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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↗
PubMed2005

Switches in bacteriophage lambda development.

The lysis-lysogeny decision of bacteriophage lambda (lambda) is a paradigm for developmental genetic networks. There are three key features, which characterize the network. First, after infection of the host bacterium, a decision between lytic or lysogenic development is made that is dependent upon environmental signals and the number of infecting phages per cell. Second, the lysogenic prophage state is very stable. Third, the prophage enters lytic development in response to DNA-damaging agents. The CI and Cro regulators define the lysogenic and lytic states, respectively, as a bistable genetic switch. Whereas CI maintains a stable lysogenic state, recent studies indicate that Cro sets the lytic course not by directly blocking CI expression but indirectly by lowering levels of CII which activates cI transcription. We discuss how a relatively simple phage like lambda employs a complex genetic network in decision-making processes, providing a challenge for theoretical modeling.

Bacteriolysis↗
PubMed2006

A novel method for evaluating free radical scavenging abilities of antioxidants using ultraviolet induction of bacteriophage lambda.

A novel biological method used to evaluate free radical scavenging abilities of antioxidants using ultraviolet (UV) induction of bacteriophage lambda in lysogenic Escherichia coli kappa12 (lambda+) has been developed. This method is based on the induction of bacteriophage lambda from lysogenic cycle to lytic cycle by ultraviolet irradiation, and formation of free radicals during the course of induction. In the experiments, 10(8)cells/ml and 30s (39J/m2) were determined as the cell density of the lysogenic bacterium and UV irradiation time, respectively. The reliability of this method was demonstrated by electron paramagnetic resonance (EPR) spectroscopy and spin trapping with DMPO. This method had good reproducibility with intra-day variations (RSD, %) of less than 4% and inter-day variations (RSD, %) of less than 5%, respectively. By this method, the free radical scavenging abilities (ID50) of well-known antioxidants such as glutathione, superoxide dismutase (SOD), catalase (CAT) and carotenoids were determined quantitatively. The results were consistent with the ones obtained by conventional methods for evaluating free radical scavenging abilities. This developed method is reliable and uses common instruments and inexpensive, stable reagents, thus it could be utilized as a routine laboratory quantitative assay to screen a large number of substances that have potential to scavenge the free radical.

Antioxidants↗
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↗
PubMed2001

Construction and use of a broad-host-range plasmid expressing the lamB gene for utilization of bacteriophage lambda vectors in the marine bacterium Vibrio harveyi.

The remarkable success of Escherichia coli as a model organism in molecular genetics was dependent, among other things, on its susceptibility to genetic manipulation. Many versatile and sophisticated genetic tools for molecular biology studies are derived from bacteriophage lambda. However, this bacteriophage is specific for E. coli, and thus lambda-based techniques have been restricted to this bacterium. Plasmids expressing the E. coli gene coding for bacteriophage lambda receptor were reported previously, and introduction of such plasmids into cells of some other bacteria made them sensitive to phage lambda infection. However, we found that these systems were not efficient for Vibrio harveyi, one of the most frequently investigated species of marine bacteria. Here we describe construction of a broad-host-range plasmid expressing the lamB gene. Introduction of this plasmid to V. harveyi cells and expression of lamB made this strain susceptible to bacteriophage lambda adsorption and lambda DNA injection. Foreign genetic material could be introduced into cells of this strain using a cosmid vector.

Journal Article↗
PubMed2001

Regulation of the switch from early to late bacteriophage lambda DNA replication.

