Proposed Ancestors of Phage Nucleic Acid Packaging Motors (and Cells)

I present a hypothesis that begins with the proposal that abiotic ancestors of phage RNA and DNA packaging systems (and cells) include mobile shells with an internal, molecule-transporting cavity. The foundations of this hypothesis include the conjecture that current nucleic acid packaging systems have imprints from abiotic ancestors. The abiotic shells (1) initially imbibe and later also bind and transport organic molecules, thereby providing a means for producing molecular interactions that are links in the chain of events that produces ancestors to the first molecules that are both information carrying and enzymatically active, and (2) are subsequently scaffolds on which proteins assemble to form ancestors common to both shells of viral capsids and cell membranes. Emergence of cells occurs via aggregation and merger of shells and internal contents. The hypothesis continues by using proposed imprints of abiotic and biotic ancestors to deduce an ancestral thermal ratchet-based DNA packaging motor that subsequently evolves to integrate a DNA packaging ATPase that provides a power stroke

Evolution and the complexity of bacteriophages

BACKGROUND: The genomes of both long-genome (> 200 Kb) bacteriophages and long-genome eukaryotic viruses have cellular gene homologs whose selective advantage is not explained. These homologs add genomic and possibly biochemical complexity. Understanding their significance requires a definition of complexity that is more biochemically oriented than past empirically based definitions. HYPOTHESIS: Initially, I propose two biochemistry-oriented definitions of complexity: either decreased randomness or increased encoded information that does not serve immediate needs. Then, I make the assumption that these two definitions are equivalent. This assumption and recent data lead to the following four-part hypothesis that explains the presence of cellular gene homologs in long bacteriophage genomes and also provides a pathway for complexity increases in prokaryotic cells: (1) Prokaryotes underwent evolutionary increases in biochemical complexity after the eukaryote/prokaryote splits. (2) Some of the complexity increases occurred via multi-step, weak selection that was both protected from strong selection and accelerated by embedding evolving cellular genes in the genomes of bacteriophages and, presumably, also archaeal viruses (first tier selection). (3) The mechanisms for retaining cellular genes in viral genomes evolved under additional, longer-term selection that was stronger (second tier selection). (4) The second tier selection was based on increased access by prokaryotic cells to improved biochemical systems. This access was achieved when DNA transfer moved to prokaryotic cells both the more evolved genes and their more competitive and complex biochemical systems. TESTING THE HYPOTHESIS: I propose testing this hypothesis by controlled evolution in microbial communities to (1) determine the effects of deleting individual cellular gene homologs on the growth and evolution of long genome bacteriophages and hosts, (2) find the environmental conditions that select for the presence of cellular gene homologs, (3) determine which, if any, bacteriophage genes were selected for maintaining the homologs and (4) determine the dynamics of homolog evolution. IMPLICATIONS OF THE HYPOTHESIS: This hypothesis is an explanation of evolutionary leaps in general. If accurate, it will assist both understanding and influencing the evolution of microbes and their communities. Analysis of evolutionary complexity increase for at least prokaryotes should include analysis of genomes of long-genome bacteriophages

A Hypothesis for Bacteriophage DNA Packaging Motors

The hypothesis is presented that bacteriophage DNA packaging motors have a cycle comprised of bind/release thermal ratcheting with release-associated DNA pushing via ATP-dependent protein folding. The proposed protein folding occurs in crystallographically observed peptide segments that project into an axial channel of a protein 12-mer (connector) that serves, together with a coaxial ATPase multimer, as the entry portal. The proposed cycle begins when reverse thermal motion causes the connector’s peptide segments to signal the ATPase multimer to bind both ATP and the DNA molecule, thereby producing a dwell phase recently demonstrated by single-molecule procedures. The connector-associated peptide segments activate by transfer of energy from ATP during the dwell. The proposed function of connector/ATPase symmetry mismatches is to reduce thermal noise-induced signaling errors. After a dwell, ATP is cleaved and the DNA molecule released. The activated peptide segments push the released DNA molecule, thereby producing a burst phase recently shown to consist of four mini-bursts. The constraint of four mini-bursts is met by proposing that each mini-burst occurs via pushing by three of the 12 subunits of the connector. If all four mini-bursts occur, the cycle repeats. If the mini-bursts are not completed, a second cycle is superimposed on the first cycle. The existence of the second cycle is based on data recently obtained with bacteriophage T3. When both cycles stall, energy is diverted to expose the DNA molecule to maturation cleavage

