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The base pairs of cosN, the site where the 12 base-long cohesive ends are generated in λ-like phages, show partial-two fold rotational symmetry. In a bioinformatic survey, we found that the cosN changes in 12 natural cosN variants are restricted to bp 6-to-12 of the cohesive end sequence. In contrast, bp 1-5 of the cohesive end sequence are strictly conserved (13/13), as are the two bp flanking the left nicking site (bp -2 and -1). The bp flanking the right nick site (bp 13 and 14) are conserved in 12 of 13 variants. Five cosN variants differing by as many as five bp were used to replace lambda's cosN. No significant effects of the cosN changes on λ's virus yield were found. In sum, bp -2 to 5 are critical cosN function, and bp 6-12 of the cohesive end sequence are not critical for terminase recognition or virus fitness.

Enterobacteriophage Mu is the best studied and paradigm member of the transposable phages. Mu-encoded proteins have been annotated in detail in UniProtKB and linked to a controlled vocabulary describing the various steps involved in the phage lytic and lysogenic cycles.Transposable phages are ubiquitous temperate bacterial viruses with a dsDNA linear genome. Twenty-six of them, that infect α, β and γ-proteobacteria, have been sequenced. Their conserved properties are described. Based on these characteristics, we propose a reorganization of the Caudovirales, to allow for the inclusion of a “Saltoviridae” family and two newly proposed subfamilies, the “Myosaltovirinae” and “Siphosaltovirinae”. The latter could temporarily be included in the existing Myoviridae and Siphoviridae families.

The α-proteobacterium Rhodobacter capsulatus is a model organism for the study of bacterial photosynthesis and the bacteriophage-like gene transfer agent. Characterization of phages that infect Rhodobacter is extremely rare, and scarce for the α-proteobacteria in general. Here, we describe the discovery of the only functional Mu-like transposing phage to have been identified in the α-proteobacteria, RcapMu, resident in the genome-sequenced R. capsulatus SB1003 strain. RcapMu packages ~ 42 kb of total DNA, including

The virulent lactococcal phage P087 was isolated from a dairy environment in 1978. This phage was then recognized as the reference member for one of the ten phage groups currently known to infect Lactococcus lactis strains. The double-stranded DNA genome of this Siphoviridae phage is composed of 60,074 bp and is circularly permuted. Five tRNA and 88 orfs were found within an uncommon genome architecture. Eleven structural proteins were also identified through SDS-PAGE and LC-MS/MS analyses. Of note, 11 translated orfs from the structural module of phage P087 have identities to gene products found in a prophage located in the genome of Enterococcus faecalis V583. The alignment of both genomic sequences suggests that DNA exchanges could occur between these two phages which are infecting low G+C bacteria found in similar ecological niches.

The virulent lactococcal phage 1706, isolated in 1995 from a failed cheese production in France, represents a new lactococcal phage species of the Siphoviridae family. This phage has a burst size of 160 and a latent period of 85 min. Its linear double-stranded DNA genome was composed of 55,597 bp with a 33.7% G+C content. Its deduced proteome (76 ORFs) shared limited similarities to other known phage proteins. SDS-PAGE coupled with LC-MS/MS analyses led to the identification of 15 structural proteins. The most striking feature of the 1706 proteome was that 22 ORFs shared similarities with proteins deduced from the genome of either Ruminococcus torques and/or Clostridium leptum. Both are Firmicutes bacteria found in the gut flora of humans. We also identified a four-gene module in phage 1706, most likely involved in host recognition that shared similarities with lactococcal prophages. We propose that the virulent phage 1706 infected another bacterial genus before picking up a lactococcal host recognition module.

