Virion Location of Most Phage Depolymerases

Stephen T. Abedon

Department of Microbiology – The Ohio State University

phage.org – phage-therapy.org – biologyaspoetry.org


 

Here is a something worth knowing about, from Pires, D.P., H. Oliveira, L.D. Melo, S. Sillankorva, and J. Azeredo. 2016. Bacteriophage-encoded depolymerases: their diversity and biotechnological applications.  Appl. Microbiol. Biotechnol. 100:2141-2151. [PubMed], (calls to figure and table excluded from quote):

Based on our search, the huge majority of phage depolymerases (126 proteins) are encoded in the same open reading frame of phage structural proteins (mostly on tail fibers, base plates, but sometimes also in the neck) or in close proximity to those genes, and were thus considered as structural proteins. Twenty other depolymerases found in this work might be soluble proteins since they are distant from any structural gene.

Depolymerases that are only soluble, that is, not virion attached, presumably are only useful in the immediate vicinity of phage-lysed bacteria, e.g., towards phage burrowing more deeply into biofilms. This perhaps means that phages don’t need depolymerases to initially infect biofilm bacteria (see here for that argument). Depolymerases that are associated with virions, by contrast, presumably are useful as well upon initial phage encounter with a biofilm bacterium.

That the majority of depolymerases are may not be soluble, but instead appear to be associated with virions, is suggestive that depolymerases are employed for the sake of initial encounter between virions and biofilm bacteteria. But this then begs the question of why more phages don’t encoded depolymerases?

Is it that we have trouble recognizing them in sequence data? Is it that bacteria are just too diverse in terms of extracellular polymers produced? (In addition to limiting utility, the latter may also interfere with our ability to detect depolymerase phenotypes during phage growth as plaques.) Is it because for the most part phages can infect biofilm bacteria sufficiently even without depolymerases? Or are there unexplored trade-offs associated with depolymerase encoding, perhaps especially when they are present as structural components of phage virions?

In the three previous paragraphs I am drawing on a tiny bit of past thought as to the role of depolymerases in phage interaction with biofilms, as can be found in my 2011 book, Bacteriophages and Biofilms. In particular, from p. 23 (of the revised pagination version, or p. 27 of the original… don’t ask…):

Ecologically, EPS depolymerases improve phage movement that occurs either adjacent to or distant from a phage’s parental infection. If distant, then movement towards bacteria will be enhanced by physical linkage between virions and depolymerases. Alternatively, for more localized movement, then soluble depolymerases may suffice, such as for phage dissemination out of biofilms [2004]. Scholl et al. [2005] thus found that efficiency of plating (EOP) was low for phages encoding a soluble EPS depolymerase when infecting a K1 capsule-producing strain and that an isogenic phage not encoding the depolymerase is “unable to form plaques on lawns of this strain” (p. 4872). This result is suggestive that though initiation of plaques occurred with low efficiency, once those infections commenced then subsequent EPS depolymerization presumably facilitated phage migration towards adjacent bacteria to complete plaque formation. In circumstances where enzymes may not be directly supplied, it should thus be advantageous for those enzymes to be carried by virion particles, if only to increase the efficiency of initial infection. That is, it should be advantageous to phages for enzymes to be present at the point of phage adsorption, by being virion attached, rather than present only immediately following the lyses of phage-infected bacteria [I then illustrate this argument with a figure…].

 

 

 

Attacking Biofilms: Another Quote, Plus Some Discussion

Stephen T. Abedon

Department of Microbiology – The Ohio State University

phage.org – phage-therapy.org – biologyaspoetry.org


 

This quote is from Lee Watkins and J. W. Costerton (1984). Growth and biocide resistance of bacterial biofilms in industrial systems. Chemical Times and Trends (October):35-40.

The article has nothing to do with viruses. The quote is interesting, however, since it speaks to the question of how exactly to employ viruses in the biocontrol of microorganisms, specifically in the biocontrol and indeed elimination of biofilms from surfaces (quotation marks in the original):

lt is important to be able to answer that old question “Shall we slug with a biocide, shall we continuously treat with a biocide or shall we soak with a biocide—what is the best deal for the situation?”

To me these three alternatives are distinguishable in terms of how we might think about treatment of bacteria or biofilms with phages, that is, phage-mediated biocontrol of bacteria, or phage therapy.

