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.     

Cloning and characterization of recA genes froM Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r

The recA genes of Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r have been isolated and introduced into Escherichia coli K-12. All the heterologous genes restore resistance to killing by UV irradiation and the mutagen 4-nitroquinoline-1-oxide in RecA- E. coli K-12 hosts. Recombination proficiency is also restored as measured by formation of Lac+ recombinants from duplicated mutant lacZ genes and the ability to propagate phage lambda derivatives requiring host recombination functions for growth (Fec-). The cloned heterologous genes increase the spontaneous induction of lambda prophage in lysogens of a recA strain. Addition of mitomycin C stimulates phage production in cells carrying the E. coli B/r and S. flexneri recA genes, but little or no stimulation is seen in cells carrying the E. carotovora and P. vulgaris recA genes. After treatment with nalidixic acid, the heterologous RecA proteins are synthesized at elevated levels, a result consistent with their regulation by the E. coli K-12 LexA repressor. Southern hybridization and preliminary restriction analysis indicate divergence among the coding sequences, but antibodies prepared against the E. coli K-12 RecA protein cross-react with the heterologous enzymes, indicating structural conservation among these proteins.     

Occurrence of the bacteriophage lambda receptor in some enterobacteriaceae.

In Escherichia coli K-12, the receptor for phage lambda is an outer membrane protein which inactivates the phage in vitro. Lambda receptor activity was found in extracts from all wild strains of E. coli tested, although most of them fail to support growth of the phage. In some cases this failure is due to a masking of the receptor in vivo, the bacteria being unable to adsorb the phage or to react with antireceptor antibodies. In other cases, adsorption does occur, and the nature of the block in phage growth was not investigated. Most Mal+ strains of Shigella have lambda receptor, whereas most Mal- strains do not have it. Synthesis of the lambda receptor in Shigella is thus presumably controlled by the positive regulator gene of the maltose regulon as is the case in E. coli K-12. Phage lambda adsorbs on many Mal+ strains of Shigella and even yields plaques on some of them, although at a low frequency. No lambda receptor activity could be found in extracts of several strains of Salmonella and Levinea.     

Effect of dark repair on ultraviolet sensitivity of bacteriophage-infected bacteria

Feiner, R. R. (Columbia University, New York, N.Y.), and R. F. Hill. Effect of dark repair on ultraviolet sensitivity of bacteriophage-infected bacteria. J. Bacteriol. 91:1239-1247. 1966.-Changes in ultraviolet (UV) sensitivity of phage-host complexes during phage development have been studied for the following systems: T1 and Escherichia coli B, T1 and E. coli K-12S, lambda and E. coli K-12S. Complexes were formed with bacterial strains differing in ability to dark-repair UV damage to deoxyribonucleic acid and, after irradiation, were plated on bacteria differing similarly. In the first half of the latent period, the resistance of complexes formed with nonrepairing bacteria increased considerably; with T1 and E. coli B hcr(-), in 4 min the resistance became the same as that of complexes formed with repairing bacteria. The repair ability of plating bacteria affected survival curves only upon irradiation in the second half of the latent period after mature phages were present in the initial complex. Use of nonrepairing bacteria both for initial infection and for plating of late complexes resulted in a series of survival curves showing for all three systems the same pattern of change originally reported for T2-E. coli B complexes. Thus, a hitherto unexplained difference between radiation survival curves for T-even and T-odd phages seems due to repair of T-odd phages by the host.     

Cloning and expression in Escherichia coli K-12 of the genes for major outer membrane protein OmpA from Shigella dysenteriae, Enterobacter aerogenes, and Serratia marcescens.

The outer membranes of many gram-negative bacteria contain a major heat-modifiable protein which shows serological cross-reactivity with the OmpA protein of Escherichia coli K-12. Using the cloned gene for the E. coli K12 protein as a DNA-DNA hybridization probe, we were able to identify the corresponding genes from Shigella dysenteriae. Enterobacter aerogenes, and Serratia marcescens. These were cloned in a phage lambda vector, and their expression in E. coli K-12 was studied. All three OmpA proteins were fully produced and correctly exported to the outer membrane. In several cases, complete or partial restoration of known function of the E. coli K-12 protein was observed.     

