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PubMed1990

Oligomerization of the bacteriophage lambda S protein in the inner membrane of Escherichia coli.

Western blot (immunoblot) analysis of cell extracts from induced bacteriophage lambda lysogens probed with S-protein-specific antibody (raised against an S--beta-galactosidase fusion protein) demonstrated that the bacteriophage lambda S protein begins to appear 10 min after phage induction and is localized to the inner membrane at all times during the lytic cycle. Between 100 and 1,000 molecules of S protein per cell were present at the time of phage-induced lysis. Western blots of chemically cross-linked membranes from induced lysogens showed a ladder of bands at 18, 24, 32, and 42 kilodaltons (the S-protein monomer ran at 8 kilodaltons) that reacted with anti-S-protein antibody. Thus, the S protein appears to reside in the inner membrane as a multimer, and the molecular weights of the cross-linked species are consistent with those of S-protein homopolymers. Sodium dodecyl sulfate-resistant dimers were also detected when S protein was purified by immunoprecipitation.

Amino Acid Sequence↗
PubMed1980

Purification and characterization of the N gene product of bacteriophage lambda.

The N protein (pN) specified by bacteriophage lambda is an antitermination factor and is required for phage development. pN can be assayed by making use of the observation that the in vitro synthesis of trp mRNA in a reaction programmed with DNA template from lambda trp transducing phage bearing N- and fed- mutations is pN dependent (Ishii et al., 1980). The assay has been used to purify pN. We have observed that pN forms a complex with E. coli protein(s) and is dissociated in the presence of urea. The complex is not formed in host bacteria bearing the nusA-nusB- mutations. pN is a basic protein and heat-stable. Using these characteristics, we have purified pN to virtual homogeneity as judged by polyacrylamide gel electrophoresis in the presence of SDS. pN is a monomeric protein and its mol. wt. is approx. 14 000. The antiterminating activity of pN appears to be enhanced by complex formation with host-encoded protein(s) depending on the nusA and/or nusB gene function.

Bacteriophage lambda↗
PubMed1979

Nicking-closing activity associated with bacteriophage lambda int gene product.

Integrative recombination of bacteriophage lambda requires the action of the protein Int, the product of the phage int gene. In this paper we show that highly purified Int relaxes supercoiled DNA. The association of this nicking-closing activity with Int is shown by: (i) the cosedimentation of nicking-closing and recombination activities of purified Int, (ii) the parallel inactivation of the two activities in purified Int by both heat and a specific antiserum, and (iii) the alteration of both activities in crude extracts of a strain expressing a mutant int gene. The nicking-closing activity of Int functions in the absence of divalent cations and in the absence of an apparent source of chemical energy. The activity displays no obvious sequence specificity and is inhibited by Mg2+, spermidine, and single-stranded DNA. Int relaxes positive as well as negative supercoils. We present a model for the mechanism of strand exchange that describes how the nicking-closing activity of Int might be used during recombination.

Bacteriophage lambda↗
PubMed1982

Lethal action of bacteriophage lambda S gene.

The functions of the bacteriophage lambda lysis genes S, R, and Rz were investigated. Different combinations of wild-type and inactive alleles of all three lysis genes were cloned into the plasmid pBH20 and were expressed under the control of a lac operator-promoter. The involvement of the Rz gene in lysis was proposed in our previous work and was confirmed by the Mg2+-dependent lysis defect of clones in which part of the Rz gene is deleted. Membrane vesicles prepared from induced S+ cells were shown to have a severely reduced capacity for active transport of glucose; this defect was detectable at least 20 min before lysis. Cell viability was also shown to decrease very soon after induction, long before physiological death and lysis; this decrease in viability is absolutely dependent on S expression and independent of R and Rz. The nonviable fraction of cells at any time after induction was demonstrated to be equal to the fraction committed to eventual lysis. Induction of an Sts clone showed that the S gene product is stable and capable of inducing lysis long after the cessation of synthesis of S gene product. A model for S action is proposed.

Bacteriophage lambda↗
PubMed1987

The functional boundaries of the Q-utilization site required for antitermination of late transcription in bacteriophage lambda.

Expression of the late genes of bacteriophage lambda requires, in addition to the host functions, the lambda p'R promoter, the antiterminator sequence qut, and the product of gene Q which interacts with the Q utilization (qut) site. In the absence of the Q function or qut site, the p'R-initiated transcription is blocked by the t'R terminator at the 194th nucleotide downstream of the start point, s'R, producing a short 6 S mRNA. In this study the position and boundaries of the qut site were deduced by constructing plasmids containing various portions of the p'R-qut region, the t'R1 terminator, and the reporter gene galK. We measured galK gene expression in response to the gamma Q gene product supplied in trans by a prophage or Q-expression plasmid. We show that among the lambda proteins, the Q gene product alone is necessary and sufficient for complete qut-mediated transcription antitermination in vivo. These antitermination experiments, employing plasmids that contain different lengths of lambda p'R-qut sequence, identified the right boundary of the qut site, which is located between +4 and +18 (for s'R = +1). The functional left boundary of qut does not extend upstream from the -26th nucleotide of the p'R promoter, as based on the following experiments. The promoter function of the truncated (-26)p'R-s'R-(+18) sequence can be restored by fusion to the complete but qut-less p'R, pp, or PLac promoter; however, no antitermination was observed for such a p-(-26)p'R-s'R-(+18)-t'R-galK plasmid. Thus we conclude that the qut site partially overlaps with the p'R promoter sequence. However, promoters that contain the -10 region of p'R, s'R, and the +1 to +18 qut sequence did mediate Q-dependent antitermination when properly fused to the homologous or heterologous -35 promoter regions. Only those transcripts that start at s'R (+1 or very near to it) and also contain at least the first 18 nucleotides (actually greater than 4 and less than or equal to 18) of 6 S RNA appear to be a target for the Q-qut-mediated transcription antitermination, which acts not only at t'R but also at other Rho-independent or Rho-dependent terminators.

