Superinfection with R Factors by Transduction in Escherichia coli and Salmonella typhimurium

Superinfection immunity is found in the conjugal transfer of R factors between two fi(+) R factors and between two fi(-) R factors (fi = fertility inhibition), as we reported previously. In contrast, no reduction in the frequencies of transduction of an fi(+) R factor 222 was caused by the presence of fi(+) R factors in the recipients in transduction systems with phage P1kc in Escherichia coli K-12 and with phage P22 in Salmonella typhimurium LT-2. The absence of superinfection immunity in transduction may be due to the difference in the route of entry of the R factor. The frequencies of transduction of an fi(+) R factor were reduced, although slightly, by the presence of fi(-) R factors in the recipients. This reduction is probably due to host-controlled restriction of the entering fi(+) R factor by the fi(-) R factors in the recipients, since transduction of an fi(+) R factor by the transducing phage propagated on the strain carrying both fi(+) and fi(-) R factors was not reduced by the presence of homologous fi(-) R factors in the recipients. The fi(+) R factor 222, when transduced to the recipient strains carrying other R factors, recombined genetically at high frequencies with these resident R factors, regardless of their fi type.     

Transduction of Various R Factors by Phage P1 in Escherichia coli and by Phage P22 in Salmonella typhimurium

R factors fi(+) and fi(-), with various combinations of drug-resistance markers and isolated from independent sources, were transduced by phage P1kc in Escherichia coli and by phage P22 in Salmonella typhimurium. Usually the entire R factor was transduced by P1kc in E. coli, as indicated by the absence of segregation of the drug-resistance markers from their conjugal transferability. In contrast, the patterns of segregation of the drug-resistance markers and their conjugal transferability differed considerably among various R factors after transduction by P22 in S. typhimurium. Transduction frequencies varied among R factors in both transduction systems.     

Episome-mediated transfer of drug resistance in Enterobacteriaceae. IX. Recombination of an R factor with F

Watanabe, Tsutomu (Keio University, Tokyo, Japan), and Chizuko Ogata. Episome-mediated transfer of drug resistance in Enterobacteriaceae. IX. Recombination of an R factor with F. J. Bacteriol. 91:43-50. 1966.-R factors can be transduced in Salmonella typhimurium with phage P-22, and a majority of the drug-resistant transductants are unable to transfer their drug resistance by cell-to-cell contact, as we have previously reported. Several exceptional types of transductants of S. typhimurium, with the markers of resistance to sulfonamide, streptomycin, and chloramphenicol, were recently obtained by transduction with phage P-22 of a four-drug-resistance R factor carrying the markers of resistance to sulfonamide, streptomycin, chloramphenicol, and tetracycline. They were exceptional in that they had low conjugal transferability of their drug resistance. When one of these exceptional transductants (38R) was transferred to an F(+) strain of Escherichia coli K-12, 38R acquired high transferability in its further transfer. This high transferability was found to be due to the recombination of 38R with F. Transductant 38R was of the fi(+) (fi = fertility inhibition) type, and did not show superinfection immunity against fi(+) and fi(-) R factors. The recombinant 38R.F was genetically very stable and resistant to elimination with acridines. It did not show superinfection immunity against fi(+) and fi(-) R factors, but did show superinfection immunity against F. Further, 38R.F did not restrict a female-specific phage (W-31), unlike wild-type F. F(-) and R(-) segregants were isolated from this recombinant 38R.F, and these segregants exhibited genetic characteristics different from the original R, its transductant 38R, and wild-type F.     

