains contained from 1 to 6 plasmids of different M _r values spanning from 1.35 · 10⁶ to 15.4 · 10⁶.
6 of the strains are commonly used as starter cultures in dry sausage and all these strains contained plasmids. The remaining 4 strains were obtained from the American Type Culture Collection and only 2 of these strains were found to harbour plasmids.     

Nickel and cobalt resistance of various bacteria isolated from soil and highly polluted domestic and industrial wastes

Abstract From enrichment cultures in the presence of 1 mM NiCl₂ 200 strains of aerobic bacteria were isolated from 50 samples collected in the metal-processing industry, waste water treatment plants and from solid waste, highly polluted by heavy metals. The strains isolated were characterized with respect to their substrate spectrum and resistance to nickel, cobalt, zinc and cadmium salts and assigned to 21 groups. One representative of each group was described with respect to cell morphology. All strains were Gram-negative, non-sporing rods or cocci. The highest concentrations of nickel, cobalt, zinc, cadmium, copper, mercury, and silver allowing growth on solid media were estimated. Two strains were able to grow at 20 mM NiCl₂ and CoCl₂, one strain tolerated 12 mM and one 7.5 mM concentrations of these salts.
Fifteen out of 21 strains contained at least one plasmid two contained two plasmids. The plasmid sizes varied between 50 and 340 kbp, except strain 10A, which contained a miniplasmid (2.6 kbp). Attempts to cure four selected strains by exposure to mitomycin C or growth at elevated temperature failed.
By helper-assisted and unassisted conjugation the plasmids of strain 31A were shown to carry nickel and cobalt resistance determinants. Alcaligenes eutrophus strains H16 and N9A and denative of strain CH34 lacking one or both of its native metal resistance plasmids were used as recipients. Both plasmids, p TOM8 and pTOM9, of strain 31A carried resistance properties which were expressed in all recipients except. A. eutrophus H16, in which only nickel resistance was expressed.
Plasmid pTOM3 residing in strain 10A could not be transferred as such. However, transconjugants derived from helper (pULB113)-assisted matings carried co-integrates of various sizes and were resistant to nickel and cobalt.     

Nucleotide sequence and structural relationships of a chloramphenicol acetyltransferase encoded by the plasmid pSCS6 from Staphylococcus aureus

M. CARDOSO AND S. SCHWARZ. 1992. The 4.6 kb chloramphenicol resistance (Cm^R) plasmid, pSCS6, isolated from a naturally occurring Staphylococcus aureus biotype C encoded an inducible chloramphenicol acetyltransferase (CAT). The respective cat gene and its regulatory region were cloned. Sequence analyses revealed two open reading frames: one for a 9-amino acid leader peptide and the other for the 215-amino acid CAT monomer. Comparisons of the predicted CAT amino acid sequences revealed a high degree of similarity between CAT from pSCS6 and the CAT variants encoded by Cm^R plasmids of the pC221 family. These close structural relationships suggested an intraspecific exchange of Cm^R-determinants between Staph. aureus of human and bovine biotype.     

The use of agarose wedge-gel electrophoresis for resolving both small and large naturally occurring plasmids

A wedge-shaped, horizontal agarose gel gave better electrophoretic resolution of both large and small plasmids than conventional linear gels. The wedge-gel increased mobility and separation of the larger plasmids whilst retarding the mobility of the smaller plasmids and tightening the bands. The linearity of the relationship between log₁₀ molecular size and log₁₀ relative mobility, for a range of plasmids from 2.1 to 221 kb, was increased. Therefore, estimations of plasmid sizes are more accurate using a wedge-gel than with conventional gels.     

Construction of derivatives of the F plasmid pOX–tra715: characterization of traY and traD mutants that can be complemented in trans

A derivative of the F plasmid, pOX38– tra715 , expresses the entire F tra operon from a foreign promoter (P_T7) derived from phage T7. A series of plasmids related to pOX38– tra715 were constructed which carry either deletion mutations or point mutations in traY . When the P_T7 promoter was induced, these plasmids expressed the F pilus but were transfer deficient unless TraY was supplied in trans from compatible plasmids. Insertion of a kanamycin-resistance cassette in the traY gene of the pOX38 plasmid, which contains the wild-type P_Y promoter, resulted in loss of F piliation and transfer ability. Introduction of TraY in trans partially restored piliation and transfer suggesting that TraY has a role in positively regulating the P_Y promoter . pOX38– tra719–traD411 , which contains a chloramphenicol-resistance cassette in place of the kanamycin-resistance cassette in pOX38– tra715 and a mutation in traD , was constructed to demonstrate the utility of this series of plasmids in studying the long (30 kb) F tra operon.     

Transmissible antibiotic resistance in strains of Escherichia coli isolated from the ovine rumen

Transmissible multiple resistance to tetracycline, ampicillin and streptomycin was found to be associated with transfer of the larger of the two plasmids (˜ 80 and ˜ 45 kbp) present in a group of tetracycline-resistant Escherichia coli strains isolated from the rumen of sheep. A second group of rumen tet^R strains, which differed in the ability to utilize several sugars, showed non-transmissible resistances to tetracycline and streptomycin.     

Phosphoenolpyruvate carboxylase in Corynebacterium glutamicum is dispensable for growth and lysine production

Abstract The symbiotic plasmid pRHc1J and the helper plasmid pJB3JI were transferred from Rhizobium "hedysari" strain RJ77 to Agrobacterium tumefaciens strain GMI9023. Transconjugants harboured recombinant plasmids (R-prime plasmids) consisting of pJB3JI carrying DNA fragments, of different sizes, surrounding the Tn 5 mob insert in pRHc1J. Two of these R-prime plasmids (pR1 and pR2) carried nod genes and were able to restore the Nod⁺ phenotype of pSym⁻ derivatives of R. "hedysari" . The R. "hedysari" nod genes harboured by both R-primes were expressed in R. leguminosarum biovar trifolii wild-type but not in a pSym⁻ derivative.