Comparative Genomics and Transduction Potential of Enterococcus faecalis Temperate Bacteriophages

To determine the relative importance of temperate bacteriophage in the horizontal gene transfer of fitness and virulence determinants of Enterococcus faecalis, a panel of 47 bacteremia isolates were treated with the inducing agents mitomycin C, norfloxacin, and UV radiation. Thirty-four phages were purified from culture supernatants and discriminated using pulsed-field gel electrophoresis (PFGE) and restriction mapping. From these analyses the genomes of eight representative phages were pyrosequenced, revealing four distinct groups of phages. Three groups of phages, ΦFL1 to 3, were found to be sequence related, with ΦFL1A to C and ΦFL2A and B sharing the greatest identity (87 to 88%), while ΦFL3A and B share 37 to 41% identity with ΦFL1 and 2. ΦFL4A shares 3 to 12% identity with the phages ΦFL1 to 3. The ΦFL3A and B phages possess a high DNA sequence identity with the morphogenesis and lysis modules of Lactococcus lactis subsp. cremoris prophages. Homologs of the Streptococcus mitis platelet binding phage tail proteins, PblA and PblB, are encoded on each sequenced E. faecalis phage. Few other phage genes encoding potential virulence functions were identified, and there was little evidence of carriage of lysogenic conversion genes distal to endolysin, as has been observed with genomes of many temperate phages from the opportunist pathogens Staphylococcus aureus and Streptococcus pyogenes. E. faecalis JH2-2 lysogens were generated using the eight phages, and these were examined for their relative fitness in Galleria mellonella. Several lysogens exhibited different effects upon survival of G. mellonella compared to their isogenic parent. The eight phages were tested for their ability to package host DNA, and three were shown to be very effective for generalized transduction of naive host cells of the laboratory strains OG1RF and JH2-2.Enterococcus faecalis is a member of the natural flora of humans and colonizes the gastrointestinal and vaginal tracts and the oral cavity. In recent years it has emerged as an important opportunistic nosocomial pathogen and is a causative agent of bacteremia, infective endocarditis, and surgical wound and urinary tract infections. The accumulation of acquired antibiotic resistance determinants, in addition to its intrinsic resistance and tenacity, has given rise to the evolution of clinical isolates of E. faecalis that are therapeutically problematic (19). Greater notoriety was afforded to this species following the observed transfer of the conjugative transposon Tn1546 to Staphylococcus aureus, imparting vancomycin resistance (11). Subsequent analysis has revealed that multiple independent E. faecalis-dependent vanA transfers had occurred in the United States prior to 2007 (50). This places enterococci in an important and dynamic position within the health care system, warranting their increased study.The specific determinants that are proposed to contribute to the virulence of E. faecalis are not universally present, and expression of the cognate genes is variable (21, 37). For example, in a recent study of 106 clonally diverse strains of E. faecalis the metallopeptidase gelatinase (GelE) was shown to be expressed in less than 60% of 106 genotypically positive isolates, whereas expression of cytolysin was less frequently observed (expression in ∼25% of isolates, with 30% being genotypically positive) (33). A proposed pathogenicity island identified with E. faecalis V583 (49) is composed of a variable gene set encoding the virulence determinants enterococcal surface protein, cytolysin, and aggregation substance. This highly variable 150-kb mobile element contains many components of unknown function that are hypothesized to facilitate survival and/or transmission in the health care setting (34, 40, 49).Two sequenced and annotated genomes of E. faecalis have been completed and published to date. These are the blood isolate and first-observed vancomycin-resistant strain V583 (40) and the oral isolate OG1RF, used as a common laboratory strain (8). A major difference between these genomes is the presence in V583 of seven regions containing phage-associated sequences. In contrast, OG1RF contains only one phage remnant, which was proposed by McBride et al. (33) to form part of the core genome, a theory supported by the presence in OG1RF of this phage remnant region together with two CRISPR loci. CRISPR sequences provide sequence-specific resistance to bacteriophages via the assembly of phage DNA sequences interspersed as spacers between repeats, in concert with associated cas genes, which collectively operate as an RNA-based gene silencing mechanism (5, 6, 28, 30, 36, 42). This elegant heritable mechanism is proposed to limit horizontal gene transfer of bacteriophage, transposable elements, and conjugative plasmids (9, 10, 32).Within the firmicute division of Gram-positive bacteria, temperate bacteriophages are key vectors for the horizontal transfer of virulence genes. In Staphylococcus aureus, bacteriophages encode and mobilize an impressive array of immune evasion genes (54, 55) and Panton-Valentine leukocidin (43). Several bacteriophage-encoded virulence determinants also contribute to pathogenesis in group A Streptococcus (2, 3, 4).The role of bacteriophages in the virulence of E. faecalis is not clear. Encoded within seven phage-related sequences of strain V583, there are multiple reported homologs of the Streptococcus mitis platelet-binding proteins PblA and PblB (7) and a ferrochelatase (40). In contrast, the absence of mobile genetic elements (MGEs) in strain OG1RF led Bourgogne et al. (8) to speculate that they did not engender virulence in E. faecalis.In this study we determined the morphology and complete genome sequences of eight induced bacteriophages purified from clinical isolates of E. faecalis. We sought to determine the potential carriage of genes that might contribute to the virulence or fitness of this organism and characterize the capacity of these phages to participate in transduction.     

Characterization of Temperate Bacteriophages of Bacillus subtilis by the Restriction Endonuclease EcoRI: Evidence for Three Different Temperate Bacteriophages

Temperate bacteriophages of Bacillus subtilis were characterized according to host range and digestion of the bacteriophage genome by endonuclease EcoRI. The three bacteriophages, 3T, SPO2, and 105, were all heteroimmune, and the DNA digests showed dissimilar patterns by agarose-ethidium bromide gel electrophoresis.     

Isolation and Characterization of Temperate Bacteriophages of Clostridium difficile

The lack of information on bacteriophages of Clostridium difficile prompted this study. Three of 56 clinical C. difficile isolates yielded double-stranded DNA phages C2, C5, C6, and C8 upon induction. Superinfection and DNA analyses revealed relatedness between the phages, while partial sequencing of C2 showed nucleotide homology to the sequenced C. difficile strain CD630.     

Intramolecular Heterogeneity of the Deoxyribonucleic Acid of Temperate Bacteriophages

Spectral changes accompanying the thermal denaturation of phage deoxyribonucleic acid suggested that λ is not unique in possessing large-scale intramolecular heterogeneity and nucleotide clustering; instead, λ seems to share this property with other enteric phages.     

Unrelatedness of Temperate Bacillus subtilis Bacteriophages SPO2 and φ105

SPO2 and phi105 are temperate Bacillus subtilis bacteriophages which have been suggested to belong to a cluster of related bacteriophages. In the present work, we show that SPO2 does not complement any of the 11 essential genes known in phi105 and that the phages do not recombine. Deoxyribonucleic acid (DNA)-DNA hybridization shows less than 10% homology between SPO2 and phi105 DNA. DNA synthesis in phi105 shows a greater dependence on host functions than does SPO2 DNA synthesis. Growth of phi105 but not of SPO2 is inhibited by the uracil analogue 6-(p-hydroxyphenylazo)-uracil. Infection of a DNA polymerase-deficient strain of B. subtilis with SPO2 leads to an increase in DNA polymerase activity in crude extracts, whereas no such increase is found after infection of this strain with phi105. It is concluded that SPO2 and phi105 are unrelated bacteriophages.     

Comparative genomics for identification of clone-specific sequence blocks in Streptococcus pneumoniae

The partial genome sequences of a serotype 3 and a serotype 2 pneumococcal strain were compared to the complete type 4 pneumococcal genome. Over 500000 and 150000 base pairs of the partial genome data, obtained from published patents, were analysed respectively. Global alignment showed that nearly the whole genome is highly conserved in accordance with data of multilocus sequence typing of housekeeping genes. The search for clone-specific genes revealed 17 new open reading frames in the type 3 strain, while no new open reading frame was detected in the type 2 strain. Allelic variation of genes was restricted by the use of crude sequence data, but still permitted identification of some new alleles and the observation that all surface proteins present in the partial genome data were highly conserved. In both strains we observed also a variety of chromosomal rearrangements and variations due to mobile genetic elements. All together, this comparative genomic approach gives a genome-based overview of strain relatedness and a prospective on what could be expected when sequencing other pneumococcal strains.     

The Morphology of Six Bacteriophages of Streptococcus thermophilus

The morphology of six phages of Streptococcus thermophilus , isolated in France from Gruyère cheese whey or yoghurt, was studied. They belong to Bradley's Group B and appear very similar. They possess a regular six-sided head (size, 49–53 nm), of which the three-dimensional structure is probably octahedral. The fairly long, non-contractile tails (length, 200–224 nm; width, 8–9 nm) are flexible and regularly striated. They appear to be the juxtaposition of 60–63 annuli packed one above the other and are assumed to contain 240–252 capsomeres. In one preparation rare short-tailed particles (tail length 130 nm) were also found. The tails have a terminal structure appearing either as a small plate, sometimes with a brush-like appearance, or a plate with short prongs provided, in five out of the six phages studied, with a more or less long central fibre.     

Electrotransformation of Streptococcus pneumoniae

Conditions of electroporation to transform encapsulated strains of Streptococcus pneumoniae with plasmid DNA have been defined. For a heavily encapsulated strain, an electroporation solution of 10–20% (v/v) glycerol and 3.2 kV cm^-1 field strength, 1000 Ω resistance and 25 μF capacitance were optimal. For lightly encapsulated or non-encapsulated strains, optimal conditions were a sucrose-based electroporation solution and 12.5 kV cm^-1 field strength, 200 Ω resistance and 25 μF capacitance.     

Comparative structural and molecular characterization of Streptococcus pneumoniae capsular polysaccharide serogroup 10

Streptococcus pneumoniae serogroup 10 includes four cross-reactive capsular polysaccharide (CPS) serotypes (10F, 10A, 10B, and 10C). In the present study, the structures of CPS10B and CPS10C were determined by chemical and high resolution NMR methods to define the features of each serotype. Both CPS10C and CPS10F had β1-6-linked Galf branches formed from the termini of linear repeating units by wzy-dependent polymerization through the 4-OH of subterminal GalNAc. The only difference between these polysaccharides was the wcrC-dependent α1-2 or wcrF-dependent α1-4 linkages between Gal and ribitol-5-phosphate. The presence of one linkage or the other also distinguished the repeating units of CPS10B and CPS10A. However, whereas these polysaccharides both had β1-3-linked Galf branches linked to GalNAc, only CPS10A had additional β1-6-linked Galp branches. These Galp branches and the reaction of a CPS10A-specific monoclonal antibody were eliminated by deletion of wcrG from the cps10A locus. In contrast, deletion of this gene from the cps10B locus had no effect on the structure of CPS10B, thereby identifying wcrG as a pseudogene in this serotype. The β1-3-linked Galf branches of CPS10A and CPS10B were eliminated by deletion of wcrD from each corresponding cps locus. Deletion of this gene also eliminated wcrG-dependent β1-6-linked Galp branches from CPS10A, thereby identifying WcrG as a branching enzyme that acts on the product of WcrD. These findings provide a complete view of the molecular, structural, and antigenic features of CPS serogroup 10, as well as insight into the possible emergence of new serotypes.     

Temperate bacteriophages of Streptococcus pneumoniae that contain protein covalently linked to the 5'' ends of their DNA.

We have characterized three temperate bacteriophages of pneumococcus (HB-3, HB-623, and HB-746). Although all the phages belong to the same family, the polypeptide composition of the virions and the DNA restriction endonuclease analysis of their DNAs revealed differences among the three phages. The genomes of these bacteriophages have been isolated as DNA-protein complexes. The protein is specifically associated with the two 5' termini of the DNA as shown by experiments carried out with exonucleases. The protein bound to the DNA in the three phages studied, iodinated in vitro with 125I, has a molecular weight of 23,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of the complexes with chaotropic agents suggested that the protein is covalently bound to the 5' termini of the DNA. Comparative pulsed-field gel electrophoresis analysis and Southern hybridization of the SmaI restriction fragments of DNAs from one lysogenic bacteria and its parental strain revealed that the prophage genome was integrated in the host chromosome.     

Comparative Genetics of the T-Even Bacteriophages

A system of amber mutants has been developed for each of the T-even bacteriophages T2 and T6, to complement those already available in T4. In T2 these mutants identify 52 genes, of which 49 are homologous with T4 genes; in T6 they identify 45 genes, of which 42 have T4 homologs, and an additional one which is homologous to a T2 gene not yet identified in T4. In both T2 and T6, recombination between mutants is characterized by considerable negative interference, which is correctable by a mapping function designed for T4. Recombinational maps of T2 and T6 constructed with these mutants have the same gene order and nearly the same gene spacings as in T4, with the exception of the tail fiber region; T2 and T6 appear to lack a localized recombinational expansion of this region found in T4. Homologous gene products from all three phages are in general interchangeable, with the exception of those from two apparently "co-adapted" tail fiber genes, 37 and 38. The general genetic similarity of all three phages suggests that they are analogous to races of higher organisms, retaining the capacity for genetic exchange despite some clear genetic differences and some incipient isolating mechanisms.     

Expression of Antisense RNA Targeted against Streptococcus thermophilus Bacteriophages

Antisense RNA complementary to a putative helicase gene (hel3.1) of a cos-type Streptococcus thermophilus bacteriophage was used to impede the proliferation of a number of cos-type S. thermophilus bacteriophages and one pac-type bacteriophage. The putative helicase gene is a component of the Sfi21-type DNA replication module, which is found in a majority of the S. thermophilus bacteriophages of industrial importance. All bacteriophages that strongly hybridized a 689-bp internal hel3.1 probe were sensitive to the expression of antisense hel3.1 RNA. A 40 to 70% reduction in efficiency of plaquing (EOP) was consistently observed, with a concomitant decrease in plaque size relative to that of the S. thermophilus parental strain. When progeny were released, the burst size was reduced. Growth curves of S. thermophilus NCK1125, in the presence of variable levels of bacteriophage κ3, showed that antisense hel3.1 conferred protection, even at a multiplicity of infection of approximately 1.0. When the hel3.1 antisense RNA cassette was expressed in cis from the κ3-derived phage-encoded resistance (PER) plasmid pTRK690::ori3.1, the EOP for bacteriophages sensitive to PER and antisense targeting was reduced to between 10⁻⁷ and 10⁻⁸, beyond the resistance conferred by the PER element alone (less than 10⁻⁶). These results illustrate the first successful applications of antisense RNA and explosive delivery of antisense RNA to inhibit the proliferation of S. thermophilus bacteriophages.     

Comparative Genomic Analyses of Streptococcus pseudopneumoniae Provide Insight into Virulence and Commensalism Dynamics

Streptococcus pseudopneumoniae (SPPN) is a recently described species of the viridans group streptococci (VGS). Although the pathogenic potential of S. pseudopneumoniae remains uncertain, it is most commonly isolated from patients with underlying medical conditions, such as chronic obstructive pulmonary disease. S. pseudopneumoniae can be distinguished from the closely related species, S. pneumoniae and S. mitis, by phenotypic characteristics, including optochin resistance in the presence of 5% CO₂, bile insolubility, and the lack of the pneumococcal capsule. Previously, we reported the draft genome sequence of S. pseudopneumoniae IS7493, a clinical isolate obtained from an immunocompromised patient with documented pneumonia. Here, we use comparative genomics approaches to identify similarities and key differences between S. pseudopneumoniae IS7493, S. pneumoniae and S. mitis. The genome structure of S. pseudopneumoniae IS7493 is most closely related to that of S. pneumoniae R6, but several recombination events are evident. Analysis of gene content reveals numerous unique features that distinguish S. pseudopneumoniae from other streptococci. The presence of loci for competence, iron transport, pneumolysin production and antimicrobial resistance reinforce the phylogenetic position of S. pseudopneumoniae as an intermediate species between S. pneumoniae and S. mitis. Additionally, the presence of several virulence factors and antibiotic resistance mechanisms suggest the potential of this commensal species to become pathogenic or to contribute to increasing antibiotic resistance levels seen among the VGS.     

喉癌的比较基因组杂交研究

寻找与喉癌发生进展的相关基因,应用比较基因组杂交技术分析了18例喉癌患者。结果显示,每例喉癌细胞染色体均有不同程度的变化,包括染色体全部或部分的扩增或丢失。平均每例有12．9处异常区域,丢失多于扩增,分别为每例7．2处和5．7处。主要为3q(78％,)、5p(61％)、11q(56％)、1q(50％,)、8p(44％)、8q(39％)和15q(39％)的扩增;3p(70％)、5q(78％)、90(67%)、13q(50％)、1p(44％)和14q(39％)的丢失。有多条染色体区带上出现特异的扩增或丢失,特别是1p13-21(8／18)、3p21-23(14／18)、5p21-22(14／18)、9p12-pter(10／18)和13q21-31(8／18)的拷贝数增加明显,而1q11-2l(11／18)、3q15-21(12／18)、8p22-24(6／18)、11q12-13(8／18)、15q21-23(7／18)和18p11(8／18)区域的丢失为喉癌的特征性变化,说明这些区域中存在与喉癌发生发展密切相关的癌基因、抑癌基因或其他相关基因。