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PubMed1972

Lytic replication of coliphage lambda in Salmonella typhosa hybrids.

Hybrids between Escherichia coli K-12 and Salmonella typhosa which conserved a continuous K-12 chromosomal diploid segment extending from pro through ara to the strA locus were sensitive to plaque formation by wild-type lambda. These partially diploid S. typhosa hybrids could be lysogenized with lambda and subsequently induced to produce infectious phage particles. When the K-12 genes were segregated from a lysogenic S. typhosa hybrid, phage-productive ability was no longer detectable due to loss of a genetic region necessary for vegetative replication of lambda. However, lambda prophage was shown to persist in a quiescent state in the S. typhosa hybrid segregant with phage-productive ability being reactivated after replacement of the essential K-12 lambda replication region. Low-frequency transduction and high-frequency transduction lysates containing the gal(+) genes of S. typhosa were prepared by induction of lambda-lysogenic S. typhosa hybrids indicating that the attlambda site is chromosomally located in S. typhosa in close proximity to the gal locus as in E. coli K-12. After propagation in S. typhosa hybrids, lambda was subject to restriction by E. coli K-12 recipients, thus establishing that S. typhosa does not perform the K-12 modification of lambda deoxyribonucleic acid. Hybrids of S. typhosa, however, did not restrict lambda grown previously on E. coli K-12. The K-12 genetic region required for lambda phage production in S. typhosa was located within min 66 to min 72 on the genetic map of the E. coli chromosome. Transfer of an F-merogenote encompassing the 66 to 72 min E. coli chromosomal region to lambda-insensitive S. typhosa hybrids enabled them to replicate wild-type lambda. The lambda-insensitive S. typhosa hybrid, WR4255, which blocks lambda replication, can be mutagenized to yield mutant strains sensitive to lambdavir and lambdaimm434. These WR4255 mutants remained insensitive to plaque formation by wild-type lambda.

Chromosome Mapping↗

PubMed1991

Halobacterium halobium strains lysogenic for phage phi H contain a protein resembling coliphage repressors.

DNA-binding proteins such as bacteriophage repressors belong to the helix-turn-helix family. Ionic interactions drive DNA binding, which means that repressors bind DNA most tightly at low salt concentrations. This raises the question of who gene expression might be regulated in obligate halophiles, which maintain internal salt concentrations of about 5 M. As a model system we have investigated the phage phi H, which infects the archaebacterium Halobacterium halobium. Previous genetic data and transcriptional mapping had suggested a region of the phage genome where a repressor might bind. A modified electrophoretic mobility shift assay was used to identify an activity, present only in lysogens, that specifically binds this region. Methylation interference and DNA sequencing were used to identify four similar binding sites, which are arranged so that two copies of a dimer might bind on one face of the DNA helix. Binding of a protein at these sites could block RNA polymerase from initiating a transcript found only during lytic growth. A nearby divergent promoter produces a lysogen-specific transcript, T6, which encodes a member of the helix-turn-helix family of DNA-binding proteins. By expressing the gene in Escherichia coli, we confirmed that T6 specifies the DNA binding activity detected biochemically. The data show that the basic DNA-binding motif of repressors can be adapted even for the unfavorable conditions of high salt concentration.

Amino Acid Sequence↗

PubMed2000

Cloning, expression, and purification of the K5 capsular polysaccharide lyase (KflA) from coliphage K5A: evidence for two distinct K5 lyase enzymes.

The Escherichia coli K5 capsular polysaccharide [-4)-betaGlcA-(1, 4)-alphaGlcNAc-(1-] is a receptor for the capsule-specific bacteriophage K5A. Associated with the structure of bacteriophage K5A is a polysaccharide lyase which degrades the K5 capsule to expose the underlying bacterial cell surface. The bacteriophage K5A lyase gene (kflA) was cloned and sequenced. The kflA gene encodes a polypeptide with a predicted molecular mass of 66.9 kDa and which exhibits amino acid homology with ElmA, a K5 polysaccharide lyase encoded on the chromosome of E. coli SEBR 3282. There was only limited nucleotide homology between the kflA and elmA genes, suggesting that these two genes are distinct and either have been derived from separate progenitors or have diverged from a common progenitor for a considerable length of time. Southern blot analysis revealed that kflA was not present on the chromosome of the E. coli strains examined. In contrast, elmA was present in a subset of E. coli strains. Homology was observed between DNA flanking the kflA gene of bacteriophage K5A and DNA flanking a small open reading frame (ORF(L)) located 5' of the endosialidase gene of the E. coli K1 capsule-specific bacteriophage K1E. The DNA homology between these noncoding sequences indicated that bacteriophages K5A and K1E were related. The deduced polypeptide sequence of ORF(L) in bacteriophage K1E exhibited homology to the N terminus of KflA from bacteriophage K5A, suggesting that ORF(L) is a truncated remnant of KflA. The presence of this truncated kflA gene implies that bacteriophage K1E has evolved from bacteriophage K5A by acquisition of the endosialidase gene and subsequent loss of functional kflA. A (His)(6)-KflA fusion protein was overexpressed in E. coli and purified to homogeneity with a yield of 4.8 mg per liter of bacterial culture. The recombinant enzyme was active over a broad pH range and NaCl concentration and was capable of degrading K5 polysaccharide into a low-molecular-weight product.

Amino Acid Sequence↗

PubMed2006

Horizontal gene transfer and the evolution of microvirid coliphage genomes.

Bacteriophage genomic evolution has been largely characterized by rampant, promiscuous horizontal gene transfer involving both homologous and nonhomologous source DNA. This pattern has emerged through study of the tailed double-stranded DNA (dsDNA) phages and is based upon a sparse sampling of the enormous diversity of these phages. The single-stranded DNA phages of the family Microviridae, including phiX174, appear to evolve through qualitatively different mechanisms, possibly as result of their strictly lytic lifestyle and small genome size. However, this apparent difference could reflect merely a dearth of relevant data. We sought to characterize the forces that contributed to the molecular evolution of the Microviridae and to examine the genetic structure of this single family of bacteriophage by sequencing the genomes of microvirid phage isolated on a single bacterial host. Microvirids comprised 3.5% of the detectable phage in our environmental samples, and sequencing yielded 42 new microvirid genomes. Phylogenetic analysis of the genes contained in these and five previously described microvirid phages identified three distinct clades and revealed at least two horizontal transfer events between clades. All members of one clade have a block of five putative genes that are not present in any member of the other two clades. Our data indicate that horizontal transfer does contribute to the evolution of the microvirids but is both quantitatively and qualitatively different from what has been observed for the dsDNA phages.

Coliphages↗

PubMed1951