Temperature Sensitivity of Maltose Utilization and Lambda Resistance in Escherichia coli B

Escherichia coli B strains that have acquired the malB region from E. coli K-12 are able to utilize maltose and to adsorb phage lambda when grown at 30 C, but when grown at 40 C they do not absorb phage lambda and are devoid of amylomaltase activity. These Mal(ts) Lam(ts) cells can be mutated or transduced to become able to grow on maltose at 40 C, but they still have no detectable amylomaltase activity nor functional lambda receptors at that temperature. This Mal(40) phenotype is governed by a gene located near or at malA. It is suggested that the temperature sensitivity of both characters results from a defect in malT. However, transduction of malA from E. coli B to E. coli K-12 results in a wild-type phenotype, whereas E. coli B cells that have acquired malA from E. coli K-12 donors are still temperature sensitive for both amylomaltase and lambda-receptor production.     

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.     

Sensitivity of Intracellular Bacteriophage λ to Colicin CA42-E2

Treatment of Escherichia coli K-12 infected by lambda CIts857 with colicin CA42-E2 resulted in partial inhibition of the infectious process. Uninfected bacteria were killed by colicin with a probability of about five times that with which similarly treated lambda-infected bacteria lose plaque-forming ability. The lambda deoxyribonucleic acid (DNA), when present in a bacterial cell either as the replicating DNA of infectious phage or as the nonreplicating DNA of superinfecting phage, was degraded to acid-soluble material after colicin treatment. Analysis of the intermediates of DNA breakdown has revealed that degradation of the DNA to acid-soluble material is preceded by endonucleolytic fragmentation of the chromosome at a limited number of sites. This is the same mechanism of degradation previously observed for E. coli DNA after colicin treatment.     

Adsorptive capacity of bacteriophage Mu on Escherichia coli K-12 strains with deletions in the attlambda region]

Escherichia coli strains with deletions in att lambda region were obtained. The comparison of the extent of deletions with the sensitivity of the corresponding mutant clones to phage Mu showed that the gene controlling the sensitivity of E. coli K-12 to the phage Mu is located in nad A-gal region of the bacterial chromosome. It is shown that the resistance of E. coli strains which had lost the region of bacterial chromosome between nad A gene and genes of gal-operon have adsorption character. Deletion of the nad A-gal region does not affect the adsorption of other phages (lambda, P1 and T4). Thus, the gene, located in this region, is responsible for the specific adsorption of the phage Mu.     

Loss of Host-controlled Restriction of λ Bacteriophage in Escherichia coli Following Methionine Deprivation

lambda Bacteriophages produced in Escherichia coli C (designated as lambda . C) are restricted in their ability to grow in E. coli K-12. The rare successful infections that arise in the K-12 population occur in "special" cells which have lost their capacity to restrict lambda . C. These infections yield modified progeny phage (designated as lambda . K) which, unlike lambda . C, plate equally well on E. coli C and E. coli K-12. When methionine, but no other amino acid, was removed from the growth medium of a mutant strain of E. coli K-12, the number of special cells rapidly increased 500- to 3,000-fold. These new special cells retain their capacity to produce modified lambda . K progeny. This conversion of restricting cells into special cells does not require the synthesis of new protein. The special cells formed when methionine was removed from the culture did not revert into restricting cells when methionine was restored. Such cells have also lost the ability to divide for at least 4 hr after methionine supplementation. When methionine was restored, the remaining restricting cells, but not the special cells, immediately resumed growth. Removing methionine from cultures of E. coli B caused a similar increase in the number of special cells able to support the growth of lambda . C and lambda . K. However, when E. coli K-12 (P1) cultures were deprived of methionine, the number of special cells increased for lambda . C but not for lambda . K. Thus, retention of the P1-restriction system, unlike the B- and the K-12-systems, does not require the presence of methionine.     

Repair promoted by plasmid pKM101 is different from SOS repair.

