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.     

Cloning and characterization of the c1 repressor of Pseudomonas aeruginosa bacteriophage D3: a functional analog of phage lambda cI protein.

We cloned the gene (c1) which encodes the repressor of vegetative function of Pseudomonas aeruginosa bacteriophage D3. The cloned gene was shown to inhibit plating of D3 and the induction of D3 lysogens by UV irradiation. The efficiency of plating and prophage induction of the heteroimmune P. aeruginosa phage F116L were not affected by the presence of the cloned c1 gene of D3. When the D3 DNA fragment containing c1 was subcloned into pBR322 and introduced into Escherichia coli, it was shown to specifically inhibit the plating of phage lambda and the induction of the lambda prophage by mitomycin C. The plating of lambda imm434 phage was not affected. Analysis in minicells indicated that these effects correspond to the presence of a plasmid-encoded protein of 36,000 molecular weight. These data suggest the possibility that coliphage lambda and the P. aeruginosa phage D3 evolved from a common ancestor. The conservation of the functional similarities of their repressors may have occurred because of the advantage to these temperate phages of capitalizing on the potential of the evolutionarily conserved RecA protein to monitor the level of damage to the host genome.     

Identification and characterization of a gene responsible for inhibiting propagation of methylated DNA sequences in mcrA mcrB1 Escherichia coli strains.

Identifying and eliminating endogenous bacterial enzyme systems can significantly increase the efficiency of propagation of eukaryotic DNA in Escherichia coli. We have recently examined one such system which inhibits the propagation of lambda DNA rescued from transgenic mouse tissues. This rescue procedure utilizes lambda packaging extracts for excision of the lambda DNA from the transgenic mouse genome, as well as E. coli cells for subsequent infection and propagation. This assay, in combination with conjugal mating, P1 transduction, and gene cloning, was used to identify and characterize the E. coli locus responsible for this difference in efficiency. It was determined that the E. coli K-12 mcrB gene when expressed on a high-copy-number plasmid can cause a decrease in rescue efficiency despite the presence of the mcrB1 mutation, which inactivates the classic McrB restriction activity. (This mutation was verified by sequence analysis.) However, this McrB1 activity is not observed when the cloned mcrB1 gene is inserted into the E. coli genome at one copy per chromosome. A second locus was identified which causes a decrease in rescue efficiency both when expressed on a high-copy-number plasmid and when inserted into the genome. The data presented here suggest that this locus is mrr and that the mrr gene product can recognize and restrict cytosine-methylated sequences. Removal of this DNA region including the mrr gene from E. coli K-12 strains allows high rescue efficiencies equal to those of E. coli C strains. These modified E. coli K-12 plating strains and lambda packaging extract strains should also allow a significant improvement in the efficiency and representation of eukaryotic genomic and cDNA libraries.     

Spontaneous lambda OR mutations suppress inhibition of bacteriophage growth by nonimmune exclusion phenotype of defective lambda prophage.

Survivor clones with defects in gene functions that participate in the replicative killing of thermally induced Escherichia coli constructs with integrated lambda N through P or cIII through P gene fragments were selected at a frequency of about 10(-6). Among the population of survivors, clones were identified that exhibited normal lambda immunity at 30 degrees C, as shown by their ability to prevent the plating of lambda wild type and to support the plating of a nearly identical heteroimmune bacteriophage lambda imm434. However, when placed at 42 degrees C to inactivate the cIts857 repressor, these survivor isolates excluded the plating of both lambda wild-type and lambda imm434 phages, a phenotype designated nonimmune exclusion (Nie). Spontaneous mutants of lambda wild type were isolated that overcame the Nie phenotype and would plaque at 42 degrees C on cell lawns of these isolates. The acquired lambda se mutations suppressed nonimmune exclusion, prevented lysogenization by interrupting repressor expression from PRM, and made the phage insensitive to replicative inhibition. The se mutations were genetically mapped and sequenced within the rightward lambda operator site.     

Development of a short-term, in vivo mutagenesis assay: the effects of methylation on the recovery of a lambda phage shuttle vector from transgenic mice.

