Lambda phage

About: Lambda phage is a research topic. Over the lifetime, 1609 publications have been published within this topic receiving 84675 citations. The topic is also known as: Enterobacteria phage lambda.

Isolation and characterization of point mutations in the Escherichia coli grpE heat shock gene.

Abstract: The Escherichia coli grpE gene (along with dnaK, dnaJ, groEL, and groES) was originally identified as one of the host factors required for phage lambda growth. The classical grpE280 mutation was the only grpE mutation that resulted from the initial screen and shown to specifically block the initiation of lambda DNA replication. Here we report the isolation of several new grpE missense mutations, again using phage lambda resistance as a selection. All mutants fall into two groups based on their temperature-dependent phenotype for lambda growth. Members of the first group (I), including grpE17 and grpE280, which was obtained again, are resistant to lambda growth at both 30 and 42 degrees C. Members of the second group (II), including grpE25, grpE66, grpE103, grpE13a, grpE57b, and grpE61, are sensitive to lambda growth at 30 degrees C but resistant at 42 degrees C. All mutations are recessive, since an E. coli grpE null mutant strain carrying these mutant alleles on low-copy-number plasmids are sensitive to infection by the lambda grpE+ transducing phage. Both group I and group II mutants are temperature sensitive for E. coli growth above 42 degrees C. The nucleotide changes were identified by sequencing analyses and shown to be dispersed throughout the latter 75% of the grpE coding region. Most of the amino acid changes occur at conserved residues, as judged by sequence comparisons between E. coli and other bacterial and yeast GrpE homologs. The isolation of these new mutations is the first step toward a structure-function analysis of the GrpE protein.

A genetic analysis of Xis and FIS interactions with their binding sites in bacteriophage lambda.

Abstract: The bacteriophage P22-based challenge-phage system was used to study the binding of Xis and FIS to their sites in attP of bacteriophage lambda. Challenge phages were constructed that contained the X1, X2, and F sites within the P22 Pant promoter, which is required for expression of antirepressor. If Xis and FIS bind to these sites in vivo, they repress transcription from Pant, allowing lysogenization to occur. Challenge phages carrying the XIX2F region in either orientation exhibited lysogenization dependent on both Xis and FIS. Neither Xis nor FIS was capable of functioning by itself as an efficient repressor in this system. This was the first time challenge phages have been constructed that require two different proteins bound simultaneously to act as a repressor. Mutations in the X1, X2, and F sites that inhibit Xis and FIS from binding were isolated by selecting mutant phages that still expressed antirepressor synthesis in the presence of Xis and FIS. DNA sequence analysis of the mutants revealed 38 unique mutations, including single-base-pair substitutions, multiple-base-pair changes, deletions, and insertions throughout the entire X1, X2, and F regions. Some of the mutations verified the importance of certain bases within the proposed consensus sequences for Xis and FIS, while others provided evidence that the DNA sequence outside of the proposed binding sites may affect the binding of the individual proteins or the cooperativity between them.

Genetic characterization of a gene for prolipoprotein signal peptidase in Escherichia coli.

Abstract: A mutation (lspA, prolipoprotein signal peptidase) rendering the prolipoprotein signal peptidase temperature-sensitive in Escherichia coli has been analyzed. The mutation was mapped in the dnaJ-rpsT-ileS-dapB region by interrupted mating with various Hfr strains and P1 phage transduction. lambda transducing phage lambda ddapB2 that carries the rpsT-ileS-dapB region was shown to complement the lspA mutation. Plasmid pLC3-13 which had been isolated from Clarke and Carbon's collection as a plasmid carrying the lspA locus was shown to carry the dnaJ and rpsT loci. Complementation analysis with plasmids carrying various DNA fragments derived from pLC3-13 showed that the lspA locus is between the rpsT and ileS loci. The wildtype allele was dominant over the lspA allele.

Interaction of bacteriophage lambda with its cell surface receptor: an in vitro study of binding of the viral tail protein gpJ to LamB (Maltoporin).

Abstract: The cell surface receptor for bacteriophage Lambda is LamB (maltoporin). Responsible for phage binding to LamB is the C-terminal part, gpJ, of phage tail protein J. To study the interaction between LamB and gpJ, a chimera protein composed of maltose binding protein (MBP or MalE) connected to the C-terminal part of J (gpJ, amino acids 684-1131) of phage tail protein J of bacteriophage Lambda was expressed in Escherichia coli and purified to homogeneity. The interaction of the MBP-gpJ chimera protein with reconstituted LamB and its mutants LamB Y118G and the loop deletion mutant LamB Delta4+Delta6+Delta9v was studied using planar lipid bilayer membranes on a single-channel and multichannel level. Titration with the MBP-gpJ chimera blocked completely the ion current through reconstituted LamB when it was added to the cis side, the extracellular side of LamB with a half-saturation constant of approximately 6 nM in 1 M KCl. Control experiments with LamB Delta4+Delta6+Delta9v from which all major external loops had been removed showed similar blocking, whereas MBP alone caused no visible effect. Direct conductance measurement with His(6)-gpJ that contained a hexahistidyl tag (His(6) tag) at the N-terminal end of the protein for easy purification revealed no blocking of the ion current, requiring other measurements for the binding constant. However, when maltoporin was preincubated with His-gpJ, MBP-gpJ could not block the channel, which indicated that also His(6)-gpJ bound to the channel. High-molecular mass bands on SDS-PAGE and Western blots, confirming the planar lipid bilayer experiment results, also demonstrated stable complex formation between His(6)-gpJ and LamB or LamB mutants. The results revealed that phage Lambda binding includes not only the extracellular loops.

Genesis and Natural History of IS-mediated Transposons

Abstract: The natural genesis of IS1-mediated transposons containing the genetic determinant cat for chloramphenicol resistance is documented. First, the small plasmid pBR325 containing the cat gene served as a target in IS1-mediated transpositional cointegration with the genome of bacteriophage P1, which was the source of the IS1. From the resulting pBR325:P1 plasmids, pBR325::IS1 segregants were isolated. Upon growth of a phage lambda derivative in the presence of this plasmid, rare plaque-forming lambda Cmr specialized transducing phages were formed. In each of six independent lambda Cmr isolates studied, the cat gene was carried between flanking IS1 elements. In one case, these IS1 elements were in the same orientation; in the other five cases, they were in opposite orientation. All of these IS1-cat-IS1 structures transposed as units to the genome of phage P1-15, pointing to stable maintenance of the transposon. However, appropriate selection allowed us to follow the decay of these transposons. Models to explain the genesis of transposons with directly and inversely repeated IS elements are discussed, as well as the evolutionary implications of these mechanisms.