Atypical incompatibility of the F-like factors of pA22-4, pAP39 and pA41 genetic transfer with the F-group incompatibility plasmids

A study was made of compatibility of three F-like factors of the genetic transfer (pAP22-4, pAP39, pAP41) identified in the cells of serologically typed E. coli strains with F-group incompatibility reference plasmids. The factors of pAP22-4 and pAP41 transfer are partly incompatible with groups FII, FIII, FIV, and FI, FIV, respectively, while the factors of pAP39 transfer are completely incompatible both with groups FI and FIV plasmids.     

Conjugativity and incompatibility of derepressed pAP11-2 plasmid

It has been demonstrated during investigation of Colplasmid pAP11-2 and its varieties labeled with transpozone (Tn1 and Tn9) that this plasmid is a derepressed one in terms of transfer functions in E. coli strain K-12 cells as well as in some of serologically typed strains of this type. The plasmid under study is incompatible with reference plasmids belonging to two different groups (FI and FIV) and is marked by a number of the properties common to the system of genetic control over Tra-functions.     

Antigenic properties of genetic recombinants of serologically typed E. coli]

The conjugation between the typed strains of E. coli belonging to various serological groups and conjugation between typed and untyped E. coli strains were studied. Genetic determinant controlling the synthesis of the O100 antigen proved to be closely linked with histidine locus. Among recombinants obtained in crossing the typed E. coli strains there were such belonging to the serological type different from the serological types of donor and recipient cells.     

Characteristics of new pKMR-plasmids detected in natural strains of Enterobacteriaceae

pKMR-plasmids controlling the antibiotic resistance and adhesive properties were isolated from clinical strains of E. coli O26 and O124, and Sh. sonnei. Two of them, i.e. pKMR 207 and pKMR 208 were conjugative. On conjugation they jointly transferred the features of the antibiotic resistance and capacity for production of the colonization antigen. The studies on transformation of E. coli K 12 802 with the plasmid DNA of E. coli O124 showed that the antibiotic resistance and colonization properties in E. coli O124 were controlled by the nonconjugative plasmid pKMR 209. It was found that plasmids pKMR 207 and pKMR 208 had the fi(-)-phenotype. None of the plasmids allotted the host cells sensitivity to the donor specific phages of the incompatibility groups F, N, P, W, and I. Probably, the plasmids did not belong to these incompatibility groups. When the cells of E. coli K 12 802 were transformed with the plasmid DNA of the wild strain to the hemolytic strain of S. typhimurium with multiple antibiotic resistance, 3 pKMR 210 plasmids with different markers of the antibiotic resistance were detected in the transformants. One of the plasmids controlled both the drug resistance and the capacity for production of hemolysin. The ability of the detected pKMR plasmids to inhibit fertility and relation to the donor specific phages was studied.     

Properties of adherence factor plasmids of enteropathogenic Escherichia coli and the effect of host strain on expression of adherence to HEp-2 cells

EPEC adherence factor (EAF) plasmids from three strains of enteropathogenic Escherichia coli (EPEC) - E2347/69 (O127:H6), E20517 (O111:H2) and E24582 (O142:H6) - were examined. The EAF plasmids were all marked with ampicillin resistance by transposition of Tn801 to give pDEP1, pDEP2 and pDEP11, respectively. All three plasmids showed incompatibility with an FIme and an FIV plasmid and had some similarity in restriction enzyme digest patterns. Plasmid pDEP1 differed from pDEP2 and pDEP11 in being autotransferring and fertility-inhibition positive. An EAF probe consisting of a 1 kb BamHI-SalI restriction endonuclease fragment of the prototype EAF-associated plasmid pMAR2 hybridized to similar-sized SalI-BamHI fragments of pDEP1 and pDEP11 but to a different-sized fragment of plasmid pDEP2. Loss of the EAF plasmids from EPEC strains resulted in a marked reduction in the ability of these strains to adhere to HEp-2 cells. The EAF-plasmid-negative variants did not express a 94 kDa outer-membrane protein (OMP). When these EAF plasmids were reintroduced into EAF-plasmid-negative EPEC strains a high level of adherence equivalent to that of the parent EPEC strains was restored and a 94 kDa OMP was usually expressed. However, when EAF plasmids were transferred into E. coli K12 or non-EPEC E. coli the host strains either did not adhere or adhered poorly to the HEp-2 cells. These transconjugants did not express a 94 kDa OMP.     

