Effect of Lysogeny on Serum Sensitivity

When Escherichia coli K-12 was infected with lambda phage and mutants of lambda characterized by the production of temperature-sensitive repressors, the lysogenic bacteria were significantly more resistant to normal serum than the uninfected organisms. Infection of E. coli K-12 with a lambdoid phage, phi80, whose prophage attachment site is different from that of lambda, did not result in a detectable change in serum resistance. Similarly, infection with certain Pseudomonas and Shigella phages caused no detectable differences in serum resistance. Finally, the well-known conversion of the Salmonella anatum serotype to S. newington by E(15) phage indicated that, despite the relatively greater roughness of S. anatum, S. newington was more sensitive to normal serum than S. anatum. Thus, the effects of lysogeny on the sensitivity of bacteria to the bactericidal action of serum mediated by the complement system may be quite variable.     

Evidence for a New Endonuclease Synthesized by λ Bacteriophage

Infection of nonlysogenic Escherichia coli CR34(S) (Thy(-)) with bacteriophage lambda C(I)857 resulted in the formation of twisted circular double-stranded phage deoxyribonucleic acid (DNA; species I). When such infected bacteria were incubated in the absence of thymine, there was a significant decrease in the amount of species I DNA after 60 min of incubation. A similar loss of species I lambda DNA during incubation in a thymine-deficient medium was also observed after infection of the endonuclease I-deficient strain, E. coli 1100(S) (Thy(-)). This destruction of twisted, circular lambda DNA in thymine-deprived cells did not occur in the presence of chloramphenicol nor in lysogenic E. coli CR34 carrying a noninducible lambda prophage. It is therefore concluded that the endonuclease which attacks this circular configuration of lambda DNA is newly synthesized after infection and is directed by the phage chromosome.     

Conditionally lethal amber mutations in the dnaA region of the Escherichia coli chromosome that affect chromosome replication.

Three amber mutations, dna-801, dna-803, and dna-806, were isolated by localized mutagenesis of the dnaA-oriC region of the chromosome from an Escherichia coli strain carrying temperature-sensitive amber suppressors. When the mutations were not suppressed at 42 degrees C, the cells did not grow and DNA synthesis was arrested. They were very closely linked to each other and to the dnaA46 mutation. The mutant phenotype of each strain was converted to the wild type by infecting the mutants with specialized transducing phase lambda i21 dnaA-2 but not with lambda i21 tna. Derivatives of lambda i21 dnaA-2, each of which carried the amber mutation dna-801 dna-803, or dna-806, converted the dnaA mutant phenotype to Dna+ but did not convert rhe amber mutants to the wild-type phenotype. E. coli uvrB cells were irradiated with ultraviolet light and infected with each of these phage strains. An analysis of proteins synthesized in the cells revealed that two proteins with molecular weights of 50,000 and 43,000 were specified by lambda i21 dnaA-2 but not by lambda i21 tna. When the ultraviolet-irradiated cells did not carry an amber suppressor, the derivative phage with the amber mutation invariably failed to produce the 43,000-dalton protein, but when the host cell carried supF (tyrT), the protein was produced. The 50,000-dalton protein was unaffected.     

An Escherichia coli gene required for bacteriophage P2-lambda interference.

The gene old of bacteriophage P2 is known to (i) cause interference with phage lambda growth; (ii) kill recB- mutants of Escherichia coli after P2 infection; and (iii) determine increased sensitivity of P2 lysogenic cells to X-ray irradiation. In all of these phenomena, inhibition of protein synthesis occurs. We have isolated bacterial mutants, named pin (P2 interference), able to suppress all of the above-mentioned phenomena caused by the old+ gene product and the concurrent protein synthesis inhibition. Pin mutations are recessive, map at 12 min on the E. coli map, and identify a new gene. Satellite bacteriophage P4 does not plate on pin-3 mutant strains and causes cell lethality and protein synthesis inhibition in such mutants. P4 mutants able to grow on pin-3 strains have been isolated.     

Alteration of active transport after bacteriophage T5 infection.

Bacteriophage T5 absorption immediately followed by injection of the first-step-transfer DNA segment produces alterations in the bacterial membrane which reduce the uptake of amino acids and of o-nitrophenyl-beta-D-galactopyranoside. Concomitantly, intracellular ATP is hydrolyzed. The extent of inhibition of uptake and of ATP hydrolysis is cooperatively increased with the multiplicity of infection. Inhibition of active transport at a high multiplicity of infection (greater than 3) is also observed after the second step of DNA injection. In contrast, at low multiplicities of infection, phage proteins are able to enhance amino acid uptake. Infection of su- bacteria with amber mutants in the first-step-transfer DNA suggests that protein A1 is implicated in this enhancement.     

