Complete genome sequence of a novel phage,vB_MoxS-ISF9, infecting methylotrophic Microbacterium: first report of a virulent Microbacterium phage

Here, we report the first genome sequence of a new virulent phage of Microbacterium oxydans, termed vB_MoxS-ISF9, which was isolated from sewage. Transmission electron microscopy showed that the isolated phage, which has a hexagonal head of about 80 nm in diameter and a long non-contractile tail of about 240 nm, belongs to the family Siphoviridae. The vB_MoxS-ISF9 DNA was completely sequenced and found to be 59,254 bp in length, with a G+C content of 62.76 % and 120 putative open reading frames (ORFs). The predicted protein products of the ORFs were identified, and their sequences were analyzed. In a comparison with all available phage genomes, vB_MoxS-ISF9 did not show any significant similarity to other previously reported bacteriophages. To the beast of our knowledge, this is the first report of the isolation and complete genomic sequencing of a virulent phage against a member of the genus Microbacterium.     

Comparative sequence analysis of the DNA packaging, head, and tail morphogenesis modules in the temperate cos-site Streptococcus thermophilus bacteriophage Sfi21.

The temperate Streptococcus thermophilus bacteriophage Sfi21 possesses 15-nucleotide-long cohesive ends with a 3' overhang that reconstitutes a cos-site with twofold hyphenated rotational symmetry. Over the DNA packaging, head and tail morphogenesis modules, the Sfi21 sequence predicts a gene map that is strikingly similar to that of lambdoid coliphages in the absence of any sequence similarity. A nearly one to one gene correlation was found with the phage lambda genes Nu1 to H, except for gene B-to-E complex, where the Sfi21 map resembled that of coliphage HK97. The similarity between Sfi21 and HK97 was striking: both major head proteins showed an N-terminal coiled-coil structure, the mature major head proteins started at amino acid positions 105 and 104, respectively, and both major head genes were preceded by genes encoding a possible protease and portal protein. The purported Sfi21 protease is the first viral member of the ClpP protease family. The prediction of Sfi21 gene functions by reference to the gene map of intensively investigated coliphages was experimentally confirmed for the major head and tail gene. Phage Sfi21 shows nucleotide sequence similarity with Lactococcus phage BK5-T and a lactococcal prophage and amino acid sequence similarity with the Lactobacillus phage A2 and the Staphylococcus phage PVL. PVL is a missing link that connects the portal proteins from Sfi21 and HK97 with respect to sequence similarity. These observations and database searches, which demonstrate sequence similarity between proteins of phage from gram-positive bacteria, proteobacteria, and Archaea, constrain models of phage evolution.     

Comparative Sequence Analysis of the DNA Packaging, Head, and Tail Morphogenesis Modules in the Temperate cos-Site Streptococcus thermophilus Bacteriophage Sfi21

The temperate Streptococcus thermophilus bacteriophage Sfi21 possesses 15-nucleotide-long cohesive ends with a 3′ overhang that reconstitutes a cos-site with twofold hyphenated rotational symmetry. Over the DNA packaging, head and tail morphogenesis modules, the Sfi21 sequence predicts a gene map that is strikingly similar to that of lambdoid coliphages in the absence of any sequence similarity. A nearly one to one gene correlation was found with the phage lambda genes Nu1 to H, except for gene B-to-E complex, where the Sfi21 map resembled that of coliphage HK97. The similarity between Sfi21 and HK97 was striking: both major head proteins showed an N-terminal coiled-coil structure, the mature major head proteins started at amino acid positions 105 and 104, respectively, and both major head genes were preceded by genes encoding a possible protease and portal protein. The purported Sfi21 protease is the first viral member of the ClpP protease family. The prediction of Sfi21 gene functions by reference to the gene map of intensively investigated coliphages was experimentally confirmed for the major head and tail gene. Phage Sfi21 shows nucleotide sequence similarity with Lactococcus phage BK5-T and a lactococcal prophage and amino acid sequence similarity with the Lactobacillus phage A2 and the Staphylococcus phage PVL. PVL is a missing link that connects the portal proteins from Sfi21 and HK97 with respect to sequence similarity. These observations and database searches, which demonstrate sequence similarity between proteins of phage from gram-positive bacteria, proteobacteria, and Archaea, constrain models of phage evolution.     