Monoclonal antibodies against O and K antigens of uropathogenic Escherichia coli O4 : K12 : H− as opsonins

Abstract Monoclonal antibodies of subclasses IgG1 and IgG2b and specific for the O4 antigen of Escherichia coli 20025 (O4 : K12 : H⁻) and the capsular K12 polysaccharide of the same strain (IgM) were obtained with the hybridoma technique using spleen cells from Balb/c mice, immunized with a crude bacterial extract, and Sp2/O-Ag8 myeloma cells. The anti-O4 antibodies reacted exclusively with the O4 lipopolysaccharide and not with those from serologically O-cross reactive E. coli . The anti-K12 antibodies recognized as epitope (part of) the KDO moiety of the capsular K12 polysaccharide. Not only anti-K12, but also anti-O4 antibodies effectively phagoopsonized encapsulated E. coli 20025. The opsonized bacteria were killed in subsequent in vitro phagocytosis by human leokocytes in the presence of human serum complement.     

Genetic mapping of the chromosomal replication origin of Salmonella typhimurium.

Two hundred strains of Escherichia coli harboring Filv+ plasmids which carry a segment of the Salmonella typhimurium chromosome were isolated independently. Among them, two strains were found to harbor F' plasmids that are able to replicate in Hfr cells of E. coli; i.e., they carry a site designated poh (permissive on Hfr) of the S. typhimurium chromosome. The poh site is presumably identical with the replication origin (oriC) of the bacterial chromosome. These two plasmids carry the dnaA-uncA-rbs-ilv-cya-metE region of the chromosome of S. typhimurium. Other F' plasmids which only carried the ilv-cya-metE region were unable to be maintained in Hfr cells. The poh site (= oriC) of S. typhimurium thus is located in the uhp-ilv region of the chromosome. The two plasmids carrying the poh site of S. typhimurium can suppress the temperature-sensitive character of an E. coli mutant that carries the temperature-sensitive dnaA46 allele, when the plasmids exist in the mutant cells. This suggests that the dnaA chromosome in place of the dnaA gene product of E. coli itself. The ability of the plasmids carrying the poh site of S. typhimurium to replicate in Hfr cells of E. coli suggests that the replication system of E. coli can recognize the Salmonella replication origin.     

Separation of the SOS-dependent and SOS-independent components of alkylating-agent mutagenesis.

Escherichia coli plasmids containing the rpsL+ gene (Strs phenotype) as the target for mutation were treated in vitro with N-methyl-N-nitrosourea. Following fixation of mutations in E. coli MM294A cells (recA+ Strs), an unselected population of mutant and wild-type plasmids was isolated and transferred into a second host, E. coli 6451 (recA Strr). Strains carrying plasmid-encoded forward mutations were then selected as Strr isolates, while rpsL+ plasmids conferred the dominant Strs phenotype in the second host. Mutation induction and reduced survival of N-methyl-N-nitrosourea-treated plasmids were shown to be dose dependent. Because this system permitted analysis and manipulation of the levels of certain methylated bases produced in vitro by N-methyl-N-nitrosourea, it afforded the opportunity to assess directly the relative roles of these bases and of SOS functions in mutagenesis. The methylated plasmid DNA gave a mutation frequency of 6 X 10(-5) (a 40-fold increase over background) in physiologically normal cells. When the same methylated plasmid was repaired in vitro by using purified O6-methylguanine DNA methyltransferase (to correct O6-methylguanine and O4-methylthymine), no mutations were detected above background levels. In contrast, when the methylated plasmid DNA was introduced into host cells induced by UV light for the SOS functions, rpsL mutagenesis was enhanced eightfold over the level seen without SOS induction. This enhancement of mutagenesis by SOS was unaffected by prior treatment of the DNA with O6-methylguanine DNA methyltransferase. These results demonstrate a predominant mutagenic role for alkylation lesions other than O6-methylguanine or O4-methylthymine when SOS functions are induced. The mutation spectrum of N-methyl-N-nitrosourea under conditions of induced SOS functions revealed a majority of mutagenic events at A . T base pairs.     

