Introduction of the mercury transposon Tn501 into Rhizobium japonicum strains 31 and 110

Abstract Transposon Tn 501 , which encodes resistance to mercuric ions, was introduced into Rhizobium japonicum 110 and 31 by conjugal transfer. The transposon donor plasmid (pMD100) was able to mobilize into R. japonicum , but could not be maintained. Hg²⁺-resistant colonies were recovered at a frequency of 1.9 × 10⁻⁸/recipient for strain 110, and 1.7 × 10⁻⁷/recipient for strain 31. Presence of Tn 501 in Hg-resistant isolates was verified by Southern analysis and demonstrating transposition of Hg resistance. Transposon mutagenesis has been used to generate auxotrophic mutations at low frequency.     

Isolation and characterization of large lactococcal phage resistance plasmids by pulsed-field gel electrophoresis

Abstract Five phage-resistant Lactococcus lactis strains were able to transfer by conjugation the lactose-fermenting ability (Lac⁺) to a plasmid-free Lac⁻ L. lactis strain. In each case, some Lac⁺ transconjugants were phage-resistant and contained one or two additional plasmids of high molecular mass, as demonstrated by pulsed-field gel electrophoresis. Plasmids pPF144 (144 kb), pPF107 (107 kb), pPF118 (118 kb), pPF72 (72 kb) and pPF66 (66 kb) were characterized: they are conjugative (Tra⁺), they confer a phage-resistant phenotype and they bear lactose-fermenting ability (Lactose plasmid) except for the last two. Plasmids pPF144, pPF107 and pPF118 resulted probably from a cointegrate formation between the Lactose plasmid and another plasmid of the donor strain, whereas pPF72, pPF66 and the Lactose plasmid were distinct in the corresponding transconjugants. Plasmids pPF72 and pPF66 produced a bacteriocin. At 30°C, the phage resistance conferred by the plasmids was complete against small isometric-headed phage and partial against prolate-headed phage, except for pPF107 whose phage resistance mechanism was totally effective against both types of phages, but was completely inactivated at 40°C. Restriction maps of four of the plasmids were constructed using pulsed-field gel electrophoresis.     

Isolation, characterization and expression of a plasmid encoding chromate resistance in Pseudomonas putida KT2441

The chromium resistance properties encoded by a natural plasmid recovered from the environment were investigated. A 200 kb plasmid was isolated by the exogenous plasmid isolation method. The plasmid conferred a chromate resistance phenotype (MIC 8 mmol l⁻¹) to a chromate susceptible strain of Pseudomonas putida KT 2441 (MIC 0·5 mmol l⁻¹). The resistant strain took up 50% less ⁵¹Cr than the isogenic susceptible strain of Ps. putida KT2441. In addition, the resistant strain expressed two new membrane proteins encoded by the plasmid, an outer membrane protein (molecular weight 60 000) and an inner membrane protein (molecular weight 35 000). The physiological significance of these proteins is under current investigation.     

Decreased cupric ion uptake as the mechanism for cupric ion resistance in Escherichia coli

Abstract Plasmid-encoded copper (Cu²⁺) resistance in Escherichia coli was due to decreased uptake of Cu²⁺. The Cu²⁺-resistant E. coli Rtsl strain contained a 60 MDa plasmid which is known to encode for both Cu²⁺ and kanamycin resistance. A plasmid-free derivative of the same organism exhibited a greater uptake of Cu²⁺, and sensitivity to Cu²⁺ in both respiration and growth studies than the E. coli Rtsl strain.     

Automated sperm morphology analysis in fishes: the effect of mercury on goldfish sperm

This study is the first to examine the morphology of fish sperm using automated sperm morphology analysis (ASMA). The technique was applied to investigate the effect of an environmental pollutant, mercury, on the sperm morphology of goldfish Carassius auratus , and the effects on sperm morphology were compared with those on sperm motility. Goldfish sperm flagellar length was significantly shortened after instant exposure to 100 mg l⁻¹ (368 µM) mercuric chloride, while curvilinear velocity (VCL) and the percentage of motile sperm were significantly decreased at mercuric chloride concentrations of 1 and 10 mg l⁻¹ (3·68 and 36·8 µM), respectively. After 24 h exposure to 0·001 mg l⁻¹ (0·0037 µM) mercuric chloride, flagellar length was significantly reduced in 38% of the spermatozoa. Following exposure to 0·1 mg l⁻¹ (0·37 µM) mercuric chloride for 24 h, however, the majority of spermatozoa (98%), had significantly shortened flagella and increased sperm head length, width and area. Sperm motility was also significantly decreased at 0·1 mg l⁻¹ (0·37 µM) mercuric chloride, probably due to the significantly reduced flagellar length at this concentration. This study shows that the morphological examination of fish sperm by ASMA provides, not only, an excellent tool for monitoring reproductive disruption caused by environmental pollution, but also has applications to other areas of fish reproductive biology, such as cryopreservation and aquaculture.     