Episome-mediated Transfer of Drug Resistance in Enterobacteriaceae X. Restriction and Modification of Phages by fi R Factors

Watanabe, Tsutomu (Keio University, Tokyo, Japan), Toshiya Takano, Toshihiko Arai, Hiroshi Nishida, and Sachiko Sato. Episome-mediated transfer of drug resistance in Enterobacteriaceae. X. Restriction and modification of phages by fi(-) R factors. J. Bacteriol. 92:477-486. 1966.-An fi(-) R factor, which restricts phages lambda, T1, and T7 without modifying them, was found to restrict and not to modify an F(-)-specific phage, W-31, in Escherichia coli K-12, but not to restrict phage P-22 in Salmonella typhimurium LT-2, whereas other fi(-) R factors restricted and modified P-22 but not W-31; fi(+) R factors did not restrict these phages. Transduction and lysogenization with phages lambda and P-22 were reduced by these fi(-) R factors in K-12 and LT-2, respectively, and the transducing phages lambda and P-22 were modified by these fi(-) R factors. Spontaneous as well as ultraviolet-induced production of phage P-22 and zygotic induction of phage lambda were not significantly affected by any R factor. Injection of the nucleic acids of phages T1 and lambda was not affected by R factors, but the injected phage nucleic acids were rapidly broken down in the bacteria carrying fi(-) R factors. The nucleic acids of the modified phages were not broken down in these bacteria. It was assumed from these results that the mechanism of restriction of phages by fi(-) R factors is due to the breakdown of the injected phage nucleic acids by a deoxyribonuclease(s), presumably located near the cell surface in the cells carrying fi(-) R factors. The deoxyribonuclease(s), formed in the cells carrying the nonmodifying fi(-) R factor, is considered to be different from that synthesized in the cells carrying the modifying fi(-) R factors. It was further shown that the average burst sizes of the unmodified as well as modified phages are slightly reduced by the presence of the fi(-) R factors.     

Recombination Between a Thermosensitive Kanamycin Resistance Factor and a Nonthermosensitive Multiple-Drug Resistance Factor

The thermosensitive kanamycin (KM) resistance factor, R(KM)(t), and a nonthermosensitive multiple-drug resistance factor, R(100), were simultaneously introduced into Escherichia coli and Salmonella typhimurium. The temperature sensitivity of both R factors remained unchanged as long as they replicated independently. Under certain conditions, however, a new thermosensitive R factor harboring resistance markers for kanamycin, streptomycin (SM), and sulfanilamide (SA) was obtained by recombination between the R(KM)(t) and R(100) factors. R factors carrying resistance markers for KM and SA, or for SM and SA, were obtained from the recombinant R(KM SA SM)(t) by spontaneous segregation. Though the R(100) factor has been known as an fi(+) (positive for F-mediated fertility inhibition of its host) type and it does not restrict any coexisting phages, the thermosensitive recombinants of R(100) with R(KM)(t) and their segregants were found to be fi(-) and to restrict the replication of all T-even phages, as does the R(KM)(t) factor. Double infection immunity was not observed between the R(KM)(t) and R(100) factors.     

Mechanisms of siderophore iron transport in enteric bacteria.

Uptake of 55Fe- and 3H-labeled siderophores and their chronic analogues have been studied in Salmonella typhimurium LT-2 and Escherichia coli K-12. In S. typhimurium LT-2, at least two different mechanisms for siderophore iron transport may be operative. Uptake of 55Fe- and 3H-labeled ferrichrome and kinetically inert lambda-cis-chromic [3H]deferriferrichrome by the S. typhimurium LT-2 enb7 mutant, which is defective in the production of its native siderophore, enterobactin, appears to occur by two concurrent mechanisms. The first mechanism is postulated to involve either rapid uptake of iron released from the ferric complex by cellular reduction without penetration of the complex or ligand or dissociation of the complex and simultaneous uptake of both ligand and iron coupled with simultaneous expulsion of the ligand. The second mechanism appears to consist of slower uptake of the intact ferric complex.     

Thermosensitive Replication of a Kanamycin Resistance Factor

A strain of Proteus vulgaris isolated from the urinary tract of a patient with postoperative pyelonephritis and resistant to sulfonamide, streptomycin, tetracycline, and kanamycin (KM) was found to transfer only KM resistance by cell-to-cell conjugation. The genetic determinant controlling the transferable KM resistance was considered to be an R factor and was designated R (KM). Successive transfer of KM resistance was demonstrated also from Escherichia coli 20S0, which received the R (KM) factor, to other substrains of E. coli K-12 or Salmonella typhimurium LT-2. The transfer of the R (KM) factor was strongly affected by the temperature at which the mating culture was kept. The transfer frequency of R (KM) at 25 C was about 10(5) times higher than at 37 C. The R (KM) factor was spontaneously eliminated from the host bacterial cells when P. vulgaris was cultured at 42 C, but no elimination occurred at 25 C. This elimination of the R (KM) factor at elevated temperature was also observed when the R (KM) factor infected E. coli and S. typhimurium. On the other hand, a normal R factor could not be eliminated from the same E. coli host strain by cultivation at the higher temperature. We consider the thermosensitive transfer and the spontaneous elimination of the R (KM) factor at higher temperature to depend upon thermosensitive replication of the R (KM) factor.     

Hemin-Deficient Mutants of Salmonella typhimurium

Nine hemin-deficient mutants of Salmonella typhimurium LT2 were isolated as neomycin-resistant colonies. Five of these mutants could be stimulated by Delta-aminolevulinic acid (Delta-ALA), thus representing hemA mutants. Since S. typhimurium LT2 is not able to incorporate hemin, the identification of the mutants not stimulated by Delta-ALA was made on the basis of the simultaneous loss of catalase activity and cytochromes. The hemA gene was mapped by conjugation in the trp region, probably in the order purB-pyrD-hemA-trp; the episome FT(71)trp does not carry the hemA gene. Transductional intercrosses by phage P22 indicate that hemA 11, 12, 13, and 37 are at very closely linked sites, whereas hemA14 is at a more distant site in the same or an adjacent gene. No joint transduction was detected between hemA and trp or pyrF. The loci affected in the other hemin-deficient mutants were linked in conjugation to the pro(+) marker (frequency of linkage, 88 to 97%), but cotransduction of the two markers could not be obtained. The episome F lac hem purE, which originates from Escherichia coli K-12, could complement these hemin-deficient mutants of S. typhimurium LT2. As a result, the sequence of the markers on the chromosome of S. typhimurium LT2 is probably pro heme purE, analogous to the sequence found in E. coli K-12. Thus, the chromosome of S. typhimurium also possesses two hem regions, with a location similar to that described in E. coli K-12.     

Transforming activity of R- and Lac-plasmids of clinical strains of Gram-negative bacteria]

The isolated plasmid DNA of clinical strains of Gram-negative bacteria were shown to have transforming activity when E. coli strain 0600 and S. typhimurium strain LT-2 were used as recipients. The frequency of transformation depended on the recipient strain and the character of the plasmids. The presence of deletion mutants was revealed among the transformants. Such mutants occurred with varying frequency, most often in S. typhimurium strain LT-20; the reason for this phenomenon is at present under discussion. The transformation of plasmids controlling lactose splitting and their conjugation transfer into recipient S. typhimurium strain LT-2 is possible only under condition of using recipient (R+). The possibility of the formation of the cointegrate (R and lac plasmids) in recipient S. typhimurium strain LT-2 is discussed.     

Comparative nucleotide sequence analysis of growth-rate-regulated gnd alleles from natural isolates of Escherichia coli and from Salmonella typhimurium LT-2.

A comparative study of gnd genes from Escherichia coli strains isolated from natural populations and laboratory strains and from Salmonella typhimurium was undertaken. In the accompanying paper (G. J. Barcak and R. E. Wolf, Jr., J. Bacteriol. 170:365-371, 1988), we showed that the growth-rate-dependent regulation of gnd expression was conserved among four natural E. coli isolates and E. coli B/r in a manner qualitatively similar to that of the gene from E. coli K-12. Here, we report the DNA sequence of the 5' regulatory region and the first 125 codons of the structural gene for the five E. coli gnd genes and the gnd gene from S. typhimurium LT-2. The sequences differed from one another by 5% on the average. All sequences defined putative secondary structures of the mRNA leader, which were previously proposed to be important in the regulation of the K-12 gene. In addition, a sequence between codons 69 and 74, which is highly complementary to the ribosome-binding site of the mRNA, was conserved in all the genes. The sequence data are discussed with respect to potential regulatory consequences.     

Mapping and characterization of the nad genes in Salmonella typhimurium LT-2.

An ampicillin enrichment technique was used to isolate 39 nicotinic acid-requiring mutants of Salmonella typhimurium LT-2. Using interrupted-mating and transductional mapping procedures, three loci, designated nadA, nadB, and nadC, were identified. These loci mapped at 33, 82, and 6 min, respectively, on the S. typhimurium linkage map. The arrangement of the loci on the Salmonella linkage map corresponded closely to the nadA, nadB, and nadC loci on the Escherichia coli K-12 linkage map, indicating that the de novo pathway to nicotinamide adenine dinucleotide and the genes governing the enzymes involved in this pathway in S. typhimurium are very similar to those in E. coli. Evidence is also presented which indicates that the product of the nadC locus in S. typhimurium LT-2 is the enzyme quinolinic acid phosphoribosyltransferase. All nadC mutants of S. typhimurium secreted between 2 and 8 mumol of quinolinic acid per 100 ml of secretion medium. In addition, none of the nadC mutants isolated were able to grow in 10(-3) M quinolinic acid, whereas all nadA and nadB mutants of S. typhimurium grew well in the presence of quinolinic acid. Transductional crosses between nadB mutants provided evidence suggestive of more than one locus in the nadB region.     

Integration of Chloramphenicol Resistance Gene of an R Factor on Escherichia coli Chromosome

An unstable mutant R factor conferring only chloramphenicol (CM) resistance was obtained by spontaneous segregation. After storage in broth culture, a stable CM-resistant mutant was obtained and its CM-resistance could not be cured by treatment with acriflavine or transduced to a recombination-deficient strain of Escherichia coli K12. Recombinational analysis indicated that the cml gene governing CM resistance had been integrated into the E. coli chromosome and closely linked with met B locus. The cml gene was co-transduced with both met and arg markers by phage P1, and the linkage order was considered to be mtl-cml-met-arg-thi. When the strain carrying this chromosomal CM-resistance was infected with a transferable R (TC) factor capable of conferring tetracycline (TC) resistance, the CM-resistance became transferable by conjugation. This mechanism is considered to account for the formation of the recombinant R (TC.CM) factor.     

Export of glutathione by some widely used Salmonella typhimurium and Escherichia coli strains.

Significant levels of extracellular glutathione (GSH) were detected in aerobically grown cultures of some strains of Salmonella typhimurium LT-2 and in Escherichia coli K-12, B, and B/r but not in cultures of nine freshly isolated clinical isolates of E. coli. Cultures of S. typhimurium generally contained less total GSH (intracellular plus external) than did E. coli cultures. S. typhimurium TA1534 contained about 2 mM intracellular GSH and exported about 30% of its total GSH. The external GSH concentration increased logarithmically during exponential growth and peaked at about 24 microM in early-stationary-phase cultures. External accumulation of GSH was inhibited by 30 mM NaN3. GSH was predominantly exported in the reduced form. Two-dimensional paper chromatography of supernatants from cultures labeled with Na2(35)SO4 confirmed the presence of GSH and revealed five other sulfur-containing compounds in the media of S. typhimurium and E. coli cultures. The five unidentified compounds were not derivatives of GSH.     

Nature of Lactose-fermenting Salmonella Strains Obtained from Clinical Sources

Six of seven lactose-fermenting (lac(+)) Salmonella strains obtained from clinical sources were found to be capable of transferring the lac(+) property by conjugation to Salmonella typhosa WR4204. All of the six S. typhosa strains which received the lac(+) property transferred it in turn to S. typhimurium WR5000 at the high frequencies typical of extrachromosomal F-merogenotes. These six lac elements were also transmissible from S. typhosa WR4204 to Proteus mirabilis and to some strains of Escherichia coli K-12; moreover, they were capable of promoting low frequency transfer of chromosomal genes from S. typhimurium WR5000 to S. typhosa WR4204. One of these lac elements was shown also to be capable of promoting low frequency chromosome transfer in E. coli K-12. E. coli K-12 strains harboring these lac elements exhibited sensitivity to the male specific phage R-17. Sensitivity to R-17 was not detected in Salmonella strains containing the elements. Examination of the lac elements in P. mirabilis by cesium chloride density gradient centrifugation showed that each element had a guanine plus cytosine content of 50%. The sizes of the elements varied from 0.8 to 3% of the total Proteus deoxyribonucleic acid. The amount of beta-galactosidase produced by induced and uninduced cultures of S. typhimurium WR5000 and S. typhosa WR4204 containing the lac elements was lower than that produced by these strains with the F-lac episome. The heat sensitivity of beta-galactosidase produced by the lac elements in their original Salmonella hosts indicated that the enzyme made by these strains differs from E. coli beta-galactosidase.     

Altered growth-rate-dependent regulation of 6-phosphogluconate dehydrogenase level in hisT mutants of Salmonella typhimurium and Escherichia coli.

In Escherichia coli, the level of 6-phosphogluconate dehydrogenase is directly proportional to the cellular growth rate during growth in minimal media. This contrasts with the report by Winkler et al. (M. E. Winkler, J. R. Roth, and P. E. Hartman, J. Bacteriol. 133:830-843, 1978) that the level of the enzyme in Salmonella typhimurium LT-2 strain SB3436 is invariant. The basis for the difference in the growth-rate-dependent regulation between the two genera was investigated. Expression of gnd, which encodes 6-phosphogluconate dehydrogenase, was growth rate uninducible in strain SB3436, as reported previously, but it was 1.4-fold growth rate inducible in other S. typhimurium LT-2 strains, e.g., SA535. Both the SB3436 and SA535 gnd genes were growth rate inducible in E. coli K-12. Moreover, the nucleotide sequences of the regulatory regions of the two S. typhimurium genes were identical. We concluded that a mutation unlinked to gnd is responsible for the altered growth rate inducibility of 6-phosphogluconate dehydrogenase in strain SB3436. Transductional analysis showed that the altered regulation is due to the presence of a mutation in hisT, the gene for the tRNA modification enzyme pseudouridine synthetase I. A complementation test showed that the regulatory defect conferred by the hisT mutation was recessive. In E. coli, hisT mutations reduced the extent of growth rate induction by the same factor as in S. typhimurium. The altered regulation conferred by hisT mutations was not simply due to their general effect of reducing the polypeptide chain elongation rate, because miaA mutants, which lack another tRNA modification and have a similarity reduced chain growth rate, had higher rather than lower 6-phosphogluconate dehydrogenase levels. Studies with genetic fusions suggested that hisT mutations lower the gnd mRNA level. The data also indicated that hisT is involved in translational control of gnd expression, but not the aspect mediated by the internal complementary sequence.     

Molecular cloning of bacterial DNA in vivo using a transposable R6K ori and a P1vir phage.

A new method of cloning in vivo using the P1vir phage and transposon Tn5-rpsL oriR6K was developed. The method relies upon recircularization of transducing DNA containing a transposon insertion in a recombination-deficient strain of Escherichia coli K-12 and subsequent stable replication of the recircularized DNA. Using this method, we were able to clone in vivo the chromosomal region located between approximately 7.1 and 9.2 min on the E. coli K-12 map in a 95-kb plasmid.     

Effect of R factors and other plasmids on ultraviolet susceptibility and host cell reactivation property of Escherichia coli

Some R factors, like some colicin factors, confer partial protection against the bactericidal effect of ultraviolet (UV) irradiation. Of 31 plasmids (17 R, 3 col, and 11 R-col factors) tested in Escherichia coli K-12, 15 protected, 11 had little or no effect, and 5 caused increased UV susceptibility. The effect of representative plasmids was qualitatively the same in K-12 of wild-type UV sensitivity, lambda-lysogenic or non-lysogenic, and in UV-sensitive mutants of classes uvrA, uvrB, uvrC, and recA (except that a sensitizing factor did not increase the sensitivity of two recA hosts). It is inferred that the UV-protecting effect of some plasmids does not result from their specifying enzymes similar to those deficient in such mutants. UV killing of multiply auxotrophic K-12, of wild-type sensitivity or recA or uvrC, was reduced by deprivation of required amino acids for 2 hr before irradiation, and further reduced if "starvation" was continued for 2 hr after irradiation. The plasmids tested in these conditions produced qualitatively the same effects as in nonstarved cells-except that in K-12 of wild-type UV sensitivity the effect of protecting plasmids was reversed (i.e. they caused decreased survival) when the cells were starved after irradiation. Two UV-protecting R factors reduced the ability of HCR(+) K-12 to support growth of irradiated phage T1.     

Genetic exchange between Escherichia coli strains in the mouse intestine

Germ-free mice contaminated with selected Escherichia coli strains were used for experiments designed to demonstrate gene transfer and recombinant formation in vivo. The well-characterized conjugation system of E. coli K-12 was examined in these experiments. Contamination of germ-free mice with a polyauxotrophic F(-) strain followed by the addition of isogenic Hfr, F', or F(+) strains resulted in the appearance of all recombinant classes at frequencies that would be expected from an in vitro mating experiment. Inheritance of unselected donor markers occurred at frequencies that were dependent on linkage relationships established in experiments in vitro. The presence of Lactobacillus had no influence on gene transfer and recombinant formation in an F' x F(-) in vivo mating. The R factor ROR-1 was transferred from E. coli strain M7-18 to an E. coli F(-) strain in the mouse intestine.     

Growth-rate-dependent expression and cloning of gnd alleles from natural isolates of Escherichia coli.

6-Phosphogluconate dehydrogenase (6PGD), encoded by gnd, is highly polymorphic among isolates of Escherichia coli form natural populations. As a means of characterizing the growth-rate-dependent regulation of the level of 6PGD, five gnd alleles, including the E. coli B/r allele, were crossed into E. coli K-12 with bacteriophage P1. In each of the isogenic strains, the level of 6PGD was two- to threefold higher in cells grown on glucose than in cells grown on acetate. The level of enzyme activity in the acetate-grown cells varied about sixfold within the set of isogenic strains. The physiological importance of these differences in enzyme level is discussed. The gnd gene was cloned from five E. coli strains and Salmonella typhimurium LT-2 and mapped with twelve restriction endonucleases. gnd was located and oriented on the chromosomal DNAs. The restriction maps of the genes were aligned at conserved restriction sites, and the relative divergence of the genes was estimated from restriction site polymorphisms. The E. coli gnd genes differed from the S. typhimurium gene by about 11%. Most of the E. coli genes differed from one another by less than 5%, but one allele differed from the others by about 10%. Only the gnd gene from E. coli K-12 had an IS5 element located nearby.     

Covert fi− R Factors in fi− R+ Strains of Bacteria

The presence of an fi(-) sex factor can be detected by propagation of the I-specific phage If1. By use of this method of detection, a high proportion of strains with fi(+) R factors were shown also to carry an fi(-) factor which was frequently a second R factor. In some doubly R(+) strains, the fi(+) and the fi(-) factor were observed to be transferred independently at conjugation.