Chromosome replication in an Hfr strain of Escherichia coli

The pattern of chromosome replication in an exponentially growing culture of an Hfr strain of Escherichia coli has been compared to that obtained with the same Hfr following a procedure which synchronizes rounds of DNA replication. The results indicate that there is significant replication from the integrated plasmid following the synchronization procedure, whereas in the exponentially growing culture replication starts most frequently from the normal origin with little, if any, replication from the sex factor, F.     

Construction of an Hfr strain useful for transferring recA mutations between Escherichia coli strains.

Strain JC10240 (Hfr PO45 srlC300::Tn10 recA56 thr-300 ilv-318 rpsE300) was constructed. On account of the close linkage of Tn10 to recA56, the latter can be moved to other Escherichia coli (and closely related) strains in transductional or conjugational crosses selecting resistance to tetracycline.     

Isolation and characterisation of dog uropathogenic Escherichia coli strains and their fimbriae

A number of Escherichia coli strains have been isolated from dogs with urinary tract infections. These strains have been characterised with respect to their O, K, H, and fimbrial antigens, colicin production, antibiotic resistance, plasmid content and their ability to haemagglutinate erythrocytes from various species. Crossed immunoelectrophoresis of fimbrial extracts, as well as the reaction of partly purified fimbriae of a number of these strains with monoclonal antibodies revealed homology or a strong crossereaction with an F12 fimbrial subunit protein of human uropathogenic E. coli strains. Unlike human F12 fimbriae producing strains, the dog isolates did agglutinate dog erythrocytes in the presence of D-mannose but not human erythrocytes, indicating that the adhesin carried by these strains is different from the adhesin on fimbriae of human uropathogenic E. coli. Similar indications were obtained from experiments with latex beads coated with the receptor for P-fimbriae. These beads were agglutinated by Escherichia coli strains from human urinary tract infections, but not by the dog isolates described here. Preliminary adhesion experiments of human and dog Escherichia coli to human bladder epithelial and canine kidney epithelial cells also showed differences in adhesion depending on the origin of the strain tested.     

Isolation of the nonviable cells produced during normal growth of recombination-deficient strains of Escherichia coli K-12

During normal growth, cultures of recombination-deficient (Rec(-)) strains contain a population of cells that do not form colonies. Such cells are not present in a culture of an isogenic Rec(+) strain. We present a procedure for isolating and studying this defective population of cells. Exposure of a growing Rec(-) or Rec(+) culture to low levels of penicillin causes the dividing cells to elongate. The size of the nonviable cells present in the Rec(-) cultures is unaffected. The nonviable cells are then separated from the elongated cells by velocity sedimentation. This isolation technique provides a convenient way of analyzing the composition, biosynthetic capacity, and enzymatic function of the nonviable cells before isolation. In this paper we present data showing that before fractionation the nonviable cells in the Rec(-) culture are defective in their ability to synthesize beta-galactosidase, whereas the Rec(-) viable cells behave like the Rec(+) cells in this regard. This observation confirms the existence of at least two classes of cells in liquid cultures of Rec(-) strains grown under normal conditions. That class of cells which is unable to synthesize beta-galactosidase is the same class that cannot form colonies when plated on solid medium.     

Plasmid replication and Hfr formation in strains of Escherichia coli carrying seg mutations.

Summary Several conditional-lethal mutations that do not permit the replication of F-factors ofEscherichia coli K-12 are located at a site calledseg. This gene is located on theE. coli chromosome betweenserB andthr. It is unrelated to other known genes involved in DNA replication. Strains carryingseg mutations were unable to replicate F-lac⁺, several F-gal⁺s, F-his⁺ and bacteriophage at 42°. However, neither phage T4, ColE1, nor any of the R factors tested were prevented from replicating at 42°C.When the kinetics of the loss of F-primes is studied inseg strains, it is found that the rate of curing depends on the size of the plasmid, larger F factors curing faster than smaller ones, and that Hfrs are formed at high frequencies. The Hfrs showed both F-genote enlargement and normal transfer of chromosomal markers. The F-genotes are unstable and segregate chromosomal markers at high frequencies. Some orthodox Hfrs were examined, and two that were known to revert to the F⁺ condition relatively frequently were found to generate enlarged F-genotes on mating, whereas two strains that were very stable with respect to reversion to the F⁺ state did not show F-genote formation.F-genote formation fromseg Hfr strains is dependent of a functionalrecA gene, as F-genote formation was not seen with aseg-2, recA-1 Hfr. This is in contrast to F-genote enlargement shown by both orthodox Hfrs and an Hfr strain constructed by integration of a temperature-sensitive F-gal⁺, whose F-genote enlargement is Rec-independent. Thus there may be more than one mechanism for the formation of enlarged F-genotes.     

