Scandal! After ingestion of hydrothermal ejaculate prokaryotes degradingly murdered by deep-sea virus!!!

Where did these atrocities take place?

Hydrothermal vents typically arise near hotspots of tectonically active mid-ocean ridges. These release super heated seawater, (as high as 400°c) into the surrounding ocean along with chemicals from the lithos’ and asthenosphere. Little if any light penetrates these depths, thus the primary production of the deep tends toward the chemosynthetic. The ‘vent environment’ is not solely restricted to the area immediately surrounding the vent; the hydrothermal ejaculate, once diluted by about “10,000 fold” cools and reaches neutral buoyancy and is hence swept laterally by currents in the surrounding seawater. In this way particulates and chemicals may spread as far as “2000km” or more.

Studies have found that abundances of chemosynthetic prokaryotes within vent fields to be higher than those found in the surrounding seawater. Viral lysis is a likely source of prokaryote mortality, and has been implicated as an affecter of carbon cycling and prokaryote community structure around the vents. However there is little knowledge on hydrothermal vent viral abundance and distribution. Compared to surface waters, viral numbers are significantly diminished by about “1000-fold”. An exemption of these maybe hydrothermal vents, where high microbial biomass can potentially lead to high lytic production.

This study examined the distribution and abundance of viruses around active vents, in an attempt to ascertain if virus mediated mortality affects the microbial dynamics within the system over a four-year period.

Samples were taken from three active sites, within these were found both viruses and prokaryotes, though viral abundance was generally higher. Results showed higher viral abundances found at active hydrothermal vents than surrounding deep sea, this is indicative that viral production was occurring and that it was a source of microbial mortality.

However within neutrally buoyant plumes, abundance of viruses and prokaryotes was lower than that of samples from active fields but were higher than non-plume samples. Though this decrease was exacerbated for viruses compared to prokaryotes. Additionally it was found that prokaryotic and viral abundances in non-hydrothermal regions were as much as 10-fold higher than in previous studies. The difference may be because this study employed the use of epifluorescent microscopy which was previously not available.

The authors suggest that viral infection may be a greater source of prokaryotic mortality than previously recognised. The results indicate that mortality of prokaryotes mediated by viruses in hydrothermal environments may be significant enough to reduce energy flow to higher trophic levels.

This study bares significance as it presents the first data on viral abundance and distribution from black smokers, additionally providing further data on the abundance of viruses and prokaryotes in hydrothermal-vent plumes, which so far has been limited.  

Alice C. Ortmann, Curtis A. Suttle, High abundances of viruses in a deep-sea hydrothermal vent system indicates viral mediated microbial mortality, Deep Sea Research Part I: Oceanographic Research Papers, Volume 52, Issue 8, August 2005, Pages 1515-1527

Available at: http://www.sciencedirect.com/science/article/pii/S0967063705001032

Lactic acid producing bacteria for use in the exploitation of seaweeds

Shobarani, Halami and Sachindra (2012) set out to determine if fermentation by marine lacticacid producing bacteria (LAB) can be used as a viable method to extract bioactive molecules from seaweeds, in particular Sargassum sp. It has been well documented that marine algae contain many biologically active compounds which pharmalogical and therapeutic uses. While these algae have been exploited extensively current methods used for extraction, while effective, are often expensive and complex. Shobarani et al (2012) examined the effect of bioactive molecule extraction using LAB fermentation of Sargassum sp. , on blood coagulation and the antioxidant capabilities.

The algae samples were collected from the west coast of India. After collection samples were thoroughly washed, dried and milled. Bacterial communities were selected from samples taken from the water column, sediments and seaweeds. Bacterial samples were plated on MRS agar containing Sargassum powder as a substitute for glucose, and bromocresol as indicator of lactic acid production. Using several characteristics LAB were selected and identified using 16S RNA sequencing, resulting in the use of four different bacterial strains for testing. 

To determine the fermentation of the Sargassum powder, a broth was made containing 1% powder. This broth was inoculated at a concentration of 10⁵ CFU ml^-1 and incubated for 18 days. At regular intervals viable cell count, pH, total titratable acidity and presence of sugars was measured. For the anticoagulation assays Shobarani et al (2012) preformed two assays, an APTT assay and PT. Multiple antioxidant assays were preformed to determine factors as reducing potential, metal chelation, DPPH scavenging, nitric oxide scavenging, hydrogen peroxide scavenging, ABTS scavenging and oxygen quenching. 