In E. coli K12 bacteria carrying plasmid pKM101, prophage lambda was induced at UV doses higher than in plasmid-less parental bacteria. UV-induced reactivation per se was less effective. Bacteria with pKM101 showed no alteration in their division cycle. Plasmid pKM101 coded for a constitutive error-prone repair different from the inducible error-prone repair called SOS repair. Plasmid pKM101 protected E. coli bacteria from UV damage but slightly sensitized them to X-ray lesions. Protection against UV damage was effective in mutant bacteria deficient in DNA excision-repair provided that the recA, lexA and uvrE genes were functional. Survival of phages lambda and S13 after UV irradiation was enhanced in bacteria carrying plasmid pKM101; phage lambda mutagenesis was also increased. Plasmid pKM101 repaired potentially lethal DNA lesions, although wild-type DNA sequences may not necessarily be restored; hence the mutations observed are the traces of the original DNA lesions.     

Ultraviolet-Sensitive Mutator Strain of Escherichia coli K-12

An ultraviolet (UV)-sensitive mutator gene, mutU, was identified in Escherichia coli K-12. The mutation mutU4 is very close to uvrD, between metE and ilv, on the E. coli chromosome. It was recessive as a mutator and as a UV-sensitive mutation. The frequency of reversion of trpA46 on an F episome was increased by mutU4 on the chromosome. The mutator gene did not increase mutation frequencies in virulent phages or in lytically grown phage lambda. The mutU4 mutation predominantly induced transitional base changes. Mutator strains were normal for recombination and host-cell reactivation of UV-irradiated phage T1. They were normally resistant to methyl methanesulfonate and were slightly more sensitive to gamma irradiation than Mut(+) strains. UV irradiation induced mutations in a mutU4 strain, and phage lambda was UV-inducible. Double mutants containing mutU4 and recA, B, or C were extremely sensitive to UV irradiation; a mutU4 uvrA6 double mutant was only slightly more sensitive than a uvrA6 strain. The mutU4 uvrA6 and mutU4 recA, B, or C double mutants had mutation rates similar to that of a mutU4 strain. Two UV-sensitive mutators, mut-9 and mut-10, isolated by Liberfarb and Bryson in E. coli B/UV, were found to be co-transducible with ilv in the same general region as mutU4.     

Genetic and biochemical analyses of pantothenate biosynthesis in Escherichia coli and Salmonella typhimurium.

Pantothenate (pan) auxotrophs of Escherichia coli K-12 and Salmonella typhimurium LT2 were characterized by enzymatic and genetic analyses. The panB mutants of both organisms and the pan-6 ("panA") mutant of S. typhimurium are deficient in ketopantoate hydroxymethyltransferase, whereas the panC mutants lack pantothenate synthetase. panD mutants of E. coli K-12 were previously shown to be deficient in aspartate 1-decarboxylase. All mutants showed only a single enzyme defect. The finding that the pan-6 mutant was deficient in ketopantoate hydroxymethyltransferase indicates that the genetic lesion is a panB allele. The pan-6 mutant therefore is deficient in the utilization of alpha-ketoisovalerate rather than the synthesis of alpha-ketoisovalerate, as originally proposed. The order of the pan genes of E. coli K-12 was determined by phage P1-mediated three-factor crosses. The clockwise order was found to be aceF panB panD panC tonA on the genetic map of E. coli K-12. The three-factor crosses were greatly facilitated by use of a closely linked Tn10 transposon as the outside marker. We also found that supplementation of E. coli K-12 auxotrophs with a high concentration of pantothenate or beta-alanine increased the intracellular coenzyme A level two- to threefold above the normal level. Supplementation with pantoate or ketopantoate resulted in smaller increases.     

Molecular cloning and characterization of the recA gene of Pseudomonas aeruginosa PAO.

The recA gene of Pseudomonas aeruginosa PAO has been isolated and introduced into Escherichia coli K-12. Resistance to killing by UV irradiation was restored in several RecA-E. coli K-12 hosts by the P. aeruginosa gene, as was resistance to methyl methanesulfonate. Recombination proficiency was also restored, as measured by HfrH-mediated conjugation and by the ability to propagate Fec-phage lambda derivatives. The cloned P. aeruginosa recA gene restored both spontaneous and mitomycin C-stimulated induction of lambda prophage in lysogens of a recA strain of E. coli K-12.     

Isolation and characterization of a lambda transducing bacteriophage carrying the cysE gene of Escherichia coli K-12

A specialized lambda transducing phage carrying the cysE and gpsA genes of E. coli K-12 has been isolated. The transducing phage has been separated from the helper phage on equilibrium gradients and has been shown to be defective. Evidence is presented that the phage kil gene is not expressed.     