Tuc2009 is a temperate bacteriophage of Lactococcus lactis subsp. cremoris UC509 which encodes a CI- and Cro-type lysogenic-lytic switch region. A helix-swap of the 0 helices of the closely related Cl-type proteins from the lactococcal phages r1t and Tuc2009 revealed the crucial elements involved in DNA recognition while also pointing to conserved functional properties of phage Cl proteins infecting different hosts. CI-type proteins have been shown to bind to specific sequences located in the intergenic switch region, but to date, no detailed binding studies have been performed on lactococcal Cro analogues. Experiments shown here demonstrate alternative binding sites for these two proteins of Tuc2009. CI2009 binds to three inverted repeats, two within the intergenic region and one within the cro(2009) gene. This DNA-binding pattern appears to be conserved among repressors of lactococcal and streptococcal phages. The Cro(2009) protein appears to bind to three direct repeats within the intergenic region causing distortion of the bound DNA. (c) 2005 Elsevier Inc. All rights reserved.

The entire double-stranded DNA genome of bacteriophage BFK20, a lytic phage of the Brevibacterium flavum CCM 251 – industrial producer of l -lysine – was sequenced and analyzed. It consists of 42,968 base pairs with an overall molar G + C content of 56.2%. Fifty-five potential open reading frames were identified and annotated using various bioinformatics tools. Clusters of functionally related putative genes were defined (structural, lytic, replication and regulatory). To verify the annotation of structural proteins, they were resolved by 2D gel electrophoresis and were submitted to N-terminal amino acid sequencing. Structural proteins identified included the portal and major and minor tail proteins. Based on the overall genome sequence comparison, similarities with other known bacteriophage genomes include primarily bacteriophages from Mycobacterium spp. and some regions of Corynebacterium spp. genomes – possible prophages. Our results support the theory that phage genomes are mosaics with respect to each other.

Complete genome sequencing of the P335 temperate Lactococcus lactis bacteriophage φLC3 (32, 172 bp) revealed fifty-one open reading frames (ORFs). Four ORFs did not show any homology to other proteins in the database and twenty-one ORFs were assigned a putative biological function. φLC3 contained a unique replication module and orf201 was identified as the putative replication initiator protein-encoding gene. φLC3 was closely related to the L. lactis r1t phage (73% DNA identity). Similarity was also shared with other lactococcal P335 phages and the Streptococcus pyogenes prophages 370.3, 8232.4 and 315.5 over the non-structural genes and the genes involved in DNA packaging/phage morphogenesis, respectively. φLC3 contained small homologous regions distributed among lactococcal phages suggesting that these regions might be involved in mediating genetic exchange. Two regions of 30 and 32 bp were conserved among the streptococcal and lactococcal r1t-like phages. These two regions, as well as other homologous regions, were located at mosaic borders and close to putative transcriptional terminators indicating that such regions together might attract recombination. The conserved regions found among lactococcal and streptococcal phages might be used for identification of phages/prophages/prophage remnants in their hosts.

The entire genome of SfV, a temperate serotype-converting bacteriophage of Shigella flexneri, has recently been sequenced (Allison, G.E., Angeles, D., Tran-Dinh, N., Verma, N.K. 2002, J. Bacteriol. 184, 1974–1987). Based on the sequence analysis, we further characterised the SfV virion structure and morphogenesis. Electron microscopy indicated that SfV belongs to the Myoviridae morphology family. Analysis of the proteins encoded by orf1, orf2, and orf3 revealed that they were homologous to small and large terminase subunits, and portal proteins, respectively; the protein encoded by orf5 showed homology to capsid proteins. Western immunoblot of the phage with anti-SfV sera revealed two antigenic proteins, and the N-terminal amino acid sequence of the 32-kDa protein corresponded to amino acids 116 to 125 of the ORF5 protein, suggesting that the capsid may be processed. Functional analysis of orf4 showed that it encodes the phage capsid protease. The proteins encoded by orfs1, 2, 3, 4, and 5 are homologous to similar proteins in the Siphoviridae phage family of both gram-positive and gram-negative origin. The capsid and morphogenesis genes are upstream and adjacent to the genes encoding Myoviridae (Mu-like) tail proteins. The organisation of the structural genes of SfV is therefore unique as the head and tail genes originate from different morphology groups.

