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PubMed1998

Amino acid-amino acid contacts at the cooperativity interface of the bacteriophage lambda and P22 repressors.

The bacteriophage lambda repressor and its relatives bind cooperatively to adjacent as well as artificially separated operator sites. This cooperativity is mediated by a protein-protein interaction between the DNA-bound dimers. Here we use a genetic approach to identify two pairs of amino acids that interact at the dimer-dimer interface. One of these pairs is nonconserved in the aligned sequences of the lambda and P22 repressors; we show that a lambda repressor variant bearing the P22 residues at these two positions interacts specifically with the P22 repressor. The other pair consists of a conserved ion pair; we reverse the charges at these two positions and demonstrate that, whereas the individual substitutions abolish the interaction of the DNA-bound dimers, these changes in combination restore the interaction of both lambdacI and P22c2 dimers.

Amino Acid Sequence↗
PubMed1988

Translational signals of a major head protein gene of bacteriophage lambda.

The D gene of bacteriophage lambda which codes for a major head protein is expressed at a high level during lytic growth. We have constructed a set of D-lacZ gene fusions in order to examine the factors determining the high efficiency of the D translational initiation signals. It was found that an integral sequence, 300 bp long and upstream of the ATG initiation codon, is required for maximal protein synthesis.

Bacteriophage lambda↗
PubMed1988

Effects of DNA heterologies on bacteriophage lambda recombination.

Previous studies of bacteriophage lambda recombination have provided indirect evidence that substantial sequence nonhomologies, such as insertions and deletions, may be included in regions of heteroduplex DNA. However, the direct products of heterology-containing heteroduplex DNA--heterozygous progeny phage--have not been observed. We have constructed a series of small insertion and deletion mutations in the cI gene to examine the possibility that small heterologies might be accommodated in heterozygous progeny phage. Genetic crosses were carried out between lambda cI- Oam29 and lambda cI+ Pam80 under replication-restricted conditions. Recombinant O+P+ progeny were selected on mutL hosts and tested for cI heterozygosity. Heterozygous recombinants were readily observed with crosses involving insertions of 4 to 19 base pairs (bp) in the cI gene. Thus, nonhomologies of at least 19 bp can be accommodated in regions of heteroduplex DNA during lambda recombination. In contrast, when a cI insertion or deletion mutation of 26 bp was present, few of the selected recombinants were heterozygous for cI. Results using a substitution mutation, involving a 26-bp deletion with a 22-bp insertion, suggest that the low recovery of cI heterozygotes containing heterologies of 26 bp or more is due to a failure to encapsulate DNA containing heterologies of 26 bp or more into viable phage particles.

Bacteriophage lambda↗
PubMed1982

Site-specific DNA condensation and pairing mediated by the int protein of bacteriophage lambda.

The int protein of bacteriophage lambda catalyzes the site-specific integrative recombination that inserts lambda DNA into the host chromosome. The attachment site region of lambda DNA required for this reaction spans 230 base pairs and includes four separable binding sites for int protein. We have used the electron microscope to determine the functional consequences of the interaction of int with its multiple binding sites. We find that int condenses a 230-base pair segment of DNA into a compact structure about 14 nm in diameter; the condensed region includes all of the four binding sites for int. Condensed segments will form paired structures between attachments sites. We suggest that a sequential cooperative interaction between bound int molecules provides for a distinct reactive DNA conformation and for pairing between substrate sites.

Bacteriophage lambda↗
PubMed1981

Retroregulation of the int gene of bacteriophage lambda: control of translation completion.

Bacteriophage lambda regulates the integration--excision reaction as a crucial aspect of the choice of pathway during lysogenic or lytic viral development. This control involves differential expression of the tightly linked, partially overlapping int and xis genes from two promoter sites: pI, positively regulated by cII/cIII proteins, and pL, positively regulated by N protein. After lambda infection, Int is synthesized from the pI transcript under cII regulation; however, very little Int is produced from the pL RNA because of the existence of a cis-acting regulatory element, sib, on the opposite side of the int gene from the pL promoter. Presumably sib serves to prevent unwanted synthesis of Int protein during the lytic response; the Int protein necessary for excisive recombination from a prophage can be supplied by pL transcription because sib is separated from int by prophage insertion. We have studied the effect of sib on nearby lambda genes by means of gel electrophoresis of labeled proteins from infected cells. Deletion of the sib region greatly enhances production of Int protein without substantial effect on Xis production; thus, sib regulation normally is highly specific for Int. When the sib region is moved past int and xis by deletion, regulation of the adjacent gene for the protein Ea22 occurs, suggesting that sib regulation can work on other genes. Although synthesis of wild-type Int is severely inhibited by sib, shorter Int protein fragments generated by nonsense mutations escape sib regulation, indicating that the regulation is translational and occurs near the completion stage of protein synthesis. Regulation by sib thus exhibits novel regulatory features: distal location, recombinational control, and regulation of the completion of protein synthesis. Because Int and Ea22 control is lost in a RNase III- host, we suggest that sib regulation might involve RNase III cleavage of a RNA duplex region that includes sib and the regulatory target (normally the int gene). We note such a potential site within int.