The results showed that during the fermentation process the viability of the cultures increased up to 12 days of incubation, and further incubation showed a further decrease in cell count. During the incubation period a significant decrease in pH was observed, this occurred together with a steady increase in lactic acid content with an optimum concentration after 12 days. Together with the increase in bacterial cell count an increase in total sugars present in the broth.  

 Two blood coagulation assays were preformed to determine which inhibition pathway would be affected, APTT assay for the intrinsic pathway and PT assay for the extrinsic pathway. For the assays the samples were used that had been incubated for the optimum of 12 days. Results showed that the all samples in the APTT assays showed higher activity compared to the PT assays and control samples, suggesting that the algae extract inhibits the intrinsic coagulation cascade. During the antioxidant assays it was noted that on average antioxidant capabilities were increased compared to control samples. A significant correlation was found between the polyphenol content of the sample and the antioxidant capabilities, with polyphenol content increasing during the fermentation period.

Overall it was suggested that, using a controlled microbial flora, can be used as a viable alternative for other bioactive molecule extraction methods and natural fermentation. As all samples showed an increase in anticoagulation activity and antioxidant capabilities. Shobarani et al. (2012) find that this study provides a basis for the application of well-characterised and controlled LAB cultures in the production of functional foods, and with further study may also be used to develop a cheap and effective method to extract and purify bioactive molecules, and possibly help in determining the functional mechanisms of these molecules for therapeutic uses. 

Shobharani P., Halami, P., M., Sachindra, N., M.. (2012). Potential of marine lactic acid bacteria to ferment Sargassum sp. for enhanced anticoagulant and antioxidant properties . Journal of Applied Microbiology. 114 (1), 96-107.

Phylodynamics and movement of Phycodnaviruses among aquatic environments

Phycodnavirus comprises of six genera that infect a range of eukaryotic algae. Therefore, it has the ability to change the dynamics of phytoplankton community structure and succession in addition to nutrient cycles. Since phytoplankton fixes around half the planet’s carbon dioxide and some algae release DMSP when lysed by the virus, it is thought that Phycodnavirus could also affect atmosphere composition. However, little is known about the evolutionary history, phylogenetics and phylodynamics of this virus, which is what this study aimed to investigate.

The Amazon River was chosen for this study because it is an ancient freshwater environment and thus may function as a reservoir for viruses to transfer to other aquatic environments. Samples were collected in July 2007 from the Solimões, Negro and Cuieiras Rivers and taken to the lab where they were immediately processed. They were filtered, the remaining particulate matter concentrated and then ultracentrifuged. The pellets were resuspended and stored in the dark at 4°c until the DNA was extracted. This involved DNA amplification via PCR, cloning and sequencing, then phylogenetic and phylodynamic analysis. The BayesTraits software was used to estimate the movement of Phycodnavirus between aquatic environments.

In total, 64 and 39 polymerase sequences were found from the Solimões and Cuieiras Rivers respectively, however none were found from the Negro River. This was the first data of Phycodnavirus collected from a tropical river and therefore allowed the first comparisons of this virus between temperate and tropical climates. All of the 104 sequences from the Amazon were aligned to 550 polymerase sequences from other studies and further analysis conducted.

The population size of Phycodnaviruses were estimated by multiplying the effective population by generation time. This was represented on a Bayesian skyline plot, displaying the population dynamics over the past 1.5 million years. This demonstrated that the Phycodnavirus dynamics have fluctuated greatly over time. A large population decrease was observed between 500-300 thousand years before present (KYBP), then an increase in lineages after a bottleneck at 300KYBP, which finally plateaued at 100KYBP.

Since they had access to data from samples from a diverse range of places, phylogenetic associations could be used to investigate the movement of the Phycodnavirus between aquatic environments. This demonstrated significant genetic transfer between rivers and lakes, but not between freshwater and marine ecosystems. Since the virus may not be transferred between all water environments this provides evidence of restricted gene flow, which could affect their evolution and dispersal. Furthermore, they conducted a multi-state character change analysis using BayesTraits, which also proposed restrictions of the virus transmission as well as the colonisation of specific freshwater systems, which could explain why the Phycodnavirus was not found in the Negro River. Other studies have also shown a dramatic decrease in freshwater viral count when seawater was added, however the same was not observed when freshwater was added to seawater. This provides evidence that freshwater viruses may be unable to tolerate changes in salinity, perhaps giving reason for the lack of movement of Phycodnavirus to marine environments.