Two putative structural genes, orf tmp (tape measure protein) and orf bpp (baseplate protein), of the temperate lactococcal phage TP901-1 were examined by introduction of specific mutations in the prophage strain Lactococcus lactic ssp. cremoris 901-1. The adsorption efficiencies of the mutated phages to the indicator strain L. lactic ssp. cremoris 3107 were determined and electron micrographs were obtained. Specific mutations in orf tmp resulted in the production of mostly phage head structures without tails and a few wild-type looking phages. Furthermore, construction of an inframe deletion or duplication of 29% in orf tmp was shown to shorten or lengthen the phage tail by approximately 30%, respectively. The orf tmp is proposed to function as a tape measure protein, TMP, important for assembly of the TP901-1 phage tail and involved in tail length determination. Specific mutations in orf bpp produced phages which were unable to adsorb to the indicator strain and electron microscopy revealed particles lacking the baseplate structure. The orf bpp is proposed to encode a highly immunogenic structural baseplate protein, BPP, important for assembly of the baseplate. Finally, an assembly pathway of the TP901-1 tail and baseplate structure is presented.

Temperate Siphoviridae from an evolutionarily related branch of low GC content gram-positive bacteria share a common genetic organization of lysogeny-related genes and the predicted proteins are linked by many sequence similarities. Their compact lysogeny modules [integrase/1–2 orfs (phage exclusion? and metalloproteinase motif proteins)/cI-like repressor/cro-like repressor/antirepressor (optional)] differ clearly from that of λ-like and L5-like viruses, the two currently established genera of temperate Siphoviridae, while they resemble those of the P2-like genus of Myoviridae. In all known temperate Siphoviridae from low GC content gram-positive bacteria the lysogeny module is flanked by the lysis module and the DNA replication module. This modular organization is again distinct from that of the known genera of temperate Siphoviridae. On the basis of comparative sequence analysis we propose a new genus of Siphoviridae: “Sfi21-like” phages. With a larger database of phage sequences it might be possible to establish a genomics-based phage taxonomy and to retrace the evolutionary history of selected phage modules or individual phage genes. The antirepressor of Sfi21-like phages has an unusual widespread distribution since proteins with high aa similarity (40%) were found not only in phages from gram-negative bacteria, but also in insect viruses.

The lambda kil gene has been shown to be responsible for premature lysis effected by addition of chloramphenicol between 15 and 20 min after thermal induction of a lambda prophage. Here, we localized the kil reading frame. The kil gene, represented by lambda orf47, overlaps genes cIII and gam. Expression of the plasmid-borne kil gene resulted in growth arrest, a reduction of colony-forming units and filament formation. However, kil-mediated cell lysis could not be triggered by chloramphenicol when the plasmid borne kil gene was expressed, suggesting that kil-induced cell lysis requires the phage context.

Global gene expression profiling of bacteriophage BFK20 infecting the industrial L-lysine producer Brevibacterium flavum CCM 251 was performed using DNA microarray. The relative gene expressions were measured in fourteen time samples collected during phage development. Phage genes were classified as early, middle, late or unassigned based on complex expression patterns during infection. Temporal classification of BFK20 genes was in concordance with previous predictions. However, proposed late regulatory genes were reclassified and new functional assignments for ORF55 were strongly suggested. Furthermore, we consider possible functions of other genes and their products regarding coexpression pattern by using “guilt-by-association” algorithm. Microarray results were validated using real-time RT-PCR. The detailed description of phage BFK20 transcriptional profile can answer the basic questions of its life cycle and it also can help to prevent phage contamination during industrial fermentation. In addition, this work presents the first complete microarray time course study of gene expression utilizing loop design.