Genomic analysis of bacteriophage ESP2949-1, which is virulent for Cronobacter sakazakii

Virulent phage ESP2949-1, which was isolated from sewage, has an icosahedral head, a contractile tail, and a double-stranded DNA genome with a length of 49,116 bp with 50.09% G+C content. Phage ESP2949-1 showed 3% similarity to enterobacteria phage TLS. Bioinformatics analysis of the phage genome revealed 43 putative open reading frames (ORFs). Predicted protein products of the ORFs were determined and described. Based on its morphology, phage ESP2949-1 can be classified as a member of the family Myoviridae. To our knowledge, this is the first report of the genomic sequence and characterization of phage ESP2949-1 isolated from sewage.     

Comparative genomics of lactococcal phages: insight from the complete genome sequence of Lactococcus lactis phage BK5-T

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.     

Nucleotide sequences involved in the neolysogenic insertion of filamentous phage Cf16-v1 into the Xanthomonas campestris pv. citri chromosome

Following a protracted carrier state in the infected cell, filamentous bacteriophage Cf16-v1 neolysogenizes Xanthomonas campestris pv. citri by inserting the phage genome into the host chromosome. The integration region in the phage and the host chromosome, respectively, and the two junctions in the lysogen chromosome were isolated and their nucleotide sequence was determined. The phage and host attachment sites shared an identical 15-bp "core," 5'-TATACATTATGCGAA-3'. Located on either side of each core were two unique arm sequences. Each of the two phage-host junctions contained an intact core flanked by a hybrid combination of phage and host arm sequences. A 10-bp symmetrical sequence arranged as inverted repeats with 1-bp spacing straddled the core sequence. A 10-bp repeated sequence, 5'-GCGCTATGGC-3', was found distal to the core in opposite orientation at the phage attachment site, while an abbreviated form of this sequence was present in the host attachment site. These sequence characteristics indicate that neolysogenic insertion of Cf16-v1 was accomplished by a site-specific recombination mechanism similar to lambda integration. However, in contrast to lambda, the phage and the host attachment sites in the Cf16-v1 system contained a high G + C nucleotide bias (except for the core sequence itself).     

Comparative genomics of the late gene cluster from Lactobacillus phages

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.     

Epitope mapping of rat neutralizing monoclonal antibody against human immunodeficiency virus type-1 by a phage peptide library: comparison with ELISA using synthetic peptides.

We generated a rat monoclonal antibody (mAb W#10) with the ability to neutralize human immunodeficiency virus type 1IIIB (HIV-1IIIB) infection. The epitope recognized by mAb W#10 was defined as R-I-Q-R-G-P-G by enzyme-linked immunosorbent assay (ELISA) with the use of synthetic peptides. The filamentous phage clones displaying random 15-amino-acid peptides on the amino terminus of the pIII coat protein reacting with mAb W#10 were identified with affinity and immunological selection procedures. Thirteen out of 16 selected phage clones contained the G-X-G-R-X-F sequence in the coat protein region representing significant homology to a part of conserved G-P-G-R-A-F sequence in the V3 loop of various HIV-1 strains. In addition, the phage clones included the G-X-G sequence in the sequence detected by synthetic peptides as the recognition site. The selected phage clones were stained by mAb W#10 specifically and were able to compete with mAb binding to cells expressing viral antigens.     

Complete genomic sequence of the Vibrio alginolyticus lytic bacteriophage PVA1

A novel Vibrio alginolyticus lytic bacteriophage was isolated from sewage samples obtained from a local aquatic market. Morphological analysis revealed that the phage, designated as PVA1, belonged to the family Podoviridae. The complete genomic sequence of phage PVA1 contained 41,529 bp with a G + C content of 43.7 % and 75 putative open reading frames. The genome was grouped into four modules, including phage structure, DNA packaging, DNA replication and regulation, and some additional functions. Further genomic comparison of the phage PVA1 with other known phages showed no significant similarities. Genes related to virulence and lysogeny were not detected in the phage genome. Our results suggest that phage PVA1 may be classified as a new Vibrio phage. We believe that these phage genomic sequence data will provide useful basic information for further molecular research on this Vibrio phage and its host as well for determining its infection/interaction mechanisms.     

Complete nucleotide sequence of Bacillus subtilis (natto) bacteriophage PM1, a phage associated with disruption of food production

“Natto”, considered a traditional food, is made by fermenting boiled soybeans with Bacillus subtilis (natto), which is a natto-producing strain related to B. subtilis. The production of natto is disrupted by phage infections of B. subtilis (natto); hence, it is necessary to control phage infections. PM1, a phage of B. subtilis (natto), was isolated during interrupted natto production in a factory. In a previous study, PM1 was classified morphologically into the family Siphoviridae, and its genome, comprising approximately 50 kbp of linear double-stranded DNA, was assumed to be circularly permuted. In the present study, the complete nucleotide sequence of the PM1 genomic DNA of 50,861 bp (41.3 %G+C) was determined, and 86 open reading frames (ORFs) were deduced. Forty-one ORFs of PM1 shared similarities with proteins deduced from the genome of phages reported so far. Twenty-three ORFs of PM1 were associated with functions related to the phage multiplication process of gene control, DNA replication/modification, DNA packaging, morphogenesis, and cell lysis. Bacillus subtilis (natto) produces a capsular polypeptide of glutamate with a γ-linkage (called poly-γ-glutamate), which appears to serve as a physical barrier to phage adsorption. One ORF of PM1 had similarity with a poly-γ-glutamate hydrolase, which is assumed to degrade the capsular barrier to allow phage progenies to infect encapsulated host cells. The genome analysis of PM1 revealed the characteristics of the phage that are consistent as Bacillus subtilis (natto)-infecting phage.     

Primary identification of Microbacterium spp. encountered in clinical specimens as CDC coryneform group A-4 and A-5 bacteria.

Over nearly two decades, 13 yellow- or orange-pigmented, fermentative gram-positive rods belonging to the genus Microbacterium were encountered in clinical specimens. All 13 strains, 10 of which came from blood cultures, were initially identified as CDC coryneform group A-4 and A-5 bacteria according to the scheme of Hollis and Weaver for the identification of gram-positive rods. The clinical isolates were compared with the type strains of the six species constituting the genus Microbacterium as well as with three Microbacterium strains isolated from hospital environments. By biochemical methods only 5 of 13 clinical isolates could be identified to species level. Peptidoglycan analysis proved to be a valuable tool for differentiation between Microbacterium spp. and related genera, whereas cellular fatty acid analysis did not allow species identification within the genus Microbacterium. The 22 Microbacterium strains studied were, in general, susceptible to antimicrobial agents used in the treatment of infections caused by gram-positive rods. This report is the first one concerning the isolation of Microbacterium strains from clinical specimens. The sources as well as the mode of transmission remain to be established.     

Isolation and characterization of APSE-1, a bacteriophage infecting the secondary endosymbiont of Acyrthosiphon pisum.

A bacteriophage infecting the secondary endosymbiont of the pea aphid Acyrthosiphon pisum was isolated and characterized. The phage was tentatively named bacteriophage APSE-1, for bacteriophage 1 of the A. pisum secondary endosymbiont. The APSE-1 phage particles morphologically resembled those of species of the Podoviridae. The complete nucleotide sequence of the bacteriophage APSE-1 genome was elucidated, and its genomic organization was deduced. The genome consists of a circularly permuted and terminally redundant double-stranded DNA molecule of 36524 bp. Fifty-four open reading frames, putatively encoding proteins with molecular masses of more than 8 kDa, were distinguished. ORF24 was identified as the gene coding for the major head protein by N-terminal amino acid sequencing of the protein. Comparison of APSE-1 sequences with bacteriophage-derived sequences present in databases revealed the putative function of 24 products, including the lysis proteins, scaffolding protein, transfer proteins, and DNA polymerase. This is the first report of a phage infecting an endosymbiont of an arthropod.     

Genome organization of membrane-containing bacteriophage PRD1

We have determined the nucleotide sequence of the late region (11 kbp) of the lipid-containing bacteriophage PRD1. Gene localization was carried out by complementing nonsense phage mutants with genomic clones containing specific reading frames. The localization was confirmed by sequencing the N-termini of isolated gene products as well as sequencing the N-termini of tryptic fragments of the phage membrane-associated proteins. This, with the previously obtained sequence of the early regions, allowed us to organize most of the phage genes in the phage genome.     

Different characteristics between mycobacteriophage Chy1 and D29, which were classified as cluster A2 mycobacteriophages

Purpose: The aim of this study was to isolate a novel mycobacteriophage and then explore its anti-tuberculosis (TB) potential. Materials and Methods: Phage was isolated from enriched soil sample. A total of 36 mycobacterial strains obtained from clinical specimens were subjected to investigate the host range of phage by the spot lysis assay. Biological characteristics were investigated through growth curve, host range and phage antimicrobial activity in vitro. Then, genome sequencing and further analysis were accomplished by using an ABI3730XL DNA sequencer and comparative genome, respectively. Results: A lytic mycobacteriophage (Chy1) was isolated and the plaque morphology was similar to D29. The genome of Chy1 was estimated to be about 47,198 base pair (bp) and strong similarity (97.4% identity) to D29, especially, the Chy1 gene 7 encoding holin which is considered as a clock controlling growth cycle of the corresponding phage, was identical (100% identity) to phage D29 gene 11, thus classifying Chy1 as a member of the cluster A2 family. However, to our surprise, Chy1 can infect a narrower range of host-mycobacterial strains than that of D29. The latent period of Chy1 was quite longer compared to D29. Moreover, Chy1 has a weaker ability to lyse Mycobacterium smegmatis compared to D29. Conclusions: The sequence of Chy1 showed 97.4% homology with the genome sequence of D29, but there was a large difference in their biological characteristics. Overall, the results of this investigation indicate that Chy1 is not an ideal candidate for developing mycobacteriophage-based anti-TB therapies but for future researches to investigate the reason why biological characteristics of Chy1 and D29 were remarkably different.     

Bacteremia Caused by Microbacterium binotii in a Patient with Sickle Cell Anemia

Microbacterium species are non-spore-forming, Gram-positive rods rarely associated with human disease. In this report, we describe the first case of bacteremia caused by Microbacterium binotii in a patient with sickle cell anemia. The utility of using 16S rRNA gene sequence analysis along with phenotypic methods for identification is shown.     

Interaction of ICAM-1 with beta 2-integrin CD11c/CD18: characterization of a peptide ligand that mimics a putative binding site on domain D4 of ICAM-1

The integrin CD11c/CD18 plays a role in leukocyte and cell matrix adhesion and is highly expressed in certain hematopoietic malignancies. To better characterize ligand binding properties, we panned random peptide phage-display libraries over purified CD11c/CD18. We identified a phage expressing the circular peptide C-GRWSGWPADL-C. C-GRWSGWPADL-C phage bound specifically to CD11c/CD18 expressing monocytes but not CD11c/CD18 negative lymphocytes and showed 5 x 10(3)-fold higher binding to purified CD11c/CD18 than control phage, without binding to CD11b/CD18. Peptide sequence analysis revealed a similar sequence in domain D5 of ICAM-1 and an alternative, phase-shifted motif in domain D4. Surface plasmon resonance experiments demonstrated direct interaction of ICAM-1 and CD11c/CD18. A soluble fusion protein containing the extracellular domain of ICAM-1 abolished C-GRWSGWPADL-C phage binding to CD11c/CD18. Moreover, synthetic monomeric circular peptide C-GRWSGWPADL-C bound specifically to CD11c/CD18 and inhibited ICAM-1 binding. Its rather low binding affinity and inability to displace pentavalent C-GRWSGWPADL-C phage from CD11c/CD18 suggests that a multimeric display of the selected peptide is essential for high affinity binding. Using ICAM-1 deletion constructs, we showed that domain D4 is required for interaction with CD11c/CD18, suggesting that C-GRWSGWPADL-C phage binds specifically to CD11c/CD18 by structurally mimicking the interaction site on D4 of ICAM-1.