Are giant viruses gene thieves?

Giant virus with a remarkable complement of genes infects marine zooplankton.

The predominant theory about the origin of giant viruses is that they have acquired most of their DNA from horizontal gene transfer with cellular organisms, however little research has actually been done into the genomes of giant viruses.
Fischer et al (2010) look at the DNA and function of CroV, a giant virus which infects the bacterivorous marine flagellate Cafeteria roenbergensis. Only one other giant virus had previously been studied at this detail, and this was a freshwater Mimivirus.
As well as mapping out the genome of CroV the researchers used fluorescent in situ hybridisation to see which genes were expressed during infection which would show which genes were functional.
Although they found that half of CroV’s genes were similar to those previously found in eukaryotes, bacteria, archaea and other giant viruses, this paper shows the importance of studying the genetics of viruses alongside their hosts, as they could not verify that eukaryotic genes found in the virus were from C. roenbergensis due to the lack of knowledge of the host genome.
Fischer et al did find that CroV is far less dependent on host cell components than smaller viruses, and has the ability to make its own tRNA and tRNA modifying enzymes. Most of the genes atypical to viruses were expressed during infection suggesting that these genes were not simply non- functional DNA from horizontal gene transfer with cellular organisms, and this virus undertakes its own processes rather than relying on the host more than other viruses.
CroV also has DNA repair genes thought to be an adaptation to the high radiation surface waters where the host lives. These included DNA repair genes primarily found in bacteria and euryarchaeotes, but thought to have been acquired from another giant virus as it is most similar to those found in the Mimivirus previously studied. Also found were regions of DNA relating to glycoprotein biosynthesis that may be needed to create a protective outer coating to the virus.
Although this paper does not answer the question of whether CroV acquired most of its huge genome from horizontal gene transfer, the impression is given that the author thinks it did not. As much of CroV's DNA is more similar to the Mimivirus previously studied than it's host, the author believes that much of CroV's DNA is viral in origin, existing in an ancestor before the line had contact with eukaryotes.
Even though it does not answer the question directly, this paper may have brought us closer to understanding the origin of viruses and transposons in eukaryotes, as more recent work done on this virus revealed the Mavirus virophage which can only survive in the presence of CroV and showed that the origin of certain types of transposons is most likely viral.
The gaps in this study such as comparisons between the virus and host genomes, the function of repetitive DNA and ubiquitin in viruses, and a more in-depth analysis of the processes occurring during infection, would make interesting further work into this and other giant viruses. Further research on the ecological relationships this giant virus may have with its host and its parasite, and how the population dynamics of one would effect the other two would also be facinating, as this is only the second known example of a virus parasitising another virus.

Fischer M.G., Allen M.J., Wilson W.H., Suttle C.A. (2010) Giant virus with a remarkable complement of genes infects marine zooplankton. Proc. Natl. Acad. Sci. U.S.A. 107(19508)
http://www.pnas.org/content/early/2010/10/15/1007615107

Fischer M.G., Suttle C.A. (2011) A virophage at the origin of large DNA transposons. Science 332(6026):231–234.

Bioluminescence: What is  its role to marine snow? 

Bacteria use a variety of mechanisms to inhabit a large diversity of environments. One of these organisms is the bioluminescent bacteria which have the ability to produce light via quorum sensing. These organisms occur in a large diversity of environments including marine snow and in the photophores of fish. It has long been hypothesised that the bacteria living on the snow glow to mark the presence of a food particle for other organisms in order to get into their guts. This is termed the bait hypothesis. So far, this hypothesis has only been supported by showing that bacteria survive passage through fish guts. Once in the gut of fish, bioluminescent bacteria gain a nutritious environment for growth and a faster moving vector for dispersal.

 The objective of this study was to test the proposed steps of the bait hypothesis:

1.        The visual attraction of zooplankton to bacterial bioluminescence

2.       Promotion of glow in zooplankton ingesting bacteria (using planktonic brine shrimps as a surrogate for zooplankton)

3.        Attraction of fish to glowing prey

4.        Survival of bacteria in the guts of both zooplankton and fish.

Zarubin's experiment was set up in a sea water tank, this was used to examine if the luminescence of Photobacterium leiognathi attracts Decapod, Mysid and Copepod zooplankton. Zarubin et al observed significant changes in the distribution of Decapods and Mysids but not in Copepods. The brine shrimp Artemia salina (the surrogate to test the promotion of glow) became luminescent after swimming in a culture of P. leiognathi. Using long-exposure photographs, the luminescence in the guts of Artemia were clearly visible, with additional glow produced by bacteria attached externally. Similarly, non glowing individual marine Mysids, Anisomysis marisrubri started to glow after contacting P. leiognathi.