Two non-inducible, but apparently complete prophages were identified in the genome of the sequenced Lactobacillus johnsonii strain NCC 533. The 38- and 40-kb-long prophages Lj928 and Lj965 represent distinct lineages of Sfi11-like pac-site Siphoviridae unrelated at the DNA sequence level. The deduced structural proteins from Lj928 demonstrated aa sequence identity with Lactococcus lactis phage TP901-1, while Lj965 shared sequence links with Streptococcus thermophilus phage O1205. With the exception of tRNA genes, inserted between DNA replication and DNA packaging genes, the transcription of the prophage was restricted to the genome segments near both attachment sites. Transcribed genes unrelated to phage functions were inserted between the phage repressor and integrase genes; one group of genes shared sequence relatedness with a mobile DNA element in Staphylococcus aureus. A short, but highly transcribed region was located between the phage lysin and right attachment site; it lacked a protein-encoding function in one prophage.

The genome of enterobacterial phage T1 has been sequenced, revealing that its 50.7-kb terminally redundant, circularly permuted sequence contains 48,836 bp of nonredundant nucleotides. Seventy-seven open reading frames (ORFs) were identified, with a high percentage of small genes located at the termini of the genomes displaying no homology to existing phage or prophage proteins. Of the genes showing homologs (47%), we identified those involved in host DNA degradation (three endonucleases) and T1 replication (DNA helicase, primase, and single-stranded DNA-binding proteins) and recombination (RecE and Erf homologs). While the tail genes showed homology to those from temperate coliphage N15, the capsid biosynthetic genes were unique. Phage proteins were resolved by 2D gel electrophoresis, and mass spectrometry was used to identify several of the spots including the major head, portal, and tail proteins, thus verifying the annotation.

For the lytic growth cycle of the temperate cos-site Streptococcus thermophilus phage Sfi21 a transcription map was developed on the basis of systematic Northern blot hybridizations. All deduced 5′ ends were confirmed by primer extension analysis. Three time classes of transcripts were observed. Early transcripts were identified in four different genome regions. One prominent early mRNA of 4.8 kb length covered a group of 12 genes located between the origin of replication and the cos-site. Two short early mRNAs represented a single gene from the direct vicinity of the cos-site and the superinfection immunity gene from the lysogeny module, respectively. A fourth early transcript covered a group of four genes located between the lysin and the integrase gene. Middle transcripts of 2.1 and 5.8 kb length covered cro-like and ant-like repressor genes and the DNA replication module, respectively. Four types of late transcripts were identified. The transcripts covered the likely DNA packaging genes, the head morphogenesis module plus the major tail gene, the remainder of the tail genes, and the putative tail fiber plus lysis genes, respectively. Only the transcript from the head morphogenesis genes yielded defined late mRNA species. The transcription map concurred with most of the in silico predictions for the genome organization of phage Sfi21 except for the separation of the DNA replication module from a possible transcription regulation module. Most 5′ ends of the transcripts determined in primer-extension experiments were not preceded by a consensus promoter sequence. The involvement of phage-encoded regulators for middle and late transcription was suggested by chloramphenicol-inhibition experiments.

The region of the bacteriophage A2 genome involved in site-specific recombination with the DNA of Lactobacillus spp. has been identified. Two orfs, transcribed from the same strand, have been found immediately upstream of the phage attachment site (attP). The orf adjacent to attP predicts a 385-amino-acid protein that presents significant similarity with site-specific recombinases of the integrase family. The other orf encodes a basic polypeptide of 76 amino acid residues. The junctions of the prophage with the genomes of its hosts have been determined, allowing the identification of the host attachment site (attB), which has a common 19-nucleotide core region with attP. The attB site is located at the 3' end of the transfer RNALeu gene (anticodon CAA). Nonreplicative plasmids containing the A2-specific recombination cassette integrate into different lactobacilli but also into unrelated Gram-positive bacteria such as Lactococcus lactis and even into Escherichia coli. In Lc. lactis, integration occurs in a previously unknown intergenic region, whereas in E. coli, it maps within the rrnD operon, 5' of rrsD gene. Comparison of the integration sites in the different hosts indicates that some flexibility is permitted in the attB sequence, since Lc. lactis and E. coli only share 13 and 11 nucleotides, respectively, with the 19-nucleotide core sequence of the lactobacilli.