Transgenic mice suitable for the in vivo assay of suspected mutagens at the chromosome level have been constructed by stable integration of a lambda phage shuttle vector. The shuttle vector, which contains a beta-galactosidase (beta-gal) target gene, can be rescued from genomic DNA with in vitro packaging extracts. Mutations in the target gene are detected by a change in lambda phage plaque color on indicator agar plates. Initial rescue efficiencies of less than 1 plaque forming unit (pfu)/100 micrograms of genomic DNA were too low for mutation analysis. We determined the cause of the low rescue efficiencies by examining primary fibroblast cultures prepared from fetuses of lambda transgenic animals. The rescue efficiency of 5-azacytidine-treated cells increased 50-fold over non-treated controls indicating that methylation was inhibiting rescue. The inhibitory role of methylation was supported by the observation that mcr deficient E. coli plating strains and mcr deficient lambda packaging extracts further improved lambda rescue efficiency. Present rescue efficiencies of greater than 2000 pfu/copy/micrograms of genomic DNA represent a 100,000-fold improvement over initial rescue efficiencies, permitting quantitative mutational analysis. The background mutagenesis rate was estimated at 1 x 10(-5) in two separate lineages. Following treatment with the mutagen N-ethyl-N-nitrosourea (EtNU), a dose dependent increase in the mutation rate was observed in DNA isolated from mouse spleen, with significant induction also observed in mouse testes DNA.     

Deoxyribonucleic acid modification by intermediate-type modification mutants of Escherichia coli K-12 and B.

The modification of bacteriophages grown on r-m+/- restriction and modification mutants of Escherichia coli K-12 or B appears to be related to the number of restriction-specific sites in the viral genome. Bacteriophage fd and its mutant U1 fd, which carry two and one B-specific sites, respectively, are not modified in vivo by rB-mB+/- mutant strains. In vitro treatment of fd RF-B+/- deoxyribonucleic acid (DNA) or U1 fd RF-B+/- DNA by endo R-Eco B results in cleavage of the substrate DNA. Lambda bacteriophage, after growth in r-m+/- mutant host strains (lambda-K+/- or lambda-B+/-), is partially protected from in vivo degradation by wild-type homospecific strains. Its efficiency of plating on these strains is approximately 10(-2). However, a hybrid phi80-lambda phage which carries only one K-specific site (sklambda-1) is not modified by rK-mK+/- strains. Labeled DNAs from lambda-B+/- and lambda-K+/- phages were used as substrates for endo R-Eco B and endo R-Eco K nucleases. Zonal centrifugation analysis of the products of the reactions indicate that rK-mK+/- mutants do not protect lambda DNA from in vitro degradation by endo R-Eco K. In contrast, rB-mB+/- mutants appear to partially protect lambda DNA from attack by endo R-Eco B.     

Influence of environmental conditions on infection of Klebsiella pneumoniae by two different types of bacteriophages

The adsorption and efficiency of plating of bacteriophages FC3-1 and FC3-9 on Klebsiella pneumoniae C3 (serotype O1:K66) cells grown at different pHs and temperatures were quantitated. Bacteriophage FC3-1, with lipopolysaccharide as its bacterial receptor, showed a large decrease in efficiency of plating on bacteria grown at low pH or low temperature. Under the same conditions, no significant decrease in efficiency of plating was found for bacteriophage FC3-9, a phage requiring capsule and lipopolysaccharide for its adsorption and carrying capsule-depolymerizing activity. We demonstrate that K. pneumoniae C3 cells grown at low pH or low temperature have less lipopolysaccharide exposed on their surface. We conclude that this is why lipopolysaccharide-specific phage FC3-1 less efficiently infects bacterial cells grown under those conditions. We propose that bacteriophage FC3-9 efficiently infects bacterial cells grown at low pH or low temperature because its enzymatic activity on the capsule makes lipopolysaccharide available to this phage.     

Clonogenicity of the progeny of surviving cells after irradiation

The clonogenic potential of the progeny of irradiated cells was tested in vitro by replating irradiated cultures after various times, allowing between five and over 25 subsequent divisions to take place after irradiation. Whereas the plating efficiency of surviving Chinese hamster cells was not decreased, in C3H10T1/2 cells a dose-dependent but slight decrease in plating efficiency was observed even after the longest follow-up period. These data do not contradict the prevalent hypothesis in radiobiology that the proliferation potential of a clonogenic cell surviving after irradiation is not significantly different from that of a non-irradiated cell.     

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.     

Analysis of plating technique for viability and drug-sensitivity determinations using Rosa cells

Rosa Paul's Scarlet'cell suspension cultures were used as a test system for working out a method of viability and drug-sensitivity determination based on plating efficiency. High plating efficiencies (80–95%) were obtained on a simple synthetic medium when aggregates of a mean size of c . 100 cells/unit from exponential phase cultures were plated at a density of 1500 units/plate in the middle layer (5 ml) of three layers of the agar-solidified medium (total = 30 ml). This 3-layer plating technique produces homogeneous colony growth and simplifies the microscopical evaluation of plating efficiencies. The reduction of plating efficiencies seen when the smaller aggregates of stationary phase cultures were plated was mainly due to low cell density and could be overcome by enriching the medium with various supplements. Reconstitution experiments using mixtures of inactivated and non-inactivated aggregates demonstrated that plating efficiency can be taken as a goodmeasure of viability. The described plating technique was found to be more sensitive and reliable compared to two other methods for determining p -fluorophenylalanine-sensitivity of Rosa cells.     