Zarubin and co found that glowing A. salina increased risk of predation by the fish Apogon annularis in dark. Almost all glowing Artemia were consumed compared with rare predation of non glowing specimens, the authors stated that this occurred only when the prey drifted by chance directly toward  the predator. Glow was detected in faeces of Artemia that fed on the bacteria which indicated that the luminescent bacteria survived the passage through the guts.

This study provides experimental evidence for some steps of the bait hypothesis. But the author’s did not differentiate between external and internal glow on the surrogates. The authors argue that both sources occur in nature, but cells attached externally are not following the proposed steps of the bait hypothesis. Therefore these results should not be used to answer steps 1 and 2 of the bait hypothesis in my opinoun. Zarubin et al used Artemia as a surrogate for zooplankton prey in this experiment because it is readily eaten by fishes, easy to handle, and lacks evasive behaviour. But I do not agree with this choice of organism because it is a freshwater organism that is being used as a model for an experiment which relates to deep sea ecology and therefore is not an appropriate choice. The authors argue that the luminesce observed in A. marisrubri had the same intensity as Artemia but they have not made it clear why A. marisrubri was not chosen in the first place. Although the experiments with P. leiognathi did not fully simulate in situ conditions I think this study provides a good basis for experimental testing of the bait hypothesis. However, experiments with real marine snow are needed to test this claim explicitly.

 

I chose to review this study due to my interest in bioluminescent bacteria. I also have a large curiosity in  marine snow ecology and how these bacteria are affecting it.

 

http://www.pnas.org/content/109/3/853.full

 

Driving microbes round the bend: Chemotaxis and sodium-ion and proton motors

Many microbial assemblages utilise chemotactic capability to detect nutrients across a gradient in tandem with a ‘run and tumble’ style of moving, whereby the bacteria will typically use a flagellum to move it around it’s medium in straight lines, a ‘run’. It will periodically ‘tumble’ by reversing the direction of the flagellum which will change its direction. When a microbe senses it is following a nutrient gradient it will perform fewer ‘tumbles’.

Within the marine environment, however, nutrients are generally patchy and being able to chemotactically find them is much more difficult, as a result, microbes have to move in excess of 200µm s^-1. To help with this many marine microbes have modified the usual ‘run and tumble’ mechanism to include a reversal where by the microbe can reverse its direction so that it can move back into a nutrient patch if it otherwise would have drifted away.

The flagella of flagellate bacteria have embedded within their cell wall and cell membrane a rotary molecular motor. There are 2 mechanisms which power these motors, sodium-ion motors and proton motors. They drive the flagella using an electrochemical gradient involving either sodium ions or protons respectively.

The research of the authors of this particular paper focused on how much of a contribution the 2 main motor mechanisms for driving the flagella in marine flagellate microbes made in terms of high speed motility. In order to do this, inhibitors of the mechanisms were introduced to microbes collected from the water column and microbes from recent isolates. This was done for realism and future applicability which allows this study to be applied in a wider context although I would be wary of doing so as comparing a natural sample of microbes to an isolate does not really give a good view of how the motor mechanisms are affected as an isolate sample will not necessarily react in the same way as a natural sample.

The inclusion of an amiloride tolerant, unidentified isolate just to provide a link and comparison to the amiloride literature seemed unnecessary as they had already identified that amiloride was not being used in preference of the sodium-ion uncoupler monensin, mainly on the grounds that the monensin was less toxic to the cells and would work at a thousandth the concentration that amiloride would. This inclusion would only really serve to show that monensin was less toxic than amiloride which had already been identified beforehand. With the isolate being tolerant to amiloride anyway i’m not entirely certain what the authors were trying to accomplish by it. However, later on in the paper it was mentioned that amiloride had indeed been used to test whether or not the sodium-ions present were responsible for the motility of the community.