The structural gene cluster and the lysis module from lytic group II Streptococcus thermophilus bacteriophage phi Sfi11 was compared to the corresponding region from other Siphoviridae. The analysis revealed a hierarchy of relatedness. phi Sfi11 differed from the temperate S. thermophilus bacteriophage phi O1205 by about 10% at the nucleotide level. The majority of the changes were point mutations, mainly at the third base position. Only a single gene (orf 695) differed substantially between the two phages. Over the putative minor tail and lysis genes, phi Sfi11 and the lytic group 1 S. thermophilus phi Sfi19 shared regions with variable degrees of similarity. Orf 1291 from phi Sfi19 was replaced by four genes in phi Sfi11, two of which (orf 1000 and orf 695) showed a complicated pattern of similarity and nonsimilarity compared with phi Sfi19. The predicted orf 695 gp resembles the receptor-recognizing protein of T-even coliphages in its organization, but not its sequence. No sequence similarity was detected between phi Sfi11 and phi Sfi19 in the region covering the major head and tail genes. Comparison of the structural gene map of phi Sfi11 with that of Siphoviridae from gram-positive and -negative bacterial hosts revealed a common genomic organization. Sequence similarity was only found between phi Sfi11 and Siphoviridae from gram-positive hosts and correlated with the evolutionary distance between the bacterial hosts. Our data are compatible with the hypothesis that the structural gene operon from Siphoviridae of the low G + C group of gram-positive bacteria is derived from a common ancestor.

Gene 1 product (G 1 P) of the related Bacillus subtilis bacteriophages SPP1, SF6, and ρ15 is essential for DNA maturation and packaging. A DNA segment containing gene 1 of phage SF6 or ρ15 origin was cloned and sequenced. SF6 and ρ15 G 1 P (both with predicted molecular mass of 16.7 kDa) share 71% identity with G 1 P of SPP1. The G 1 P of all three phages contains three conserved segments (I, II, and III). Within segments I and II helix-turn-helix DNA binding and nucleotide binding motifs were predicted. G 1 P of both SPP1 and SF6 origin was purified. SPP1 G 1 P protein (20.7 kDa), purified from cells overexpressing the cloned gene, purifies together with another polypeptide, having a molecular mass of about 13 kDa. The 13-kDa polypeptide results from a translation start signal within gene 1 , and hence was termed SPP1 G 1 P * . G 1 P of both SPP1 and SF6 binds specifically to a pac -containing DNA fragment, whereas G 1 P * , which lacks segment I, does not. Chimeric G 1 P protiens were obtained by domain swapping between gene 1 of SPP1 and SF6. The results presented here suggest that the G 1 P DNA binding motif lies in segment I and the major determinant for G 1 P::G 1 P interaction might lie in segment II. Segment III and the extended C-terminal part of SPP1 G 1 P are dispensable. The G 1 P::G 2 P interacting region remains uncharacterized.

We have studied the assembly of bacteriophage lambda head proteins on the phage phi 29 connector to produce in vitro chimeric proheads, whose ability to package different types of DNA depends on the physical integrity of the phi 29 connector. Terminal protein-free phi 29 as well as nonviral DNAs have been shown to be efficiently packaged by this hybrid system. An RNA, that can be provided by any of the extracts used in the complementation mixture, was required for DNA packaging, both by the hybrid system as well as by the homologous lambda system. The DNA-packaging activity of RNase-treated proheads can be restored by adding a mixture of ribosomal RNAs. There is also a requirement for a minimal length of DNA to be stably packaged. The packaging protein p16 of phi 29 can replace the lambda terminase complex in the in vitro packaging system, both with the chimeric as well as genuine lambda proheads.