Contributions of traT and iss genes to the serum resistance phenotype of plasmid ColV2-K94

Abstract A genetic determinant for serum resistance, designated iss , has been found previously on the colicinogenic plasmid ColV2-K94. In this work we have identified a second serum resistance gene, traT , on ColV2-K94. The serum resistance mediated by derivatives of ColV2-K94 was due to presence of one or both of the iss and traT genes. Plasmid pWS12 (TraT⁺ Iss⁺) contained the kanamycin (Km) resistance transposon Tn 903 inserted near the origin of replication of ColV2-K94, and plasmids pWS15 (TraT⁺), pWS16 (TraT⁺) and pWS18 (TraT⁺ Iss⁺) were deletion derivatives of pWS12 constructed in vitro and in vivo. pWS12 and pWS18 conferred a 20-fold increase in relative resistance to 20% guinea pig serum when introduced into the serum-susceptible, genetically defined recA strain of Escherichia coli K-12, AB2463. Plasmids pWS15 and pWS16, from which iss had been deleted, still conferred 5-fold increases in relative resistance on AB2463. The level of resistance conferred on this strain by the antibiotic resistance plasmid R100–1 (which expresses the traT serum resistance gene) was comparable to that of plasmids pWS15 and pWS16. The 25-kDa traT gene product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the outer membrane proteins of strain AB2463 carrying ColV2-K94. This protein cross-reacted immunologically with the traT protein expressed by F or R100–1. Our results indicated that both traT and iss are capable of mediating serum resistance in ColV2-K94.     

Plasmid and pathogenicity in Xanthomonas oryzae pathovar oryzae, the bacterial blight pathogen of Oryza sativa

Xanthomonas oryzae pv. oryzae , the causative agent for bacterial leaf blight of rice, comprises diverse groups of strains differing in biochemical and pathological characteristics. A collection of X.o . pv. oryzae strains differing in geographical origin was screened for the presence of plasmids. Out of 17 isolates of X.o. pv. oryzae , 14 harboured plasmids of which two isolates (XoP5, XoC26) had two plasmids each and one isolate (XoR20) had three. The remaining isolates contained a single plasmid of identical mobility. Finger print analysis of plasmids was carried out using Eco RI for 10 isolates. The restriction fragment pattern was distinct for each isolate. They were classified under three groups based on cluster analysis using the unweighted pair group method with averages (UPGMA). Of the 18 plasmids, the plasmid pMA36 ( X.o. pv. oryzae XoC36) was further characterized. This plasmid was cured by acridine orange at the frequency rate of 10%. The cured strain was transformed with pMA36 at a frequency of 2.3 times 10² transformants μg^-1 of plasmid DNA. The plasmid-cured strain was virulent on rice but symptom development was delayed when compared to wild and transformed strains. The wild type strain ( X.o. pv. oryzae XoC36) was resistant to ampicillin, carbenicillin and rifampicin whereas the cured strain was resistant to carbenicillin and rifampicin but sensitive to ampicillin. The transformant was resistant to the three antibiotics indicating that the plasmid pMA36 codes for ampicillin resistance. The plasmid influenced the pathogenicity of X.o. pv. oryzae.     

Variation in chlorobenzoate catabolism by Pseudomonas putida P111 as a consequence of genetic alterations.

