Richard Calendar

Complete nucleotide sequence, molecular analysis and genome structure of bacteriophage A118 of Listeria monocytogenes : implications for phage evolution

A118 is a temperate phage isolated from Listeria monocytogenes. In this study, we report the entire nucleotide sequence and structural analysis of its 40 834 bp DNA. Electron microscopic and enzymatic analyses revealed that the A118 genome is a linear, circularly permuted, terminally redundant collection of double‐stranded DNA molecules. No evidence for cohesive ends or for a terminase recognition (pac) site could be obtained, suggesting that A118 viral DNA is packaged via a headful mechanism. Partial denaturation mapping of DNA cross‐linked to the tail shaft indicated that DNA packaging proceeds from left to right with respect to the arbitrary genomic map and the direction of genes necessary for lytic development. Seventy‐two open reading frames (ORFs) were identified on the A118 genome, which are apparently organized in a life cycle‐specific manner into at least three major transcriptional units. N‐terminal amino acid sequencing, bioinformatic analyses and functional characterizations enabled the assignment of possible functions to 26 ORFs, which included DNA packaging proteins, morphopoetic proteins, lysis components, lysogeny control‐associated functions and proteins necessary for DNA recombination, modification and replication. Comparative analysis of the A118 genome structure with other bacteriophages revealed local, but sometimes extensive, similarities to a number of phages spanning a broader phylogenetic range of various low G+C host bacteria, which implies relatively recent exchange of genes or genetic modules. We have also identified the A118 attachment site attP and the corresponding attB in Listeria monocytogenes, and show that site‐specific integration of the A118 prophage by the A118 integrase occurs into a host gene homologous to comK of Bacillus subtilis, an autoregulatory gene specifying the major competence transcription factor.

Mode of DNA packing within bacteriophage heads.

Abstract Electron micrographs of five different DNA bacteriophages, as prepared by drying in thin films of negative stain, frequently show their heads to be disrupted and flattened. In such cases DNA strands, no larger than 2.5 nm in diameter, become visible, either contained within partially ruptured capsids or completely ejected from severely ruptured ones. Seen in either aspect, the strands appear with circular outline; in some cases a set of concentric circles (or a tightly wound spiral) is evident. Two alternative models of DNA packing within phage heads are proposed. Both are consistent with the electron microscopic observations and, as applied specifically to T4 phage heads, they are also consistent with available data from birefringence studies. One model proposes that the DNA, in simple double-helix form, is wound into a ball. The other suggests that the DNA is wound like a spool, with a greater number of turns in the central region than at the two ends and with the spool axis perpendicular to the axis of the phage particle. The available evidence does not permit a choice to be made between the two models.

Genome and proteome of Listeria monocytogenes phage PSA: an unusual case for programmed + 1 translational frameshifting in structural protein synthesis

PSA is a temperate phage isolated from Listeria monocytogenes strain Scott A. We report its complete nucleotide sequence, which consists of a linear 37 618 bp DNA featuring invariable, 3′‐protruding single stranded (cohesive) ends of 10 nucleotides. The physical characteristics were confirmed by partial denaturation mapping and electron microscopy of DNA molecules. Fifty‐seven open reading frames were identified on the PSA genome, which are apparently organized into three major transcriptional units, in a life cycle‐specific order. Functional assignments could be made to 33 gene products, including structural proteins, lysis components, DNA packaging proteins, lysogeny control functions and replication proteins. Bioinformatics demonstrated relatedness of PSA to phages infecting lactic acid bacteria and other low G + C Gram‐positives, but revealed only few similarities to Listeria phage A118. Virion proteins were analysed by amino acid sequencing and mass spectrometry, which enabled identification of major capsid and tail proteins, a tape measure and a putative portal. These analyses also revealed an unusual form of translational frameshifting, which occurs during decoding of the mRNAs specifying the two major structural proteins. Frameshifting yields different length forms of Cps (gp5) and Tsh (gp10), featuring identical N‐termini but different C‐termini. Matrix‐assisted laser‐desorption ionization mass spectrometry (MALDI‐MS) and electrospray ionization mass spectrometry (ESI‐MS) of tryptic peptide fragments was used to identify the modified C‐termini of the longer protein species, by demonstration of specific sequences resulting from + 1 programmed translational frameshifting. A slippery sequence with overlapping proline codons near the 3′ ends of both genes apparently redirects the ribosomes and initiates the recoding event. Two different cis‐acting factors, a shifty stop and a pseudoknot, presumably stimulate frameshifting efficiency. PSA represents the first case of + 1 frameshifting among dsDNA phages, and appears to be the first example of a virus utilizing a 3′ pseudoknot to stimulate such an event.

Characterization of REP-mutants and their interaction with P2 phage

Abstract Three rep 5 mutations in Escherichia coli have been mapped between metE and ilv. rep is recessive. Using REP− strains derived by transduction, we have obtained no evidence that three rep mutations confer recombination deficiency, although they do do appear to confer a slight increase in UV sensitivity. A number of temperate coliphages cannot use REP− mutants as a host in lytic multiplication. The interaction of REP− mutants and the non-inducible temperate phage P2 is examined in detail, with the following findings: (1) P2 can inject its DNA into REP− hosts, and the phage DNA can circularize, but replication of the DNA is blocked. (2) P2 can lysogenize REP− mutants at approximately normal frequency. (3) REP− mutants are resistant to killing by P2.