Multiplicity reactivation of ultraviolet-inactivated T1 can be detected using suitably irradiated bacteria as host cells. Under certain conditions the amount of multiplicity reactivation is independent of the dose on the bacteria, even when the dose is reduced to zero so that normal cells are infected. The results are consistent with the idea that in normal cells some other reactivation phenomenon masks multiplicity reactivation of T1.

When Shigella dysenteriae is lysogenized by the phage P1, it becomes immune to infection with “ordinary” T1 phage (“restricted T1” or “rT1”). A host-controlled variant of T1 (“unrestricted T1” or “uT1”) can multiply in the lysogenic S. dysenteriae. Three-factor crosses were done between suitably marked strains of rT1 and uT1 in both lysogenic and nonlysogenic S. dysenteriae. When lysogenic cells are infected with both rT1 and uT1, one finds some recombinants among the progeny, but few, if any phage bearing the genotype of the restricted parent. The proportion of recombinants in the total yield is about one-eighth to one-twelfth that in control crosses (in nonlysogenic hosts) and is independent of the order of addition of either parent and of the time elapsing between the addition of each parent. We infer, then, that (1) the genome of rT1 enters the lysogenic cells; (2) it is neither replicated nor is it destroyed; and (3) it can “mate” with genomes of uT1 which may be present. The proportions of the various recombinant classes are unusual. Complementary classes are not equal; certain markers may be recovered together with a high frequency, whereas certain others, more closely linked, are seldom found together. Certain “double crossover” classes are more frequent than certain “single crossover” classes. These observations can be explained in terms of a “copy choice” mechanism of recombination by assuming that the rT1 genome contains at least two “bad spots,” the location of which can be determined approximately and which force a “switch” in replication from the restricted genome to the unrestricted genome.

Early stages of invasion by T5 st can be studied through changes in the response of phage-bacterium complexes to a calcium-deficient buffer called challenge buffer . T5 st , as free phage, and uninfected Escherichia coli F are each stable in challenge buffer. Upon adsorption, phage-bacterium complexes remain resistant to challenge buffer until a certain event, called sensitization , occurs. The plaque-forming ability of sensitized complexes rapidly decays in challenge buffer at either 37.5° or 0°; decay can be arrested by simple addition of calcium to the medium. A second postadsorptive event, called desensitization , occurs in calcium-supplemented nutrient medium, but not in calcium-supplemented buffer; desensitized complexes are resistant to challenge buffer. A tentative interpretation of some of these phenomena is proposed.

The capacity of Escherichia coli B/r to yield phages T1 and T3 is more ultraviolet (UV)-sensitive than is the capacity to yield phage T2. The capacity to yield phage from UV-irradiated T1 or T3 is about ten times as UV-sensitive as the capacity for unirradiated phage. The capacity of UV-irradiated host cells for UV-irradiated T2 is the same as for normal T2. Ultraviolet-irradiated T1 is reactivated on UV-irradiated host cells. When unirradiated T1 is grown on UV-irradiated cells, the frequency of plaquemorphology mutants is the same as the spontaneous frequency. When UV-irradiated T1 is grown on UV-irradiated cells, there is about a sevenfold increase in mutant plaque frequency over the spontaneous frequency. Thirty phenotypically distinct plaque-morphology mutants of T1 have been found.

Mutational alterations of density in phage T5 are described. The changes of density are correlated with changes in temperature sensitivity of the phage. Genetic recombination has been observed between density markers and a plaque-type marker, but not between pairs of density mutants. No phenotypic mixing of density characters has been found.

