Cell–gel interactions of in-gel propagating bacteria

Serwer, Philip; Hunter, Barbara; Wright, Elena T.

doi:10.1186/s13104-018-3811-x

Cited by 4 publications

(11 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…During formation of a bacteriophage plaque in the typical 0.5-0.7% agar plaque-supporting gels, bacterial host cells push aside the gel-forming fibers while the cells are elongating and dividing. This hypothesis has been found accurate by electron microscopy of thin sections of 0.6% agarose gel-embedded, propagating, uninfected Gram-positive and Gram-negative cells [26]. Furthermore, the gel-breaking pressure exerted by elongating bacterial cells results in counter pressure on cells.…”

Section: Ramificationsmentioning

confidence: 81%

“…Furthermore, the gel-breaking pressure exerted by elongating bacterial cells results in counter pressure on cells. This counter pressure is in the range of pressures that cause change in state of the bacterial cytoplasm [26], a change that, in turn, might explain an observed [30] altering of phage T3 infection in-gel.…”

Section: Ramificationsmentioning

confidence: 96%

Section: Fundamentalsmentioning

confidence: 99%

“…Our host for phage G has recently been found to be a Lysinibacillus, although the host was originally labeled Bacillus megaterium (Unpublished data of J.A. Thomas, bacterial size-confirmed in [26]). Furthermore, agarose is the least electrically charged sub-fraction of agar; the net charge of agar sub-fractions is negative [27,28].…”

mentioning

confidence: 99%

See 3 more Smart Citations

In-Gel Isolation and Characterization of Large (and Other) Phages

Serwer

Wright

2020

Viruses

Self Cite

View full text Add to dashboard Cite

We review some aspects of the rapid isolation of, screening for and characterization of jumbo phages, i.e., phages that have dsDNA genomes longer than 200 Kb. The first aspect is that, as plaque-supporting gels become more concentrated, jumbo phage plaques become smaller. Dilute agarose gels are better than conventional agar gels for supporting plaques of both jumbo phages and, prospectively, the even larger (>520 Kb genome), not-yet-isolated mega-phages. Second, dilute agarose gels stimulate propagation of at least some jumbo phages. Third, in-plaque techniques exist for screening for both phage aggregation and high-in-magnitude, negative average electrical surface charge density. The latter is possibly correlated with high phage persistence in blood. Fourth, electron microscopy of a thin section of a phage plaque reveals phage type, size and some phage life cycle information. Fifth, in-gel propagation is an effective preparative technique for at least some jumbo phages. Sixth, centrifugation through sucrose density gradients is a relatively non-destructive jumbo phage purification technique. These basics have ramifications in the development of procedures for (1) use of jumbo phages for phage therapy of infectious disease, (2) exploration of genomic diversity and evolution and (3) obtaining accurate metagenomic analyses.Here, we both review and augment data projected to help answer the above questions. We propose that data of this type are critical to efficient isolation of large phages, which include those with genomes longer than 200 Kb (jumbo phages [4][5][6]19]) and even larger phages with genome longer than 520 Kb (mega-phages [20,21]). The importance of these answers is illustrated by the fact that no mega-phage has ever been isolated [20,21], although even larger eukaryotic viruses have been isolated [22]. The existence of mega-phages is surmised from assembly of metagenomic sequences [20,21]. Host Bacteria In-Gel: Values of P E for Agarose Gels FundamentalsThe following data indicate that Gram-negative and Gram-positive phage hosts do not fit in the spaces of the 0.5-0.7% agar gels typically used to form phage plaques. This conclusion is derived, first, from the measurement of P E for agarose gels. This measurement begins by determining vs. sphere radius the minimal agarose gel concentration that excludes a sphere during electrophoresis [23]. These P E values are then used to anchor P E values determined from the sieving (gel-induced retardation) of spheres during gel electrophoresis. The most advanced study of the latter [24] yields the following relationship for underivatized, low-electro-osmosis agarose gels cast in 0.025 M sodium phosphate, pH 7.4, 0.001 M MgCl 2 at 25 • C: P E = 148A −0.87 nm; A is the weight percentage of agarose.A 0.7% agarose gel, if characterized by this relationship, has a P E of 202 nm, not large enough to admit a bacterial cell, such as the host for phage G. Our phage G host is~500 nm in radius and 2000-5000 nm in length, as is Escherichia coli [25], a representative of the ...

