Bacteriophage-based synthetic biology for the study of infectious diseases

An interesting new short paper out of Tim Lu’s synthetic biology lab showcasing what bacteriophage can bring to the table,

Bacteriophage-based synthetic biology for the study of infectious diseases

Robert J Citorik, Mark Mimee, and Timothy K Lu Published 2014 in Curr. Opin Microbiol. DOI: 10.1016/j.mib.2014.05.022

Since their discovery, bacteriophages have contributed enormously to our understanding of molecular biology as model systems. Furthermore, bacteriophages have provided many tools that have advanced the fields of genetic engineering and synthetic biology. Here, we discuss bacteriophage-based technologies and their application to the study of infectious diseases. New strategies for engineering genomes have the potential to accelerate the design of novel phages as therapies, diagnostics, and tools. Though almost a century has elapsed since their discovery, bacteriophages continue to have a major impact on modern biological sciences, especially with the growth of multidrug-resistant bacteria and interest in the microbiome.

•Multidrug-resistant infections have sparked a renewed interest in bacteriophages.
•Synthetic biology has enabled technologies for next-generation phage engineering.
•Engineered phages and derived parts constitute a new antimicrobial paradigm.
•Bacteriophage-based reporters permit detection of specific pathogens.
•Components from phage form a core set of parts in the synthetic biology toolbox.

Bacteriophage - Synthetic Biology



A new study led by researchers at San Diego State University has found that more than half the world’s population is host to a newly described virus, named crAssphage, which infects one of the most
common gut bacterial species, Bacteroides. This bacterium is thought to be connected with obesity, diabetes and other gut-related diseases.

Big fleas have little fleas, Upon their backs to bite ’em, And little fleas have lesser fleas, and so, ad infinitum.

One of the many problems farmers of various kinds of legumes need to deal with is the pea aphid, which reproduce incredibly fast and live by sucking the sap out of the plants. However, while they are terrifying parasites of legumes, they have their own yet more horrific parasites, a parasitoid wasp. Below is a really nice close up picture of one doing its thing, here is a video of the act, and here is a brain meltingly horrific video of a dissection of the mummified aftermath 8 days later. Essentially, these wasps deposit their eggs in a pea aphid and the growing larva feeds on it, developing there for about a week, and then consuming the host from the inside out like a Xenomorph. When it’s done, the wasp larva dries the aphid’s cuticle into a papery brittle shell and an adult wasp emerges from the aphid mummy. Legume farmers love these wasps as much as they despise the aphids that destroy their crops, however, when farmers noticed that the wasps didn’t work as effectively on all of aphids infestations, Nancy Moran’s group at the University of Texas in Austin went to work figuring out why. It turns out that all aphids have a primary bacterial endosymbiont living inside their cells, in addition to and much like a mitochondria, and that many have some combination of five other known secondary endosymbionts. Interestingly, two of those other five, Hamiltonella defensa and Serratia symbiotica have been shown to confer varying levels of resistance to the parasitoid wasp, allowing the aphid to survive infection. However, it turns out that there is yet one more layer to this story,