In vivo and in vitro functional alterations of the bacteriophage lambda receptor in lamB missense mutants of Escherichia coli K-12

lamB is the structural gene for the bacteriophage lambda receptor in Escherichia coli K-12. In vivo and in vitro studies of the lambda receptor from lamB missence mutants selected as resistant to phage lambda h+ showed the following. (i) Resistance was not due to a change in the amount of lambda receptor protein present in the outer membrane but rather to a change in activity. All of the mutants were still sensitive to phage lambda hh*, a two-step host range mutant of phage lambda h+. Some (10/16) were still sensitive to phage lambda h, a one-step host range mutant. (ii) Resistance occurred either by a loss of binding ability or by a block in a later irreversible step. Among the 16 mutations, 14 affected binding of lambda h+. Two (lamB106 and lamB110) affected inactivation but not binding; they represented the first genetic evidence for a role of the lambda receptor in more than one step of phage inactivation. Similarly, among the six mutations yielding resistance to lambda h, five affected binding and one (lamB109) did not. (iii) The pattern of interactions between the mutated receptors and lambda h+ and its host range mutants were very similar, although not identical, in vivo and in vitro. Defects were usually more visible in vitro than in vivo, the only exception being lamB109. (iv) The ability to use dextrins as a carbon source was not appreciably affected in the mutants. Possible working models and the relations between phage infection and dextrins transport were briefly discussed.     

Discovery of a protein phosphatase activity encoded in the genome of bacteriophage lambda. Probable identity with open reading frame 221.

Infection of Escherichia coli with phage lambda gt10 resulted in the appearance of a protein phosphatase with activity towards 32P-labelled casein. Activity reached a maximum near the point of cell lysis and declined thereafter. The phosphatase was stimulated 30-fold by Mn2+, while Mg2+ and Ca2+ were much less effective. Activity was unaffected by inhibitors 1 and 2, okadaic acid, calmodulin and trifluoperazine, distinguishing it from the major serine/threonine-specific protein phosphatases of eukaryotic cells. The lambda phosphatase was also capable of dephosphorylating other substrates in the presence of Mn2+, although activity towards 32P-labelled phosphorylase was 10-fold lower, and activity towards phosphorylase kinase and glycogen synthase 25 50-fold lower than with casein. No casein phosphatase activity was present in either uninfected cells, or in E. coli infected with phage lambda gt11. Since lambda gt11 lacks part of the open reading frame (orf) 221, previously shown to encode a protein with sequence similarity to protein phosphatase-1 and protein phosphatase-2A of mammalian cells [Cohen, Collins, Coulson, Berndt & da Cruz e Silva (1988) Gene 69, 131-134], the results indicate that ORF221 is the protein phosphatase detected in cells infected with lambda gt10. Comparison of the sequence of ORF221 with other mammalian protein phosphatases defines three highly conserved regions which are likely to be essential for function. The first of these is deleted in lambda gt11.     

Behavior of coliphage lambda in hybrids between Escherichia coli and Salmonella

Salmonella typhosa hybrids able to adsorb lambda were obtained by mating S. typhosa recipients with Escherichia coli K-12 donors. After adsorption of wild-type lambda to these S. typhosa hybrids, no plaques or infective centers could be detected. E. coli K-12 gal(+) genes carried by the defective phage lambdadg were transduced to S. typhosa hybrids with HFT lysates derived from E. coli heterogenotes. The lysogenic state which resulted in the S. typhosa hybrids after gal(+) transduction differed from that of E. coli. Ability to produce lambda, initially present, was permanently segregated by transductants of the S. typhosa hybrid. S. typhosa lysogens did not lyse upon treatment for phage induction with mitomycin C, ultraviolet light, or heat in the case of thermoinducible lambda. A further difference in the behavior of lambda in Salmonella hybrids was the absence of zygotic induction of the prophage when transferred from E. coli K-12 donors to S. typhosa. A new lambda mutant class, capable of forming plaques on S. typhosa hybrids refractory to wild-type lambda, was isolated at low frequency by plating lambda on S. typhosa hybrid WR4254. Such mutants have been designated as lambdasx, and a mutant allele of lambdasx was located between the P and Q genes of the lambda chromosome. Plaques were formed also on the S. typhosa hybrid host with a series of lambda(i21) hybrid phages which contain the N gene of phage 21. The significance of these results in terms of Salmonella species as hosts for lambda is discussed.     