Bacteriophage lambda↗
PubMed1986

Mutational analysis of bacteriophage lambda lysis gene S.

A plasmid carrying the bacteriophage lambda lysis genes under lac control was subjected to hydroxylamine mutagenesis, and mutations eliminating the host lethality of the S gene were selected. DNA sequence analysis revealed 48 single-base mutations which resulted in alterations within the coding sequence of the S gene. Thirty-three different missense alleles were generated. Most of the missense changes clustered in the first two-thirds of the molecule from the N terminus. A simple model for the disposition of the S protein within the inner membrane can be derived from inspection of the primary sequence. In the first 60 residues, there are two distinct stretches of predominantly hydrophobic amino acids, each region having a net neutral charge and extending for at least 20 residues. These regions resemble canonical membrane-spanning domains. In the model, the two domains span the bilayer as a pair of net neutral charge helices, and the N-terminal 10 to 12 residues extend into the periplasm. The mutational pattern is largely consistent with the model. Charge changes within the putative imbedded regions render the protein nonfunctional. Loss of glycine residues at crucial reverse-turn domains which would be required to reorient the molecule to reenter the membrane also inactivate the molecule. Finally, a number of neutral and rather subtle mutations such as Ala to Val and Met to Ile are found, mostly within the putative spanning regions. Although no obvious explanation exists for this subtle and heterogeneous class of mutations, it is noted that all of the changes result in a loss of alpha-helical character as predicted by Chou-Fasman theoretical analysis. Alternative explanations for some of these changes are also possible, including a reduction in net translation rate due to substitution of a rare codon for a common one. The model and the pattern of mutations have implications for the probable oligomerization of the S protein at the time of endolysin release at the end of the vegetative growth period.

Alleles↗
PubMed1985

Functional and physical characterization of transcription initiation complexes in the bacteriophage lambda OR region.

We have used transcriptional activity assays and DNase I footprinting techniques to examine in vitro the binding of Escherichia coli RNA polymerase and lambda repressor protein to the bacteriophage lambda rightward promoter-operator region. For the lambda PR promoter, the activity and physical binding results determined at several repressor concentrations correlated very well. Good agreement was also found for repression of PRM, which occurred at higher repressor concentrations; however, our results indicate that at low repressor concentrations, RNA polymerase can physically occupy PRM in a transcriptionally inactive form. These inactive complexes formed with a binding constant similar to that previously measured for "closed complexes" at PRM. A kinetic study of PR open complex formation on an OR2-template in the presence of lambda repressor showed that decreased initiation frequency from this promoter was due largely to a decrease in KB. The kinetically determined inhibition constant for repressor (Ki = 4 nM) was similar to the dissociation constant (Kd approximately 2 nM) determined from the footprinting studies.

Bacteriophage lambda↗
PubMed1982

Properties of the N gene transcription antitermination protein of bacteriophage lambda.

The product of the N gene of bacteriophage lambda prevents the termination of lambda early transcription. Here we describe the physical properties of pure lambda N protein. N protein is small and very basic. The apparent Mr of N protein during electrophoresis in the presence of sodium dodecyl sulfate is 12,500. It contains 22 mol % (arginine plus lysine) and only one methionine. The methionine residue is at the blocked NH2 terminal since N protein is not detectably shortened by reaction with cyanogen bromide. When use is made of the DNA sequence of the N gene region of lambda DNA (Franklin, N. C., and Bennett, G. N. (1979) Gene 8, 107-119), the lack of an internal methionine residue, the size, and the amino acid composition of N protein can be used to predict that N protein contains 107 amino acids (calculated Mr = 12,241) and that its coding sequence begins at position 223 of the lambda pL operon mRNA. N protein can be assayed by its ability to stimulate endolysin synthesis in vitro in a reaction programmed with lambda N- DNA. N protein activity is heat-stable and trypsin-sensitive. Its sedimentation velocity in a sucrose gradient and its Stokes' radius indicate that N protein is an extremely asymmetric monomer (f/fmin = 1.6). The relationship between this high degree of molecular asymmetry and the sequence which N protein must recognize in lambda nucleic acid is discussed.

Amino Acids↗
PubMed1979

Recombination of bacteriophage phi X174 by the red function of bacteriophage lambda.