Bacteriophage lambda↗
PubMed1999

The geometry of a synaptic intermediate in a pathway of bacteriophage lambda site-specific recombination.

Bacteriophage lambda uses site-specific recombination to move its DNA into and out of the Escherichia coli genome. The recombination event is mediated by the phage-encoded integrase (Int) at short DNA sequences known as attachment ( att ) sites. Int catalyzes recombination via at least four distinct pathways, distinguishable by their requirements for accessory proteins and by the sequence of their substrates. The simplest recombination reaction catalyzed by Int does not require any accessory proteins and takes place between two attL sites. This reaction proceeds through an intermediate known as the straight-L bimolecular complex (SL-BMC), a stable complex which contains two attL sites synapsed by Int. We have investigated the orientation of the two substrates in the SL-BMC with respect to each other using two independent direct methods, a ligation assay and visualization by atomic force microscopy (AFM). Both show that the two DNA substrates in the complex are arranged in a tetrahedral or nearly square planar alignment skewed towards parallel. The DNA molecules in the complex are bent.

Attachment Sites, Microbiological↗
PubMed1991

Genetic analysis of Escherichia coli integration host factor interactions with its bacteriophage lambda H' recognition site.

The bacteriophage P22-based challenge phage system was used to study the binding of integration host factor (IHF) to its H' recognition site in the attP region of bacteriophage lambda. We constructed challenge phages that carried H' inserts in both orientations within the P22 Pant promoter, which is required for antirepressor synthesis. We found that IHF repressed expression of Pant from either challenge phage when expressed from an inducible Ptac promoter on a plasmid vector. Mutants containing changes in the H' inserts that decrease or eliminate IHF binding were isolated by selecting challenge phages that could synthesize antirepressor in the presence of IHF. Sequence analysis of 31 mutants showed that most changes were base pair substitutions within the H' insert. Approximately one-half of the mutants contained substitutions that changed base pairs that are part of the IHF consensus binding site; mutants were isolated that contained substitutions at six of the nine base pairs of the consensus site. Other mutants contained changes at base pairs between the two subdeterminants of the H' site, at positions that are not specified in the consensus sequence, and in the dA + dT-rich region that flanks the consensus region of the site. Taken together, these results show that single-base-pair changes at positions outside of the proposed consensus bases can weaken or drastically disrupt IHF binding to the mutated site.

Bacterial Proteins↗
PubMed1998

High-level expression of soluble heterologous proteins in the cytoplasm of Escherichia coli by fusion to the bacteriophage lambda head protein D.

The bacteriophage Lambda head protein D (gpD) is a small major capsid protein (110aa; 11.6kDa; pI=5.68, devoid of cysteine residues) that is essential for stable head morphogenesis. We found that a His-tagged derivative of gpD (gpHD) is a monomeric protein with efficient expression properties and high resistance towards thermally induced irreversible aggregation. In addition, gpHD can be used as a fusion partner for high-level expression of soluble heterologous proteins in the cytoplasm of Escherichia coli. Its broad utility is illustrated by the production of various mammalian proteins by fusion to its C-terminus. As a fusion partner, gpHD is thought to mediate optimal translation initiation while reducing inclusion body formation and protein degradation. In addition, it provides a His-tag for simple purification. gpHD may act as a 'cytoplasmic anchor' by keeping its unfolded fusion partner in solution, thereby providing more time for proper folding. An ever-increasing number of open reading frames (ORFs) are being identified in the various genome sequencing programs. gpHD has the potential to be harnessed for the development of highly efficient cytoplasmic expression systems that might contribute to the production and characterization of these novel polypeptides. Protein D is also an established fusion partner for phage display. It thus presents the attractive opportunity of coupling the selection of heterologous proteins from a phage library to their subsequent high-level expression.

Bacteriophage lambda↗
PubMed1998

Defining the structural and functional roles of the carboxyl region of the bacteriophage lambda excisionase (Xis) protein.

The bacteriophage lambda excisionase (Xis) protein is required for excisive site-specific recombination. Xis is composed of 72 amino acids and binds cooperatively to two DNA sites (X1 and X2) that are arranged as direct repeats. Alternatively, Xis binds cooperatively with the host-encoded factor for inversion stimulation (FIS) protein at the X1 and F sites, respectively. Here we analyzed the effects of missense substitutions from codon 57 to the carboxyl end of the protein and nonsense mutations that truncate the protein at various positions from residues 60 to 69. We find that all of the mutant proteins promote excision to some extent and interact cooperatively with FIS. Some mutants have no detectible phenotype while others are altered in their abilities to promote excision or to interact cooperatively with integrase (Int). Computer modeling predicts that amino acids from residues 59 to 65 are in an alpha-helix conformation. Mutants with substitutions on one side of the helix at residues 57, 60, 63 and 64 as well as truncated mutants containing 60, 61 or 63 amino acids, fail to interact cooperatively with Int suggesting that this region of the protein forms the interface with Int. Mutants with substitutions at other positions in the putative helix have no detectible phenotype. Residues 66 to 68 may form a reverse turn and the last four amino acids (69 to 72) may not be crucial for the structure or function of the protein.

Amino Acid Sequence↗
PubMed1985

Extent of sequence homology required for bacteriophage lambda site-specific recombination.

Bacteriophage lambda integration and excision occur by reciprocal recombination within a 15-base homologous core region present in the recombining attachment (att) sites. Strand exchange within the core occurs at precise nucleotide positions, which define an overlap region in which the products of recombination contain DNA strands derived from different parents. In order to define the role of sequence homology during recombination we have constructed point mutations within the core and assayed their effects in vivo and in vitro on site-specific recombination. Two of the mutations are located at position -3 of the core, which is one base-pair outside of the overlap region where strand exchange occurs. These mutations do not affect integrative or excisive recombination, thereby suggesting that homology outside the overlap region is not required for recombination. Two other mutations are located at position -2 of the core, which is one base-pair within the overlap region. These mutations show severely depressed integrative and excisive recombination activities in vitro and in vivo when recombined against wild-type att sites. However, the -2 mutations show normal recombination activity when recombined against att sites containing the homologous mutation, thereby suggesting that homology-dependent DNA interactions are required within the overlap region for effective recombination. In vitro recombination between homoduplex attP sites and heteroduplex attB sites demonstrated that the DNA interactions require only one strand of the attB overlap region to be homologous to attP in order to promote recombination.

Attachment Sites, Microbiological↗
PubMed1995

S gene expression and the timing of lysis by bacteriophage lambda.

The S gene of bacteriophage lambda encodes the holin required for release of the R endolysin at the onset of phage-induced host lysis. S is the promoter-proximal gene on the single lambda late transcript and spans 107 codons. S has a novel translational initiation region with dual start codons, resulting in the production of two protein products, S105 and S107. Although differing only by the Met-1-Lys-2... N-terminal extension present on S107, the two proteins are thought to have opposing functions, with the shorter polypeptide acting as the lysis effector and the longer one acting as an inhibitor. The expression of wild-type and mutant alleles of the holin gene has been assessed quantitatively with respect to the scheduling of lysis. S mRNA accumulates during the late gene expression period to a final level of about 170 molecules per cell and is maintained at that level for at least the last 15 min before lysis. Total S protein synthesis, partitioned at about 2:1 in favor of the S105 protein compared with the other product, S107, accumulates to a final level of approximately 4,600 molecules per cell. The kinetics of accumulation of S is consistent with a constant translational rate of less than one S protein per mRNA per minute. Mutant alleles with alterations in the translational initiation region were studied to determine how the translational initiation region of S achieves the proper partition of initiation events at the two S start codons and how the synthesis of S105 and S107 relates to lysis timing. The results are discussed in terms of a model for the pathway by which the 30S ribosome-fMet-tRNA complex binds to the translational initiation region of S. In addition, analysis of the relationship between lysis timing and the levels of the two S gene products suggests that S107 inhibits S105, the lethal lysis effector, by a stoichiometric titration.

Amino Acid Sequence↗
PubMed2005

A quantitative description of the binding states and in vitro function of antitermination protein N of bacteriophage lambda.

The N protein of bacteriophage lambda activates transcription of genes that lie downstream of termination sequences by suppressing transcription termination. N binds to specific (boxB) and non-specific sites on the transcript RNA and contacts RNA polymerase via cis-RNA looping, resulting in "antitermination" of transcription. To find the effect of N-boxB binding on antitermination, we quantitatively relate binding measurements made in isolation to in vitro antitermination activity. We measure binding of N to boxB RNA, non-specific single-stranded RNA, and non-specific double-stranded DNA fluorimetrically, and use an equilibrium model to describe quantitatively the binding of N to nucleic acids of Escherichia coli transcription elongation complexes. We then test the model by comparison with in vitro N antitermination activity measured in reactions containing these same elongation complexes. We find that binding of N protein to the nucleic acid components of transcription elongation complexes can quantitatively predict antitermination activity, suggesting that antitermination in vitro is determined by a nucleic acid binding equilibrium with one molecule of N protein per RNA transcript being sufficient for antitermination. Elongation complexes contain numerous overlapping non-specific RNA and DNA-binding sites for N; the large number of sites compensates for the low N binding affinity, so multiple N proteins are expected to bind to elongation complexes. The occupancy/activity of these proteins is described by a binomial distribution of proteins on transcripts containing multiple non-specific sites. The contribution of specific (boxB) binding to activity also depends on this distribution. Specificity is not measured accurately by measurements made in the presence and in the absence of boxB. We find that antitermination is inhibited by non-productive binding of N to non-specific sites on template DNA, and that NusA protein covers RNA sites on the transcript, limiting N access and activity. The activity and specificity of regulatory proteins that loop from high-affinity binding sites are likely modulated by multiple non-specific binding events; in vivo activity may also be regulated by the modulation of non-specific binding.

Bacteriophage lambda↗
PubMed1992

The purification and properties of the scaffolding protein of bacteriophage lambda.

The Nu3 gene of bacteriophage lambda resides within a cluster of genes that specify structural components of the bacteriophage head. Previous experiments indicate that the Nu3 gene product (gpNu3) is associated with immature proheads but is not detectable in mature proheads or bacteriophage particles, hence its classification as a scaffolding protein. The Nu3 gene has been cloned and overexpressed, and its protein product has been purified. The purified protein is biologically active, as demonstrated by its ability to complement a gpNu3-deficient extract in an in vitro assembly reaction. The sequence of the amino terminus of the protein indicates that translation of Nu3 starts at nucleotide position 5,342 on the standard lambda DNA sequence, yielding a protein with a calculated Mr of 13,396. A combination of gel exclusion chromatography and velocity sedimentation gradient data indicates that gpNu3 possesses an unusually elongated shape.

Amino Acid Sequence↗
PubMed1977

A map of the cleavage sites for endonuclease AvaI in the chromosome of bacteriophage lambda.

The linear order of nine fragments generated by the action of endonuclease AvaI on the DNA of bacteriophage lambda was determined from the altered fragmentation patterns of bacteriophages containing known deletions and of hybrids of bacteriophages lambda and phi80. Digestion of 5'-terminally 32P-labelled bacteriophage-lambda DNA was used to identify the terminal fragments. Measurement of relative fragment lengths permitted rough mapping of the endonuclease-AvaI cleavage sites relative to the ends of the bacteriophage-lambda chromosome. The fragment order was confirmed and the map refined by analysis of the fragmentation of derivative phages containing single cleavage sites for endonuclease EcoRI.

Chromosome Mapping↗
PubMed1991

Damage and mutagenesis of bacteriophage lambda induced by high pH.

Bacteriophage lambda-Escherichia coli complexes exhibited remarkable sensitivity to alkaline pH 10.0 at 37 degrees C. The decline in plaque forming units after alkali treatment was more pronounced in complexes with some of the radiation repair defective mutants of E. coli K-12, i.e. uvrArecA, recA, rer and lexA mutants as compared to those of uvrA, recB and wild-type strains. The red gene of lambda phage and recA gene of E. coli seem to have a complementary effect on the alkali induced lesions. Alkaline treatment to lysogenic lambda phage was also found to be mutagenic. An enhanced level of mutagenesis was observed when treated phage particles were allowed to adsorb on treated wild-type bacteria. Moreover, the alkali treatment to lysogen (lambda cI857-E. coli) resulted in prophage induction in nutrient broth even at 32 degrees C. Thus on the basis of these results the role of error prone SOS repair systems in the repair of alkali induced lesions in lysogenic bacteriophage lambda has been suggested.