Phage Folac: an Folac plasmid-dependent bacteriophage

By enriching sewage with Escherichia coli or Salmonella typhimurium strains harbouring the plasmid EDP208, a constitutive pilus-producing derivative of plasmid F olac, a phage was isolated which plated on these two organisms but not on isogenic strains without the plasmid. The phage was named F olac; it has a hexagonal outline with a diameter of 28 nm, contained RNA, was resistant to chloroform, and probably adsorbed preferentially to the sides of EDP208 pili very near the tip. Phage multiplication could be demonstrated on E. coli or S. typhimurium strains carrying the plasmid F olac, but an increase in titre did not occur on E. coli strains carrying plasmids of the F complex. Results of phage multiplication experiments on strains carrying the depressed pilus-producing plasmids R71 or TP224-Tc, which determine pili serologically related to those of EDP208, were inconclusive. Phage F olac was found to be serologically related to phage UA-6, another isolate specific for EDP208.     

Plasmid recombination stimulated by restriction endonuclease EcoRI in vivo: formation of recombinant plasmids in recA+-cells of E. coli

The possible participation of restriction endonuclease EcoRI in recombination of compatible nonhomologous plasmids in E. coli cells has been studied. To study the process, plasmids RP4 and R245 have been transferred by conjugation into the recipient cells of E. coli harbouring one of isogenic plasmids, pSA14 and pSA25, different for the genes coding restriction endonuclease EcoRI. The genetic analysis of transconjugant phenotypes, coded by the plasmids, has permitted to register the recombinant plasmids after compatibility of parent plasmids in E. coli cells. Recombination of plasmid RP4 with the plasmid pSA14, carrying EcoRI genes, has been registered in E. coli cells, producing the restriction endonuclease, while plasmid recombination has not been found in the cells harbouring plasmid pSA25, isogenic for all genes, except for EcoRI genes, with plasmid pSA14. Restriction endonuclease EcoRI is concluded to stimulate site specific recombination of nonhomologous compatible plasmids in vivo. EcoRI-mediated recombination of plasmid R245 with plasmid pSA14 is discussed.     

Changes in the nature of the sensitivity of an E. coli M strain carrying Inc-group R plasmids, to coliphages T3 and T7

After the transfer of prototype plasmids R6K (IncX), R387 (IncK), R27 (IncH1) and T (IncN) to E. coli M nalr the appearance of histidine-dependent mutants (R27, T), histidine-leucine-dependent mutants (R6K), methionine-proline-dependent mutants (R387) was observed among the resulting transconjugates. The mutations of E. coli M nalr R+ cells induced by the introduction of the plasmids were accompanied by the transformation of the cells from the S-form into the R-form. In contrast to the prototrophs E. coli M nalr, the auxotrophs carrying plasmids R6K, R27, T acquired sensitivity to phage T7, and the methionine-proline-dependent mutant became sensitive to phages T and T7. The above-mentioned plasmids rendered E. coli M cells capable of synthetizing the donor pili. But the adsorption of phages T3 and T7 on the auxotrophic cells, both with and without plasmids, occurred due to their interaction with the cell-wall receptors.     

Transfer of multiple drug resistance plasmids between bacteria of diverse origins in natural microenvironments

Plasmids harboring multiple antimicrobial-resistance determinants (R plasmids) were transferred in simulated natural microenvironments from various bacterial pathogens of human, animal, or fish origin to susceptible strains isolated from a different ecological niche. R plasmids in a strain of the human pathogen Vibrio cholerae O1 E1 Tor and a bovine Escherichia coli strain were conjugated to a susceptible strain of the fish pathogenic bacterium Aeromonas salmonicida subsp. salmonicida in marine water. Conjugations of R plasmids between a resistant bovine pathogenic E. coli strain and a susceptible E. coli strain of human origin were performed on a hand towel contaminated with milk from a cow with mastitis. A similar conjugation event between a resistant porcine pathogenic E. coli strain of human origin was studied in minced meat on a cutting board. Conjugation of R plasmids between a resistant strain of the fish pathogenic bacterium A. salmonicida subsp. salmonicida and a susceptible E. coli strain of human origin was performed in raw salmon on a cutting board. R plasmids in a strain of A. salmonicida subsp. salmonicida and a human pathogenic E. coli strain were conjugated to a susceptible porcine E. coli strain in porcine feces. Transfer of the different R plasmids was confirmed by plasmid profile analyses and determination of the resistance pattern of the transconjugants. The different R plasmids were transferred equally well under simulated natural conditions and under controlled laboratory conditions, with median conjugation frequencies ranging from 3 x 10(-6) to 8 x 10(-3). The present study demonstrates that conjugation and transfer of R plasmids is a phenomenon that belongs to the environment and can occur between bacterial strains of human, animal, and fish origins that are unrelated either evolutionarily or ecologically even in the absence of antibiotics. Consequently, the contamination of the environment with bacterial pathogens resistant to antimicrobial agents is a real threat not only as a source of disease but also as a source from which R plasmids can easily spread to other pathogens of diverse origins.     