Although this paper does have very good applications in the research of chemotactic ability in flagellate bacteria, and in showing that many bacteria can maintain the speeds required for chemotaxis within the marine environment by use of 2 particular motors, I feel that this paper should not have drawn inferences or conclusion from inconclusive data concerning the toxicity of the uncouplers for the sodium-ion motors and its effects.

Overall this paper has highlighted some interesting areas for future research in areas such as microbial physiology and the interactions it has with the marine environment and exactly how marine microbe assemblages manage to maintain the high speeds they do within the viscous media that they inhabit.

Mitchell J.G., Barbara G.M., 1999, High speed marine bacteria use sodium-ion and proton driven motors, Aquatic Microbial Ecology, 18, 227-233

SAR86, what’cha metabolising there?

SAR86 is a ubiquitous clade of 16S rRNA which is highly resistant to cultivation in the lab. In previous studies the genomes of some sub-classes have been shown to include genes for rhodopsin, allowing the bacteria to use light to supplement ATP production. However no work has been done on the metabolic capabilities of the clade.

SAR86 genomes were sequenced by combination of flowcytometry and PCR, and then gene fragments from across the clade were compared to genetic libraries to investigate their metabolism. This revealed four main assemblies of genes for the clade, named A, B, C and D. The genomes of A and B were 90% complete, whereas the genomes for C and D were roughly 50% complete. Comparing gene segments to the GOS metagenomic library (sampled from around the world) revealed ecotypes for each group; A segments were found in the open ocean, B segments were found in warm coastal waters, gene fragments from both C and D were found in the colder coastal sites. This is similar to what Colin was teaching, a small streamlined genome reducing the overall maintenance of the genes, but reducing the adaptability of the bacteria leaving a narrow environment in which it can live.

Dupont et al. (2012) found SAR86 to be free living aerobic heterotrophs with a buffering capacity of phototrophic ATP (via proteorhodopsin). All SAR86 were found to be largely auxotrophic in regards to vitamins and some animo acids (except from sub-class B having a Vitamin B1 biosynthesis pathway). However the authors point out that this could be due to either; the use of alternative biosynthetic routes yet undiscovered or the genes coding for the proteins were on fragments lost in analysis.

Two clades which recruited the largest portions of GOS were SAR86 and SAR11 which are often found in the same environment, so the authors made comparisons between the two most abundant bacterial clades. SAR11 uses protein compounds as primary energy source, whereas SAR86 utilises fats and carbohydrates as primary energy source. Both SAR11 and SAR86 contained sulphur reducing metabolic pathways, but SAR86 has a putative transporter for glutathione and ϒ-glutamyl transferases (allows the break down of glutathione into cysteine) which means SAR86 has a relatively larger pool of organic sulphur to metabolise than SAR11.

To conclude Dupont et al. (2012) draws attention to the relatively small overlap between the niches of both SAR11 and SAR86, and ties the abundance of both Clades to the ratios of protein, carbohydrate and lipids found within the sea.

Genomic insights to SAR86, an abundant and uncultivated marine bacterial lineage

Coral-mucus Vibrio Integrons are Evolutionary Hotspots

Coral-mucus-associated Vibrio integrons in the Great Barrier Reef: genomic hotspots for environmental adaptation

Aside from the well-known symbiotic associations between corals and dinoflagellate zooxanthellae (corals being largely dependent on zooxanthellae for certain nutrients), the role of prokaryotes in the coral microbiodome has been the target of recent study. The authors of this study (Boucher et al, 2011) focus on Vibrionaceae, a family of proteobacteria, which they obtained from the mucus of the scleractinian coral Pocillopora damicornis from the Great Barrier Reef and investigate their associations within the coral microbiodome.

Some corals exhibit an apparent immunity to pathogens, although this is not the same adaptive immunity observed in vertebrates, and the hypothesis aiming to explain this immunity is known as the coral probiotic hypothesis. The authors make use of the ideas behind this hypothesis to explore the relationship between Pocillopora damicornis and its associated Vibrios.

The coral probiotic hypothesis suggests that the prokaryotic microbiome provides pathogen resistance (notably to pathogens that may cause coral bleaching) to the host coral and that this immunity insinuates a dynamic pattern of antimicrobial production by non-pathogenic (commensal) bacteria, enabled by horizontal gene transfer.