The relationship these endosymbionts have with the aphid, as well as the primary endosymbiont, is hard to classify as they confer a fitness cost in the absence of the wasp but a significant fitness boost when the wasps are around and trying to infect the aphids. At least for H. defensa, the reason why some strains are fully parasitic and provide no protection against the wasps while others are at least plausibly commensal and do provide protection, is a bacterial virus that infects the endosymbiont, even while it is inside the eukaryotic aphid cell. To understand why it will require a bit of knowledge of how some bacteriophages work. Most bacterial viruses, also known as bacteriophages, have a clear dividing line between two strategies. The simplest and most virulent phages will always immediately shut down their host’s metabolism upon infection and replace it with their own. Within a short period of time, generally between 20 and 80 minutes, the phage will have used the host cell to replicate its genome, build new viral particles, packed those particles with the genome and lysed the cell; setting loose 30-3000 new inert infectious particles. These are known as obligately lytic phages. Most phages however, use a mix of this strategy and another one known as lysogeny. These temperate phages will, at the beginning, decide to either virulently infect, producing particles at the total expense of the host, or hide in the host’s genome and inactivate all of its many host lethal genes. Generally it does this by expressing a transcriptional repressor that prevents expression of everything but the repressor, which incidentally protects the host from subsequent infection by related phages. However, some temperate phages will allow for expression of a genomic cassette that will perform some function of benefit to their host – they might as well since they are completely dependent on their host’s wellbeing while in this stage of their life cycle.
It turns out that there is a temperate bacteriophage called APSE, which is common in H. defensa populations in the aphids, that encodes for a cassette of genes that causes H. defensa to attack the wasp larvae with vicious toxins while the phage hides in the genome of its bacterial host. This makes for a really fascinatingly complex system of interdependencies for each of the agents involved. The phage, the bacterial symbiont, and the aphid are all each united in their interdependent need to combat the wasp that kills all three when it succeeds. However, at the same time, both the phage and the bacteria are dependent on the wasp to apply pressure on the aphid to keep them around – otherwise the aphid would cure itself of both creatures that would then be free-loading. Additionally, the wasp the bacteria, and the phage are all completely dependent on the aphid’s sap sucking ability to sustain them, and the aphid is totally dependent on the farmer to continue growing legumes in massive vulnerable monocultures. Furthermore the farmer and the legumes are dependent on the wasp to combat the aphid and largely helpless against the bacteria and the phage. At the same time, despite all of the interlocking incentives toward cooperation, there are also incentives towards each of these agents cheating each other. The farmer has an incentive to ‘cheat’ and save money by neglecting to buy aphid mummies every so often, because they still benefit from the fitness cost caused by the aphid not rejecting the bacteria or the bacteria rejecting the phage. Similarly, the aphid has an incentive to cheat both the bacteria and the phage to cure itself of them and bet on the farmer not buying aphid mummies full of wasps that year. The bacteria also has an incentive to cheat the aphid by curing itself of the phage, and also bet on the farmer not buying mummies that year itself.
What I love most about this story is that complex series of interdependent yet competing evolutionary interests, which forms as an emergent property of the Siphonaptera – this blog’s namesake,

Big fleas have little fleas, Upon their backs to bite ’em, And little fleas have lesser fleas, and so, ad infinitum. And the great fleas, themselves, in turn Have greater fleas to go on; While these again have greater still, And greater still, and so on.

It is much like another wonderful paper, where a woman’s eye is a big flea bitten the smaller flea of an Acanthamoeba polyphaga parasite,  which is in turn bitten by its Mimiviridae (Lentille virus), which is bitten by its virophage (Sputnik 2), which is itself in a sense bitten by its mobile genetic elements. We have a situation where the farmer is a big flea bitten by their legume, which is bitten by its aphid, which is then bitten by both its parasitoid wasp and its secondary endosymbiont, which is in turn then bitten by its temperate bacteriophage.

This post is deeply indebted to one made on Moselio Schaechter’s excellent blog Small Things Considered, which is slightly more technical and no doubt more clearly written.

Bacteriophages encode factors required for protection in a symbiotic mutualism

Oliver KM, Degnan PH, et al. Published 2009 in Science doi: 10.1126/science.1174463  [REQUIRES FREE REGISTRATION]

Bacteriophages are known to carry key virulence factors for pathogenic bacteria, but their roles in symbiotic bacteria are less well understood. The heritable symbiont Hamiltonella defensa protects the aphid Acyrthosiphon pisum from attack by the parasitoid Aphidius ervi by killing developing wasp larvae. In a controlled genetic background, we show that a toxin-encoding bacteriophage is required to produce the protective phenotype. Phage loss occurs repeatedly in laboratory-held H. defensa–infected aphid clonal lines, resulting in increased susceptibility to parasitism in each instance. Our results show that these mobile genetic elements can endow a bacterial symbiont with benefits that extend to the animal host. Thus, phages vector ecologically important traits, such as defense against parasitoids, within and among symbiont and animal host lineages.

The players in a mutualistic symbiosis: insects, bacteria, viruses, and virulence genes.

