Isolation of Bacteriophages T2 and T4 Attached to the Outer Membrane of Escherichia coli

Phage T2 or T4 was adsorbed to Escherichia coli, and the outer (L) membrane was then isolated with the phage still attached in their usual postinjection appearance. T2 was readily inactivated by isolated cell walls but very poorly by purified L membrane. T4 was inactivated by neither.     

Effect of Osmotic Shock and Low Salt Concentration on Survival and Density of Bacteriophages T4B and T4Bo1

Measurements of survival and buoyant densities of bacteriophages T4B, T4Bo(1), and T4D have demonstrated the following: (a) After suspension in a concentrated salt solution, T4B and T4D are sensitive both to osmotic shock and to subsequent exposure to low monovalent salt concentrations. (b) Sensitivity of T4B to dilution from a concentrated salt solution is dependent on dilution rate, that of T4D is less dependent, and that of T4Bo(1) is independent. (c) Sensitivity of all three phages to low salt concentrations depends on initial salt concentrations to a variable extent. (d) Density gradient profiles indicate that nearly half of osmotically shocked T4B retain their DNA. Similar analysis demonstrates that few, if any, T4Bo(1) lose DNA when subjected to a treatment causing 90% loss of infectivity. (e) The effective buoyant densities of T4B and T4Bo(1) depend significantly on the dilution treatments to which the phages are subjected prior to centrifugation in CsCl gradients. These data are explicable in terms of the different relative permeabilities of the phages to water and solutes, and of alterations in the counterion distribution surrounding the DNA within the phage heads.     

Exclusion of Bacteriophages by T2 Ghosts

T2 ghosts do not exclude T4, T7, or lambda-induction in Escherichia coli which survive ghost infection. Latent periods are extended, probably by the temporary inhibition of protein synthesis.     

Phospholipid Synthesis in Escherichia coli Infected with T4 Bacteriophages

After infection of Escherichia coli with T4 phage, phospholipid synthesis continued but at a reduced rate. The same phospholipid components were synthesized as in uninfected cells; however, the relative rates of (32)P(i) incorporation into phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) were altered. This alteration was most pronounced during the first 10 min after infection. Under these conditions, the isotope incorporated into PG equaled or exceeded that found in PG from uninfected cells. Chloramphenicol (CM) added before, but not 5 min after, infection inhibited the relative increase in PG synthesis, and CM added at different times after infection indicated that a protein synthesized between 3 and 6 min was required for this change to occur. Supplies of exogenous l-serine or l-alpha-glycerol-P failed to affect the relative rates of (32)P(i) incorporation into PG and PE by infected or uninfected cells. Phospholipid synthesis was somewhat higher after infection with T4rII mutants than after infection with wild-type phage. After infection with these mutants or several amber mutants, the relative synthesis of PG and PE was characteristic of T4r(+)-infected cells. The phospholipid synthesized after infection did not rapidly turn over, but infection accelerated the loss of PG synthesized prior to infection.     

Brochothrix thermosphacta Bacteriophages Feature Heterogeneous and Highly Mosaic Genomes and Utilize Unique Prophage Insertion Sites

Brochothrix belongs to the low-GC branch of Gram-positive bacteria (Firmicutes), closely related to Listeria, Staphylococcus, Clostridium, and Bacillus. Brochothrix thermosphacta is a nonproteolytic food spoilage organism, adapted to growth in vacuum-packaged meats. We report the first genome sequences and characterization of Brochothrix bacteriophages. Phage A9 is a myovirus with an 89-nm capsid diameter and a 171-nm contractile tail; it belongs to the Spounavirinae subfamily and shares significant homologies with Listeria phage A511, Staphylococcus phage Twort, and others. The A9 unit genome is 127 kb long with 11-kb terminal redundancy; it encodes 198 proteins and 6 tRNAs. Phages BL3 and NF5 are temperate siphoviruses with a head diameter of 56 to 59 nm. The BL3 tail is 270 nm long, whereas NF5 features a short tail of only 94 nm. The NF5 genome (36.95 kb) encodes 57 gene products, BL3 (41.52 kb) encodes 65 products, and both are arranged in life cycle-specific modules. Surprisingly, BL3 and NF5 show little relatedness to Listeria phages but rather demonstrate relatedness to lactococcal phages. Peptide mass fingerprinting of viral proteins indicate programmed −1 translational frameshifts in the NF5 capsid and the BL3 major tail protein. Both NF5 and BL3 feature circularly permuted, terminally redundant genomes, packaged by a headful mechanism, and integrases of the serine (BL3) and tyrosine (NF5) types. They utilize unique target sequences not previously described: BL3 inserts into the 3′ end of a RNA methyltransferase, whereas NF5 integrates into the 5′-terminal part of a putative histidinol-phosphatase. Interestingly, both genes are reconstituted by phage sequence.Brochothrix thermosphacta is a Gram-positive, rod-shaped, nonmotile, non-spore-forming, facultative anaerobic, and psychrotrophic organism frequently involved in the spoilage of prepacked and vacuum-packaged meat or meat products (23). The conditions found in these foods selectively favor development of B. thermosphacta due to its ability to grow at 4°C under O₂ depletion and in the presence of elevated CO₂ concentrations (58). B. thermosphacta frequently constitutes the dominant portion of the spoilage microflora of aerobically and anaerobically stored meats (7), where it produces undesired volatile compounds such as acetoin, diacetyl (aerobic growth), or lactic acid and ethanol (anaerobic growth) (28, 58, 66). The taxonomic standing of the genus Brochothrix has been in dispute for a long time (65), until 16S rRNA sequences revealed its close phylogenetic relationship to the genus Listeria (16, 52), which led to the affiliation of Brochothrix, together with the genus Listeria into the Listeriaceae family. The two genera feature a similar GC content and other common characteristics, such as the same major fatty acids and menaquinones, meso-diamino pimelic acid in the peptidoglycan, production of catalase, and more (65).The potential of bacteriophages and phage-encoded lytic enzymes for control of specific pathogens in foods has been intensively investigated in recent years (26, 31, 48). As an example, the application of phage to foods has turned out to be a useful approach in the control of Salmonella, Campylobacter, Listeria, and Escherichia coli (reviewed in reference 31). Another possibility is the direct application of purified bacteriophage endolysins to foods or raw products (reviewed in reference 48). These approaches might also be suitable for the biocontrol of B. thermosphacta to prevent food spoilage. Previous work has shown that Brochothrix phage A3 was able to limit off-odor formation and increase the storage life of pork adipose tissue (28).Unfortunately, only very limited data and no genome sequences are available for bacteriophages infecting Brochothrix. Ackermann et al. classified 21 Brochothrix phages by electron microscopy and grouped them into three species based on morphology (1): species A19 contains 5 myoviruses with long tails that undergo extensive rearrangements upon contraction, species NF5 consists of 14 siphoviruses with rigid tails and complex baseplate structures with appendages, and species BL3 contains only 1 member of the siphovirus morphology. Due to the close phylogenetic relatedness of their hosts, phages of the genus Brochothrix were expected to share similarities with Listeria phages also on the molecular level.We report here the complete genome sequences and molecular characterization of three bacteriophages infecting B. thermosphacta, namely, A9, BL3, and NF5. Their physical genome structures and attachment loci of the temperate bacteriophages were determined and revealed novel prophage insertion sites. Programmed translational frameshifts in structural genes and lytic activity of bacteriophage-encoded endolysin gene products were demonstrated. In addition, intra- and interspecies comparative genomics led to the placement of phage A9 into the recently proposed Spounavirinae subfamily, and indicated relatedness of phages BL3 and NF5 to the P335 quasispecies of Lactococcus phages.     

菱叶唇柱苣苔的组织培养和快速繁殖

1植物名称菱叶唇柱苣苔（Chirita subrhomboidea W.T.Wang）。2材料类别幼叶。3培养条件（1）诱导不定芽分化培养基：MS＋6-BA1.0mg·L-1（单位下同）＋NAA0.1;（2）生根培养基：1/2MS＋0.5%活性炭。上述培养基均加入3.0%蔗糖和0.6%琼脂,pH5.8。培养温度为（26±2）℃;     

尖萼唇柱苣苔的组织培养和快速繁殖

1植物名称尖萼唇柱苣苔(Chirita pungentisepala W.T.Wang)。2材料类别花梗和带苞片的花蕾。3培养条件(1)愈伤组织诱导和不定芽分化培养基:MS 6-BA0.1mg·L-1(单位下同) NAA0.1;     

三苞唇柱苣苔的组织培养与快速繁殖

1植物名称三苞唇柱苣苔（Chirita tribracteata W.T.Wang）。2材料类别幼叶。3培养条件（1）诱导培养基：MS＋6-BA0.5mg·L-1（单位下同）＋NAA0.1;（2）增殖培养基：MS＋6-BA0.1＋NAA0.1;（3）生根培养基：1/2MS＋NAA0.1。上述培养基均加入3.0%蔗糖和0.5%琼脂,pH5.8。     

桂林唇柱苣苔的组织培养和快速繁殖

1植物名称桂林唇柱苣苔（Chirita guilinensis W．T．Wang）。 2材料类别幼嫩叶片。 3培养条件（1）诱导培养基：MS＋6-BA0．2mg·L-1（单位下同）＋IBA0．1＋3％蔗糖;     

多齿吊石苣苔的组织培养与快速繁殖

1植物名称多齿吊石苣苔（Lysionotus denticulosus W.T.Wang）。 2材料类别中上部叶片。 3培养条件（1）诱导培养基：MS＋6-BA1.0mg·L^-1（单位下同）＋IBA0.1＋3%蔗糖; （2）增殖培养基：MS＋6-BA0.5～1.0＋IBA0.05＋3%蔗糖;     

The Cotransduction of pET System Plasmids by Mutants of T4 and RB43 Bacteriophages

The study of the cotransduction of the plasmid pairs pET-3a–pLysE and pET-3a–pLysS by the mutant phage T4alc7 showed that the antibiotic resistance markers of the plasmids were cotransduced with a high frequency. The analysis of the plasmid DNA of cotransductants and cotransformants showed that the mutant phage T4alc7 can be used for obtaining the monomeric and oligomeric forms of plasmids and for the cotransduction of two-plasmid overproduction systems into E. coli strains. The plaque mutants RB43-03 and RB43-13 derived from bacteriophage RB43 were found to be able to cotransduce the antibiotic resistance markers of pET-3a and pLysE plasmids.     

Induction of P1 Prophage and Superinfection by Bacteriophage T1

Induction of the resident prophage did not permit restricted phage T1 to replicate freely in P1-lysogenic hosts. A few induced cells did become infectible.     

Comparative Genome Analysis of Listeria Bacteriophages Reveals Extensive Mosaicism,Programmed Translational Frameshifting,and a Novel Prophage Insertion Site

The genomes of six Listeria bacteriophages were sequenced and analyzed. Phages A006, A500, B025, P35, and P40 are members of the Siphoviridae and contain double-stranded DNA genomes of between 35.6 kb and 42.7 kb. Phage B054 is a unique myovirus and features a 48.2-kb genome. Phage B025 features 3′ overlapping single-stranded genome ends, whereas the other viruses contain collections of terminally redundant, circularly permuted DNA molecules. Phages P35 and P40 have a broad host range and lack lysogeny functions, correlating with their virulent lifestyle. Phages A500, A006, and B025 integrate into bacterial tRNA genes, whereas B054 targets the 3′ end of translation elongation factor gene tsf. This is the first reported case of phage integration into such an evolutionarily conserved genetic element. Peptide fingerprinting of viral proteins revealed that both A118 and A500 utilize +1 and −1 programmed translational frameshifting for generating major capsid and tail shaft proteins with C termini of different lengths. In both cases, the unusual +1 frameshift at the 3′ ends of the tsh coding sequences is induced by overlapping proline codons and cis-acting shifty stops. Although Listeria phage genomes feature a conserved organization, they also show extensive mosaicism within the genome building blocks. Of particular interest is B025, which harbors a collection of modules and sequences with relatedness not only to other Listeria phages but also to viruses infecting other members of the Firmicutes. In conclusion, our results yield insights into the composition and diversity of Listeria phages and provide new information on their function, genome adaptation, and evolution.The opportunistic pathogen Listeria monocytogenes is ubiquitous in nature and can become endemic in food processing environments, causing contamination of dairy products, meats, vegetables, and processed ready-to-eat food (14). L. monocytogenes is the causative agent of epidemic and sporadic listeriosis. The risk of infection is markedly increased among immunocompromised patients, newborns, pregnant women, and the elderly and is associated with a mortality rate of about 20 to 30% (37).Although all strains of L. monocytogenes are considered potentially pathogenic, epidemiological evidence has shown that certain serovars are more frequently associated with both sporadic cases and larger food-borne outbreaks. However, genetic variation within the virulence genes of wild-type strains appears to be limited and could not be directly linked to differences in pathogenicity (30) or environmental distribution.It is becoming increasingly clear that bacteriophages have an important role in bacterial biology, diversity, and evolution, as indicated by the advances in genome sequencing which revealed a high incidence of phage-related sequences in bacterial genomes. Many phages have been described for the genus Listeria, and lysogeny appears to be widespread (28). Availability of the genome sequences of different L. monocytogenes and L. innocua strains also revealed the presence of several different putative prophages in the genomes of these bacteria, constituting up to 7% of the coding capacities of the genomes (15, 32). Although the genomes of L. monocytogenes were found to be essentially syntenic, a significant portion of sequence variability is apparently based upon phage insertions and subsequent rearrangements. Investigations of prophage contributions to population dynamics in Salmonella suggest that prophages can improve the competitive fitness of the lysogenized host strains (5). This type of selective pressure also results in diversification and generation of new strains by lysogenic conversion. In the case of Listeria, however, the potential influence of prophages on their host strains, such as phenotypic variation or provision of selective benefits, has not been investigated. To gain more insight in bacteriophage-host interactions and the molecular biology and characteristics of Listeria phages, more information on the structure, information content, and variability of different Listeria phage genomes is required.Although a number of Listeria phages have been isolated and described (25, 27, 42), only limited information was available at the sequence level for phages PSA, A118, A511, and P100 (9, 19, 26, 41). As the result of a comprehensive study to determine the diversity of this group of bacterial viruses, we here report the complete nucleotide sequences of a representative set of six different Listeria phages from the Siphoviridae (A006, A500, B025, P35, and P40) and Myoviridae (B054) families in the order Caudovirales. In addition to molecular and in silico analyses, we also determined the physical genome structures and attachment loci of the temperate phages, and we describe integration of the B054 prophage into a highly conserved elongation factor gene. Another interesting finding is the unusual decoding used by some of the phages, which use programmed frameshifting to generate C-terminally modified structural proteins required for assembly of the capsid and tail.     

