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PubMed1985

Integration of bacteriophages lambda and phi 80 in wild-type Escherichia coli at secondary attachment sites. II. Genetic structure and mechanism of polylysogen formation for lambda, phi 80 and the lambda att80 hybrid.

The frequency of occurrence and the genetic structure of polylysogens were studied for phages lambda, phi 80 and lambda att80. In the case of lambda, frequency of polylysogenization is high (0.20 to 0.41) with a tandem integration of prophages at the primary att site (att lambda). With phi 80 and lambda att80, this frequency is about 10 times lower, and usually one prophage becomes integrated at the primary att site (att80-I) while another (sometimes two others) integrates at one of the secondary sites. At least four secondary att80 sites have been found in wild-type Escherichia coli, two of which (near the his and tolC loci) are preferred. The frequency of secondary integration of phi 80 and lambda att80 does not differ significantly in the wild-type host and in that deleted for the primary att site (0.041 and 0.045, respectively, among surviving cells at an MOI of 10). Homoimmune superinfection has revealed a constitutive cI-independent expression of the phi 80 int gene in the prophage state. The only phi 80 tandem detected proved to be unstable. With the phi 80int- mutant, we observed stabilization of phi 80 tandems; as a consequence, their frequency of occurrence during coinfection with phi 80int+ was up to the lambda level and no nontandem insertions were found. A model is proposed for the phi 80 and lambda att80 nontandem integration.

Bacteriophage lambda↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1987

Inhibition of streptomycin-dependent mutation in Escherichia coli on the lytic growth of bacteriophage lambda.

Spontaneous streptomycin-dependent mutants (StrDA) were isolated from Escherichia coli C600. On C600 StrD, the lytic growth of phage lambda Nam and lambda cI857 was inhibited. After E. coli lysogenic strain 1.1485 (lambda cI857) mutated to StrDA, induction of lambda was decreased greatly. On StrDA of E. coli strains C600 and 1.1485 (lambda cI857), the plating efficiencies and burst sizes of phage T4 and T7 remained normal. Since StrDA is a mutation in the structural gene for ribosomal protein S12, the results obtained in the present study suggest that ribosomes of the StrDA mutants inhibit the lytic growth of lambda phage. The possibility that StrDA ribosomes inhibit the expression of lambda N gene is discussed based on the comparison of the genetic background of lambda cI857 and lambda Nam.

Bacteriophage lambda↗
PubMed1986

Characterization of the DNA binding domain of bacteriophage lambda O protein.

The lambda O and P gene products are required for the initiation of lambda DNA replication. In order to study the biochemistry of this process, we have constructed plasmids that carry the lambda O gene, P gene, and half of the O gene coding for the amino-terminal half of the O protein. Each is under the control of the inducible lambda promoter, PL. We have purified these three proteins from induced cells carrying the plasmids. Our results show that the amino-terminal portion of the O protein binds to the lambda origin of replication in a manner similar to the intact lambda O protein, demonstrating that the amino-terminal portion of O protein contains the DNA binding domain. Using chromatographic procedures, we have isolated a complex of lambda O and P proteins with lambda dv DNA. The amino-terminal portion of the O protein does not complex with P protein under the same conditions. This suggests that the specificity of the lambda O protein for P protein resides in the carboxyl-terminal half of the lambda O protein. Our results also show that, while the intact O protein is active in in vitro replication of lambda dv plasmid DNA, the amino-terminal portion of the O protein is inactive and is a competitive inhibitor of the lambda O protein in this reaction. These results confirm previous genetic observations that were interpreted as indicating a bifunctional structure for the lambda O protein with the amino-terminal domain recognizing the lambda origin of replication and the carboxyl-terminal domain interacting with the lambda P protein.

Bacterial Proteins↗
PubMed1989

Translesion synthesis is the main component of SOS repair in bacteriophage lambda DNA.

Agents that interfere with DNA replication in Escherichia coli induce physiological adaptations that increase the probability of survival after DNA damage and the frequency of mutants among the survivors (the SOS response). Such agents also increase the survival rate and mutation frequency of irradiated bacteriophage after infection of treated bacteria, a phenomenon known as Weigle reactivation. In UV-irradiated single-stranded DNA phage, Weigle reactivation is thought to occur via induced, error-prone replication through template lesions (translesion synthesis [P. Caillet-Fauquet, M: Defais, and M. Radman, J. Mol. Biol. 117:95-112, 1977]). Weigle reactivation occurs with higher efficiency in double-stranded DNA phages such as lambda, and we therefore asked if another process, recombination between partially replicated daughter molecules, plays a major role in this case. To distinguish between translesion synthesis and recombinational repair, we studied the early replication of UV-irradiated bacteriophage lambda in SOS-induced and uninduced bacteria. To avoid complications arising from excision of UV lesions, we used bacterial uvrA mutants, in which such excision does not occur. Our evidence suggests that translesion synthesis is the primary component of Weigle reactivation of lambda phage in the absence of excision repair. The greater efficiency in Weigle reactivation of double-stranded DNA phage could thus be attributed to some inducible excision repair unable to occur on single-stranded DNA. In addition, after irradiation, lambda phage replication seems to switch prematurely from the theta mode to the rolling circle mode.