Moran NA, Degnan PH, et al. Published 2005 in PNAS USA, doi:10.1073/pnas.0507029102

Aphids maintain mutualistic symbioses involving consortia of coinherited organisms. All possess a primary endosymbiont, Buchnera, which compensates for dietary deficiencies; many also contain secondary symbionts, such as Hamiltonella defensa, which confers defense against natural enemies. Genome sequences of uncultivable secondary symbionts have been refractory to analysis due to the difficulties of isolating adequate DNA samples. By amplifying DNA from hemolymph of infected pea aphids, we obtained a set of genomic sequences of H. defensa and an associated bacteriophage. H. defensa harbors two type III secretion systems, related to those that mediate host cell entry by enteric pathogens. The phage, called APSE-2, is a close relative of the previously sequenced APSE-1 but contains intact homologs of the gene encoding cytolethal distending toxin (cdtB), which interrupts the eukaryotic cell cycle and which is known from a variety of mammalian pathogens. The cdtB homolog is highly expressed, and its genomic position corresponds to that of a homolog of stx (encoding Shiga-toxin) within APSE-1. APSE-2 genomes were consistently abundant in infected pea aphids, and related phages were found in all tested isolates of H. defensa, from numerous insect species. Based on their ubiquity and abundance, these phages appear to be an obligate component of the H. defensa life cycle. We propose that, in these mutualistic symbionts, phage-borne toxin genes provide defense to the aphid host and are a basis for the observed protection against eukaryotic parasites.

It Sometimes Rains Nonsense After Hurricanes

Today I want to talk about a paper that looks incredibly cool in a whole bunch of different ways in the abstract, introduction, and discussion but for whom much of that awesomeness falls apart under closer inspection of the results, methods, and context.  In it, the authors report their results having flown in NASA’s venerable old DC-8 across the US and down the west coast as well as through a couple of hurricanes with a filter designed to capture bacteria sized particles hanging out the side.  They then took the filter and analyzed it with fluorescent dyes and microscopes as well as genomically to see what was there.  In their paper they appear to arrive at five different major conclusions: that viable bacterial cells represented on average around 20% of the total particles in the 0.25- to 1-μm diameter range; that 60 to 100% of the 1.5 × 105 cells m−3 they saw were viable; that bacteria are at least two orders of magnitude more abundant than fungal cells in the troposphere; and that fecal coliforms represent a significant amount of the microbiota of hurricanes after landfall. Additionally, what has made the most splash though, is their speculation that because some of the taxa they determined were present by small subunit rRNA sequencing had been shown to metabolize oxalic acid, a major chemical component of clouds, it was plausible that there was active bacterial metabolism happening in the clouds they analyzed.  Unfortunately, despite the journal it is published in and glowing praise from excellent blogs like Not Exactly Rocket ScienceClimate CentralWiredMetafilter, and The Scientist, the speculation is pretty foolish and each of these conclusions is either inherently false, actively misleading, or very difficult to support with their data.

SnoopyWhat I’d love to do with the Authors’ platform


Here is the paper:

Microbiome of the upper troposphere: Species composition and prevalence, effects of tropical storms, and atmospheric implications

N DeLeon-Rodriguez, TL Lathem, LM Rodriguez-R, et al. Published 2013 in PNAS. doi: 10.1073/pnas.1212089110
The composition and prevalence of microorganisms in the middle-to-upper troposphere (8–15 km altitude) and their role in aerosol-cloud-precipitation interactions represent important, unresolved questions for biological and atmospheric science. In particular, airborne microorganisms above the oceans remain essentially uncharacterized, as most work to date is restricted to samples taken near the Earth’s surface. Here we report on the microbiome of low- and high-altitude air masses sampled onboard the National Aeronautics and Space Administration DC-8 platform during the 2010 Genesis and Rapid Intensification Processes campaign in the Caribbean Sea. The samples were collected in cloudy and cloud-free air masses before, during, and after two major tropical hurricanes, Earl and Karl. Quantitative PCR and microscopy revealed that viable bacterial cells represented on average around 20% of the total particles in the 0.25- to 1-μm diameter range and were at least an order of magnitude more abundant than fungal cells, suggesting that bacteria represent an important and underestimated fraction of micrometer-sized atmospheric aerosols. The samples from the two hurricanes were characterized by significantly different bacterial communities, revealing that hurricanes aerosolize a large amount of new cells. Nonetheless, 17 bacterial taxa, including taxa that are known to use C1–C4 carbon compounds present in the atmosphere, were found in all samples, indicating that these organisms possess traits that allow survival in the troposphere. The findings presented here suggest that the microbiome is a dynamic and underappreciated aspect of the upper troposphere with potentially important impacts on the hydrological cycle, clouds, and climate.