Draft Genome Sequences of Actinomyces timonensis Strain 7400942T and Its Prophage

A draft genome sequence of Actinomyces timonensis, an anaerobic bacterium isolated from a human clinical osteoarticular sample, is described here. CRISPR-associated proteins, insertion sequence, and toxin-antitoxin loci were found on the genome. A new virus or provirus, AT-1, was characterized.     

Construction and Characterization of T7 Bacteriophages Harboring Apidaecin-Derived Sequences

The global spread of multi- and pan-resistant bacteria has triggered research to identify novel strategies to fight these pathogens, such as antimicrobial peptides and, more recently, bacteriophages. In a proof-of-concept study, we have genetically modified lytic T7Select phages targeting Escherichia coli Rosetta by integrating DNA sequences derived from the proline-rich antimicrobial peptide, apidaecin. This allowed testing of our hypothesis that apidaecins and bacteriophages can synergistically act on phage-sensitive and phage-resistant E. coli cells and overcome the excessive cost of peptide drugs by using infected cells to express apidaecins before cell lysis. Indeed, the addition of the highly active synthetic apidaecin analogs, Api802 and Api806, to T7Select phage-infected E. coli Rosetta cultures prevented or delayed the growth of potentially phage-resistant E. coli Rosetta strains. However, high concentrations of Api802 also reduced the T7Select phage fitness. Additionally, plasmids encoding Api802, Api806, and Api810 sequences transformed into E. coli Rosetta allowed the production of satisfactory peptide quantities. When these sequences were integrated into the T7Select phage genome carrying an N-terminal green fluorescent protein (GFP-) tag to monitor the expression in infected E. coli Rosetta cells, the GFP–apidaecin analogs were produced in reasonable quantities. However, when Api802, Api806 and Api810 sequences were integrated into the T7Select phage genome, expression was below detection limits and an effect on the growth of potentially phage-resistant E. coli Rosetta strains was not observed for Api802 and Api806. In conclusion, we were able to show that apidaecins can be integrated into the T7Select phage genome to induce their expression in host cells, but further research is required to optimize the engineered T7Select phages for higher expression levels of apidaecins to achieve the expected synergistic effects that were visible when the T7Select phages and synthetic Api802 and Api806 were added to E. coli Rosetta cultures.     

Arrangement on the Chromosome of the Known Pre-Early Genes of Bacteriophages T5 and BF23

Genetic crosses between mutants defective in the known pre-early genes of T5 and BF23 and the detection of putative N-terminal fragments have allowed the determination of the order of genes along the initially transferred 8% section of DNA of these phages.     

Effect of Nalidixic Acid on the Growth of Deoxyribonucleic Acid Bacteriophages

The effect of nalidixic acid on the growth of various deoxyribonucleic acid (DNA) bacteriophages has been investigated by one-step growth experiments. The Escherichia coli bacteriophages T5, lambda, T7 and phiR are strongly inhibited by nalidixic acid, whereas T4 and T2 are only partially inhibited. The Bacillus subtilis bacteriophages SP82, SP50, and phi29 are relatively unaffected by nalidixic acid. There is no correlation between those bacteriophages which can grow in the presence of nalidixic acid and the presence of an unusual base in the phage DNA.     

Effect of Zn2+ on the Adsorption of Male-Specific Filamentous Deoxyribonucleic Acid and Isometric Ribonucleic Acid Bacteriophages

By means of both electron microscopy and plaque assay techniques, 10(-3)m Zn(2+) has been shown to reduce the adsorption rate of male-specific filamentous deoxyribonucleic acid bacteriophages to the tips of F pili. In contrast, 10(-3)m Zn(2+) did not affect the adsorption of male-specific ribonucleic acid phages to the sides of F pili.