The first alternative I interpret as the application of large amounts of biocide over short periods, perhaps in a single dose, i.e., slugging, or what we might describe as passive treatment in the case of phage therapy. Keep in mind that passive treatment should mean that for every bacterium targeted not only should at least 10 phages be added but at least 10 phages should be adsorbing, per adsorbable bacterium.

Continuous application, to me, would imply the application of lower but still minimally effective concentrations of biocide over longer periods. Continuous application represents an extreme of multiple dosing, i.e., where the time gap between applications is reduced to zero. Key here is that something other than overwhelming amounts of biocide is being applied, what many (unfortunately) would describe as something other than high multiplicities of infection (MOI) in the case of phage application. One can view such continuous application a preventive, or prophylactic.

Lastly there is soaking, which could also be viewed as continuous application, though this is an application that takes place over a relatively short period, i.e., days or weeks rather than months or years. This would be equivalent to the application of phages by soaking bandages, soaking various absorbent material (one sees mention of “tampons” in various places in the phage therapy literature, though it’s important to realize that the word has a medical definition), or instead via the application of, e.g., Phage Bioderm (for example, as discussed here).

What’s missing, of course, are any assumptions that the biocide will replicate in situ, i.e., so-called active treatment, which is typically considered to be a hallmark of phage-mediated biocontrol/phage therapy. That absence, though, is not unexpected given that this is from a discussion of chemical and physical anti-biofilm biocides rather than of phages.

Still, it once again is nice to see that there really is little that is new under the sun. When dealing with bacterial infections, particularly chronic ones which are associated with biofilms, it can be important to keep in mind these ideas:

  1. We can hit them very hard (literally overkill) over short periods,
  2. We can hit them less hard (minimally adequate biocide concentrations) but over long periods, perhaps particularly towards prevention, or
  3. We can soak the infections over intermediate periods, presumably with periodic re-invigoration of dosing, using antibacterial levels which, also presumably, are somewhat in excess of what might be viewed as minimally adequate.

Any other approach, unless backed by hard data, should be considered to represent mostly wishful thinking.

Some additional reading:

Abedon, S.T. 2016. Bacteriophage exploitation of bacterial biofilms: phage preference for less mature targets?  FEMS Microbiol. Lett. 363:fnv246. [PubMed]

Abedon, S.T. 2016. Commentary: phage therapy of staphylococcal chronic osteomyelitis in experimental animal model.  Front. Microbiol. 7:1251. [PubMed]

Abedon, S.T. 2016. Phage therapy dosing: The problem(s) with multiplicity of infection (MOI).  Bacteriophage 6:e1220348. [PubMed]

Abedon, S.T. 2014. Bacteriophages as drugs: the pharmacology of phage therapy., p. 69-100. In J. Borysowski, R. Miedzybrodzki, and A. Górski (eds.), Phage Therapy: Current Research and Applications. Caister Academic Press, Norfolk, UK.

Abedon, S. 2011. Phage therapy pharmacology: calculating phage dosing.  Adv. Appl. Microbiol. 77:1-40. [PubMed]

Abedon, S.T., S.J. Kuhl, B.G. Blasdel, and E.M. Kutter. 2011. Phage treatment of human infections.  Bacteriophage 1:66-85. [PubMed]

Abedon, S.T. and C. Thomas-Abedon. 2010. Phage therapy pharmacology.  Curr. Pharm. Biotechnol. 11:28-47. [PubMed]

Bacterial Lawns as Biofilm-Like Environments: A New Old Quotation

Stephen T. Abedon

Department of Microbiology – The Ohio State University

phage.org – phage-therapy.org – biologyaspoetry.org


 

Way back in 2010 we (Abedon and Thomas-Abedon) suggested that the growth of phage plaques within bacterial lawns could serve as mimics of bacteriophage interaction with bacterial biofiolms. In fact, we made a rather extensive argument with six Roman numeraled points: (i) constraint of bacterial movement, (ii) bacterial growth within lawns as microcolonies, (iii) inhibition of phage movement, (iv) plaque-like phage growth within actual biofilms, (v) possible temporary shielding of bacteria within lawn microcolonies from phage attack, and (vi) variation in bacterial physiologies again as found within microcolonies within lawns and as potentially equivalent to bacterial microcolonies within biofilms. We concluded that, “Given these similarities, phage plaques as a facile laboratory model therefore could enrich our understanding of phage-bacterial interrelations as they may occur during the phage therapy of biofilm-producing bacterial infections.”