I will start with the most sensational aspect of the paper speculated about by the authors, their assertion that their results indicate it is plausible there is active bacterial life growing in clouds in the troposphere.  While the idea that clouds are themselves life forms, ice seeded by cells and cells fed by oxalic acid attracted by the ice, which the authors go to great lengths to speculate on, is very attractive they neglect to mention the temperature readings taken during the flights anywhere in the paper or even supplementary information, which should have pretty much immediately dismissed all of it.  The troposphere that the authors were sampling is typically between -50 and -70°C, which approaches the kinds of temperatures I use in my lab to keep bacterial cells in immortal suspended animation.   Indeed, even the most extreme psychrophiles don’t grow much below -12°C and even then only really in the presence of a large amount of salt that helps them keep the water they’re in from freezing.  The bacteria that they saw may not have been dead exactly when they were sampled, but they certainly weren’t living, which brings us to their next sexy conclusion.

The authors claim that viable bacterial cells represented on average around 20% of the total particles in the 0.25- to 1-μm diameter range and that 60 to 100% of the 1.5 × 105 cells m−3 they saw were viable. This implies that even if the bacteria involved were not actively living, many of them were at least only mostly dead, in a Princess Bride sense. Unlike ordinary humans, bacteria can generally quite happily be frozen in place and exist suspended indefinitely, coming back to life should they thaw in a favorable situation. To determine just how dead the bacteria they were looking at were, the authors used a set of fluorescent dyes sold by Invitrogen that stains DNA green if it is surrounded by an intact membrane, or red if not, on particles they liberated from their filters.   However, while the promotional materiel for the various kinds of kits they could have used from their description talk a big game about determining viability, that is not something they can do, at least not for these authors.  All that those dyes can measure is whether cells are intact or not, which in some well characterized kinds of systems can be an excellent proxy for whether or not they are viable, but the authors have no idea if it is for them and it is in fact really profoundly unlikely.  Even setting aside the likelihood that a significant portion of what they were staining was not DNA to begin with, the upper atmosphere is an incredibly hostile place that one would expect to leave cells a lot more than just mostly dead, even while leaving them intact.  Ultraviolet radiation from the sun would kink the DNA of anything up there causing the same kinds of damage as a sunburn, while the dry conditions should dessicate all but the hardiest spores.  This is not to say that it isn’t plausible that there truly are viable bacteria up there, but it is to say that the authors do not measure viability in a remotely meaningful way and cannot really contribute to our understanding of how viable exactly those bacteria might be.


Fig. 1. Quantification of bacterial and fungal cells in samples from high altitudes in the atmosphere. Concentration of bacterial (A) and fungal (B) cells based on qPCR analysis of SSU rRNA gene copies in the samples. Note that samples are ordered by the collection time on the x axis except for blank samples, which are shown at the rightmost part of the graphs in light gray. (C) Live/dead microscopy image of two samples from the California coast and transit flights. Green-stained cells represent cells with viable/intact membrane (e.g., cell indicated by left arrow), and red/yellow-stained cells represent cells with a damaged membrane (e.g., cell indicated by right arrow). Credit: (DeLeon-Rodriguez et al., 2013)

The authors also conclude based on the data shown above that bacteria are at least two orders of magnitude more abundant than fungal cells in the troposphere, and that is indeed what their data plausibly shows. This is to say that even though all of the qPCR data they are relying on is very weak having massive amounts of contamination in their control blanks (just look at the scale), they are blind to that contamination having failed to sequence it, they have no replicates to do statistics with, and they get results that are inconsistent with their other data by two orders of magnitude – they still make a decent case that they see less fungal DNA than bacterial DNA in their filters and that that means fewer fungal cells than bacterial cells. They do however also neglect to mention anywhere in the paper that fungi could be reasonably expected to be less abundant in the late summer when they were measuring, particularly in relation to the early Spring or to a lesser extent mid Fall when fungi tend to sporulate.