Isolation of Circular Deoxyribonucleic Acid from Salmonella typhosa Hybrids Obtained from Matings with Escherichia coli Hfr Donors

Heterozygous, partial diploid Salmonella typhosa hybrids obtained from matings with Escherichia coli K-12 Hfr strains were observed to contain supercoiled, circular deoxyribonucleic acid (DNA) when examined by the dye-buoyant density method. Examination of one such S. typhosa hybrid after its loss, by segregation, of the inherited E. coli genetic markers revealed a concurrent loss of its supercoiled circular DNA. Subsequent remating of this segregant with various E. coli Hfr strains resulted in the reappearance of the circular DNA. Molecular weight determinations of circular DNA molecules isolated from a number of S. typhosa partial diploid hybrids were made by sucrose density gradient ultracentrifugation and electron microscopy. These studies revealed a range of molecular sizes among the various hybrids examined, but each hybrid exhibited only a single characteristic size for its contained circular DNA. The range of size is consistent with the presence in each hybrid of a different length of E. coli chromosome. It was concluded that the E. coli Hfr genetic segments transferred to these S. typhosa hybrids were conserved, in the diploid state, in the form of supercoiled, circular DNA molecules.     

Stability of a plasmid F Trim in populations of a recombination-deficient strain of Escherichia coli in continuous culture

Populations of a recA derivative of Escherichia coli AB1157 containing the plasmid F Trim were grown in carbon-limited continuous culture at dilution rates of 0.1 h^-1 to 0.4 h¹. The plasmid was lost after a lag, except in fermenter-experienced populations when it wwas retained. These results can be explained in terms of non-specific competition.     

Activation of RecA protein in recombination-deficient strains of Escherichia coli following DNA-damaging treatments.

Activation of the RecA protein following UV-irradiation or bleomycin (BM) treatment was measured in rec mutants of E. coli by monitoring beta-galactosidase activity. We provide evidence here that the defect in the recN mutant results in high constitutive and induced levels of activated RecA protein. In all rec mutants studied, with the exception of the recN mutant, induction of enzyme activity, following DNA-damaging treatments, was reduced relative to the wild type. The kinetics of induced sfiA expression indicates that the DNA-unwinding activity of the RecBCD enzyme plays a major role in SOS-signal formation. The RecF protein is not needed for BM induction in strains with a functional RecBCD pathway of recombination. However, a functional product of recF gene is implied in the formation of an efficient inducing signal after UV-irradiation, as well as in the additional processing of BM-induced lesions after exposure to the drug. A fully expressed RecF pathway of recombination does not provide a high level of activated RecA protein following DNA-damaging treatments.     

Isolation and characterization of Hfr strains of Erwinia amylovora

Hfr strains (Hfr 159 and its derivatives, Hfr 160 and Hfr 161) were constructed from Erwinia amylovora ICPB EA178 by introducing an Escherichia coli F'his+ plasmid and then selecting for integration of F'his+ after treatment with acridine orange. The Hfr strains were relatively stable upon repeated transfers on nonselective media. Interrupted mating experiments and analyses of inheritance of unselected markers showed that his+ is transferred by Hfr 159 as the proximal marker at a relatively high frequency (about 5 x 10(-4) recombinants per input donor cell), followed by ilv+, orn+, arg+, pro+, rbs+, met+, trp+, leu+, ser+, and thr+ (not necessarily in that precise order). The donor strains, previously constructed in E. amylovora by integration of F'lac+ from E. coli transfer cys+ as the proximal marker followed by ser+. Further analysis of one of those earlier donor strains, Hfr99, showed that ser+ is followed by arg+, orn+, met+, pro+, leu+, ilv+, rbs+, his+, trp+, and thr+ (not necessarily in that precise order). Thus, the Hfr strains constructed by integration of F'his+ are different, in terms of origin and direction of transfer, from those derived from integration of F'lac+. The applicability of these Hfr strains to mapping the genes on the E. amylovora chromosome is indicated.     