Bacteriophage lambda↗
PubMed1987

Overproduction of an antisense RNA containing the oop RNA sequence of bacteriophage lambda induces clear plaque formation.

We have constructed an IPTG-inducible plasmid which overexpresses oop RNA sequences in Escherichia coli. Infection of these transformed E. coli cells (SB221/pOOP5) with lambda+ phage produced clear plaques, whereas lambda+ infection of cells transformed with the plasmid vector (SB221/pJDC406) or the plasmid expressing the oop RNA transcript in the other orientation (SB221/pOOP9) gave rise to turbid plaques characteristic of lambda+. Calculations of the percentage of infected cells forming lysogens show a 6-fold decrease in the absence of isopropyl beta-D-thiogalactoside (IPTG) and a 20-fold decrease in the presence of IPTG for SB221/pOOP5 as compared to both SB221/pJDC406 and SB221/pOOP9. We have thus shown that the overexpression of oop RNA favors the lytic mode of lambda development.

Bacteriophage lambda↗
PubMed1980

Mapability of very close markers of bacteriophage lambda.

Recombinant frequency was compared with nucleotide distance in crosses involving markers in either the PRM or the cy region of phage lambda. For each pair of markers, we performed reciprocal four-factor crosses of the following types: (I) A+m1+m2-B- X A-m1-m2+B+; and (II) A+m1-m2+B- X A-m1+m2-B+. In crosses of type I, the frequency of A+m1+m2+B+ recombinants among total (selected) A+B+ progeny was directly proportional to nucleotide distance between m1 and m2 in the range from 3 to 160 nucleotides. When less than three nucleotides separated m1 and m2, the measured yields of m1+m2+ recombinants were significantly depressed. We also found that the frequency of A+m1+m2+B+ recombinants among total A+B+ progeny was significantly lower (about 10-fold on the average) in crosses of type II than in the corresponding crosses of type I. Since mismatch correction should yield A+m1+m2+B+ recombinants with approximately equal frequencies in type I and II crosses, we suggest: (1) that most m1+m2+ recombinants produced in type I crosses must arise from the formation of heteroduplex structures with a discontinuity (in the source of genetic information) between sites m1 and m2, and (2) that mismatch correction is not a major pathway for production of recombinants for close markers in normal lambda infection.

Bacteriophage lambda↗
PubMed1992

Host factor requirements for processive antitermination of transcription and suppression of pausing by the N protein of bacteriophage lambda.

The N protein of phage lambda prevents termination of transcription by Escherichia coli RNA polymerase at Rho-dependent and -independent terminators in the lambda early operons. The modification of RNA polymerase by N requires an N-utilization (nut) site, present in each lambda early operon, and involves the E. coli factors NusA, NusB, NusG, and ribosomal protein S10. We show that, in the presence of NusA, N inhibits pausing by RNA polymerase and Rho-dependent termination in vitro at three sites in the lambda terminator tR1 which are located less than 100 base pairs downstream from nutR. NusA is also sufficient for partial antitermination at sites located farther downstream from nutL and nutR if there is a high concentration of N in the reaction. At low concentrations of N, the additional factors NusB, S10, and NusG are essential for antitermination at distal sites. In these conditions, the presence of NusA, NusB, S10, and NusG in the reaction enables N-modified RNA polymerase to elongate efficiently and processively through Rho-dependent and -independent terminators over distances as great as 7 kilobases downstream from the lambda nut sites. This substantial processivity of antitermination in vitro also occurs in vivo and probably reflects the stable association of N, NusA, NusB, S10, and NusG with RNA polymerase and nut site RNA in elongation complexes transcribing the lambda chromosome.

Bacterial Proteins↗
PubMed1979

DNA from recombinogenic lambda bacteriophages generated by arl mutant of Escherichia coli is cleaved by single-strand-specific endonuclease S1.

When propagated on arl strains (a subclass of Escherichia coli hyper-rec mutants), lambda "Red-" duplication phages accumulated an enhanced potential for recombination. The physical properties of the recombinogenic phages thus obtained ("Arl-" phages) were similar to those of phages grown on arl+ bacteria. However, Arl- phage DNA was cleaved by endonuclease S1 under conditions such that the nuclease is specific for single-stranded DNA;DNA from control phages was S1-resistant. The number of S1 sites (defined by the apparent decrease in single-strand molecular weight) reached a maximum (seven to nine sites per strand of lambda DNA) after five or six rounds of growth on arl bacteria. Similarly, the recombinogenicity of Arl- phages reached a limiting value (recombination frequency, 15%) that was 5 times that of Arl+ phages. Recombinogenicity and S1 susceptibility were accumulated concomitantly during growth on arl+ bacteria. If all increased recombination occurred at the S1 sites, then these regions (about 40 bases each) were about 300 times as recombinogenic as normal DNA regions of the same size, and 1.5 times as recombinogenic as UV-induced lesions. Chromosomal DNA and plasmid DNA (pBR322) from arl cells were more susceptible to nuclease S1 than was DNA from arl+ bacteria. Analysis of the cleavage products suggests that the S1 sites on Arl- lambda phage DNA are located randomly.

Bacteriophage lambda↗