Pseudomonas putida P111 is able to utilize a broad range of monochlorinated, dichlorinated, and trichlorinated benzoates. The involvement of two separate dioxygenases was noted from data on plasmid profiles and DNA hybridization. The benzoate dioxygenase, which converts 3-chlorobenzoate (3-CB), 4-CB, and benzoate to the corresponding catechols via reduction of a dihydrodiol, was shown to be chromosomally coded. The chlorobenzoate-1,2-dioxygenase that converts ortho-chlorobenzoates to the corresponding catechols without the need of a functional dioldehydrogenase was shown to be encoded on plasmid pPB111 (75 kb). Cured strains were unable to utilize ortho-chlorobenzoates for growth. DNA hybridization data indicated that catabolism of the corresponding chlorocatechols was coded on chromosomal genes. Maintenance of plasmid pPB111 was dependent on the presence of ortho-chlorobenzoates in the growth media. A unique variant of P111 (P111D), able to grow on 3,5-dichlorobenzoate (3,5-DCB), was obtained by continuous subculturing from media containing progressively lower and higher concentrations of 3-CB and 3,5-DCB, respectively. The low frequency of segregants able to grow on 2,5-DCB, 2,3-DCB, and 2,3, 5-trichlorobenzoate was evident by lag periods greater than 200 h. Continued subculture on 3,5-DCB resulted in the formation of new plasmid pPH111 (120 kb), which was homologous to pPB111. A probe from the clc operon, which encodes for the chlorocatechol pathway, hybridized to plasmid pPH111 and to the chromosome of the wild-type strain P111 but not to its plasmid pPB111 nor to the chromosome of strain P111A, which had lost the ability to utilize chlorobenzoates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Segregational and structural instability of recombinant plasmid carrying genes for naphthalene degrading pathway

The stability of recombinant plasmid carrying genes for naphthalene mineralization was determined. A strain of Pseudomonas putida capable of mineralizing naphthalene (Nap⁺) via salicylate (Sal⁺) was isolated, and all regulatory and structural genes for the whole pathway were found to be encoded on a 25 kb Eco RI fragment of an approximately 83 kb plasmid present in this strain. The 25 kb Eco RI fragment was cloned into a tetracycline-resistant (Tc^R) cloning vector pLAFR3 and the recombinant plasmid, pRKJ3 (Nap⁺, Sal⁺, Tc^R), thus obtained was transferred into the plasmid-free strain Pseudomonas putida KT2442 in order to test the stability of the plasmid. Plasmid pRKJ3 was found to be segregationally and/or structurally unstable, depending on the growth conditions. Two types of novel derivative strains having the phenotypes Nap⁻, Sal⁺, Tc^R and Nap⁻, Sal⁻, Tc^R with specific deletions of approximately 2 kb and 18 kb, respectively, were obtained.     

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.     

Transfer of broad host-range plasmids to sulphate-reducing bacteria

Abstract The broad-host-range, IncQ, plasmid R300B (Sm, Su) has been stably transferred to two strains of sulphate-reducing bacteria ( Desulfovibrio sp. 8301 and Desulfovibrio desulfuricans 8312), using the IncP1 transfer system of the helper plasmid pRK2013 and cocultivation of sulphate-reducing bacteria with facultative anaerobes in media provided with sulphate and nitrate ions as electron acceptors. R300B was transferred at a frequency of 10⁻² to 1 per acceptor cell. The Sm^R marker was expressed in both sulphate-reducing bacteria strains while the Su^R was expressed only in strain 8301. R300B can also be transferred back to E. coli strains provided with IncP1 plasmids taking advantage of the retrotransfer ability of these plasmids. This occurs at a frequency up to 10⁻⁴ by recipient E. coli cell.     

Subdivision of Salmonella enteritidis PT 4 by pulsed-field gel electrophoresis: Potential for epidemiological surveillance

Abstract The capacity to reduce nitrate (NIT⁺ character) of wild-type and mutant strains of the purple nonsulfur bacterium Rhodobacter capsulatus was analysed by the methods of plasmid genetics and by DNA-DNA hybridization techniques. By conjugative introduction of the endogenous 115-kb plasmid of strain AD2 into a plasmid-free NIT⁻ mutant of the same strain the missing assimilatory nitrate reductase activity was restored. By analogous experimental techniques, the capacity to reduce nitrate was also temporarily established in the Rb. capsulatus NIT⁻ wild-type strain B10. DNA-DNA hybridization experiments with the narGHIJ operon of Escherichia coli and napA of Alcaligenes eutrophus yielded positive signals with an 11-kb Eco RI fragment of the AD2 plasmid.     