Spontaneous emergence of an Hfr strain with a cit plasmid from natural isolates of citrate-positive Escherichia coli bovine origin.

From citrate-utilizing (Cit+) Escherichia coli strain C53 of bovine origin, strains C53A and C53B were obtained. Upon mating with recA+ but not with recA mutant recipients of K-12, C53A produced chromosomal recombinants at quite high frequencies, leading to the following conclusions: (i) C53A is an Hfr strain; (ii) the site of integration of the Cit plasmid (IncH1) is between metA (89 min) and ara (1 min); (iii) the direction of chromosome transfer is clockwise; and (iv) the plasmid-associated determinants are transferred as the terminal markers. A transductant of a dnaA(Ts) strain, CRT46, which acquired Cit determinants from a recombinant, SG13, was also an Hfr strain similar to SG13, and thermoresistant due to suppressive integration. On the other hand, unstable C53B did not produce recombinants, but the frequency of RecA-independent transfer of the Cit plasmid was high, indicating that the Cit plasmid (IncH1) exists autonomously in C53B. Attempts to isolate an Hfr strain from C53B failed.     

Isolation and characteristics of sorbitol-fermenting Escherichia coli O157 strains from cattle

Cattle can be a reservoir of sorbitol-fermenting Escherichia coli O157 (SF E. coli O157) and a source of human diseases. In this study, six strains of SF E. coli O157 were isolated and characterized from cattle using an immunomagnetic separation procedure. PCR analysis of the SF E. coli O157 virulence markers showed that all six isolates tested positive for sfpA, rfbE and eaeA, and negative for terA, ureA, katP and espP. Two of the isolates contained the stx genes. Four isolates tested positive for enterohemorrhagic E. coli hlyA (EhlyA) by PCR but were nonhemolytic on the blood agar. Five isolates tested positive for the cdtA gene. The possession of these virulence factors was an indication of their pathogenic potential. The random amplified polymorphic DNA patterns, which were generated by the arbitrarily primed PCR of the SF E. coli O157 isolates from the cattle, were significantly different from those of the non-sorbitol-fermenting E. coli O157 (NSF E. coli O157) strains originating from cattle or humans. GelCompar analysis showed that the SF E. coli O157 isolates had only a 57% genetic similarity with the NSF E. coli strains. The minimal inhibitory concentration assay showed that imipenem inhibited the growth of the six isolates at a concentration of <4 microg/ml.     

New types of Escherichia coli recombination-deficient mutants.

A set of Escherichia coli mutants deficient in intramolecular recombination and different from those previously found is described. All have temperature-sensitive lethal mutations. The mutants have been characterized with respect to the following properties: the Pap phenotype, deoxyribonucleic acid synthesis, sensitivity to ultraviolet light, ability to support the growth of phage lambda, filament formation, and mutation frequency.     

Conservation and transfer of Escherichia coli genetic segments by partial diploid Hfr strains of Salmonella typhosa

Heterozygous, partial diploid hybrids were obtained in a Salmonella typhosa Hfr strain by using it as the recipient in a mating with the Escherichia coli Hfr donor WR2004 (O...proA...leu). Three of these S. typhosa Hfr hybrids were observed to mobilize and transfer the diploid E. coli genes, at high frequencies, to an E. coli recipient. The gradient of transfer frequencies of E. coli markers from these S. typhosa Hfr hybrids was similar to that observed with E. coli Hfr WR2004, from which they were derived. Interrupted matings with one of these S. typhosa Hfr hybrids, designated WR4272, showed the entry times for the proA, thr(-)leu, and argB E. coli diploid markers to be identical to the times obtained for these markers with E. coli Hfr WR2004. Also, the pattern of unselected inheritance of the diploid E. coli markers of S. typhosa Hfr hybrid WR4272 was similar to that observed with the chromosomal markers of E. coli Hfr WR2004. It was concluded that S. typhosa Hfr hybrid WR4272 contains, in addition to its Salmonella genome, a physically continuous E. coli chromosomal segment which is genetically complete from proA to at least the strA locus. The two other S. typhosa Hfr hybrids, on the basis of transmission frequency gradients, appeared to contain a continuous E. coli diploid segment complete from proA through the fuc locus. Other classes of S. typhosa Hfr hybrids, derived from mating with E. coli Hfr WR2010 (O...tna...xyl), were also observed to transfer E. coli genes at high frequency.