Aggregates of bacteriophage 0305φ8-36 seed future growth

Lytic bacteriophage 0305φ8-36 forms visually observed aggregates during plaque formation. Aggregates intrinsically lower propagation potential. In the present study, the following observations indicate that lost propagation potential is regained with time: (1) Aggregates sometimes concentrate at the edge of clear plaques. (2) A semi-clear ring sometimes forms beyond the plaques. (3) Formation of a ring is completely correlated with the presence of aggregates at the same angular displacement along the plaque edge. To explain this aggregate-derived lowering/raising of propagation potential, the following hypothesis is presented: Aggregation/dissociation of bacteriophage of 0305φ8-36 is a selected phenomenon that evolved to maintain high host finding rate in a trade-off with maintaining high rate of bacteriophage progeny production. This hypothesis explains ringed plaque morphology observed for other bacteriophages and predicts that aggregates will undergo time-dependent change in structure as propagation potential increases. In support, fluorescence microscopy reveals time-dependent change in the distance between resolution-limited particles in aggregates

Comparative genomics of Bacillus thuringiensis phage 0305φ8-36: defining patterns of descent in a novel ancient phage lineage

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Phage Capsids as Gated, Long-Persistence, Uniform Drug Delivery Vehicles

Over the last 25 years, cancer therapies have improved survivorship. Yet, metastatic cancers remain deadly. Therapies are limited by inadequate targeting. Our goal is to develop a new drug delivery vehicle (DDV)-based strategy that improves targeting of drug delivery to solid tumors. We begin with a capsid nanoparticle derived from bacteriophage (phage) T3, a phage that naturally has high persistence in murine blood. This capsid has gating capacity. For rapidly detecting loading in this capsid, here, we describe procedures of native agarose gel electrophoresis, coupled with fluorescence-based detection of loaded molecules. We observe the loading of two fluorescent compounds: the dye, GelStar, and the anticancer drug, bleomycin. The optimal emission filters were found to be orange and green, respectively. The results constitute a first milestone in developing a drug-loaded DDV that does not leak when in blood, but unloads its cargo when in a tumor

Propagating the missing bacteriophages: a large bacteriophage in a new class

The number of successful propagations/isolations of soil-borne bacteriophages is small in comparison to the number of bacteriophages observed by microscopy (great plaque count anomaly). As one resolution of the great plaque count anomaly, we use propagation in ultra-dilute agarose gels to isolate a Bacillus thuringiensis bacteriophage with a large head (95 nm in diameter), tail (486 × 26 nm), corkscrew-like tail fibers (187 × 10 nm) and genome (221 Kb) that cannot be detected by the usual procedures of microbiology. This new bacteriophage, called 0305φ8-36 (first number is month/year of isolation; remaining two numbers identify the host and bacteriophage), has a high dependence of plaque size on the concentration of a supporting agarose gel. Bacteriophage 0305φ8-36 does not propagate in the traditional gels used for bacteriophage plaque formation and also does not produce visible lysis of liquid cultures. Bacteriophage 0305φ8-36 aggregates and, during de novo isolation from the environment, is likely to be invisible to procedures of physical detection that use either filtration or centrifugal pelleting to remove bacteria. Bacteriophage 0305φ8-36 is in a new genomic class, based on genes for both structural components and DNA packaging ATPase. Thus, knowledge of environmental virus diversity is expanded with prospect of greater future expansion

PCR-Directed Formation of Viral Hybridsin Vitro

AbstractWhen constructing viruses that have desired hybrid phenotypes, anticipated difficulties include the nonviability of many, possibly most, of the hybrid genomes that can be constructed by incorporation of DNA fragments. Therefore, many different hybrid genomes may have to be constructed in order to find one that is viable. To perform this combinatorial work in a single experiment, we have used bacteriophage T7-infected cell extracts to transfer DNAin vitro.In an extract, we have incubated T7 DNA, together with DNA obtained by polymerase chain reaction (PCR) amplification of the gene (gene 17) for the tail fiber of the T7-related bacteriophage, T3. Afterin vitropackaging of DNA in the extract, hybrid progeny bacteriophage were detected by probing with a T3-specific oligonucleotide; hybrids are found at a frequency of 0.1%. By determination of the nucleotide sequence of the entire gene 17 of 14 independently isolated hybrids, both right and left ends of the PCR fragment are found to be truncated in all hybrids. For all 14 hybrids, the right end is in the same location; the left end is found at 3 different locations. The nonrandom location of the ends is explained by selection among different inserts for viability; that is, most of the hybrid genomes are nonviable. Some hybrids acquire from T3 the desirable phenotype of nonadherence to agarose gels during agarose gel electrophoresis

Evidence for bacteriophage T7 tail extension during DNA injection

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens