Phage P1kc Research Articles

GIV002 Modulation of Helicobacter pylori specific pathogenicity profile by gastric epithelial cells and granulocytes

Phage P1kc transduced nutritional and motility characters, at frequencies of 10−6 to 8 × 10−5/PFU, to some Salmonella typhimurium LT2 recipients making incomplete core LPS, viz all tested mutants of classes galE (deficient of UDPgalactose-epimerase), rfaH and rfaG (deficient of galactosyl- or glucosyl-LPS transferase) and galU (deficient of UDPglucose pyrophosphorylase) and one only of several mutants making complete core LPS. The rate of cotransduction of H1 with flaC, motA, and motB was 45–65% for P1kc, compared with 0.01–5% for P22. P1kc cotransduced hisC and flaD at about 1%. The numbers of trails obtained from certain fla motA recipients showed that most of the chromosomal fragments carrying motA which are present in P1kc transducing particles also carry flaA and flaB, compared with <0.4% of them in the case of P22 transducing particles. Phage P1kc grown on E. coli K12 or Shigella dysenteriae 16 was applied to restriction-negative S. typhimurium recipients making galactose-deficient LPS because of galE mutation. Complete transduction of trpB+ and metA+ was observed at frequencies ca. 1300 (for E. coli lysates) and ca. 110 (for Shigella lysates), of those obtained in a control intraspecies cross. Although no swarms were obtained the production of trails from nonmotile recipients showed that E. coli has functional equivalents of flaA, B, C, Q, K, F and other, unidentified, fla loci and also of motA and B of S. typhimurium, whereas strain 16 has only flaF (or, at least, a part of it); this suggests that in Shigella the main cluster of motility genes may be deleted.

Involvement of gntS in the control of GntI, the main system for gluconate metabolism in Escherichia coli.

The initial steps of gluconate metabolism in E. coli, transport and phosphorylation, occur through duplicate activities. These activities have been included in two systems designated as GntI (main) and GntII (subsidiary), encoded by differently regulated operons located at the 76.4-77 and 95.3-96.9 regions on the map respectively. Despite recent molecular advances related to genetics and physiology of these systems, there is no information about the coordination of their expression when E. coli grows on gluconate. Under these conditions, the subsidiary gluconokinase (gntV gene, min 96.8) as well as the GntI activities are expressed in inducible form. Therefore it was of interest to find out if GntS, the positive regulator of gntV has a similar effect on GntI activities expression. Our results agree with this hypothesis. GntS, in addition to its regulatory action on the gntV gene, seems to assist, direct or indirectly, the expression of the GntI activities. A gntS E. coli mutant does not grow on gluconate but spontaneously pseudoreverts to a gluconate growing phenotype at high rate per cell generation when cultivated in rich media with or without gluconate or mineral medium containing any other suitable carbon source. In the pseudorevertants, the thermosensitive gluconokinase remains repressed while the GntI activities are inducibly expressed. At present, the location and nature of the gntS suppressor mutation are not known. Phage P1Kc mediated transductions have ruled out that it alters the gntT gene. This is the first report on GntI activities alteration due to a lesion located out of the bioH-asd region.

Genetic Localization of the Resistance to Fosfomycin

The possible existence of a transferable resistance to fosfomycin was studied in 50 strains of enterobacteria that were isolated in our hospital and which showed a resistance to fosfomycin of more than 256 mug/ml. E. coli K12 E711 was used as a receptor strain in the conjugation -- the times of conjugation were 20 min and 18 h. We found a transferable resistance in 28% of the strains. In all but one of the cases, only the resistance to fosfomycin was transferred. The recombinant which received the resistance to fosfomycin together with three other resistances was used as the donor for the transduction with the phage P1Kc to the receptor strain which was E. coli UTH 1038. A study was carried out on the influence of the radiation of the transducing phages with ultraviolet light on the frequency of transduction. The findings demonstrated a linear decrease in the frequency in proportion to the increase in the time of radiation.

Integration, at hag or elsewhere, of H2 (phase-2 flagellin) genes transduced from Salmonella to Escherichia coli.