When plated directly on Escherichia coli B, a UV-irradiated T1 population reveals two fractions of different sensitivities. The fraction with lower sensitivity (about 30% of the phage-bacterial complexes) is believed to result from a reactivation by host bacteria of UV lesions in the phage genome (host-cell reactivation = HCR). Using bacteria starved prior to phage infection, we obtain HCR in 100% of the phage-bacterial complexes. Also, synchronized bacteria all perform HCR during short periods before and after cell division, not during the remaining 70% of their division cycle. When DNA synthesis is stopped by thymine withdrawal in a thymine-requiring strain of E. coli, HCR occurs in every phage-bacterial complex. These findings explain the shape of the T1 survival curve obtained with direct plating of UV-irradiated phage. In a growing bacterial culture about 70% of the T1 particles presumably infect bacteria in which synthesis of bacterial DNA prevents HCR, whereas 30% infect bacteria without DNA synthesis and can undergo HCR. In bacteria that promote HCR, multiplicity reactivation (MR) of UV-irradiated T1 can be demonstrated. The efficiency of MR is the same as in bacteria that do not perform HCR. Multiplicities above two do not increase the chance of survival of T1 multicomplexes, a fact suggesting a limited participation of T1 in MR.

Lactococcus lactis phage BK5-T and Streptococcus thermophilus phage Sfi21, two cos-site temperate Siphoviridae with 40-kb genomes, share an identical genome organization, sequence similarity at the amino acid level over about half of their genomes, and nucleotide sequence identity of 60% over the DNA packaging and head morphogenesis modules. Siphoviridae with similarly organized genomes and substantial protein sequence similarity were identified in several genera of low-GC-content Gram-positive bacteria. These phages demonstrated a gradient of relatedness ranging from nucleotide sequence similarity to protein sequence similarity to gene map similarity over the DNA packaging and head morphogenesis modules. Interestingly, the degree of relatedness was correlated with the evolutionary distance separating their bacterial hosts. These observations suggest elements of vertical evolution in phages. The structural genes from BK5-T shared no sequence relationships with corresponding genes/proteins from lactococcal phages belonging to distinct lactococcal phage species, including phage sk1 (phage species 936) that showed a closely related gene map. Despite a clearly distinct genome organization, lactococcal phages sk1 and c2 showed nine sequence-related proteins. Over the early gene cluster phage BK5-T shared nine regions of high nucleotide sequence similarity, covering at most two adjacent genes, with lactococcal phage r1t (phage species P335). Over the structural genes, the closest relatives of phage r1t were not lactococcal phages belonging to other phage species, but Siphoviridae from Mycobacteria (high-GC-content Gram-positive bacteria). Evidence for recent horizontal gene transfer between distinct phage species was obtained for dairy phages, but these transfers were limited to phages infecting the same bacterial host species.

Using a variety of genomic (BLASTN, ClustalW) and proteomic (Phage Proteomic Tree, CoreGenes) tools we have tackled the taxonomic status of members of the largest bacteriophage family, the Siphoviridae. In all over 400 phages were examined and we were able to propose 39 new genera, comprising 216 phage species, and add 62 species to two previously defined genera (Phic3unalikevirus; L5likevirus) grouping, in total, 390 fully sequenced phage isolates. Many of the remainders are orphans which the Bacterial and Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) chooses not to ascribe genus status at the time being.

Alt, encoded by the lactococcal phage TP901-1, is needed for late transcription. We identify Alt as a DNA-binding protein, and footprint analysis shows that Alt binds to a region containing four imperfect direct repeats (ALT boxes) located −76 to −32 relative to the P late transcriptional start site. The importance of the ALT boxes was confirmed by deletion of one or two ALT boxes and by introducing mutations in ALT boxes 1 and 4. Alt is proposed to act as a tetramer or higher multimer activating transcription of TP901-1 late genes by binding to the four ALT boxes, and bending of the DNA may be important for transcriptional activation of P late . Furthermore, our results suggest that DNA replication may be required for late transcription in TP901-1. Additionally, we identify gp28 of the related lactococcal phage Tuc2009 as an activator and show that the activators required for late transcription in TP901-1 and Tuc2009 are interchangeable.