Indeed, we noted as well that Gallet et al. (2009) described phage formation of plaques also as phage growth within a “biofilm-like environment”.

Here I provide a quote from an earlier publication which serves to further these arguments. From Gilbert and Brown (1995) [Mechanisms of the protection of bacterial biofilms from antibacterial agents, p. 118-130. In J. W. Costerton and H. Lappin-Scott (ed.), Microbial biofilms. Cambridge University Press, Cambridge, UK.], p. 119:

The most simple in vitro method of generating biofilms to study antimicrobial sensitivity is to inoculate the surface of an agar plate to produce a confluent growth. Such cultures, whilst not fully duplicating the in vivo situation, have been suggested to model the close proximity of individual cells to one another and the various gradients found in biofilms. In this respect, colonies grown on agar may bе representative of biofilms at solid-air interfaces.

Of course, one cannot claim that bacteria growing within soft agar overlays are perfect representations of naturally occurring biofilm structures. Nonetheless, as we’ve noted previously, e.g., Abedon and Yin (2009), plaque formation within them is a lot more complex than people otherwise may realize.

Phage Tails as Polymeric Substance Probes: Arguments For and Against

Stephen T. Abedon

Department of Microbiology – The Ohio State University

phage.org – phage-therapy.org – biologyaspoetry.org


 

Recently I suggested that:

Phage tails, to the extent that they display smaller diameters than phage virions as a whole, might contribute to nonenzymatic virion translocation into EPS [Extracellular Polymeric Substance, i.e. as associated with biofilm matrix], perhaps with longer, narrower tails permitting deeper or faster local penetration to biofilm-surface located bacteria.

Perhaps not unexpectedly, I now find that this was not an entirely original thought. From Wilkinson (1958), p. 68:

Physical blocking of the surface receptor. Can the presence of a capsule protect the cell simply because of its physical properties? Presumably an infective phage must be able to inject its DNA through the cytoplasmic membrane and, therefore, the main body of the phage must be at a distance from the cytoplasmic membrane smaller than the length of the phage tail (rarely longer than 150 mµ [meaning 150 nm]). Therefore, any layer outside the cytoplasmic membrane which is greater in thickness than 150 mµ and is impermeable to phages will act as a nonspecific phage inhibitor. It has already been shown that a capsule is by definition greater than 150 mµ in thickness and that
it is probably impermeable to particles of the size of a phage head (about 100 mµ). An additional barrier to the phage might be the high negative charge of the polysaccharide capsular surface. In confirmation of this role, capsulate bacteria have been reported to be generally phage resistant…

In my defense, my suggestion pointed specifically to longer phage tails, and the general thrust of my article was that it is especially less mature aspects of biofilms which may be more vulnerable to phages. Less maturity, in other words, might be associated with less thick biofilm matrix, e.g., as perhaps associated with new growth on biofilm surfaces.

Indeed, Wilkinson discusses further an article, unfortunately which is not in English, suggesting that when polymeric substance material is thinner then successful phage adsorption may be more likely (as continuing directly from the previous quote):

Thus, Kauffmann and Vahlne (82) found that most capsulate strains of E. coli were resistant to phage and that when capsulate strains were attacked, there was a proportionality between the thickness of the capsule and the resistance.

One could speculate therefore that it could be, conversely, that shorter tails would be less able to penetrate thicker “capsulate”.

It should be noted that I am making no claims that phage tails exist solely for the sake of penetrating extracellular polymers towards adsorption of bacteria, though it is entirely possible that such penetration could serve as a benefit of possessing especially “longer, narrower tails”.

Freezing Selects for Phage T7 Deletion Mutations… Not!

Stephen T. Abedon

Department of Microbiology – The Ohio State University

phage.org – phage-therapy.org – biologyaspoetry.org


 

Myth: Freezing phage T7 can select for deletion mutations…

From Clark and Geary (1973), with emphasis added:

ATCC freeze-dries phages for distribution (Clark and Geary, 1969). Davis and Hyman (1971) have reported the existence of at least two authentically different T7 phage strains, T7M (Meselsohn) and T7L (Luria) (ATCC 11303-B7). They also stated that T7L lyophilized stock from ATCC contained a high percentage of deletions in the DNA molecule, and they attributed this to lyophilization selecting for DNA deletions. However, since this publication, ATCC prevailed upon these authors to test the ATCC broth stock of T7L, which had been maintained at 4° C unlyophilized or unfrozen since its deposit in the Collection by Luria in 1952. They reported in a letter to W. A. Clark that the T7L stock before freeze-drying contained the same deletions as the freeze-dried product.