Genetic characteristics of plasmid pAP53 derepressed in transfer functions detected in the cells of an opportunistic E. coli strain

A study was made of plasmid pAP53 derepressed as regards transfer functions (Tra-functions) detected in E. coli strain cells, serogroup 0128, after its labeling with transpozones Tn1 and Tn9. The compatibility tests demonstrated that the plasmid belongs to the incompatibility group FIII and is partially incompatible with the group FII reference-plasmid. Plasmid pAP53 is unable to inhibit Tra-functions of plasmid F'lac and is not inhibited by the fin type genetic regulation on the OP group plasmids under study. At the same time Tra-functions of plasmid pAP53 are inhibited in the presence of pAP41 plasmid, which indicates that this plasmid has a special type of genetic regulation.     

Transfer of multiple drug resistance plasmids between bacteria of diverse origins in natural microenvironments.

Plasmids harboring multiple antimicrobial-resistance determinants (R plasmids) were transferred in simulated natural microenvironments from various bacterial pathogens of human, animal, or fish origin to susceptible strains isolated from a different ecological niche. R plasmids in a strain of the human pathogen Vibrio cholerae O1 E1 Tor and a bovine Escherichia coli strain were conjugated to a susceptible strain of the fish pathogenic bacterium Aeromonas salmonicida subsp. salmonicida in marine water. Conjugations of R plasmids between a resistant bovine pathogenic E. coli strain and a susceptible E. coli strain of human origin were performed on a hand towel contaminated with milk from a cow with mastitis. A similar conjugation event between a resistant porcine pathogenic E. coli strain of human origin was studied in minced meat on a cutting board. Conjugation of R plasmids between a resistant strain of the fish pathogenic bacterium A. salmonicida subsp. salmonicida and a susceptible E. coli strain of human origin was performed in raw salmon on a cutting board. R plasmids in a strain of A. salmonicida subsp. salmonicida and a human pathogenic E. coli strain were conjugated to a susceptible porcine E. coli strain in porcine feces. Transfer of the different R plasmids was confirmed by plasmid profile analyses and determination of the resistance pattern of the transconjugants. The different R plasmids were transferred equally well under simulated natural conditions and under controlled laboratory conditions, with median conjugation frequencies ranging from 3 x 10(-6) to 8 x 10(-3). The present study demonstrates that conjugation and transfer of R plasmids is a phenomenon that belongs to the environment and can occur between bacterial strains of human, animal, and fish origins that are unrelated either evolutionarily or ecologically even in the absence of antibiotics. Consequently, the contamination of the environment with bacterial pathogens resistant to antimicrobial agents is a real threat not only as a source of disease but also as a source from which R plasmids can easily spread to other pathogens of diverse origins.     

Use of multiple primers in RAPD analysis of clonal organisms provides limited improvement in discrimination.

Randomly amplified polymorphic DNA (RAPD) analysis using two or more primers has been reported to provide additional discriminatory ability over one primer used individually. This may be of particular application in epidemiological typing of clonal organisms, such as Shiga toxin-producing E. coli O157, where strain differentiation can be difficult. Using four arbitrary primers individually, and in all possible permutations, E. coli O157 isolates and other arbitrarily chosen E. coli strains were typed using RAPD analysis. For most nonclonal strains, the use of two primers resulted in increased differentiation between isolates; however, more than two primers did not increase further the discriminatory capacity. E. coli O157 isolates that produced virtually identical profiles using one primer did not show increased differentiation when using two or more primers, demonstrating that in some cases, where strains of an organism are highly related, there is limited advantage to using more than one primer in RAPD analysis.     