There are two modes of bacteriophage lambda DNA replication following infection of its host, Escherichia coli. Early after infection, replication occurs according to the theta (theta or circle-to-circle) mode, and is later switched to the sigma (sigma or rolling-circle) mode. It is not known how this switch, occurring at a specific time in the infection cycle, is regulated. Here it is demonstrated that in wild-type cells the replication starting from orilambda proceeds both bidirectionally and unidirectionally, whereas in bacteria devoid of a functional DnaA protein, replication from orilambda is predominantly unidirectional. The regulation of directionality of replication from orilambda is mediated by positive control of lambda p(R) promoter activity by DnaA, since the mode of replication of an artificial lambda replicon bearing the p(tet) promoter instead of p(R) was found to be independent of DnaA function. These findings and results of density-shift experiments suggest that in dnaA mutants infected with lambda, phage DNA replication proceeds predominantly according to the unidirectional theta mechanism and is switched early after infection to the sigma mode. It is proposed that in wild-type E. coli cells infected with lambda, phage DNA replication proceeds according to a bidirectional theta mechanism early after infection due to efficient transcriptional activation of orilambda, stimulated by the host DnaA protein. After a few rounds of this type of replication, the resulting increased copy number of lambda genomic DNA may cause a depletion of free DnaA protein because of its interaction with the multiple DnaA-binding sites in lambda DNA. It is proposed that this may lead to inefficient transcriptional activation of orilambda resulting in unidirectional theta replication followed by sigma type replication.

Bacterial Proteins↗
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↗
PubMed1984

Bacteriophage lambda preconnectors. Purification and structure.

The morphogenesis of bacteriophage lambda proheads is under the control of the four phage genes B, C, Nu3 and E, and the two Escherichia coli genes groEL and groES . It has been shown previously that extracts prepared from cells infected with a lambda C-E- mutant accumulate a gpB polymer, which behaves as a biologically active intermediate in prohead assembly. This gpB activity has been called a preconnector , as it is probably a precursor to the head-tail connector. We now report the partial purification of biologically active preconnectors and the characterization of its structure. In the electron microscope, preconnectors appear as donut -like structures composed of several subunits displaying radial symmetry. Optical filtration of periodic arrays of preconnectors showed that the structure has 12-fold rotational symmetry. Side views of the preconnector reveal that it resembles an asymmetrical dumbell . This information has been used to construct a three-dimensional model of the preconnector . The implications of this structure for prohead shape and function, and for DNA packaging are discussed.

Bacteriophage lambda↗
PubMed1968

Loss of host-controlled restriction of lambda 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.

Coliphages↗
PubMed2003

Stationary phase-like properties of the bacteriophage lambda Rex exclusion phenotype.

The rex genes of bacteriophage lambda were found to protect lysogenic Escherichia coliK host cells against killing by phage T4 rII, when compared in parallel to isogenic Rex(-) lysogens and nonlysogens. This protective effect was abrogated upon mutation of the host stationary-phase sigma factor RpoS. Rex(+) lysogens infected by T4 rII contracted, formed aggregates and shed flagella, thus resembling cells entering stationary phase. These phenotypes were accentuated in nonlysogenic cells carrying multicopy plasmids expressing rexA-rexB: cells were about two-fold contracted in length, expressed membrane-bound and detached flagella, were insensitive to infection by a variety of phages and clumped extensively; in addition, cultures of these cells were odorous. Our observations support the hypothesis that the Rex system can cause a stationary-phase-like response that protects the host against infection by T4 rII.

Bacterial Proteins↗
PubMed1999

Complementation and characterization of the nested Rz and Rz1 reading frames in the genome of bacteriophage lambda.

Transposon insertions in the Rz gene of bacteriophage lambda block lysis if the medium contains divalent cations at concentrations greater than 5 mM, but otherwise cause no change in phenotype. The Rz protein is thought to have an endopeptidase activity, previously reported in lambda lysates, which might be involved in cleavage of oligopeptide crosslinks between glycosidic strands in the peptidoglycan and the Lpp lipoproteins of the outer bacterial membrane. Recently, a small lipoprotein has been reported as the product of a short reading frame, designated Rz1, in the +1 register within Rz. This protein has been detected in membranes of induced lambda lysogens. To determine whether Rz1 has a function in the lambda vegetative cycle, amber nonsense alleles of Rz and Rz1 have been constructed by site-directed mutagenesis and used for complementation and suppression analysis. Both Rzam and Rz1am alleles have phenotypes identical to those of the original Rz insertion alleles, and complement and are fully suppressed in a supE host, indicating that the two genes are independent, trans-acting genes encoding proteins required for lysis in the presence of cations. Moreover, supF suppresses Rzam but not the Rz1am mutation, and the defective Rz1am product in the supF host shows a partially dominant character and significantly retards lysis even in the absence of additional cations in the medium. Rz and Rz1 represent a unique example of two genes located in different reading frames in the same nucleotide sequence, which encode different proteins that are both required in the same physiological pathway.