In other words, the evidence is not consistent with freezing selecting for deletion mutations in phage T7…

REFERENCES

Clark, W. A., and D. Geary. 1969. The collection of bacteriophages at the American Type Culture Collection, p. 179-187. In T. Nei (ed.), Freezing and Drying Microorganisms. University Park Press, Baltimore.

Clark, W. A., and D. Geary. 1973. Preservation of bacteriophages by freezing and freeze-drying. Cryobiology 10:351-360.

Davis, R. W., and R. W. Hyman. 1971. A study of evolution: The DNA base sequence homology between coliphages T3 and T7. J. Mol. Biol. 62:287-301.

Cages of phages: improved production of hydrogen by enzymes encapsulated in phage capsids

A guest post by Dr. Paul Hyman,
Ashland University, Ashland, OH, USA
phyman@ashland.edu

Bacteriophage capsids have been studied as frameworks for the development of new materials. In part this is an outgrowth of phage display which allows the precise placement of binding elements on the phage surface. But another approach is to use the native capsids and to nonspecifically attach conductive metals to create wire-like structures or to capture and encapsulate molecules in the capsids as the capsids assemble. In a recent paper in Nature Chemistry, Trevor Douglas’ group at Indiana University and colleagues have shown that the efficiency of an enzyme that reduces protons to form hydrogen gas is more efficient and stable when enclosed in a bacteriophage capsid.

The bacteriophage they used is the Salmonella phage P22.  P22’s capsid is an icosahedron composed of a major coat protein assembled onto a scaffold protein. Coat protein and scaffold protein self-assemble into a procapsid that during a normal infection is then packaged with the phage DNA.  The researchers fused the two subunits of a hydrogenase protein to separate scaffold protein genes.  When expressed together with coat protein, procapsid self-assembled with the heterodimeric hydrogenase protein inside as outlined in this figure.

Hyman blog post

(modified from Jordan 2015)

When they tested for hydrogenase activity, the highest efficiency was found if the scaffold protein/hydrogenase subunits were expressed several hours before the coat protein (line one in the table).  This pre-encapsulation period presumably allowed the two hydrogenase subunits to assemble into the active heterodimer before being constrained by the coat protein.

pH 5 pH 8
P22 encapsulated with sequential expression 6118 nmol H2/mg min 3218 nmol H2/mg min
P22 encapsulated with simultaneous expression 757 nmol H2/mg min
Unencapsulated hydrogenase + scaffold without coat protein 46 nmol H2/mg min 12.6 nmol H2/mg min
Free hydrogenase 12-38 nmol H2/mg min (pH not specified)

Data from Jordan 2015

Additional experiments showed that the encapsulation also partially protected the hydrogenase against trypsin, heat denaturation (60°C for 45 min.) and air exposure.

The reason for the increased enzymatic activity is not entirely clear.  The increased activity and protection results suggest that the enzyme’s quaternary structure is stabilized in some way in the capsid.  It may also be that enzyme efficiency is higher when several hundred copies of the enzyme are in close proximity to each other in some sort of synergistic effect.

Overall, this paper demonstrates another way that phages can be used in non-biological technologies as well as biological.  Independent of the phage aspect, an improved catalyst for production of hydrogen gas could prove quite valuable as alternative fuels, such as hydrogen, are increasingly sought after.

Reference: Paul C. Jordan, Dustin P. Patterson, Kendall N. Saboda, Ethan J. Edwards, Heini M. Miettinen, Gautam Basu, Megan C. Thielges, and Trevor Douglas, “Self-Assembling Biomolecular Catalysts for Hydrogen Production”, Nature Chemistry doi:10.1038/nchem.2416, published on-line December 21, 2015.

The ecology of viruses that infect eukaryotic algae

The world of algal viruses has only gotten more interesting in recent years as it has become increasingly clear just the sheer quantity of mortality they cause and how much that drives global nutrient cycling in ways we’ve been largely blind to.