Effect of bacteriophage lambda infection on synthesis of groE protein and other Escherichia coli proteins.

We used two-dimensional gel electrophoresis to quantitate the changes in rates of synthesis that follow phage lambda infection for 21 Escherichia coli proteins, including groE and dnaK proteins. Although total protein synthesis and the rates of synthesis of most individual E. coli proteins decreased after infection, some proteins, including groE protein, dnaK protein, and stringent starvation protein, showed increases to rates substantially above their preinfection rates. Infection by lambda Q- affected host synthesis in the same way as infection by gamma+, whereas infection by lambda N- showed no detectable effect on host synthesis. Deletion of the early genes between att and N abolished the effect, and shorter deletions in this region gave intermediate effects. By this sort of deletion mapping, we show that a large part, though not all, of the effect of lambda infection on host protein synthesis can be ascribed to the early region that contains phage genes Ea10 and ral. We compared the changes in protein synthesis after infection with the changes that occur in uninfected cells upon heat shock or amino acid starvation. The spectrum of changes that occurred on infection was very different from that seen after heat shock but quite similar to that seen during amino acid starvation. Despite this similarity of the effects of lambda infection and starvation, we did not detect any increase in the level of guanosine tetraphosphate during infection. We show that the groE protein is the same protein as B56.5 of Lemaux et al. (Cell 13:427-434, 1978) and A protein of Subramanian et al. (Eur. J. Biochem. 67:591-601, 1976).     

SOS induction in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of complementary-strand DNA synthesis.

We report that the SOS response is induced in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of the complementary (minus)-strand synthesis. One such mutant, R377, which lacks the entire region of the minus-strand origin, failed to synthesize any detectable amount of primer RNA for minus-strand synthesis. In addition, the rate of conversion of parental single-stranded DNA of the mutant to the double-stranded replicative form in infected cells was extremely slow. Upon infection, R377 induced the SOS response in the cell, whereas the wild-type phage did not. The SOS induction was monitored by (i) induction of beta-galactosidase in a strain carrying a dinD::lacZ fusion and (ii) increased levels of RecA protein. In addition, cells infected with R377 formed filaments. Another deletion mutant of the minus-strand origin, M13 delta E101 (M. H. Kim, J. C. Hines, and D. S. Ray, Proc. Natl. Acad. Sci. USA 78:6784-6788, 1981), also induced the SOS response in E. coli. M13Gori101 (D. S. Ray, J. C. Hines, M. H. Kim, R. Imber, and N. Nomura, Gene 18:231-238, 1982), which is a derivative of M13 delta E101 carrying the primase-dependent minus-strand origin of phage G4, did not induce the SOS response. These observations indicate that single-stranded DNA by itself induces the SOS response in vivo.     

Bacteriophage T4 RNA ligase: preparation of a physically homogeneous, nuclease-free enzyme from hyperproducing infected cells.

Infection of Escherichia coli by a bacteriophage T4 regA, gene 44 double mutant leads to about a 7-fold increase in the amount of RNA ligase obtained after infection by wild-type phage. Using cells infected by the double mutant, RNA ligase was purified to homogeneity with a 20% yield. Unlike previous preparations of this enzyme, the ligase is free of contaminating nuclease and is therefore suitable for intermolecular ligation of DNA substrates. In the course of these studies it was discovered that adenylalation of the enzyme--a step in the reaction pathway--markedly decreased the electrophoretic mobility of RNA ligase through polyacrylamide gels containing sodium dodecyl sulfate. This behavior allows identification of RNA ligase among a mixture of proteins and was used to demonstrate that virtually all of the purified protein is enzymatically active.     

The rep mutation. VI. Purification and properties of the Escherichia coli rep protein, DNA helicase III.

The protein product of the rep gene of Escherichia coli is required for the replication of certain bacteriophage genomes (phi X174, fd, P2) and for the normal replication of E. coli DNA. We have used a specialized transducing phage, lambda p rep+, which complements the defect of rep mutants, to identify the rep protein. The rep protein has been purified from cells infected with lambda p rep+ phage; it has a molecular weight of about 70 000 and appears similar to the protein found in normal cells. Stimulation of phi X174 replicative form DNA synthesis in vitro was observed when highly purified rep protein was supplied to a cell extract derived from phi X-infected E. coli rep cells and supplemented with replicative form DNA. The purified protein has a single-stranded DNA-dependent ATPase activity and is capable of sensitizing duplex DNA to nucleases specific for single-stranded DNA. For this reason we propose the enzyme be called DNA helicase III. We infer that the rep protein uses the energy of hydrolysis of ATP to separate the strands of duplex DNA; the E. coli DNA binding protein need not be present. The rep3 mutant appeared to make a limited amount of active rep protein.     