Recombination of bacteriophage phi X174 was effectively promoted when the Red function of lambda was supplied by either co-infection with lambda or induction of lambda lysogens. Mutations in red alpha and red beta genes of lambda abolished recombination nearly completely, whereas a mutation in gam gene reduced it only slightly. The Red-promoted recombination of phi X174 occurred in recA, recB, and polA mutants as well as in wild-type strains of Escherichia coli. It was further stimulated when phi X174 mutants were irradiated with UV light before infection.

Coliphages↗
PubMed1977

Insertion sequence IS2 associated with int-constitutive mutants of bacteriophage lambda.

We have examined mutations in bacteriophage lambda called int-c, which confer elevated constitutive expression on the int gene for prophage integration. One class of mutations, which map between the b538 and bio386 endpoints, does not appear to be associated with any major chromosomal modification, whereas the second class has the IS2 insertion sequence in orientation II within the region between gene int and the b538 endpoint, All int-c mutations are within gene xis, with the possible exception of int-c548, which might be located between int and xis. The present data are most consistent with the following notion: (1) the point mutations of class one inactivate the tI terminator signal of the pI-tI leader RNA for gene int and thus render int expression independent of the antiterminating action of the cII and cIII products, and (2) the second class of int-c mutants is constitutive for Int because the IS2 insertion, when strategically located between int and tI, provides a new constitutive promoter for int transciption.

Coliphages↗
PubMed1987

A novel in vitro DNA packaging system demonstrating a direct role for the bacteriophage lambda FI gene product.

A new in vitro bacteriophage lambda DNA packaging system is described in which all the proteins necessary for head morphogenesis are supplied by extracts of plasmid-transformed cells. This assay is used to demonstrate that the lambda FI gene product (gpFI) is necessary for maximal packaging efficiency when proheads and terminase are present in limiting amounts. A 100- to 200-fold decrease in packaging is seen when gpFI is omitted. gpFI is shown to act at and/or after the stage in packaging where proheads bind to the DNA:terminase complex.

Bacteriophage lambda↗
PubMed1987

Deletion analysis of the DNA sequence required for the in vitro initiation of replication of bacteriophage lambda.

Supercoiled DNA containing the replication origin of bacteriophage lambda can be replicated in vitro. This reaction requires purified lambda O and P replication proteins and a partially purified mixture of Escherichia coli proteins (Tsurimoto, T., and Matsubara, K. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7639-7643; Wold, M. S., Mallory, J.B., Roberts, J. D., LeBowitz, J. H., and McMacken, R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6176-6180). The lambda origin region has four repeats of a 19-base pair sequence to which O protein binds. To the right of these sites on the lambda map is a 40-base pair region that is rich in adenine and thymine, followed by a 28-base pair palindromic sequence. To define more precisely the boundaries of the lambda origin, we cloned a 358-base pair piece of lambda DNA containing the origin region into M13mp8 in both orientations. In vitro replication of RF I DNAs prepared from cells infected with these two M13 ori lambda phage was dependent on lambda O and P proteins and a crude protein fraction from uninfected E. coli; with these conditions there was no replication of M13mp8 RF I DNA. We made deletions from the left and the right ends of the lambda origin DNA and determined the deletion end points by DNA sequencing. We have tested RF I DNAs prepared from cells infected with phage carrying ori lambda deletions for their ability to function as templates for O- and P-dependent replication in vitro. Our results show that lambda DNA between nucleotide positions 39072 and 39160 is required for efficient O- and P-dependent replication. This 89-base pair piece of DNA includes only two of the four 19-base pair O protein-binding sites (the two right-most) and the adjoining adenine- and thymine-rich region to the right of the O-binding sites.

Bacteriophage lambda↗
PubMed1984

Length determination in bacteriophage lambda tails.

We have isolated viable mutants of bacteriophage lambda that have in-frame deletions in gene H, which codes for a minor tail protein. They produce correspondingly smaller but active gene H protein products and assemble shorter-tailed phage particles. The deficiency in tail length for each mutant corresponds to the calculated shortening of the gene H protein caused by the deletion. These results show that the H protein determines tail length and argue strongly for a scheme in which the H protein is a ruler or template that measures length during tail assembly.

Bacteriophage lambda↗
PubMed1987

An elongation control particle containing the N gene transcriptional antitermination protein of bacteriophage lambda.

The N gene transcriptional antitermination protein of bacteriophage lambda is incorporated in vitro into transcriptional elongation complexes containing the E. coli proteins NusA and NusB. The binding of NusA to elongating RNA polymerase is sequence-independent and follows the release of sigma 70. Incorporation of N into the elongation complex requires an N utilization site (nut site) on the DNA template. Incorporation of NusB into the complex requires NusA, ribosomal protein S10, and the boxA component of the nut site. T1 RNAase releases N, but not NusB, from the elongation complex. We therefore propose that an N-modified termination-resistant elongation complex includes an elongation control particle (ECP) containing at least NusA, NusB, S10, N, and an RNA transcript of the nut site.

Bacterial Proteins↗