Genetic properties of the Salmonella enteritidis R404 plasmid aggregate. IV. Reconstruction in R404 plasmid aggregate and separation of twelve genetically distinct derivative forms.

R404 plasmid aggregate is composed of two conjugative and two nonconjugative plasmids. Plasmid aggregate reconstructed from separated plasmids had the same genetic properties as the original R404 plasmid aggregate. It was found that plasmids of R404 factor could be transferred in conjugation in twelve different sets. These twelve genetically distinct classes of transconjugants formed only six groups differing in phenotypic characters.     

Genetic properties of the Salmonella enteritidis R404 plasmid aggregate I. Conjugational separation of different derivative plasmid aggregates and their genetic properties.

Conjugational transfer of R404 factor, found in Salmonella enteritidis strain, was examined. Six derivative forms differing in resistance pattern were isolated. The manner of conjugational separation during transfer of R404 factor from E. coli to E. coli strain was the same as from the original strain of S. enteritidis to E. coli strain. During following conjugation, all isolated forms except one, could give forms differing in a resistance pattern from the parenteral ones. This one form, from which different conjugational forms could not be separated, was assumed to be a single plasmid and was designated pCK2. All other forms, including the original R404 factor were assumed to be plasmid aggregates.     

Genetic properties of the Salmonella enteritidis R404 plasmid aggregate. III. Entry exclusion features of its separative forms.

The genetic relationship of four plasmids separated from R404 plasmid aggregate has been characterized by investigating entry exclusion and intracellular incompatibility. It has been found that the four plasmids exist in an autonomous state in cells carrying the aggregate. They are compatible and the two conjugative plasmids possess different traS genes.     

Genetic map of the virulence plasmid of Salmonella enteritidis and nucleotide sequence of its replicons

Partial sequencing of a genomic library of the virulence plasmid of Salmonella enteritidis has been used to localize in the restriction map of this plasmid the genetic loci already described in other Salmonella plasmids. The comparison of the vestigial tra region with the corresponding genes in the F plasmid allowed us to define the extent of the deletions that the S. enteritidis plasmid should have suffered. The putative replicons of the plasmid, repB and repC, were isolated and both proved to be functional in Escherichia coli, but repC was segregationally unstable. The nucleotide sequence of repB showed the typical organization of RepFIIA replicons, although the similarity was lower than usual in this group of replicons. The highest homology was found with the replicon of the virulence plasmid pYVe439-80 from Yersinia enterocolitica (72.5%). Replicon repC also showed a maximum identity of 72.6% with known replicons, namely the RepFIB of pColV-K30 and P307, both virulence plasmids isolated from E. coli. We conclude that the S. enteritidis plasmid could arise from the S. typhimurium plasmid through deletions, and that they are evolutionary distant from other IncFI and IncFII plasmids.     

Genetic transformation of Rhizobium leguminosarum by plasmid DNA.

We demonstrated the genetic transformation of Rhizobium leguminosarum by R68.45 plasmid DNA by freezing and thawing cell suspensions in the presence of R68.45 plasmid DNA and 20 mM MgCl2. Clones resistant to kanamycin and tetracycline were recovered at a frequency of 10(-8) per recipient cell. No colonies that were doubly drug resistant were recovered in parallel control experiments.     

Genetic transformation of Rhodopseudomonas sphaeroides by plasmid DNA.

A broad-host-range cloning vector, pUI81, was constructed in vitro from plasmids RSF1010 and pSL25 (a pBR322 derivative) and used to assay for transformation in Rhodopseudomonas sphaeroides. Washing cells with 500 mM Tris was an effective means of inducing competence for DNA uptake. Transformation frequencies as high as 10(-5) (transformants per viable cell) have been achieved by incubating Tris-treated cells with plasmid DNA, 100 mM CaCl2, and 20% polyethylene glycol 6000. Maximum frequencies were obtained when recipient cells were spread onto selective media after a 6.5-h outgrowth period in antibiotic-free medium. The structure (open circular versus closed, covalent circular), size, and concentration of plasmid DNA all significantly affected the transformation frequency. Four different plasmids, all small and suitable as cloning vectors, have been introduced by transformation into several different R. sphaeroides strains. Recombinant DNA carried on small, nonconjugative plasmids with broad host ranges can now be directly transferred to R. sphaeroides by this method.     

