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PubMed2001

Crystal structure of the lytic transglycosylase from bacteriophage lambda in complex with hexa-N-acetylchitohexaose.

The three-dimensional structure of the lytic transglycosylase from bacteriophage lambda, also known as bacteriophage lambda lysozyme, complexed to the hexasaccharide inhibitor, hexa-N-acetylchitohexaose, has been determined by X-ray crystallography at 2.6 A resolution. The unit cell contains two molecules of the lytic transglycosylase with two hexasaccharides bound. Each enzyme molecule is found to interact with four N-acetylglucosamine units from one hexasaccharide (subsites A-D) and two N-acetylglucosamine units from the second hexasaccharide (subsites E and F), resulting in all six subsites of the active site of this enzyme being filled. This crystallographic structure, therefore, represents the first example of a lysozyme in which all subsites are occupied, and detailed protein-oligosaccharide interactions are now available for this bacteriophage lytic transglycosylase. Examination of the active site furthermore reveals that of the two residues that have been implicated in the reaction mechanism of most other c-type lysozymes (Glu35 and Asp52 in hen egg white lysozyme), only a homologous Glu residue is present. The lambda lytic transglycosylase is therefore functionally closely related to the Escherichia coli Slt70 and Slt35 lytic transglycosylases and goose egg white lysozyme which also lack the catalytic aspartic acid.

Acetylglucosamine↗
PubMedDate not supplied

Sequence-induced DNA curvature at the bacteriophage lambda origin of replication.

DNA replication in bacteriophage lambda begins at a unique origin between residues 39,000 and 39,200 of the lambda genome. This segment of DNA serves a dual function since it also lies within the coding sequence of the lambda replication initiator protein O which binds origin DNA. The lambda origin sequence contains four 19-base-pair (bp) segments (iterons) which have dyad symmetry, followed by a 40-bp A + T-rich zone of highly asymmetrical base composition. It was noted earlier that lambda origin DNA exhibits an anomalous electrophoretic mobility on gels; that is, the length of DNA as determined by DNA sequencing is approximately 20% less than is predicted from electrophoretic mobility. Recent studies of kinetoplast minicircle DNA (K-DNA) from the protozoan Leishmania tarentolae have led to the proposal that sequence-induced DNA curvature could account for such electrophoretic anomalies by alteration of the shape of the DNA molecule. We now present evidence that the lambda origin contains a static curve.

Bacteriophage lambda↗
PubMed1982

Transcriptional termination sites in the b2 region of bacteriophage lambda that are unresponsive to antitermination.

A bacteriophage lambda cloning vector carrying the trp/lacW205 substitution is described. The vector facilitates the fusion in vitro of genetic control signals to the lacZ structural gene of Escherichia coli. This system was used to define transcriptional termination sites in the lambda b2 region. This region contains termination sites that are unresponsive to the lambda antiterminating proteins pQ and pN.

Bacteriophage lambda↗
PubMed1984

Lambda placMu: a transposable derivative of bacteriophage lambda for creating lacZ protein fusions in a single step.

We isolated a plaque-forming derivative of phage lambda, lambda placMu1 , that contains sequences from bacteriophage Mu enabling it to integrate into the Escherichia coli chromosome by means of the Mu transposition system. The Mu DNA carried by this phage includes both attachment sites as well as the cI, ner (cII), and A genes. Lambda placMu1 also contains the lacZ gene, deleted for its transcription and translation initiation signals, and the lacY gene of E. coli, positioned next to the terminal 117 base pairs from the S end of Mu. Because this terminal Mu sequence is an open reading frame fused in frame to lacZ, the phage can create lacZ protein fusions in a single step when it integrates into a target gene in the proper orientation and reading frame. To demonstrate the use of this phage, we isolated lacZ fusions to the malB locus. These showed the phenotypes and regulation expected for malB fusions and could be used to isolate specialized transducing phages carrying the entire gene fusion as well as an adjacent gene (malE). They were found to be genetically stable and rarely (less than 10(-7] gave rise to secondary Lac+ insertions. We also isolated insertions into high-copy-number plasmids. The physical structure of these phage-plasmid hybrids was that expected from a Mu-dependent insertion event, with the lambda placMu prophage flanked by the Mu attachment sites. Lac+ insertions into a cloned recA gene were found at numerous positions and produced hybrid proteins whose sizes were correlated with the position of the fusions in recA.