Three prophage sequences were identified in the Lactobacillus johnsoni strain NCC533. Prophage Lj965 predicted a gene map very similar to those of pac-site Streptococcus thermophilus phages over its DNA packaging and head and tail morphogenesis modules. Sequence similarity linked the putative DNA packaging and head morphogenesis genes at the protein level. Prophage Lj965/S. thermophilus phage Sfi11/Lactococcus lactis phage TP901-1 on one hand and Lactobacillus delbrueckii phage LL-H/Lactobacillus plantarum phage phig1e/Listeria monocytogenes phage A118 on the other hand defined two sublines of structural gene clusters in pac-site Siphoviridae from low-GC Gram-positive bacteria. Bacillus subtilis phage SPP1 linked both sublines. The putative major head and tail proteins from Lj965 shared weak sequence similarity with phages from Gram-negative bacteria. A clearly independent line of structural genes in Siphoviridae from low-GC Gram-positive bacteria is defined by temperate cos-site phages including Lactobacillus gasseri phage adh, which also shared sequence similarity with phage D3 infecting a Gram-negative bacterium. A phylogenetic tree analysis demonstrated that the ClpP-like protein identified in four cos-site Siphoviridae from Lactobacillus, Lactococcus, Streptococcus, and Pseudomonas showed graded sequence relationships. The tree suggested that the ClpP-like proteins from the phages were not acquired by horizontal gene transfer from their corresponding bacterial hosts.

The bacteriophage phiSASD1, isolated from a failed industrial avermectin fermentation, belongs to the Siphoviridae family. Its four predominant structural proteins, which include the major capsid, portal and two tail-related proteins, were separated and identified by SDS-PAGE and N-terminal sequence analysis. The entire double-stranded DNA genome of phiSASD1 consists of 37,068bp, with 3'-protruding cohesive ends of nine nucleotides. Putative biological functions have been assigned to 24 of the 43 potential open reading frames. Comparative analysis shows perfect assembly of three “core” gene modules: the morphogenesis and head module, the tail module and the right arm gene module, which displays obvious similarity to the right arm genes of Streptomyces phage phiC31 in function and arrangement. Meanwhile, structural module flexibility within phiSASD1 suggests that assignment of phage taxonomy based on comparative genomics of structural genes will be more complex than expected due to the exchangeability of functional genetic elements.

Lysis inhibition has been demonstrated with a small-plaque mutant of T5, designated M12. This appears to be the first example of the phenomenon outside the T-even system. M12 and T5 appear identical in host range, adsorption rate, serum neutralization, and injection rate, but differ in minimal latent period in single infection (45–50 minutes for M12 as against 37 minutes for T5) and in plating efficiency under various conditions. Evidence of lysis inhibition in multiply infected cultures was obtained with M12, but not T5, by observations on the clearing of turbid cultures, one-step growth experiments, and microscopic examination. The best demonstrations in one-step growth experiments were obtained when young bacterial cultures (Escherichia coli strain F, smooth derivative of FCb) were multiply infected with M12 in a single input (multiplicity of 6–8 adequate) and further incubated undiluted for 30–40 minutes, or diluted earlier into sonicated extracts of B 5 . The latter do not affect the lysis of cultures singly or multiply infected with T5 or singly infected with M12. One-step growth experiments suggest multistep release of phage in lysisinhibited cultures. A possible explanation, actual multiplication of the infected bacteria, received no support from microscopic studies.

The metabolism of nucleic acid in Escherichia coli infected with phage T5 has been studied using antibiotics, amino acid analogs, and auxotrophic bacterial strains. Comparison with other phage infections shows that the metabolism of E. coli infected with phage T5 is similar to that of E. coli infected with phage T2, T4, or T6.