Bacteriophage lambda↗
PubMed2000

Formation and stability of the bacteriophage lambda replication complexes in UV-irradiated Escherichia coli.

Bacteriophage lambda replication complex, containing the phage-encoded O initiator protein protected from proteases by other elements of this complex, is a stable structure that can be inherited by one of the two daughter lambda DNA copies after a replication round in Escherichia coli. In normal growth conditions in bacteria bearing a plasmid derived from bacteriophage lambda, such a complex may be stable for many cell generations. However, it was found that this stable structure is disassembled under certain conditions, namely, after heat shock. Therefore, we asked whether other environmental stresses may cause disassembly of the lambda replication complex. We found that UV irradiation of the host cells prevented formation of the stable lambda replication complex (though not preventing phage replication), while the same UV doses did not affect the stability of the replication complex assembled prior to the irradiation. These results indicate that the stable lambda replication complex, although sensitive to heat shock, is resistant to some other environmental stresses and that formation of at least two types of lambda replication complexes is possible. Both stable and unstable lambda replication complexes are functional because replication of lambda DNA under conditions preventing formation of the stable complex proceeds efficiently.

Bacteriolysis↗
PubMed1986

Role of RecA protein in untargeted UV mutagenesis of bacteriophage lambda: evidence for the requirement for the dinB gene.

Untargeted UV mutagenesis of bacteriophage lambda--i.e., the increased recovery of lambda mutants when unirradiated lambda infects UV-irradiated Escherichia coli--is thought to be mediated by a transient decrease in DNA replication fidelity, generating mutations in the newly synthesized strands. Using the bacteriophage lambda cI857----lambda c mutation system, we provide evidence that the RecA protein, shown previously to be required for this mutagenic pathway, is no longer needed when the LexA protein is inactivated by mutation. We suggest that the error-prone DNA replication responsible for UV-induced untargeted mutagenesis is turned on by the presence of replication-blocking lesions in the host cell DNA and that the RecA protein is required only to derepress the relevant din gene(s). This is in contrast to mutagenesis of irradiated bacteria or irradiated phage lambda, in which activated RecA protein has a second role in mutagenesis in addition to the cleavage of the LexA protein. Among the tested din genes, the dinB gene product (in addition to the uvrA and uvrB gene products) was found to be required for untargeted mutagenesis of bacteriophage lambda. To our knowledge, a phenotype associated with the dinB gene has not been reported previously.

Bacterial Proteins↗
PubMedDate not supplied

ClpP/ClpX-mediated degradation of the bacteriophage lambda O protein and regulation of lambda phage and lambda plasmid replication.

The O protein is a replication initiator that binds to the orilambda region and promotes assembly of the bacteriophage lambda replication complex. This protein, although protected from proteases by other elements of the replication complex, in a free form is rapidly degraded in the host, Escherichia coli, by the ClpP/ClpX protease. Nevertheless, the physiological role of this rapid degradation remains unclear. Here we demonstrate that the copy number of plasmids derived from bacteriophage lambda is significantly higher in wild-type cells growing in rich media than in slowly growing bacteria. However, lambda plasmid copy number in bacteria devoid of the ClpP/ClpX protease was not dependent on the bacterial growth rate and in all minimal media tested was comparable to that observed in wildtype cells growing in a rich medium. Contrary to lambda plasmid replication, the efficiency of lytic growth of bacteriophage lambda was found to be dependent on the host growth rate in both wild-type bacteria and clpP and clpX mutants. The activities of two major lambda promoters operating during the lytic development, p(R) and p(L), were found to be slightly dependent on the host growth rate. However, when p(R) activity was significantly decreased in the dnaA mutant, production of phage progeny was completely abolished at low growth rates. These results indicate that the O protein (whose level in E. coli cells depends on the activity of ClpP/ClpX protease) is a major limiting factor in the regulation of lambda plasmid replication at low bacterial growth rates. However, this protein seems to be only one of the limiting factors in the bacteriophage lambda lytic development under poor growth conditions of host cells. Therefore, it seems that the role of the rapid ClpP/ClpX-mediated proteolysis of the O protein is to decrease the efficiency of early DNA replication of the phage in slowly growing host cells.

ATPases Associated with Diverse Cellular Activitie↗