show abstract

Section: Ramificationsmentioning

confidence: 81%

Section: Ramificationsmentioning

confidence: 96%

Section: Fundamentalsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

In-Gel Isolation and Characterization of Large (and Other) Phages

Serwer

Wright

2020

Viruses

Self Cite

View full text Add to dashboard Cite

show abstract

“…In-plaque (in-gel) analysis is used because (1) in-gel phage propagation occurs in the wild [14,15] and (2) in-gel propagation results in a gel fiber-induced increase in pressure on the T3 host [16]. The increased pressure may alter both cytoplasm and DNA packaging-generated particles, as discussed below.…”

Section: Introductionmentioning

confidence: 99%

Electron Microscopy of In-Plaque Phage T3 Assembly: Proposed Analogs of Neurodegenerative Disease Triggers

2020

Self Cite

View full text Add to dashboard Cite

Increased knowledge of virus assembly-generated particles is needed for understanding both virus assembly and host responses to virus infection. Here, we use a phage T3 model and perform electron microscopy (EM) of thin sections (EM-TS) of gel-supported T3 plaques formed at 30 °C. After uranyl acetate/lead staining, we observe intracellular black particles, some with a difficult-to-see capsid. Some black particles (called LBPs) are larger than phage particles. The LBP frequency is increased by including proflavine, a DNA packaging inhibitor, in the growth medium and increasing plaque-forming temperature to 37 °C. Acidic phosphotungstate-precipitate (A-PTA) staining causes LBP substitution by black rings (BRs) that have the size and shape expected of hyper-expanded capsid containers for LBP DNA. BRs are less frequent in liquid cultures, suggesting that hyper-expanded capsids evolved primarily for in-gel (e.g., in-biofilm) propagation. BR-specific A-PTA staining and other observations are explained by α-sheet intense structure of the major subunit of hyper-expanded capsids. We hypothesize that herpes virus triggering of neurodegenerative disease occurs via in-gel propagation-promoted (1) generation of α-sheet intense viral capsids and, in response, (2) host production of α-sheet intense, capsid-interactive, innate immunity amyloid protein that becomes toxic. We propose developing viruses that are therapeutic via detoxifying interaction with this innate immunity protein.

show abstract

Additions to Alpha-Sheet Based Hypotheses for the Cause of Alzheimer’s Disease

Serwer¹,

Wright²,

Hunter³

2022

JAD

View full text Add to dashboard Cite

Protein amyloid-β (Aβ) oligomers with β-sheet-like backbone (β-structured) form extracellular amyloid plaques associated with Alzheimer’s disease (AD). However, the relationship to AD is not known. Some investigations suggest that the toxic Aβ component has α-sheet-like backbone (α-structured) subsequently detoxified by intracellular α-to-β conversion before plaque formation. Our objective is to compare this latter hypothesis with observations made by electron microscopy of thin sections of AD-cerebral cortex. We observe irregular, 200–2,000 nm, intracellular, lipofuscin-like inclusions. Some are light-staining and smooth. Others are dark-staining and made granular by fibers that are usually overlapping and are sometimes individually seen. Aspects unusual for lipofuscin include 1) dark and light inclusions interlocking as though previously one inclusion, 2) dark inclusion-contained 2.6 nm thick sub-fibers that are bent as though α-structured, and 3) presence of inclusions in lysosomes and apparent transfer of dark inclusion material to damaged, nearby lysosomal membranes. These data suggest the following additions to α-structure-based hypotheses: 1) Lipofuscin-associated, α-structured protein toxicity to lysosomal membranes is in the chain of AD causation; 2) α-to-β detoxification of α-structured protein occurs in lipofuscin and causes dark-to-light transition that, when incomplete, is the origin of cell-to-cell transmission essential for development of AD.

show abstract

Cell–gel interactions of in-gel propagating bacteria

Cited by 4 publications

References 22 publications

In-Gel Isolation and Characterization of Large (and Other) Phages

In-Gel Isolation and Characterization of Large (and Other) Phages

Electron Microscopy of In-Plaque Phage T3 Assembly: Proposed Analogs of Neurodegenerative Disease Triggers

Additions to Alpha-Sheet Based Hypotheses for the Cause of Alzheimer’s Disease

Contact Info

Product

Resources

About