A fla mutant of E. coli K12 was given fla+ and H1-i by phage P1kc cotransduction from S. typhimurium, then made Fla- by transduction of ah1 from S. typhimurium. Motile clones expressing a Salmonella phase-2 antigen, e,n,x or 1,2, were obtained from the K12 i ah1 (therefore Fla-) line by P1kc transduction of flagellin-specifying genes, H2-e,n,x or H2-1,2, from Salmonella donors. Of eighteen such transductants sixteen failed to show phase variation, and on transduction back to Salmonella each structural gene for a phase-2 flagellin (or at least for its antigenically determinant part) now behaved as an allele of H1, presumably in consequence of incorporation in the hag region of the K12 recipient, in place of H1-i ah1. The e,n,x- and 1,2-specifying genes were shown to have been integrated in the K12 chromosome without the linked H1-repressor gene or the adjacent vh2 gene (controlling rate of phase-variation) and they responded to the repressing activity of an H2 allele elsewhere in the cell, in this respect resembling H1 alleles of Salmonella or hag alleles of E. coli. Two K12 e,n,x transductants had flagellin-specifying genes which when transduced back to Salmonella were integrated at H2; they are inferred to have resulted from integration of H2-e,n,x in the K12 chromosome elsewhere than the hag region. These two clones showed phase variation, between a Fla+ phase, with antigen e,n,x, and a Fla- phase (with e,n,x determinant in the nonactive state and the determinant of antigen i inactivated by ah1). The two integrated e,n,x genes when in the "active" state retained the ability to repress expression of exogenote H1 alleles, which indicates that the closely linked H1-repressor gene also was integrated. One of the two exceptional transductants derived its e,n,x gene from a Salmonella donor with the linked vh2- gene, which in Salmonella almost entirely prevents change of phase, and transduction of this e,n,x gene back to Salmonella recipients proved that vh2- had been incorporated into the E. coli chromosome along with the e,n,x determinant and the H1-repressor gene. The high frequency of change of phase (Fla+ in equilibrium Fla-) in the K12 e,n,x vh2- transductant concerned suggests that vh2- fails to prevent frequent change of state of the phase-determined part of H2 when vh2- and H2 are incorporated in the E. coli chromosome.

Transduction by phage P1 kc in Salmonella typhimurium

Phage P1 kc transduced nutritional and motility characters, at frequencies of 10 −6 to 8 × 10 −5/PFU, to some Salmonella typhimurium LT2 recipients making incomplete core LPS, viz all tested mutants of classes galE (deficient of UDPgalactose-epimerase), rfaH and rfaG (deficient of galactosyl- or glucosyl-LPS transferase) and galU (deficient of UDPglucose pyrophosphorylase) and one only of several mutants making complete core LPS. The rate of cotransduction of H1 with flaC, motA, and motB was 45–65% for P1 kc, compared with 0.01–5% for P22. P1 kc cotransduced hisC and flaD at about 1%. The numbers of trails obtained from certain fla motA recipients showed that most of the chromosomal fragments carrying motA which are present in P1 kc transducing particles also carry flaA and flaB, compared with <0.4% of them in the case of P22 transducing particles. Phage P1 kc grown on E. coli K12 or Shigella dysenteriae 16 was applied to restriction-negative S. typhimurium recipients making galactose-deficient LPS because of galE mutation. Complete transduction of trpB + and metA + was observed at frequencies ca. 1 300 (for E. coli lysates) and ca. 1 10 (for Shigella lysates), of those obtained in a control intraspecies cross. Although no swarms were obtained the production of trails from nonmotile recipients showed that E. coli has functional equivalents of flaA, B, C, Q, K, F and other, unidentified, fla loci and also of motA and B of S. typhimurium, whereas strain 16 has only flaF (or, at least, a part of it); this suggests that in Shigella the main cluster of motility genes may be deleted.

Transduction of resistance determinants and R factors of the transfer systems by phage Plkc.

Transduction by P1ke shows that Δ-mediated R factors fall into two groups: those in which the resistance and the Δ transfer factor are transduced as a single unit; and those in which the resistance determinant is transduced independently of the transfer factor. The first group is exemplified by the T-Δ R factor, which is transferable after transduction. An example of the second group is the SSu,Δ R factor, in which the SSu determinant is transduced independently to recipient cells. The SSu resistance is therefore not transferable until Δ is introduced into these recipients. These observations support the postulate, originally based on conjugational observations, that R factors are of two classes. In Class 1 the resistance determinant and the transfer factor form a single covalently bonded complex which is transferred intact to recipient cells; T-Δ belongs to this class. In Class 2 the resistance determinant and the transfer factor are separate plasmids. Although the transfer factor is necessary for transfer of the determinant in this class, independence of the plasmids is maintained in new hosts, and the nature of the association between the respective plasmids during transfer requires clarification.