The genetic element behind this immunity and its transfer (and also facilitating antimicrobial resistance among pathogens), known as the integron, is a system consisting of an integrase gene (intI) and an associated integration site (attI), where an integrase protein (IntI) catalyzes the insertion and removal of gene cassettes. Gene cassettes typically consist of a single gene and recombination site (attC) and there may be up to 100 cassettes per integron array.

Integrons are vital in the spread of both antimicrobials and antimicrobial resistance among commensal bacteria and pathogenic bacteria. Their importance is evidenced by a rise in proportion of Vibrio 16s rRNA genes sequenced by both commensal and pathogenic bacteria during coral bleaching events.

The authors cultivated coral mucus samples and amplified, cloned and sequenced the genes present in the samples using PCR. They then acquired datasets from which to draw comparisons via a variety of methods including screening the colonies for IntI, constructing fosmid libraries, sequencing Vibrio housekeeping genes, performing a taxonomic assignment of Vibrio cultivars by recA phylogeny and more as detailed in the article.

            Their main findings were that a diverse variety of Vibrio species were contained within the mucus of P. damicornis and that the mucus Vibrio-cassette arrays were found to be highly dynamic (in that around 90% of their integron associated gene cassettes were being actively shared, leaving around 10% at most in common between cultivars). This meant that mucus Vibrio-cassette arrays could evolve more rapidly in comparison to chromosomal genes, and the high mobility of genes allows the integrons of mucus Vibrio to be viewed as strong evolutionary hotspots in genomes. Comparisons to free living Vibrios exemplified this observed high gene mobility. A direct link between the Vibrio cultivars, Vibrio coral pathogens and human pathogens was demonstrated by the exchange of a subset of integron associated gene cassettes (associated with antimicrobial resistance), exemplifying the extensive scale of cassette sharing between microbial niches. This link may be useful in further understanding how resistance antimicrobial and antibacterial drugs spreads in a medical context.

            The diversity of cassette genes discovered in the coral mucus may show a cooperative sharing of resources and a mutually beneficial association within the coral microbiodome. This diversity and association allows quick adaptation in response to threats from pathogens that may be viewed as immunity as mentioned earlier. While beneficial to the coral microbiodome, this rapid adaptation to threats does act as a selective pressure to pathogens, facilitating an evolutionary arms race on both sides.

Boucher, Y. et al., 2011. Coral-mucus-associated Vibrio integrons in the Great Barrier Reef: genomic hotspots for environmental adaptation. ISME Journal (2011) 5, pp.962–972. Available at: http://www.nature.com/ismej/journal/v5/n6/full/ismej2010193a.html

Viruses have proteorhodopsin genes too!

Genomic techniques can reveal information on the evolution of proteorhodopsin

As Colin explained in our lecture this week, viral genomes can encode proteins which are not directly part of virus reproduction.  These extra proteins can modify functional systems of the infected cell to somehow aid, or boost, virus reproduction; a well-documented example of this being cyanophage which contain genes for photosynthesis. Viruses are thought to get these extra genes via horizontal gene transfer.

Yutin & Koonin (2012) have very recently found evidence that giant marine viruses have genes for proteorhodopsin, another light-dependant system with two different functions. The first being a light-driven proton pump which generates ATP, the second being a light-sensitive signal receptor potentially involved in phototaxis. It is useful to note at this point that it was only in the last decade that proteorhodopsins were discovered to be widespread in a diverse range of marine microbes and recognised as an important metabolic process in the oceans.

The authors analysed alignment of conserved sequence blocks in the rhodopsin super family (across viral, bacterial, archaeal and eukaryotic forms of the protein) and found that the viral sequence is highly conserved; however, it does not include the conservation of a proton donor. Therefore they conclude that without a proton donor, the function of the protein in the viral infected host is likely to be for sensory purposes, such as phototaxis, as opposed to being used as a light-driven proton pump. The alignment data then was used to construct a phylogenetic tree based on sequence similarity, authors were able to visualise distinct clades which infer that the giant virus acquired proteorhopsin from bacteria, or more likely eukaryotes, via horizontal gene transfer.