Bacteriophage lambda↗
PubMed1993

Expression of the Rz gene and the overlapping Rz1 reading frame present at the right end of the bacteriophage lambda genome.

The Rz lysis gene of bacteriophage lambda was cloned into the expression vectors, pT7-3 and pT7-7. The recombinant plasmids expressed either a protein of an unexpected 6.5-kDa size (pT7-3H and pSB54) or two proteins of 6.5 and 17.2 kDa (pBH21). The 6.5-kDa protein alone did not complement the lysis defect of the lambda Rz mutant; hence, this protein was not the Rz gene product. Complementation observed as a result of pBH21 expression thus can be ascribed to the 17.2-kDa protein, which agrees with the size based on the nucleotide sequence of Rz. The 6.5 kDa is a product of an open reading frame entirely encompassed within the Rz sequence and denoted by us Rz1. Both proteins were detectable only by autoradiography, which may mean that the genes are expressed at low rates. Polyclonal anti-Rz antibodies (Ab) were obtained by rabbit immunization with a synthetic polypeptide corresponding to an antigenic determinant of Rz defined by a computer program. The Ab reacted with the 17.2-kDa protein resulting from pBH21 expression, as well as with the 17.2-kDa protein present in the induced Escherichia coli W3350(lambda cI857Sam7) lysate.

Amino Acid Sequence↗
PubMed2001

Facilitation of bacteriophage lambda DNA injection by inner membrane proteins of the bacterial phosphoenol-pyruvate: carbohydrate phosphotransferase system (PTS).

Infection of Escherichia coli by bacteriophage lambda depends on two membrane protein complexes: (i) maltoporin (LamB) in the outer membrane for adsorption and (ii) the IIC(Man)-IID(Man) complex of the mannose transporter in the inner membrane for DNA penetration. IIC(Man) and IID(Man) are components of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) which together with the IIAB(Man) subunit mediate transport and phosphorylation of sugars. To identify structural determinants important for penetration of lambda DNA, the homologous IIC-IID complexes of E. coli, K. pneumoniae and B. subtilis, and chimeric complexes between the IIC and IID were characterized. All three complexes support sugar transport in E. coli. Only IIC-IID of E. coli and B. subtilis also support bacteriophage lambda infection. The six chimeric complexes had lost transport activity, but three containing IIC of E. coli or B. subtilis continue to support bacteriophage lambda infection. Complexes containing IIC(Man) and fusion proteins between truncated IID(Man) and alkaline phosphatase or beta-galactosidase support penetration of lambda DNA if less than 100 residues are missing from the C-terminus of IID(Man). Truncation of IIC(Man) renders the complex unstable. Taken together, these results suggest, that IIC is the major specificity determinant for lambda infection but that the IIC subunit is stably expressed only in a complex with the IID subunit. Lambda DNA in transit across the periplasmic space, but not transforming plasmid DNA, is inaccessible to the non-specific nuclease NucA of Anabaena sp. targeted to the periplasmic space either in soluble form or as a fusion protein to the C-terminus of IID(Man).

Bacillus subtilis↗
PubMed2004

In vivo recombineering of bacteriophage lambda by PCR fragments and single-strand oligonucleotides.

We demonstrate that the bacteriophage lambda Red functions efficiently recombine linear DNA or single-strand oligonucleotides (ss-oligos) into bacteriophage lambda to create specific changes in the viral genome. Point mutations, deletions, and gene replacements have been created. While recombineering with oligonucleotides, we encountered other mutations accompanying the desired point mutational change. DNA sequence analysis suggests that these unwanted mutations are mainly frameshift deletions introduced during oligonucleotide synthesis.

Bacteriophage lambda↗
PubMed1992

Bacteriophage lambda PaPa: not the mother of all lambda phages.

The common laboratory strain of bacteriophage lambda--lambda wild type or lambda PaPa--carries a frameshift mutation relative to Ur-lambda, the original isolate. The Ur-lambda virions have thin, jointed tail fibers that are absent from lambda wild type. Two novel proteins of Ur-lambda constitute the fibers: the product of stf, the gene that is disrupted in lambda wild type by the frameshift mutation, and the product of gene tfa, a protein that is implicated in facilitating tail fiber assembly. Relative to lambda wild type, Ur-lambda has expanded receptor specificity and adsorbs to Escherichia coli cells more rapidly.

Adsorption↗
PubMed1987

Pseudogene in the genome of bacteriophage lambda?

We find a region in the non-coding part of bacteriophage lambda genome that codes for the conserved fold which repressors and other proteins use for specific DNA binding. The region is involved in a long open reading frame exceeding one kilobase and is read in the same frame as gene A in the opposite strand. The putative translation product of this open reading frame has a highly ordered secondary structure with a predominance of alpha helices, which is typical of repressors. In addition, codon usage in this frame suggests a protein-coding region. However, there is a TGA stop codon located between the putative gene start point and the region coding for the DNA binding fold. It thus appears that bacteriophage lambda had one more DNA binding protein, perhaps repressor, in the past that was inactivated by a mutation.

Amino Acid Sequence↗
PubMedDate not supplied

Resolution of synthetic att-site Holliday structures by the integrase protein of bacteriophage lambda.

Site-specific recombination of the bacteriophage lambda genome into and out of the host bacterial genome is postulated to involve the formation of Holliday structure intermediates by reciprocal single-strand exchanges. Synthetic analogues of the predicted recombination intermediates are resolved in vitro by the protein product of the lambda int gene. Some of the structural features and reaction conditions for this genetic recombination can now be defined.

Bacteriophage lambda↗
PubMed1988

The Nu1 subunit of bacteriophage lambda terminase.

The maturation and packaging of bacteriophage lambda DNA are catalyzed by the phage terminase enzyme. Terminase is composed of two protein subunits, gpNu1 and gpA. The holoenzyme is multifunctional in vitro; it binds to and cleaves lambda DNA at the cos site (where cos represents cohesive-end site), packages DNA into lambda proheads, and is also a DNA-dependent ATPase. The genes of the two subunits have been cloned separately into powerful expression vectors which allow for very high levels of protein overproduction. The gpNu1 protein has been purified to homogeneity and has a monomeric molecular weight of 21,200, in close agreement with the Mr of 20,444 expected from its amino acid sequence. Both gel filtration and sedimentation velocity centrifugation indicate that the native gpNu1 protein exists as a Mr greater than 500,000 aggregate. The sequence of the first 20 amino acids and the overall composition both match those predicted by the nucleotide sequence of the Nu1 gene. Purified gpNu1 is able to complement gpA-containing extracts in both lambda DNA packaging and cos cleavage assays. The Nu1 gene amino acid sequence predicts DNA binding by the protein, and gpNu1 does show specific binding to lambda DNA by filter binding assays. Also, as predicted from its sequence, gpNu1 exhibits ATPase activity; but in contrast to the holoenzyme, this activity is DNA-independent.

Amino Acids↗
PubMed1991

Isolation and characterization of mutations in the bacteriophage lambda terminase genes.

The terminase enzyme of bacteriophage lambda is a hetero-oligomeric protein which catalyzes the site-specific endonucleolytic cleavage of lambda DNA and its packaging into phage proheads; it is composed of the products of the lambda Nul and A genes. We have developed a simple method to select mutations in the terminase genes carried on a high-copy-number plasmid, based on the ability of wild-type terminase to kill recA strains of Escherichia coli. Sixty-three different spontaneous mutations and 13 linker insertion mutations were isolated by this method and analyzed. Extracts of cells transformed by mutant plasmids displayed variable degrees of reduction in the activity of one or both terminase subunits as assayed by in vitro lambda DNA packaging. A method of genetically mapping plasmid-borne mutations in the A gene by measuring their ability to rescue various lambda Aam phages showed that the A mutations were fairly evenly distributed across the gene. Mutant A genes were also subcloned into overproducing plasmid constructs, and it was determined that more than half of them directed the synthesis of normal amounts of full-length A protein. Three of the A gene mutants displayed dramatically reduced in vitro packaging activity only when immature (uncut) lambda DNA was used as the substrate; therefore, these mutations may lie in the endonuclease domain of terminase. Interestingly, the putative endonuclease mutations mapped in two distinct locations in the A gene separated by a least 400 bp.

Bacteriophage lambda↗
PubMed1988

Translation initiation controls the relative rates of expression of the bacteriophage lambda late genes.

The late operon of bacteriophage lambda contains the genes encoding the morphogenetic proteins of the phage. These genes are transcribed equally from the single late promoter. Although the functional half-lives of the mRNA for the various genes of this operon vary less than 2-fold, their relative rates of expression have been shown to vary by nearly 1000-fold. This variation could result from differing rates of translation initiation, from overlapping upstream translation, or from differential elongation rates due to the presence of codons for which the corresponding tRNAs are rare. To distinguish between these possibilities, we have cloned sequences surrounding the initiator codons of several of these genes and measured their ability to drive synthesis of hybrid lambda-beta-galactosidase proteins. The rates of expression of the hybrid genes thus produced correlate very well with the natural rates of expression of the corresponding phage genes, suggesting that the rate of initiation of translation controls the relative expression rates of these genes.

Bacteriophage lambda↗
PubMed1984

Proteinase sensitivity of bacteriophage lambda tail proteins gpJ and pH in complexes with the lambda receptor.

Previous studies have shown that bacteriophage lambda initially binds to liposomes bearing its receptor protein by the tip of the tail fiber (type 1 complex). It then associates more directly so that the hollow tail tube is in direct contact with the membrane (type 2 complex). DNA can be injected across the lipid bilayer into the liposome from type 2 complexes. We show here that gpJ, the tail fiber protein, becomes more sensitive to proteolytic degradation in type 2 complexes, indicating that the tail fiber does not pass into the liposome and that the tail fiber may undergo a conformational change in type 2 complexes. Another bacteriophage protein, pH, is sensitive to proteolytic degradation in free bacteriophage, type 1 complexes, or type 2 complexes formed with free receptor, but is resistant to proteinases in type 2 complexes formed with liposomes. This finding suggests that pH associates with the membrane. We suggest that this association is part of the mechanism by which a transmembrane hole for DNA entry is formed.

Bacterial Outer Membrane Proteins↗
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↗
PubMed1999

The incorporation of a non-natural amino acid (aza-tryptophan) may help to crystallize a protein and to solve its crystal structure. Application to bacteriophage lambda lysozyme.

Until now, wild-type bacteriophage lambda lysozyme had been impossible to crystallize. This difficulty could be overcome by the replacement of the four tryptophan residues by aza-tryptophans. Analysis of the intermolecular and intramolecular contacts in this modification allows understanding of the differences in behaviour between the native and modified molecules. Furthermore, this mutation was very useful for the creation of new heavy-atom binding sites and for the solution of the non-crystallographic symmetry, which is extremely important for phase improvement. This procedure seems to be generally applicable, at least in the search for new possibilities for heavy-atom binding sites.

Aza Compounds↗
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↗