Participation of Escherichia coli K-12 groE gene products in the synthesis of cellular DNA and RNA.

Cells having the temperature-sensitive mutation groES131(Ts) were isolated from Escherichia coli K-12 strain C600T by thymineless death selection at 44 degrees C. This conditionally expressed mutation affected both cellular DNA and RNA syntheses at nonpermissive temperature, in addition to rendering cells unable to propagate phage lambda at permissive temperature.     

Inactivation of DNA-binding activity of repressor in extracts of lambda-lysogen treated with mitomycin C

Summary Crude extracts from -lysogens treated with mitomycin C were prepared, and immunity repressor levels in the extracts were assayed by the binding activity specific to DNA immunity region. It has been shown that while the repressor levels in the extracts from C600(+) are reduced after mitomycin C treatment, the levels in C600recA(+) and C600 C72(+) which have defects in lifting the immunity are not affected by the treatment. The repressor levels in the extracts prepared from C600 T44(+) after temperature shift up, whose prophage is inducible at high temperature, are also reduced. From the study of chloramphenicol effect, it was indicated that de novo protein synthesis is required for the inactivation of the repressor in C600(+) by mitomycin C, but not in T44(+) by high temperature.     

Methylated bases in the host-modified deoxyribonucleic acid of Escherichia coli and bacteriophage lambda

Gough, Michael (Brown University, Providence, R.I.), and Seymour Lederberg. Methylated bases in the host-modified deoxyribonucleic acid of Escherichia coli and bacteriophage lambda. J. Bacteriol. 91:1460-1468. 1966.-The deoxyribonucleic acid (DNA) from strains of Escherichia coli and phage lambda was examined to determine whether the types or amounts of methionine-derived methylated bases present correlated with the host-specific modification of that DNA. The DNA of strain C600 (which has K-12 modification specificity) and of a modificationless mutant of C600 are similar in their content of 5-methylcytosine and 6-methylaminopurine. Strains Bc251 and its P1-lysogen differ in P1-controlled specificity, but they have the same content of 6-methylaminopurine, and both lack 5-methylcytosine in their DNA. Phage lambda contains the same methylated bases as its host of origin, but in reduced amounts and in different proportions. Although minor amounts of these methylated bases may have importance as a result of their location, the presence of the majority of these methylated bases is irrelevant to the specificity of host modification of DNA.     

The biological activity of bacteriophage DNA, prepared by the cationic detergent dilution technique

The preparation of phage lambda DNA infecting E. coli K 12 with cationic detergent is described. This DNA infects E. coli spheroblasts with the same efficiency as DNA prepared by phenol methods.     

Molecular cloning of a human immunoglobulin lambda chain variable sequence

We have cloned a human V lambda cDNA sequence from an Ig lambda-producing human Burkitt lymphoma cell line (BL2) by taking advantage of a cloned constant region gene as a primer for cDNA synthesis instead of an oligo(dT) primer. The amino acid sequence deduced from the nucleotide sequence of V lambda clones is highly related to that of the NEW V lambda protein of subgroup I. Southern blot hybridization of human DNAs with the V lambda I probe showed at least 12 hybridizing V lambda fragments. These fragments are amplified in K562 cells which derive from a case of chronic myelogenous leukemia and contain an amplified c-abl oncogene and amplified C lambda sequences.     

Characteristics of bacteriophages for Micromonospora purpurea.

Chemical and physical stabilities of bacteriophages ?UW 21 and ?UW 51 infecting Micromonospora purpurea ATCC 15835 were examined. Both phages were stable over the pH range of 5 to 8 and to heating at temperatures up to 50 degrees C and especially stable in buffer containing magnesium ion. Exposure to 1 M Ca(NO3)2 inactivated both phages, and phage ?UW 51 was also susceptible to 1 M CaCl2, 0.1 M tris(hydroxymethyl)aminomethane, and 0.3% H2O2. Phage plating efficiency was highest on the cultures at logarithmic phase and sometimes much influenced by host growth. Phage ?UW 51 has a latent period of 2 h at 34 degrees C and a burst size between 35 and 40. The latent period for phage ?UW 21 is about 12 h, and the burst size is smaller than 30.     