Specific deletion of a large segment of pRA500: a 3,5-xylenol degradative plasmid

3,5-Xylenol degradative plasmid, pRA500 (approximately 500 kilobase pairs, kb) carried by Pseudomonas putida NCIB 9869, also encodes resistance to inorganic mercuric ions (Hg^r). Following growth of Ps. putida on benzoate, p -cresol, p -hydroxybenzoate or protocatechuate, the frequency of loss of the 3,5-xylenol phenotype was 75–90%. The deletion of a large segment of approximately 350 kb from pRA500, to give rise to archetypal plasmid designated pRA502, occurred in such 3,5-xylenol-negative derivatives. During the conjugational transfer of pRA500 following selection of transconjugants for Hg^ronly, frequency of loss of 3,5-xylenol phenotype was approximately 60%. A high number of these 3,5-xylenol-negative transconjugants carried pRA502. A site-specific deletion is suggested in the formation of archetypal plasmid pRA502 by growth of Ps. putida on the above compounds and during transfer of pRA500.     

Isolation of cadmium sensitive mutants in Chlamydomonas reinhardtii by transformation/insertional mutagenesis

Abstract An arg7, cw15, mt⁺ strain of Chlamydomonas reinhardtii (CC1618) was transformed with pARG7.8, a plasmid containing the wild-type ARG7 gene. Over 2300 arg⁺ transformants were selected on TAP media. Upon subsequent analysis on TAP plus cadmium plates, five of the transformants failed to grow at a level of 400 μM cadmium and were designated as cadmium sensitive (Cd^s) mutants. Hybridization data indicated that vector (pBR329) sequences were present in these five mutants, but not in the untransformed parental strain. Two of the mutants have been back crossed to an arg7, cw15, Cd⁺, mt⁻ strain (CC425) and found to have progeny which always cosegregate the arg⁺ and Cd^s phenotypesin these two mutants results from the insertion of the plasmid pARG7.8 into a gene involving cadmium detoxification, and it provides a method by which to clone the interrupted gene(s).     

Transfer of a miniplasmid determining bacteriocin production and bacteriocin immunity in Streptococcus faecium

Abstract Streptococcus faecium strain 3 produced a bacteriocin (enterococcin Sf3) and contained a homogeneous species of plasmid DNA (pJK3) with an M _r of 3.5 · 10⁶. Plasmid pJK3 was transferable in a filter mating procedure to S. faecium M16. The non-bacteriocinogenic strain M16 was susceptible to enterococcin Sf3 and harboured a non-selftransferable 19.1 MDa plasmid, which was responsible for erythromycin resistance. Transcipient cells of S. faecium M16 contained the 19.1 MDa and the pJK3 plasmid, produced the enterococcin Sf3 and were resistant against the inhibitory action of this bacteriocin.     

Role of plasmids in mercury transformation by bacteria isolated from the aquatic environment

Eight mercury-resistant bacterial strains isolated from the Chesapeake Bay and one strain isolated from the Cayman Trench were examined for ability to volatilize mercury. Mercury volatilization was found to be variable in the strains tested. In addition, plasmids were detected in all strains. After curing, two of the bacterial strains lost mercury resistance, indicating that volatilization is plasmid mediated in these strains. Only two cultures demonstrated ability to methylate mercuric chloride under either aerobic or anaerobic conditions. Methylation of mercury, compared with volatilization, appears to be mediated by a separate genetic system in these bacteria. It is concluded that mercury volatilization in the estuarine environment can be mediated by genes carried on plasmids.     

Role of plasmids in mercury transformation by bacteria isolated from the aquatic environment.

Eight mercury-resistant bacterial strains isolated from the Chesapeake Bay and one strain isolated from the Cayman Trench were examined for ability to volatilize mercury. Mercury volatilization was found to be variable in the strains tested. In addition, plasmids were detected in all strains. After curing, two of the bacterial strains lost mercury resistance, indicating that volatilization is plasmid mediated in these strains. Only two cultures demonstrated ability to methylate mercuric chloride under either aerobic or anaerobic conditions. Methylation of mercury, compared with volatilization, appears to be mediated by a separate genetic system in these bacteria. It is concluded that mercury volatilization in the estuarine environment can be mediated by genes carried on plasmids.