Heat-labile antigens of Salmonella enteritidis. II. Mouse-protection studies

Collins, F. M. (University of Adelaide, Adelaide, South Australia), and Margaret Milne. Heat-labile antigens of Salmonella enteritidis. II. Mouse-protection studies. J. Bacteriol. 92:549-557. 1966.-A number of extracts prepared from a virulent and an avirulent strain of Salmonella enteritidis were used to immunize mice. Living and alcohol-killed whole-cell vaccines were also used to compare the relative protective value of the various preparations. All mice were challenged intravenously with 100 to 1,000 ld(50) of S. enteritidis. Daily counts of the liver, spleen, and blood populations of vaccinated and control mice revealed that the challenge organism was rapidly eliminated only in those mice which had been immunized with a living vaccine. Immunization with extracts resulted in rapid clearance of S. enteritidis from the blood, but, after a delay of 24 to 48 hr, the bacterial populations increased until a maximal liver and spleen population of approximately 5 x 10(8) was reached. Between 55 and 100% of the immunized animals died, compared with 95 to 100% of the controls. With all four extracts, it was only the first antigenic fraction eluted from diethylaminoethyl cellulose which had any detectable effect on host resistance. The ineffectiveness of vaccines prepared with the various extracts or with whole killed bacteria relative to the protection observed after immunization with living organisms is discussed.     

Genetic transformation of Streptococcus pneumoniae by heterologous plasmid deoxyribonucleic acid.

A number of heterologous plasmid deoxyribonucleic acids (DNAs) coding for erythromycin, tylosin, lincomycin, tetracycline, or chloramphenicol resistance have been introduced into Streptococcus pneumoniae via genetic transformation with frequencies that varied between 10(-5) to as high as 5 x 10(-1) per colony-forming unit. Transformation with plasmid DNA required pneumococcal competence, was competed by chromosomal DNA, and showed a saturation at about 0.5 micrograms/ml (with a recipient population of 3 x 10(7) colony-forming units of competent cells per ml). Plasmid transformation did not occur with a recipient strain, 410, defective in endonuclease I activity and in chromosomal genetic transformation. All erythromycin-resistant transformants examined contained covalently closed circular DNA with the same electrophoretic mobility on agarose gels as the donor DNAs, and when examined in detail the plasmid reisolated from the transformants had the same restriction patterns and the same specific transforming activity as the donor DNA. In the cases of two plasmids examined in detail--pAM77 and pSA5700 Lc9--most of the transforming activity was associated with DNA monomers; DNA multimers present in pSA5700 Lc9 also had biological activity. An unexpected finding was the demonstration of transformation (2 x 10(-5) per colony-forming unit) with plasmid DNAs linearized by treatment with S1 nuclease or with restriction endonucleases.     

Serum survival and plasmid possession by strains of Salmonella enteritidis, Salm. typhimurium and Salm. virchow

Strains of Salmonella enteritidis, Salm. typhimurium and Salm. virchow , carrying different numbers of plasmids, were examined for the ability to multiply in sera. Viable counts were performed to monitor the kinetics of growth of bacteria when in human, chicken and turkey sera. The presence of plasmids in Salm. enteritidis, Salm. typhimurium and Salm. virchow reduced considerably the ability of strains of these serotypes to multiply in serum. SDS-PAGE was used to show that growth of Salm. enteritidis in serum did not involve changes in outer membrane proteins or lipopolysaccharide. It was concluded that the carriage of plasmids may be disadvantageous for the survival in serum of certain common salmonella serotypes.     

Computer-assisted restriction endonuclease analysis of plasmid DNA in field strains of Salmonella enteritidis.

Computer-assisted restriction endonuclease analysis of plasmid DNA in field strains of Salmonella enterica serovar Enteritidis (S. enteritidis) is described. The procedure consists of plasmid DNA purification, its digestion with restriction endonuclease TaqI, electrophoresis, charge-coupled device camera scanning of the gels, and an analysis of the restriction patterns with the software Gel Manager. The system allowed us to analyse, in detail, results of plasmid profiling in more than 600 field strains of S. enteritidis. In addition to plasmid-free and virulence plasmid only containing strains, 15 additional plasmid types were detected. All the images and detailed protocols are available at the Web site http://www.clark.cz/vri/salmon.htm.     

Identification of contemporary plasmid virulence genes in ancestral isolates of Salmonella enteritidis and Salmonella typhimurium

Six epidemiologically distinct ancestral strains of Salmonella enteritidis and 5 of S. typhimurium from the pre-antibiotic era were examined for plasmid content, and for presence of plasmid genes implicated in mouse-virulence. Five sizes of plasmid were detected in S. enteritidis varying from 1 to 60 MDa. Two sizes of plasmid were found in S. typhimurium, 28 and 60 MDa. Plasmids of the same size were not common to both serovars. The HindIII restriction patterns of 3 of the ancestral S. enteritidis plasmids were identical to the modern 38 MDa plasmid, while all contained identical bands of 3.5, 2.7 and 1.9 kb. All the 60-MDa S. typhimurium plasmids, ancestral and contemporary, had an identical restriction pattern. Three different sized S. enteritidis plasmids and one size S. typhimurium plasmid contained a 3.5-kb DNA fragment carrying the virulence locus VirA. The VirB virulence locus was located on a 2.7-kb DNA fragment in S. enteritidis and on a 2.5-kb fragment in S. typhimurium. Both loci were precisely conserved between the ancestral strains and the modern representatives of both serovars.     

High frequency of yeast transformation by plasmids carrying part or entire 2-micron yeast plasmid.

By using two chimeric plasmids containing yeast ura3 gene and 2-micron yeast DNA linked to the bacterial plasmid pCR1, yeast transformation of a high frequency has been achieved. The first plasmid is such that the 2-micron DNA part, in which the ura3 gene is incorporated, can be removed in one step and thus the 2-micron-ura3 sequence can be considered as a "transposable" block. In contrast, the second one bears the entire 2-micron plasmid and the ura3 gene is inserted in the bacterial plasmid part. As shown through hybridization experiments and genetic studies, the ura3 gene was maintained as a cytoplasmic element. Plasmids recovered from the yeast transformants were used to transform Escherichia coli. Their analysis by EcoRI showed that in many cases the vector had recombined with the endogenous 2-micron DNA of the recipient strain. The specific activity of orotidine 5'-monophosphate decarboxylase (coded by ura3) in yeast transformants was 10- to 30-fold higher than in the wild type.     

Characterization of fibronectin binding to Salmonella enteritidis strain 27655R

Abstract The optimal conditions for the binding of fibronectin to Salmonella enteritidis strain 27655R, and the cell-surface components involved in the binding, were identified. Cultivation on colonisation factor antigen (CFA) agar or in CFA broth at 33°C for 24 h were found to be optimal for the expression of fibronectin binding. Such cultures exhibited 88% and 70% binding of ¹²⁵I-labelled fibronectin and its 29-kDa N-terminal domain, respectively. The fibronectin binding was reversed by the addition of unlabelled fibronectin or its 29-kDa fragment. Scatchard plot analysis of the binding showed that the strain possessed one high-affinity ( K _d= 5.8 × 10⁻¹⁰ M) and one low-affinity ( K _d= 2 × 10⁻⁸ M) binding site. The fibronectin-binding could be inhibited by cell surface components of S. enteritidis 27655R released by 30 min treatment at 65°C or 95°C. Inhibition could also be achieved using purified fimbriae. A non-fimbriated mutant of strain 27655R showed a much reduced binding of fibronectin (15%). Electron microscopic analysis showed association of the gold-labelled 29-kDa N-terminal fragment with S. enteritidis 27655R fimbriae. In conclusion, the findings suggest that S. enteritidis (strain 27655R) possesses fibronectin-binding fimbriae.     

Fimbriation genes of Salmonella enteritidis.

From a cosmid clone, a 5.3-kilobase (kb) HindIII fragment of Salmonella enteritidis DNA containing the fimA gene was subcloned into bacteriophage T7 promoter vectors in both orientations. Predominantly mature fimbrin (14,000 Mr) was produced by clones containing the 5.3-kb insert, whereas the original cosmid clone predominantly accumulated a prefimbrin precursor (16,500 Mr). T7 RNA polymerase-dependent expression of the 5.3-kb insert and of a series of nested deletions revealed several membrane-localized polypeptides (80,000, 40,000, 29,000, 25,000, and 16,500 Mrs) transcribed in the same direction as fimA as well as a single polypeptide (9,000 Mr) transcribed in the opposite direction. Mini-Mu and TnphoA (Tn5 IS50L::phoA) transposon mutagenesis was used to identify a 2- to 3.5-kb region downstream of fimA that affected fimbrin production and processing. A more distant region (greater than 7 kb), revealed by Tn10 and Mu dI mutagenesis, was also required for fimbriation but did not hybridize with the 5.3-kb fragment. Yet another distant region did hybridize to the 5.3-kb fragment, suggesting the existence of other fimbriation-related genes.     

Genetic instability of R plasmids in relation to the shift of drug resistance patterns in Salmonella johannesburg

Observation of the resistance of Salmonella johannesburg to the six drugs ampicillin (A), streptomycin (S), tetracycline (T), chloramphenicol (C), kanamycin(K) and sulphadiazine (Su) was made over the 7 years from 1973 to 1979. Strains with ASTCKSu- and ASCKSu- resistance patterns predominated in the years 1973-1975 and 1976-1979, respectively. These resistances were found to be mediated by autotransferring plasmids belonging to the incompatibility group FIme. The ASTCKSu-resistance plasmids were unstable, giving rise to deletion variants at a much higher frequency than ASCKSu-resistance plasmids either of natural origin or derived in vitro from the ASTCKSu-resistance plasmids. Thus, the ASCKSu-resistance plasmid might be a deletion variant of the ASTCKSu-resistance plasmid. This is supported by the extensive similarity of their cleavage patterns produced by specific restriction endonucleases.     

Starvation survival of Salmonella enteritidis.

Washed cells of Salmonella enteritidis harvested from a defined medium during logarithmic growth were subjected to starvation in pH 7 phosphate buffer at 37 C. Viability was measured by slide cultures and plate counts. The survival of cell suspensions equivalent to 1 to 10 mg (dry wt)/ml was influenced by cryptic growth. The rate of cryptic growth, assessed by plate counts, increased with cell density and could not be alleviated by starvation with dialysis. Dialysis of the starving culture did retard the onset of cryptic growth but did not eliminate it, indicating that the major substrates for regrowth were relatively large cellular components. In phosphate buffer, 6.7 homologous heat-killed cells allowed for the doubling of one S. enteritidis cell. Cryptic growth was not observed when cells were starved on the surface of membrane filters or in suspensions equivalent to 20 mug (dry wt)/ml (105 cells/ml). Similar half-life survival times were calculated for both these populations, but the shape of their survival curves differed significantly. These differences were attributed to stress factors encountered during cell preparation and during starvation. The half-life survival time of S. enteritidis starved at 20 mug (dry wt)/ml was 140 h in phosphate buffer, 82 h in 3,6-endomethylene-1,2,3,-6-tetrahydrophthalic acid buffer, and 77 h in tris(hydroxymethyl)aminomethane buffer.     

Survey of plasmids and resistance factors among veterinary isolates of Salmonella enteritidis in Malaysia

Thirty-five veterinary isolates of Salmonella enteritidis were characterized by their susceptibility to 10 antimicrobial agents and by their plasmid profiles on agarose gel electrophoresis. All were susceptible to carbenicillin, chloramphenicol and nalidixic acid but 89% were resistant to tetracycline. When examined, 91% of the isolates harboured plasmids, with sizes ranging from 9.8 to 60 MDa. However, it was only possible to associate the presence of plasmids with tetracycline resistance; plasmids occurring in 90% of the tetracycline-resistant isolates. In conjugation experiments, with Escherichia coli K12 Nal^r as recipient, the tetracycline resistance in three selected S. enteritidis isolates was observed to transfer at frequencies of 3.0×10^-3 to 1.0×10^-2/donor cell. The concomitant transfer of a 56-MDa or 60-MDa plasmid in these three S. enteritidis isolates was also detected.R. Son. A. Ansary and I. Salmah are with the Department of Genetics and Cellular Biology. University of Malaya, 59100 Kuala Lumpur, Malaysia