Cell Division and Prophage Induction in Escherichia coli: Studies of Nucleotide Levels

Cell division and prophage repression in the Escherichia coli mutant, T-44, are very sensitive to the levels of certain purine and pyrimidine derivatives in the media. The hypothesis that a change in the level of an adenine derivative in the small molecule pool of this strain was responsible for prophage induction and filament formation was tested. The nucleoside triphosphate pools in T-44 and C-600 nonlysogenic and lysogenic strains were labeled in experiments with (32)P and (33)P. Cultures were mixed, and the nucleotides were isolated. When adenine was present, the level of adenosine triphosphate (ATP) in T-44 compared to C-600 (as indicated by the isotope ratio) was increased up to twofold. Most of the other nucleotides increased but not to the same degree. In the lysogenic strain guanosine triphosphate and deoxycytidine triphosphate showed increases comparable to ATP, whereas increases noted in the deoxynucleotides in T-44 +/- lambda with adenine present were less. In experiments where T-44 and C-600 were incubated with (3)H- and (14)C-adenine, the levels of several compounds, including ATP, were slightly elevated in T-44. The combined data suggest that cultures of T-44 +/- lambda, grown in the presence of adenine, show a preferential increase in the level of ATP when compared to C-600 +/- lambda, but the increase in relation to the other nucleotides is less than twofold. In the experiment with (3)H- and (14)C-adenine, the level of inosine was found to be increased in T-44 relative to C-600. Cyclic AMP, when added to cultures of T-44 under various conditions, had no effect on prophage induction. Intracellular and extracellular levels of cyclic AMP in T-44 compared to C-600, incubated with had-acidin, guanosine, and cytidine (HGC) or with HGC plus adenine, were not significantly different. No compelling evidence for altered nucleotide metabolism in T-44 +/- lambda as a cause of prophage induction or filament formation was obtained.     

Influence of the recF143 mutation of Escherichia coli K12 on prophage lambda induction.

Prophage lambda induction in a recF143 mutant of E. coli K12 was studied. The recF143 (lambda) lysogen was inducible by UV irradiation or treatment with mitomycin C. However, the time required for the onset of derepression brought about by these treatments was longer in the recF143 mutant than in rec+ strains, suggesting that the induction pathway was altered in the recF143 mutant. The recF143 (lambda) lysogen was induced at very low doses of UV irradiation or mitomycin C treatment. Moreover, the presence of the recF143 mutation increased the sensitivity to thermal induction of a tif strain.     

Temperature-dependent photoinhibition and recovery of photosynthesis in the green alga Chlamydomonas reinhardtii acclimated to 12 and 27°C

Photoinhibition of photosynthesis and subsequent recovery were studied in cultures of the unicellular green alga Chlamydomonas reinhardtii L. (wt strain 137 c mating type +) acclimated at high (27°C) and low (12°C) temperature, Photoinhibition was assayed by fluorescence kinetics (77K) and oxygen evolution measurements under growth temperature conditions Inhibition of 50% was obtained by exposing cultures acclimated at high temperature to a photosynthetic photon flux density (PPFD) of 1 600 μmol m⁻² S⁻¹ at. 27°C. and cultures acclimated at low temperature to a PPFD of 900 μmol m⁻² s⁻¹ at 12°C When the photoinhibitory conditions were shifted it was revealed that algae acclimated at low temperature had acquired an increased resistance to photoinhibition at both 12 and 27°C. Furthermore, acclimation at low temperature increased the capacity to recover from 50% photoinhibition at both 12 and 27°C Studies of photoinhibition in the presence of the protein synthesis inhibitor, chloramphenicol, revealed that in response to acclimation at low temperature during growth the algae became more dependent on protein synthesis to avoid photoinhibition. It is suggested that acclimation at low temperature rendered C. reinhardtii an increased resistance to photoinhibition by. increasing the rate of turnover of photodamaged proteins in photosystem II (PS II). However, we cannot exclude the possibility that the increased resistance to photoinhibition of C. reinhardtii acclimated at low temperature also involves modifications of the mechanism of photoinhibition.     

Restriction-modification systems of type II in Shigella strains

Two restriction-modification systems specified by two plasmids are discovered in the clinical species of Shigella. The plasmids are designated pKMR114 and pKMR115. Both are of 60.800 bp and belong to the IncN incompatibility group. The EcoRI, EcoRV, HindIII restriction patterns of both plasmid DNAs are identical. As shown by efficiency of plating of bacteriophage lambda vir on the strains harbouring plasmids encoding EcoRI, EcoRII, EcoRIII, EcoRIV, EcoRV systems and plasmids studied, the discovered plasmids control synthesis of EcoRII specificity enzymes. The main distinctive feature of the pKMR114 is the ability to decrease efficiency of plating of bacteriophage T4 having glycolised DNA.