Isolation of specialized transducing bacteriophage lambda carrying genes of the L-arabinose operon of Escherichia coli B/r.

A heat-inducible lysis-defective phage lambda (lambdacI857S7) has been integrated at multiple sites within the L-arabinose region (araCOIBAD) of a strain of Escherichia coli K-12 deleted for the normal lambda attachment site (lambdaattdelta). The lambda phage has become integrated with opposite orientations at two different loci within the aratb gene and with the "normal" orientation (clockwise N-RA-J) at a single site in the araC gene. The burst size, spontaneous-curing frequencies, and number of prophage harbored by each of the ara secondary-site lysogens have been determined. From these secondary-site lysogens it has been possible to generate plaque-forming ara-transducing phage (lambdapara) and defective ara-transducing phage (lambdadara), as well as defective leucine-transducing particles (lambdadleu). The construction and characterization of these lambdaara-transducing phage and their derivatives which carry genetically defined portions of the L-arabinose region are presented.     

Packaging of ColE1 DNA having a lambda phage cohesive end site.

The mechanism of lambda phage-mediated transduction of hybrid colicin E1 DNAs of various lengths was studied, and factors influencing the formation of these transducing particles were investigated. The results were as follows: 1. The presence of a cohesive end site of lambda phage (coslambda) on colicin E1 DNA was essential for packaging of the DNA. 2. Packaging of colicin E1 DNAs, which carry coslambda with molecular sizes corresponding to 68% of that of lambda phage DNA, was observed in the absence of all known recombination functions of E. coli K-12 and of lambda phage. 3. Hybrid colicin E1 DNAs having coslambda with molecular sizes corresponding to 28% of that of lambda phage DNA were packaged within lambda phage particles as trimers; hybrid DNAs with coslambda of 40 and 47% of the length of lambda phage DNA were packaged as dimers; and those with molecular sizes of 68% of that of lambda phage DNA were packaged mostly as monomers. These results demonstrated that two factors are essential for the packaging of DNAs within lambda phage particles; the presence of coslambda on the DNA molecule and an appropriate size of DNA.     

Isolation and Characterization of a New Generalized Transducing Bacteriophage Different from Pl 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.     

Regulation of glutamine synthetase formation in Escherichia coli: characterization of mutants lacking the uridylyltransferase.

A lambda phage (lambdaNK55) carrying the translocatable element Tn10, conferring tetracycline resistance (Tetr), has been utilized to isolate glutamine auxotrophs of Escherichia coli K-12. Such strains lack uridylyltransferase as a result of an insertion of the TN10 element in the glnD gene. The glnD::Tn10 insertion has been mapped at min 4 on the E. coli chromosome and 98% contransducible by phage P1 with dapD. A lambda transducing phage carrying the glnD gene has been identified. A glnD::Tn10 strain synthesizes highly adenylylated glutamine synthetase under all conditions of growth and fails to accumulate high levels of glutamine synthetase in response to nitrogen limitation. However, this strain, under nitrogen-limiting conditions, allows synthesis of 10 to 20 milliunits of biosynthetically active glutamine synthetase per mg of protein, which is sufficient to allow slow growth in the absence of glutamine. The GlnD phenotype in E. coli can be suppressed by the presence of mutations which increase the quantity of biosynthetically active glutamine synthetase.     

Behavior of Coliphage Lambda in Shigella flexneri 2a

The insensitivity of wild-type Shigella flexneri 2a to coliphage lambda is a consequence of its native genetic defect in the malA gene cluster. The "smooth" S. flexneri 2a lipopolysaccharide layer affects the efficient adsorption of lambda. Derivatives, capable of serving as functional hosts for lambda, were obtained by repairing the malA lesion, enabling the expression of the malB-lambdarcp region of S. flexneri. Introduction of a mutation into S. flexneri causing a "rough" lipopolysaccharide character resulted in more efficient adsorption of lambda. Such S. flexneri hosts can be stably lysogenized and upon induction yield gal(+)-transducing lysates. Lambda propagated on a malA(+) rough S. flexneri host was restricted by Escherichia coli K-12 and E. coli B, but not by E. coli C. This S. flexneri host did not restrict lambda grown on these E. coli strains.