Chlorovirus

The ecology of viruses that infect eukaryotic algae

Because viruses of eukaryotic algae are incredibly diverse, sweeping generalizations about their ecology are rare. These obligate parasites infect a range of algae and their diversity can be illustrated by considering that isolates range from small particles with ssRNA genomes to much larger particles with 560 kb dsDNA genomes. Molecular research has also provided clues about the extent of their diversity especially considering that genetic signatures of algal viruses in the environment rarely match cultivated viruses. One general concept in algal virus ecology that has emerged is that algal viruses are very host specific and most infect only certain strains of their hosts; with the exception of viruses of brown algae, evidence for interspecies infectivity is lacking. Although some host–virus systems behave with boom-bust oscillations, complex patterns of intraspecies infectivity can lead to host–virus coexistence obfuscating the role of viruses in host population dynamics. Within the framework of population dynamics, host density dependence is an important phenomenon that influences virus abundances in nature. Variable burst sizes of different viruses also influence their abundances and permit speculations about different life strategies, but as exceptions are common in algal virus ecology, life strategy generalizations may not be broadly applicable. Gaps in knowledge of virus seasonality and persistence are beginning to close and investigations of environmental reservoirs and virus resilience may answer questions about virus inter-annual recurrences. Studies of algal mortality have shown that viruses are often important agents of mortality reinforcing notions about their ecological relevance, while observations of the surprising ways viruses interact with their hosts highlight the immaturity of our understanding. Considering that just two decades ago algal viruses were hardly acknowledged, recent progress affords the optimistic perspective that future studies will provide keys to unlocking our understanding of algal virus ecology specifically, and aquatic ecosystems generally.

Data Storage and Standard Parts

Genetic data storage, scaleable cell-cell communication, and still-better gene expression, all thanks to phage!

Cross-talk between Diverse Serine Integrases

Abstract
Phage-encoded serine integrases are large serine recombinases that mediate integrative and excisive site-specific recombination of temperate phage genomes. They are well suited for use in heterologous systems and for synthetic genetic circuits as the attP and attB attachment sites are small (< 50 bp), there are no host factor or DNA supercoiling requirements, and they are strongly directional, doing only excisive recombination in the presence of a recombination directionality factor. Combining different recombinases that function independently and without cross-talk to construct complex synthetic circuits is desirable, and several different serine integrases are available. However, we show here that these functions are not reliably predictable, and we describe a pair of serine integrases encoded by mycobacteriophages Bxz2 and Peaches with unusual and unpredictable specificities. The integrases share only 59% amino acid sequence identity and the attP sites have fewer than 50% shared bases, but they use the same attB site and there is non-reciprocal cross-talk between the two systems. The DNA binding specificities do not result from differences in specific DNA contacts but from the constraints imposed by the configuration of the component half-sites within each of the attachment site DNAs.

New Applications for Phage Integrases

Within the last 25 years, bacteriophage integrases have rapidly risen to prominence as genetic tools for a wide range of applications from basic cloning to genome engineering. Serine integrases such as that from ϕC31 and its relatives have found an especially wide range of applications within diverse micro-organisms right through to multi-cellular eukaryotes. Here, we review the mechanisms of the two major families of integrases, the tyrosine and serine integrases, and the advantages and disadvantages of each type as they are applied in genome engineering and synthetic biology. In particular, we focus on the new areas of metabolic pathway construction and optimization, biocomputing, heterologous expression and multiplexed assembly techniques. Integrases are versatile and efficient tools that can be used in conjunction with the various extant molecular biology tools to streamline the synthetic biology production line.

21st Biennial Evergreen International Phage Meeting!

the-evergreen-state-college

You can register for the 21st Biennial Evergreen International Phage Meeting Aug. 2-7 on the Evergreen web site: www.evergreen.edu/phage.  If you register by April 30, you will qualify for the “Early Registration” rate: $600 for academics, $700 for Corporate Rate, $450 for graduate and undergraduate students and guests.  This covers all meeting costs, including room and board.  (If you choose to stay off campus, it will be $150 less, but still include meals.)  Some registration assistance is potentially available.

You will then have until May 24 to at least pay a $100 nonrefundable deposit, apply for assistance, or tell us that you are waiting for a visa.  If you register after May 1, the rates will be $100 more in each category.  Further meeting information is on the web site and will be regularly updated there.