The Terminally Redundant, Nonpermuted Genome of Listeria Bacteriophage A511: a Model for the SPO1-Like Myoviruses of Gram-Positive Bacteria

Only little information on a particular class of myoviruses, the SPO1-like bacteriophages infecting low-G+C-content, gram-positive host bacteria (Firmicutes), is available. We present the genome analysis and molecular characterization of the large, virulent, broad-host-range Listeria phage A511. A511 contains a unit (informational) genome of 134,494 bp, encompassing 190 putative open reading frames (ORFs) and 16 tRNA genes, organized in a modular fashion common among the Caudovirales. Electron microscopy, enzymatic fragmentation analyses, and sequencing revealed that the A511 DNA molecule contains linear terminal repeats of a total of 3,125 bp, encompassing nine small putative ORFs. This particular genome structure explains why A511 is unable to perform general transduction. A511 features significant sequence homologies to Listeria phage P100 and other morphologically related phages infecting Firmicutes such as Staphylococcus phage K and Lactobacillus phage LP65. Equivalent but more-extensive terminal repeats also exist in phages P100 (approximately 6 kb) and K (approximately 20 kb). High-resolution electron microscopy revealed, for the first time, the presence of long tail fibers organized in a sixfold symmetry in these viruses. Mass spectrometry-based peptide fingerprinting permitted assignment of individual proteins to A511 structural components. On the basis of the data available for A511 and relatives, we propose that SPO1-like myoviruses are characterized by (i) their infection of gram-positive, low-G+C-content bacteria; (ii) a wide host range within the host bacterial genus and a strictly virulent lifestyle; (iii) similar morphology, sequence relatedness, and collinearity of the phage genome organization; and (iv) large double-stranded DNA genomes featuring nonpermuted terminal repeats of various sizes.

P2 phage amber mutants: Characterization by use of a polarity supressor

Abstract Seventy amber mutants of temperate phage P2 have been characterized, using an Escherichia coli strain which suppresses polarity. Two new genes involved in tail synthesis were defined. The eighteen known P2 late genes can be divided into four transcription units. One of these is transcribed in the opposite direction from the other three. The largest transcription unit contains four head genes, a gene involved in cell lysis, and two tail genes.

Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage φC31 site-specific recombination system

Abstract . The site-specific recombination system used by the Streptomyces bacteriophage φC31 was tested in the fission yeast Schizosaccharomyces pombe. A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to a ura4+ marker, and a second plasmid expressing the φC31 integrase gene. High-efficiency transformation to the Ura+ phenotype occurred when the integrase gene was expressed. Southern analysis revealed that the attB-ura4+ plasmid integrated into the chromosomal attP site. Sequence analysis showed that the attB×attP recombination was precise. In another approach, DNA with a ura4+marker flanked by two attB sites in direct orientation was used to transform S. pombe cells bearing an attP duplication. The φC31 integrase catalyzed two reciprocal cross-overs, resulting in a precise gene replacement. The site-specific insertions are stable, as no excision (the reverse reaction) was observed on maintenance of the integrase gene in the integrant lines. The irreversibility of the φC31 site-specific recombination system sets it apart from other systems currently used in eukaryotic cells, which reverse readily. Deployment of the φC31 recombination provides new opportunities for directing transgene and chromosome rearrangements in eukaryotic systems.

Bacteriophage P2 head morphogenesis: Cleavage of the major capsid protein

Abstract The protein products of four P2 head genes have been identified, and their molecular weights estimated by migration in SDS polyacrylamide gels. The product of the N gene is a protein with a molecular weight of 44,000, which is cleaved to give the major capsid protein, N∗, with a molecular weight of 36,000. Two other protein components of the head may also be cleavage products of the N protein. Coupled to the cleavage of the N protein is the cleavage of the O gene product from a 30,000 molecular weight protein to material of less than 17,000 molecular weight. The P gene product has been identified and is bound to rapidly sedimenting material, possibly membrane. The products of genes M, O, and P are not detected in mature phage heads. Mutants in head genes L, M, P, and Q can synthesize head-like structures under nonpermissive conditions, but mutants in genes O and N cannot. A model of P2 head assembly is presented in which the O and N gene products assemble and are cleaved to give a semistable head precursor. This precursor is then acted upon by the four other head gene products to form the mature head.

Interactions between a satellite bacteriophage and its helper

Abstract P4 is a satellite phage which relies on a helper such as P2 to supply the gene products necessary for particle construction and cell lysis (Six, 1975). P4 can activate the expression of late genes from a P2 helper phage, using a mechanism different from that employed by the helper. In the presence of P4, replication of P2 DNA is not required for late gene expression (Six & Lindqvist, 1971), and the polar effects of P2 amber mutations are suppressed. Despite its small size P4 codes for two late proteins as well as two early proteins. One of the P4 early proteins is that the product of gene α. The expression of P4 late genes is stimulated by the helper phage. Thus the P2 and P4 chromosomes exhibit reciprocal transactivation. The presence of the P4 genome causes the P2 head proteins to form a head smaller than that found after infection by P2 (Gibbs et al., 1973). P4 late proteins associate with head-like structures and may determine the small size of P4 heads.

Identification of a mutation within the structural gene for the a subunit of DNA-dependent RNA polymerase of E. coli

An E. coli mutant rpoA109 unable to support the growth of phage P2 produces DNA-dependent RNA polymerase with an altered alpha subunit. Histidine is substituted for leucine in one tryptic peptide from the mutant alpha subunit. The existence of only one rpoA gene within the E. coli chromosome is indicated.