Isolation and characterization of a new generalized transducing bacteriophage different from P1 in Escherichia coli.

A new generalized transducing bacteriophage in the Escherichia coli system was isolated and characterized. This phage, designated D108, makes clear plaques on E. coli K-10, K-12, K-12(P1kc), K-12(D6), B/r, C, and 15 T(-), and Shigella dysenteriae. The plaque of phage D108 is larger in size than that of phage P1kc. Electron-microscopic observation revealed that phages D108 and P1kc are morphologically different from each other, suggesting that phage D108 belongs to a phage group different from phage P1. The fact that all of the 10 markers tested were transduced by phage D108 indicates that this phage is a generalized transducing phage in the E. coli system. The transduction frequency by phage D108 of chromosomal markers and of a drug resistance factor (R factor) ranged from 2 x 10(-6) to 3 x 10(-8) and 3 x 10(-9) to 6 x 10(-10) per phage, respectively. The cotransduction frequency of the thr and leu markers was 2.8% for phage P1kc and 1.5% for phage D108. The CM and TC markers (chloramphenicol-resistant and tetracycline-resistant markers, respectively) of the R factor were not cotransduced by phage D108, but the markers were generally cotransduced by phage P1kc. The results suggest that the transducing particle of phage D108 contains a smaller amount of host deoxyribonucleic acid than does phage P1kc.

Biochemical and genetic studies with nitrate reductase C-gene mutants of Escherichia coli.

Twenty-eight narC (chlC) mutants of Escherichia coli were isolated and characterised by their resistance to chlorate, inability to use nitrate as terminal electron acceptor and positive gas reaction. The extent of gas production by the majority of mutants was almost normal but quantitative differences ranging from 40 to 100% of wild-type activity were found. Biochemical studies showed that all the mutants lacked nitrate reductase, decreasing gas production was correlated with a simultaneous decrease in formate dehydrogenase activity and the lowest gas production was due to deficiencies in formate dehydrogenase and hydrogenase. The position of narC relative to other loci was determined as: purB ... hemA ... narC ... supIII,C ... galU ... attΦ80 ... tonB ... trp ... cysB by transduction analysis, and the mutant sites of 6 strains representing the complete range of gas reactions were clustered at this position. It is suggested that narC is the structural gene for nitrate reductase and the variations in phenotype may be due to polarity effects on neighbouring genes specifying components of the formate hydrogenlyase system. Transduction of narC by Φ80 could not be detected but an effect of galU− on phage P1kc susceptibility was demonstrated.

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.

Superinfection with R Factors by Transduction inEscherichia coliandSalmonella 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.

Physical evidence for the integration of prophage P1 into the Escherichia coli chromosome

The relationship of the phage P1 chromosome to the chromosome in lysogenic bacteria has been investigated. 32P-labeled bacterial DNA isolated from logphase Escherichia coli K12 W3110, either lysogenic or non-lysogenic for phage P1kc, was sedimented through a sucrose gradient (5 to 20%) and fractions were removed for DNA-DNA hybridization on P1 DNA-agar. Approximately 2% of any fraction tested was specifically bound to P1 DNA. The binding specificity was demonstrated by using both W3110 and W3110 (P1) DNA, and by the interference exhibited by P1 DNA, and not W3110 DNA, for the binding of W3110 (P1) DNA to P1 DNA-agar. The finding that a constant percentage of the W3110 (P1) DNA, recovered from any part of the sucrose gradient binds specifically to P1 DNA is interpreted to mean that the P1 prophage DNA is an integrated part of the bacterial chromosome. The approximate 2% specific hybridization suggests that there is one P1 prophage associated with each chromosome in a lysogenic bacterium.

The genetic constitution of the radiation-sensitive mutant Escherichia coli Bs-1.

Abstract The radiation-sensitive strains Escherichia coli B s−1 and B s−1 are both unable to repair UV and X-ray damage in their DNA; in addition the B s−1 strain is also unable to repair certain UV-irradiated bacteriophages. In this respect the latter strain resembles Hcr − mutants of E. coli K12 strains, which are also UV sensitive, but, in contrast to E. coli B s−1 , X-ray resistant. By means of transduction analysis with phage P1kc it is shown that strain B s−1 is in fact a double mutant, carrying an Hcr − -like mutation, located on the chromosome near the gal locus, and a second mutation near met 3 , denoted exr − , which is responsible for the X-ray sensitivity. Both mutations influence the UV sensitivity of the B s−1 strain. The B s−2 mutant contains the exr − mutation only, which also accounts for the UV sensitivity of this strain.

Loss and repair of conjugal fertility and infectivity of the resistance factor and sex factor in Escherichia coli.

Hirota, Yukinori (University of Osaka, Osaka, Japan), Toshio Fujii, and Yukinobu Nishimura. Loss and repair of conjugal fertility and infectivity of the resistance factor and sex factor in Escherichia coli. J. Bacteriol. 91:1298-1304. 1966.-The drug-resistance factor, R, and the sex factor, F, have homologous traits, including contagious transmission, mediation of sexuality of the host cell, and autonomous replication in their host bacteria. Cooperation between F and R factors was found with a mutant R factor, which is nontransmissible in F(-) bacteria, becoming transmissible when introduced into bacteria carrying F. Conversely, the chromosome of a sterile male strain carrying the mutant sex factor, F(r), becomes transmissible when an R factor is introduced into the cell. The genetic determinants of R factors have been analyzed by isolation of mutant R factors, by sexual conjugation of the host bacteria, and by transduction of R factors with phage P1kc. The fertility determinant of the R factor, m, is inseparable from the determinant for its infectivity, but can be separated from the loci for autonomous replication of the R factor. R and F thus carry genetic determinants governing the same functions.

Transducing fragments in generalized transduction by phage P1: I. Molecular origin of the fragments

Bromouracil-labelled Escherichia coli thy− was infected with a virulent mutant of phage P1kc and incubated in a medium containing thymine until lysis occurred. The analysis of the phage yield by CsCl density-gradient centrifugation showed that the transducing particles carrying various chromosomal genes have a density and band profile similar to those prepared on bromouracil-labelled bacteria in a medium containing bromouracil. When the phage particles were prepared on the bromouracil-labelled bacteria in a medium containing thymine and 32PO4, radioactivity was not found in fractions which contained transducing particles. These reults indicate that most of the transducing particles lack phage genome and carry only fragments of the bacterial chromosome existing at the time of infection. The results show interruption of the replication of bacterial chromosome by phage infection. The replication of λ prophage and F′ lac was also interrupted by infection. On the other hand, the replication of an R factor was not arrested. In contrast to the behaviour of the virulent mutant, the original P1kc phage did not arrest the replication of bacterial chromosome. When the virulent mutant was grown on [3H]thymidine-labelled bacteria in a medium containing 32PO4, infective particles and transducing particles could be differentially labelled with 32P and 3H, respectively. The results of the analysis of the phage particles showed that the transducing particles comprised 0·3% of the total phage particles. Physical properties of DNA's of infective and transducing particles were studied. The density of P1 DNA is 1·706 g cm−3, which corresponds to a guanine–cytosine content of 46%. The molecular weights of DNA's of infective and transducing particles are the same, namely, 6 × 107 daltons. Homology between PI DNA and E. coli DNA is rare.

Effect of Integrated Sex Factor on Transduction of Chromosomal Genes in Escherichia coli

Pittard, James (Yale University, New Haven, Conn.). Effect of integrated sex factor on the transduction of chromosomal genes in Escherichia coli. J. Bacteriol. 89:680-686. 1965.-Generalized transducing phage P1kc can contransduce the markers tna and ilv in Escherichia coli K-12 at a frequency of approximately 25%. When sex factor is integrated into the donor chromosome between tna and ilv, as in Hfr AB313, the entire sequence tna F ilv is not cotransduced at a detectable frequency, nor is F cotransduced with either tna or ilv. The frequency with which ilv is transduced from Hfr AB313 to female recipients is approximately one-eighth the frequency obtained when either F(+) or female strains are used as donors. This reduction of transduction frequency does not occur if the recipient is an Hfr male similar to AB313. The reduction is attributed to pairing difficulties which occur when the transduced fragment carrying the ilv genes also carries sex-factor material. When ilv(+) is transduced from a female to an ilv(-) derivative of AB313, 70% of the transductants are converted to F(+) males. A model is proposed to account for all of these observations.

EPISOME-MEDIATED TRANSFER OF DRUG RESISTANCE IN ENTEROBACTERIACEAE VIII

Watanabe, Tsutomu (Keio University School of Medicine, Tokyo, Japan), Chizuko Ogata, and Sachiko Sato. Episome-mediated transfer of drug resistance in Enterobacteriaceae. VIII. Six-drug-resistance R factor. J. Bacteriol. 88:922-928. 1964.-The multiple-drug-resistant Escherichia coli strain isolated by Lebek in 1963 was found to transfer resistance to sulfonamide, streptomycin, chloramphenicol, tetracycline, kanamycin, and neomycin together by conjugation, as well as by transduction with phage P1kc, suggesting that these drug-resistance markers are carried by a single R factor (R(6)). The results of transductional and spontaneous segregations of the drug-resistance markers of R(6) have shown that R(6) has independent genetic determinants for sulfonamide, streptomycin, chloramphenicol, tetracycline, and kanamycin-neomycin resistance. Resistance to kanamycin and neomycin is probably controlled by a single gene, because no segregation was observed between these two. The resistance transfer factor of R(6) was found to be of the fi(+) type.

Episome-mediated transfer of drug resistance in Enterobacteriaceae. III. Transduotion of resistance factors.

Watanabe, Tsutomu (Keio University, Tokyo, Japan), and Toshio Fukasawa. Episome-mediated transfer of drug resistance in Enterobacteriaceae. III. Transduction of resistance factors. J. Bacteriol. 82:202-209. 1961.-Transmissible multiple drug resistance of Enterobacteriaceae, which includes resistance to streptomycin (SM), chloramphenicol (CM), tetracycline (TC), and sulfonamide (Su), was found to be transduced in the systems of Salmonella typhimurium strain LT-2 with phage P-22 and Escherichia coli strain K-12 with phage P1kc. No linked chromosomal markers to the resistance factors have so far been found in the substrains of K-12. The transductants of K-12 were all found to be able to transfer their resistance factors by conjugation, whereas a majority of those of LT-2 could not transfer their resistance factors by conjugation. The resistance factors were segregated rarely in K-12 and consistently in LT-2. Defective resistance factors could be transferred by conjugation to the transductants of LT-2. The transductants thus made resistant to the four drugs were able to transfer only the resistance factors which had been introduced by conjugation and none of those which had been transduced. The patterns of segregation of the resistance factors by transduction suggested that they are located in the following sequence; Su-----Sm-----Cm-----Tc, and they were assumed to be carried by an episome "resistance transfer factor" which is considered to be located at the distal end of Tc.

Transduction and recombination study of linkage relationships among the genes controlling tryptophan synthesis in Escherichia coli

The relationship between gene sequence and the sequence of biochemical reactions in the biosynthesis of tryptophan was investigated in the K12 strain of Escherichia coli. Transduction with phage P1 kc was employed for genetic mapping. All the tryptophan auxotrophs examined were found to be mutant in a small region of genetic material (>80% joint transduction of the most distal markers). One strain with a block in the synthesis of several aromatic compounds, including tryptophan, was found to be mutant at a site weakly linked to the other tryp genes. Two cysteine auxotrophs were also mutant at sites linked to the tryp region. The transduction tests employed suggested the following order of the tryp genes and the cysteine markers: cys … tryp 4. tryp 3. tryp 1. tryp 2. This order differs from the gene order established with the corresponding mutants of Salmonella typhimurium only with respect to the last two tryp genes, tryp 1 and tryp 2. However, since the proteins controlled by these genes are both involved in the terminal reaction in tryptophan synthesis, and thus cannot be assigned a biosynthetic order, the results obtained indicate that the clustered tryp genes of E. coli are arranged in the same relative order as the steps in the biosynthesis of tryptophan which they control. An examination of the category of tryptophan auxotrophs which acquired a requirement for tryptophan simultaneously with mutation to resistance to phage T1 suggested that these strains arose as a result of deletions including the V 1 r locus and one or more tryp genes. It was found that in strain B, where mutations of this type are fairly frequent, most of the V 1 r tryp mutants lack all the clustered tryp genes. In strain K12, where V 1 r tryp mutants are rare among V 1 r isolates, the few strains obtained did not lack all the clustered tryp genes. Studies with hybrids formed between strains K12 and B suggested that genic material at or near the tryp region in the two strains determines the type of mutation pattern when cells are exposed to T1. The deletion from one V 1 r tryp mutant was transferred by transduction and 60% of the phage carrying the tryp 4 marker were found to carry the entire tryp region and thus were capable of converting tryp deletion mutants to tryptophan-independent strains.