The authors quite rightly conclude that viral proteorhodopsins, regardless of their speculated function (signalling or proton pump), could be major players in virus-host ecology in the ocean. Looking at the bigger picture, maybe what these findings could potentially tell us about the evolution and conservation of proteorhodopsins is more interesting… It is clear that rhodopsins are an important protein as they are so highly conserved across different lineages, from the human eye, to bacterial proton pumps and now also found in viruses. Whilst it is likely that this protein evolved independently in both the eukaryotes and bacteria (and then diverged via horizontal gene transfer), more comparative and experimental work is needed in order to follow the evolution of this system; questions over its independent origins (or lack of) remain to be answered.

Yutin, N., & Koonin, E. V. (2012). Proteorhodopsin genes in giant viruses. Biology direct, 7(1), 34.

N.B. this paper has only just been accepted and is still "in press", only a provisional copy is available.

http://www.biology-direct.com/content/7/1/34/abstract

 

 

The Aftermath of Iron Fertilization from a Microbial Perspective

The idea of being able to decrease atmospheric CO₂ by boosting phytoplankton seemed promising when the idea of iron fertilization first emerged in the 1990s. Several in situ experiments have shown since then that iron is the limiting factor for phytoplankton growth in high nutrient, low chlorophyll regions (HNLC) and that the addition of iron results in vast plankton blooms. However, a concomitant controversy on the efficiency as carbon sink and on the ecological consequences was unavoidable. 

Thiele et al. (2012) focused on the very central part of the biological pump, namely the microbes, and monitored the response of microbial communities to iron fertilization, by looking especially for the characteristic succession patterns observed during natural plankton blooms. It is unquestionable that this knowledge is essential to understand the fate of fixed carbon following iron fertilization.

Water samples from different depths, at time intervals from 4-5 days were taken using Niskin bottles from inside and outside the fertilization area during the LOHAFEX experiment in the Southern Atlantic Ocean. Thymidine and leucine incorporation rates, into DNA or proteins respectively, were used to assess microbial productivity, whereas CARD-FISH (remember that is the enzyme catalysed version of fluorescence in situ hybridization) allowed to quantify and identify community members, using both general and clade-specific probes. 

The iron addition caused a phytoplankton bloom stretching over 300 km² mainly composed of Prymnesiophytes (containing the maybe better known group coccolithophorids) and not of diatoms, because of silicate depleted waters.  The data analysis showed a significant increase in total microbial cell number inside the fertilization patch, along with significant increases in thymidine and leucine uptakes. Moreover the CARD-FISH revealed that the Bacteria, rather than the Crenarchaea, were responsible for the significant increase in cell numbers within the fertilized patch. In fact, the SAR11 clade accounted for 50 % of total cell counts and increased significantly at day 18, then remained stable. Also Roseobacter and Bacteroidetes were significantly more abundant within the fertilized area than outside. However, no changes were observed for Gammaproteobacteria. 

Three different community richness indices were applied: whereas Chao-1 values decreased till day 9 before increasing till the end of the experiment, this trend was not reflected by Shannon and Simpson indices. 

So far, so good.  But then I read their discussion, where after having listed innumerous results of significant increases, the authors conclude “total cell numbers of bacterioplankton and of the major clades are rather constant”. What does “rather constant” even mean, relative to what!? Moreover, they state that their results are concordant with similar studies showing that iron fertilization is not followed by a change in microbial communities. If they had made reference to the usual scale of community shifts during other plankton blooms, the reader would maybe be able to come to the same conclusion. Instead, the three diversity indices are not even mentioned again in the conclusion, nor a possible explanation for the observed trend in the Chao-1 values.  The increase in cell numbers might have been small, but it was statistically significant, still no biological explanations are proposed. Instead the authors relate the apparently constant numbers of cells to flagellate grazing and top-down control, an idea that had emerged from previous iron fertilization experiments. 

By no means am I saying that the results and the conclusion of this study are wrong, but I think the paper is lacking some essential biological information to be able to come to the same conclusion, and too many assumptions were made (suddenly an hypothesised pre-experiment plankton bloom makes its appearance in the discussion to explain inconvenient results) . In my opinion, the authors should have made an advance and tested the hypothesis of a negative correlation between bacterial abundance and abundance of heterotrophic nanoflagellates “hinted” by previous studies, instead of basing their results on the same assumptions as their colleagues.

Thiele, S., Fuchs, B., Ramaiah, N., Amann, R., 2012. Microbial community response during the iron fertilization experiment LOHAFEX. Applied and environmental microbiology, (October). Available at: http://www.ncbi.nlm.nih.gov/pubmed/23064339