Functional Interaction of the tonA/tonB Receptor System in Escherichia coli

Host range mutants of phage T1 (T1h), which productively infected tonB mutants of Escherichia coli, were isolated. The phage mutants were inactivated by isolated outer membranes of E. coli in contrast to the wild-type phage, which only adsorbed reversibly. For the infection process, the tonB function is apparently only required for the irreversible adsorption of the phage T1, but not for the transfer of the phage DNA through the outer membrane and the cytoplasmic membrane of the cell. Mutants of the tonA gene expressing normal amounts of outer membrane receptor proteins were isolated and found to be partially sensitive to phage T5 and resistant to the phages T1 and T1h, colicin M, and albomycin and unable to take up iron as a ferrichrome complex. One tonA mutant remained partially sensitive to T5, colicin M, and albomycin and supported growth of T1h (not of T1) with the same plating efficiency as the parent strain. Only a small region of the tonA receptor protein seems to function for all the very different substrates. A newly isolated host range mutant of T5 (T5h) adsorbed faster to tonA(+) cells than did wild-type T5 and infected tonA missense mutants resistant to wild-type T5. The interplay of the tonA with the tonB function was observed with phage T5 infection, although T5 required only the tonA receptor. Ferrichrome inhibited plaque formation of T5 only when plated on tonB mutants. Adsorption of T5 to cells in liquid medium was influenced by ferrichrome as follows: complete inhibition by 0.1 muM ferrichrome with tonB mutants, not more than 35% inhibition by 1 to 100 muM ferrichrome with the tonB(+) parent strain in the presence of glucose as energy source, and 90% inhibition by 1 muM ferrichrome with partially starved parent cells. We conclude that there exist different functional states of the receptor protein that depend on the energy state of the cell and the tonB function. The latter seems to be required only for translocation processes with outer membrane proteins involved.     

Recovery of the accumulation ability of thiomethyl-beta-galactoside in Escherichia coli after bacteriophage T4 infection.

Effects of UV-irridiated and unirradiated T4 phage infection on the beta-galactoside accumulation ability in Eschericia coli have been examined by the use of 14C-labeled thiomethyl-beta-galactoside (TMG). Under conditions where a synchronous adsorption of phage takes place, the cellular ability for TMG accumulation is found to be largely inhibited immediately after phage adsorption, but it recovers with time to a new level, which is dependent on the multiplicity of infection. When cells are infected with UV-irradiated T4 at the same multiplicity as that of unirradiated phage, the cellular accumulation ability is more severely inhibited and there is no recovery from the inhibition. The recovery process in T4-infected cells is mostly sensitive to puromycin. These results suggest that the initial inhibition of the TMG accumulation ability is probably caused by the adsorption of phage coats, and the subsequent restoration occurs through the action of a phage-directed protein(s). In the recovery process, no new transport system appears to be involved. The restored ability of TMG accumulation is resistant to the action of superinfecting UV phage. However, different mechanisms appear to be operating in T4-infected cells for the establishment of resistance to ghosts and for the recovery from the phage coat-induced inhibition.     

Repression of lambda-Associated Enzyme Synthesis After lambda(vir) Superinfection of Lysogenic Hosts.

Lisio, Arnold L. (National Institutes of Health, Bethesda, Md.), and Arthur Weissbach. Repression of lambda-associated enzyme synthesis after lambda(vir) superinfection of lysogenic hosts. J. Bacteriol. 90:661-666. 1965.-Phage lambda(vir) is a multiple mutant of lambda which is capable of overcoming the immunity of a host lysogenic for lambda, and initiating normal vegetative replication of the superinfecting phage genome. Superinfection of Escherichia coli K-112 (lambda(22)) with lambda(vir) results in a normal phage yield, lysis time, and H(3)-thymine incorporation compared with infection of the sensitive host, K-112 (S). However, the production of the lambda phage-specific early protein, lambda-exonuclease, after superinfection of E. coli K-112 (lambda(22)) with lambda(vir) is only 25 to 50% of that obtained from corresponding infection of a nonlysogenic host, E. coli K-112 (S). This repression of lambda-exonuclease synthesis is dependent on the C(1) cistron of the prophage and is overcome if the lysogenic host cells are induced prior to superinfection. The data are interpreted as evidence for partial repression of lambda(vir) by the host immunity.     

Genetic and physiological control of host cell lysis by bacteriophage lambda.

