Nucleotide sequence of the right early region of Bacillus subtilis phage PZA completes the 19366-bp sequence of PZA genome. Comparison with the homologous sequence of phage phi 29

We have sequenced the rightmost 2079 bp of the Bacillus subtilis phage PZA genome. This region encompasses the right early region. We compared it with the homologous region of phage phi 29. Six open reading frames (ORFs) were found in this region of PZA and one of them was assigned to gene 17. Analysis of putative ribosome-binding sites and comparison with phi 29 ORFs indicate that at least some of the remaining ORFs could encode proteins. Corresponding genes were not identified so far by genetic methods. Promoter candidates in the right early region of PZA were found and compared to phi 29 promoters. The sequenced region together with previously determined sequences [Paces et al., Gene 38 (1985) 45-56 and 44 (1986) 107-114] completes the entire 19,366-bp sequence of phage PZA genome.     

Nucleotide sequence of the right early region of Bacillus phage phi 15 and comparison with related phages: reorganization of gene 17 during evolution

The rightmost 2016 bp of the Bacillus subtilis phage phi 15 genome were sequenced. The nucleotide sequence was compared with the homologous regions of the related phages PZA and phi 29. There are six open reading frames (ORFs) in this region of the phi 15 genome; all of them are present in the PZA and phi 29 genomes. One of the ORFs was assigned to gene 17, which is involved in the replication of the phage DNA. Gene 17 has undergone reorganization during the evolution of this phage family. Comparison of the nucleotide sequence of its mRNA-like strand in phi 15, PZA and phi 29 showed that deletions in its central and 3'-end-proximal parts are tolerated and do not interfere with the gene 17 product function. It seems that the only portion of gene 17 that has to be conserved to encode the functional product is its 5'-end-proximal part.     

Organization of the early region of bacteriophage phi 80. Genes and proteins

An EcoRI segment containing the early region of bacteriophage phi 80 DNA that controls immunity and lytic growth was identified as a segment whose presence on a plasmid prevented growth of infecting phi 80cI phage. The nucleotide sequence of the segment (EcoRI-F) and adjacent regions was determined. Based on the positions of amber mutations and the sizes of some gene products, the reading frames for five genes were identified. From the relative locations of these genes in the genome, the properties of some isolated gene products, and the analysis of the structures of predicted proteins, the following phi 80 to lambda analogies are deduced: genes cI and cII to their lambda namesakes; gene 30 to cro; gene 15 to O; and gene 14 to P. An amber mutation by which gene 16 was defined is a nonsense mutation in the frame for gene 15 protein, excluding the presence of gene 16. An amber mutation in gene 14 or 15 inhibits phage DNA synthesis, as is the case with their lambda analogues, gene O or P. Some characteristics of proteins from the early region predicted from their primary structures and their possible functions are discussed.     

Nucleotide sequence of the immunity region of Bacillus subtilis bacteriophage phi 105: identification of the repressor gene and its mRNA and protein products

A gene involved in the regulation of lysogeny in the temperate Bacillus subtilis phage phi 105 has been identified and isolated. A plasmid, pDC4, was constructed that contains a 740-bp HindIII-PvuII fragment that is derived from the phi 105 immunity region and is capable of rendering B. subtilis immune to infection by phi 105. Three different hybrid plasmids that contain the 740-bp fragment, pAG101 [Cully and Garro, J. Virol. 34 (1980) 789-791], pDC1 and pDC2, were found to synthesize a common 18-kDal polypeptide in B. subtilis minicells and Escherichia coli maxicells. The nucleotide (nt) sequence of this region revealed three open reading frames (ORFs) that predict proteins with Mrs of 16521, 7332, and 5516. In vivo synthesized phi 105 prophage RNA was mapped by primer extension and shown to be transcribed from the DNA strand coding for the Mr 16521 protein. The 5' end of the phi 105 lysogen RNA was mapped to a region that contains conserved sequences for RNA polymerase recognition.     