Amino Acid Sequence↗
PubMed2002

The solution structure of the bacteriophage lambda head-tail joining protein, gpFII.

The bacteriophage lambda FII protein (gpFII) is a 117 residue structural protein found in the phage particle that is required for the joining of phage heads and tails at the last step of morphogenesis. We have performed biophysical experiments to show that gpFII is stable, monomeric, and reversibly folded. We have also determined the atomic resolution structure of gpFII using NMR spectroscopy. gpFII is shown to possess a novel fold consisting of seven beta-strands and a short alpha-helix. It also displays two large unstructured regions at the N terminus (residues 1-24) and in a large loop near the middle of the protein (residues 46-62). We speculate that these unstructured regions become structured when gpFII assembles into the phage particle, and that these conformational changes play an important role in regulating the assembly pathway. Alignment of the gpFII sequence with those of homologues from other lambdoid phages has allowed us to putatively identify distinct surfaces on the gpFII structure that mediate binding to the phage head and tail.

Amino Acid Sequence↗
PubMed1980

DNA sequence of regulatory region for integration gene of bacteriophage lambda.

The cII and cIII proteins specified by bacteriophage lambda direct the lysogenic response to infection through the coordinate establishment of repression and integration of the viral DNA. The regulatory activity of cII/cIII involves positive regulation of two promoter sites: the p(E) promoter, turning on expression of the cI protein that maintains lysogeny, and the p(I) promoter, activating synthesis of the Int protein for integrative recombination. Regulation of the p(I) promoter provides for differential expression of the Int protein with respect to the excision-specific Xis protein from the closely linked int and xis genes. We have determined the DNA sequence of the p(I) promoter region for wild-type lambda DNA and for two classes of mutations: intc mutations, which result in a high rate of Int synthesis in the absence of cII, and deletion mutations, some of which eliminate cII-activated expression of the int gene. We find a sequence with considerable homology (11 of 15 bases) to a "typical" (computer-generated) promoter sequence, adjacent to a region with striking homology (11 of 14 bases) to part of the p(E) promoter region. This presumed p(I) sequence overlaps the start of the xis gene and includes the site of two intc point mutations. A cII-insensitive xis(+) deletion partially removes the proposed p(I) sequence; a deletion that leaves the p(I) sequence intact but terminates 21 bases upstream does not interfere with cII activation of the int gene. From our results and the analysis of the p(E) region, we suggest that cII acts in the promoter -35 recognition region to facilitate binding by RNA polymerase at the -10 interaction region. Differential expression of the int and xis genes results because the p(I) transcript lacks the initiation codon for Xis protein synthesis.

Bacteriophage lambda↗
PubMed2000

Characterization of bacteriophage lambda excisionase mutants defective in DNA binding.

The bacteriophage lambda excisionase (Xis) is a sequence-specific DNA binding protein required for excisive recombination. Xis binds cooperatively to two DNA sites arranged as direct repeats on the phage DNA. Efficient excision is achieved through a cooperative interaction between Xis and the host-encoded factor for inversion stimulation as well as a cooperative interaction between Xis and integrase. The secondary structure of the Xis protein was predicted to contain a typical amphipathic helix that spans residues 18 to 28. Several mutants, defective in promoting excision in vivo, were isolated with mutations at positions encoding polar amino acids in the putative helix (T. E. Numrych, R. I. Gumport, and J. F. Gardner, EMBO J. 11:3797-3806, 1992). We substituted alanines for the polar amino acids in this region. Mutant proteins with substitutions for polar amino acids in the amino-terminal region of the putative helix exhibited decreased excision in vivo and were defective in DNA binding. In addition, an alanine substitution at glutamic acid 40 also resulted in altered DNA binding. This indicates that the hydrophilic face of the alpha-helix and the region containing glutamic acid 40 may form the DNA binding surfaces of the Xis protein.

Amino Acid Sequence↗