The timing of host cell lysis at the end of the lytic cycle of phage lambda is under complex control. The lambda S protein stimulates lysis. Another physiological system, the lysis regulator, inhibitis lysis from occurring prematurely. The effects of a series of phage and bacterial mutations on these controls are described. They show that the lambda rex gene plays a role in regulating lysis under suboptimal growth conditions. In certain mutant cells, and especially under anaerobic culture conditions, the rex gene aids in the scheduling of host cell lysis. The data also suggest that the lysis regulator may control the transition of the lambda S protein from an inactive to an active state.     

Studies on energy supply for genetic processes. Requirement for membrane potential in Escherichia coli infection by phage T4

In this study the hypothesis considering the requirement for an electrochemical proton gradient in the injection of phage T4 DNA into Escherichia coli cell has been verified experimentally. The phage caused a reversible depolarization of cell membrane, while phage 'ghosts' induced an irreversible depolarization. The phage infection was strictly dependent on E. coli membrane potential value when phage/cell ratio was 5 and higher. When the ratio was close to 1, the decrease in the membrane potential up to -100 mV caused practically no effect on the phage infection. The infection inhibition was observed when the membrane potential was lowered below this 'threshold' value. On the other hand, the decrease in the membrane potential caused no effect on the phage infection under conditions promoting a concomitant increase in the value of the transmembranous pH gradient. The phage DNA transfer through the membrane of ATPase-deficient cells was reversibly inhibited by switching off the respiratory chain - the sole generator of a protonmotive force in these mutant cells. The membrane should be kept in the energized state during the phage DNA entrance into the cell. Adsorption of the phage on E. coli was followed by the reversible release of the respiratory control. Thus the results presented here indicate the requirement of the electrochemical proton gradient across the plasma membrane for injection of phage T4 DNA into E. coli. They support the concept postulating an expenditure of host cell metabolic energy for phage T4 DNA transfer through the membrane.     

Substrate of a UV-induced repair system providing for W-reactivation of lambda phage]

Escherichia coli uvrA, polA and uvrD cells carrying non-UV-inducible prophage lambdac1857ind- were infected with 3H-thymidine labelled homoimmune phage lambdac1857, and the effect of UV-irradiation of super-infecting phage and lysogenic bacterial cells on the content of intracellular covalently-closed lambda DNA circles (cccDNA) and pyrimidine dimer content in lambda DNA are studied. UV-irradiation of host cells results in two-fold increase of relative content of cccDNA of UV-irradiated phage lambda in uvrD mutant, while there is no such an effect in uvrA and polA mutants. In UV-irradiated or intact uvrA lysogens cccDNA molecules, forming after the infection with UV-irradiated phage lambda, contain pyrimidine dimers, but in uvrD mutant cccDNA in free of dimers. The data indicate that the repair system induced by UV-irradiation of uvrA and polA cells acts exclusively on the DNA defects appearing after (or in the course) of phage genomes replication. UV-inducible repair system in uvrD mutant can operate also on some intermediates of abortive excision repair, possibly on long single straided excision gaps.     

Evidence for common binding sites for ferrichrome compounds and bacteriophage phi 80 in the cell envelope of Escherichia coli.

Mutants ton A and ton B of Escherichia coli K12, known to be resistant to bacteriophage phi80, were found to be insensitive as well to albomycin, an analogue of the specific siderochrome ferrichrome. Ferrichrome at micromolar concentrations strongly inhibited plaque production by phi80. Preincubation with ferrichrome did not inactivate the phage. At a concentration at which ferrichrome allowed 90% inhibition of plaque formation, the chromium analogue of ferrichrome showed no detectable activity. Similarly, ethylenediaminetetraacetic acid, ferrichrome A, and certain siderochromes structurally distinct from ferrichrome, such as ferrioxamine B, schizokinen, citrate, and enterobactin, did not show detectable inhibitory activity. However, rhodotorulic acid showed moderate activity. A host range mutant of phi80, phi80h, was also inhibited by ferrichrome, as was a hybrid of phage lambda possessing the host range of phi80. However, phage lambdacI- and a hybrid of phi80 possessing the host range of lambda were not affected by ferrichrome. Finally, ferrichrome and chromic deferriferrichrome were shown to inhibit adsorption of phi80 to sensitive cells, ferrichrome giving 50% inhibition of adsorption at a minimal concentration of 8 nM. It is suggested that a component of the ferrichrome uptake system may reside in the outer membrane of E. coli K12 and may also function as a component of the receptor site for bacteriophage phi80, and that ferrichrome inhibition of the phage represents a competition for this common site.