Fig. 3. Habitat of origin of the SSU rRNA gene sequences recovered in the GRIP samples. Sequences were assigned to a habitat (see key) based on the source of isolation of their best match in the GreenGenes database. The graph represents the relative abundance of each habitat (vertical axis) for each sample (x axis). Numbers on the top denote the fraction of sequences that were assignable to a habitat for each sample. Credit: (DeLeon-Rodriguez et al., 2013)

The last major finding of this paper, that fecal coliforms represent a significant amount of the microbiota of hurricanes after they make landfall is a really cool one, but if the authors can demonstrate that this is the case, they don’t do it convincingly in this paper.  Even setting aside the question of how viable the fecal coliforms they saw were, there is still the very tricky question of the DNA contamination they saw.  While I’m sure we can trust that this wasn’t contamination with lab strains from say the autoclave they used for sterilization, which they speculated might be a source in the paper as that would show up very obviously as having little to no diversity, the amount of coliforms they saw is still well within the levels of contamination they know they have.  This could all plausibly come from say a livestock operation next to the airport they left from and returned to or some other artifact of the post landfall expeditions they made.

Flight trajectories

Figure S1.  Flight trajectory maps.  (A) Flights conducted in the west coast (red) and across the USA (blue).  (B) Flights conducted in the area of the Caribbean Sea and the mid-western Atlantic Ocean. The route of each flight is color-coded (see figure key). The trajectory of Hurricane Earl and Karl are colored-coded based on the intensity of the hurricane at each time point (scale bar). Credit: (DeLeon-Rodriguez et al., 2013)

I think it is a particular shame that this research has been sexed up as a microbiota paper that it is not, or at least is not yet, because it still looks like a very interesting climate and meteorological paper.  They still are able to convincingly show that that intact bacterial cells in the atmosphere, and particularly within hurricanes, are at least within the same order of magnitude as particles from non-biological origins – this is a really cool and, at least as far as I can tell, novel finding outside of dust from the Sahara.    Even if they say silly things about how alive those cells are, have nothing really they could say about how only mostly dead those cells could be, and are limited in what they could say about what kinds of cells they see, that doesn’t mean they couldn’t use stronger techniques in the future.  Really to say the kinds of things they want to say, the authors would absolutely need to use culture dependent methods to look at their filters.  You just cannot have anything meaningful to say about a bacteria’s ability to grow without actually growing some bacteria.  Now, culture dependent methods do have their weaknesses, namely that using them, we only seem to be able to grow about 1% of the bacteria present on Earth, but with the kinds of huge numbers of bacteria the authors are throwing around that shouldn’t scare them.  It would also help them to do something I would find really cool: look for viruses.

If they’re actually serious about the contention that there are critters actually actively alive up there, there would be nothing stopping bacteriophages from infecting them, and showing their presence would go a long way to demonstrating that there is ecology going on in clouds. The idea that hurricanes could spread viable cells through the troposphere would go a long way towards explaining why we routinely do things like find in American belly buttons a bacteria that had previously only been found once in Japanese soil; finding viable (by which I strictly mean culturable) bacteriophages in the troposphere would go a long way towards explaining why we see the same thing with them. Now all I need to do is convince the NSF to let me look!

See also a letter to PNAS and its response in the next issue:

Inadequate methods and questionable conclusions in atmospheric life study (Smith & Griffin, 2013)


Reply to Smith and Griffin: Methods, air flows, and conclusions are robust in the DeLeon-Rodriguez et al. study (DeLeon-Rodriguez et al., 2013)