Molecular cloning and genetic analysis of the rfb region from Shigella flexneri type 6 in Escherichia coli K-12

The rfb gene cluster which determines the biosynthesis of the Shigella flexneri serotype 6 O-antigen specificity has been cloned in pHC79, generating plasmids pPM3115 and pPM3116. These plasmids mediate expression, in Escherichia coli K-12, of lipopolysaccharides (LPS) immunologically similar to the S. flexneri type 6 LPS as judged by SDS-PAGE and Western-immunoblot analysis using S. flexneri type 6 specific antisera. Thus, unlike other S. flexneri serotypes, no additional loci are required for serotype specificity. This expression is independent of E. coli K-12 rfb genes. Southern-hybridization analysis using the 16.2-kb BglII probe from S. flexneri type 6 rfb region detected very little sequence homology in S. flexneri serotypes 1-5, however, some homology was detected with E. coli O2 and O18, but not in E. coli 0101 strains, Salmonella and Vibrio cholerae.     

Detection of F-like plasmids in serologically typed E. coli by the reactions of the donor-specific phage titre increase]

The authors studied 130 strains of serologically typed E. coli for the capacity to provide reproduction of donor-specific phages MS2, Qbeta, and f1; positive increase of the donor-specific phages reaction was found in 12 cases. Some of the detected L-like plasmides bore determinants of colicinogenicity or drug resistance. Such plasmides were transmissive and capable of inhibiting donor properties controlled by factor F'-lac+, i.e. they served as plasmides of type fi+.     

On the serological specificity of the Escherichia coli O8 and O9 antigens.

The O8 and O9-specific lipopolysaccharides of Escherichia coli lost their serological activity during liberation of the polysaccharide moieties (alpha-mannans) by mild acid hydrolysis, as tested by passive haemagglutination and haemagglutination inhibition. The serological activities and specificities were restored by substitution of the polysaccharides with 1 to 2 stearoyl groups per polysaccharide chain. The mannans obtained by biosynthesis in vitro were serologically active only when bound to the membrane-associated hydrophobic carrier molecule. Liberation of the polysaccharides from the carrier by treatment with aqueous phenol resulted in loss of the serological activity. The O8- and O9-specific mannans of E. coli are thus serologically active when they are part of an amphiphilic molecule and not as free polysaccharides.     

Molecular heterogeneity of heat-stable toxin-producing and non-producing Escherichia coli O25 strains isolated in Japan

Escherichia coli O25 strains that produce heat-stable toxin (ST) have been recently isolated in Japan, and epidemiological study of this type of enterotoxigenic E. coli is required. In this study the heterogeneity of 16 ST-producing and non-producing strains of E. coli O25 was investigated. All eight ST-producing strains were shown to have STIb gene, and seven of them had similar profiles of plasmids, ladder-banding of LPS in SDS-polyacrylamide gel electrophoresis, and chromosomal DNA digestions in pulsed-field gel electrophoresis (PFGE). In contrast, ST-non-producing strains were more heterogeneous in all parameters examined. PFGE of the digested chromosomal DNA with several restriction enzymes was proved to be an effective procedure to compare the closely related strains of E. coli O25.     

Transfer and expression of the genetic determinants for O and K antigen synthesis in Escherichia coli O9:K(A)30 and Klebsiella sp. O1:K20, in Escherichia coli K12

Escherichia coli serotype O9:K(A)30 and Klebsiella O1:K20 produce thermostable capsular polysaccharides or K antigens, which are chemically and serologically indistinguishable. Plasmid pULB113 (RP4::mini-Mu) has been used to mediate chromosomal transfer from E. coli O9:K30 and Klebsiella O1:K20 to a multiply marked, unencapsulated, E. coli K12 recipient. Analysis of the cell surface antigens of the transconjugants confirmed previous reports that the genetic determinants for the E. coli K(A) antigens are located near the his and rfb (O antigen) loci on the E. coli linkage map. The Klebsiella K20 capsule genes were also found to be in close proximity to the his and rfb loci. Electron microscopy revealed significant differences in the structural organization of capsular polysaccharides in these two microorganisms and the morphological differences were also readily apparent in transconjugants expressing the respective K antigens. These results are consistent with the interpretation that at least some of the organizational properties of capsular polysaccharides may be genetically determined, rather than being a function of the outer membrane to which the capsular polysaccharides are ultimately attached.