Determining the repertoire of immunodominant proteins via whole-genome amplification of intracellular pathogens

Culturing many obligate intracellular bacteria is difficult or impossible. However, these organisms have numerous adaptations allowing for infection persistence and immune system evasion, making them some of the most interesting to study. Recent advancements in genome sequencing, pyrosequencing and Phi29 amplification, have allowed for examination of whole-genome sequences of intracellular bacteria without culture. We have applied both techniques to the model obligate intracellular pathogen Anaplasma marginale and the human pathogen Anaplasma phagocytophilum, in order to examine the ability of phi29 amplification to determine the sequence of genes allowing for immune system evasion and long-term persistence in the host. When compared to traditional pyrosequencing, phi29-mediated genome amplification had similar genome coverage, with no additional gaps in coverage. Additionally, all msp2 functional pseudogenes from two strains of A. marginale were detected and extracted from the phi29-amplified genomes, highlighting its utility in determining the full complement of genes involved in immune evasion.     

Nucleotide sequence of the genome of the bacteriophage alpha 3: interrelationship of the genome structure and the gene products with those of the phages, phi X174, G4 and phi K.

The complete nucleotide sequence of the genome of the circular single-stranded DNA (isometric) phage alpha 3 has been determined and compared with that of the related phages phi X174 and G4. The alpha 3 genome consists of 6087 nucleotides, which is 701 nucleotides longer than the nucleotide sequence of the phi X174 genome and 510 nucleotides more than that of the G4 genome. The results demonstrated that the three phage species have 11 homologous genes (A, A*, B, C, K, D, E, J, F, G and H), the order of which is fundamentally identical, suggesting that they have evolved from a common ancestor. The sequence of some genes and untranslated intergenic regions, however, differs significantly from phage to phage: for example, the degree of amino acid sequence homology of the gene product is averaged at 47.7% between alpha 3 and phi X174 and 46.9% between alpha 3 and G4, and alpha 3 has a remarkable longer intergenic region composed of 758 nucleotides between the genes H and A compared with the counterparts of phi X174 and G4. Meanwhile, in vivo experiments of genetic complementation showed that alpha 3 can use none of the gene products of phi X174 and G4, whereas the related phage phi K can rescue alpha 3 nonsense mutants of the genes B, C, D and J. These sequencing and in vivo rescue results indicated that alpha 3 is closely related to phi K, but distantly remote from phi X174 or G4, and supported an evolutional hypothesis which has been so far proposed that the isometric phages are classified into three main groups: the generic representatives are phi X174, G4 and alpha 3.     

The complete nucleotide sequence of phi CTX, a cytotoxin-converting phage of Pseudomonas aeruginosa: implications for phage evolution and horizontal gene transfer via bacteriophages

phi CTX is a cytotoxin-converting phage isolated from Pseudomonas aeruginosa. In this study, we determined the complete nucleotide sequence of the phi CTX phage genome. The precise genome size was 35,538 bp with 21 base 5'-extruding cohesive ends. Forty-seven open reading frames (ORFs) were identified on the phi CTX genome, including two previously identified genes, ctx and int. Among them, 15 gene products were identified in the phage particle by protein microsequencing. The most striking feature of the phi CTX genome was an extensive homology with the coliphage P2 and P2-related phages; more than half of the ORFs (25 ORFs) had marked homology to P2 genes with 28.9-65.8% identity. The gene arrangement on the genome was also highly conserved for the two phages, although the G + C content and codon usage of most phi CTX genes were similar to those of the host P. aeruginosa chromosome. In addition, phi CTX was found to share several common features with P2, including the morphology, non-inducibility, use of lipopolysaccharide core oligosaccharide as receptor and Ca(2+)-dependent receptor binding. These findings indicate that phi CTX is a P2-like phage well adapted to P. aeruginosa, and provide clear evidence of the intergeneric spread and evolution of bacteriophages. Furthermore, comparative analysis of genome structures of phi CTX, P2 and other P2 relatives revealed the presence of several hot-spots where foreign DNAs, including the cytotoxin gene, were inserted. They appear to be deeply concerned in the acquisition of various genes that are horizontally transferred by bacteriophage infection.     

Structure and assembly of phage phi29.

Bacteriophage phi29 is a small, morphologically complex, virus with a DNA of molecular mass 12 X 10(6). The most likely structure of the head of phi29 consists of two fivefold symmetric end-caps based on T = 1 icosahedral symmetry, separated by an equatorial row of 5 hexamers. The eighteen genes identified in phi29 genome have been mapped and, in some cases, the gene products have been identified. Five linked genes, four coding for structural proteins (G, A, E, H) and one coding for a non-structural protein (J), are essential to determine the normal shape of the capsid. Protein pJ may be a scaffolding protein. An account of the effects of mutations in phi29 genes is given.