It must be noted that this study was based on the transmission of the virus between freshwater and marine environments from a limited number of samples. Moreover, the PCR required highly degenerate primers, which may have caused biased sampling, meaning not all Phycodnaviruses were detected. Nonetheless, this would not invalidate the findings concerning the phylodynamics and evidence of restricted transmission of the virus between aquatic environments. Overall, the study was important in demonstrating the long term fluctuations of the Phycodnavirus dynamics and the restriction of gene flow between freshwater and marine environments. Further studies would be valuable in determining whether the restriction in salinity tolerance is a limiting factor in Phycodnavirus survival and therefore a potential reason for the lack of transition between freshwater and marine ecosystems during its evolution.  

Manuela V Gimenes¹, Paolo M de A Zanotto¹, Curtis A Suttle², Hillândia B da Cunha³ and Dolores U Mehnert¹

The ISME Journal (2012) 6, 237–247; doi:10.1038/ismej.2011.93; published online 28 July 2011

http://www.nature.com/ismej/journal/v6/n2/full/ismej201193a.html

Hydrothermal Vent Symbioses and Hydrogen: A Recently Discovered Source of Energy for Marine Chemosynthetic Ecosystems

Free-living, chemosynthetic, hydrothermal vent bacteria are known to utilise many energy sources, including ammonium, ferrous iron, hydrogen and manganese.  However until recently only two energy sources had been shown to fuel primary production in symbiotic bacteria crucial to the metazoans which inhabit hydrothermal vents: reduced sulphur compounds and methane. Yet the energy yield from hydrogen oxidation is much higher than that for the oxidation of methane or sulphur, making it a favourable election donor.  Furthermore, some hydrothermal vents produce fluids with very high hydrogen concentrations, due seawater interaction with mantle-derived rock; these vents also tend to have high methane concentrations and low hydrogen sulphide (H₂S) concentrations, whereas vents in basalt rock are characterised by fluids high in H₂S, and low in hydrogen and methane.  

Abundant, vent-inhabiting Bathymodiolus mussels host methane-oxidising and chemoautotrophic sulphur-oxidising bacteria in their gills; Peterson et al. (2011) investigated whether hydrogen was being used as an energy source in the autotrophy of these mussel symbionts. Hydrogen metabolism is known to involve enzymes which channel electrons from hydrogen, into the quinone pool, thus linking hydrogen oxidation to energy production; the large subunit of these key, membrane-bound, uptake hydrogenases are coded by the hupL gene.  The authors amplified and sequenced this gene from symbiont-containing gill-tissues of Bathymodiolus mussel species collected from both hydrogen-rich and hydrogen–poor vent sites, thus showing the genetic potential for hydrogen oxidation in symbionts from both types of vent.  Peterson et al. (2011) went on to examine the actual uptake of hydrogen by symbiont-containing mussel gill tissues.

The authors recorded rapid hydrogen consumption by symbiont-containing Bathymodiolus gills, in contrast to symbiont-free foot tissue, which matched other negative controls; additionally hydrogen consumption rates were found to be 20- to 30-fold lower in mussel gill tissue from hydrogen-poor sites compared to tissue from hydrogen-rich environments.  In order to confirm that hydrogen uptake was coupled to CO₂ fixation in Bathymodiolus symbionts, Peterson et al. (2011) incubated mussel gill tissues collected from a hydrogen-rich site, in seawater containing ¹⁴C-bicarbonate, with hydrogen, or with either sulphide or no electron source (controls).  Carbon fixation rates in mussel gill tissue incubated with hydrogen were comparable to those incubated with sulphide, suggesting that not only does hydrogen fuel autotrophy, providing energy for mussel biomass production, but that at hydrogen-rich sites, hydrogen is as important as sulphide in this process.

Lastly, as both sulphur-oxidising bacteria and methane-oxidising bacteria are known to be associated with Bathymodiolus, Peterson et al. (2011) explored which type of chemosynthetic symbiont was using hydrogen; symbiont Identity was linked to function via both DNA and protein based methods.   Metagenomic analysis revealed that a single genome fragment contained all the genetic components necessary for hydrogen uptake, alongside key genes for sulphur oxidation and CO₂ fixation, leading the authors to suggest that the fragment came from a sulphur-oxidising symbiont.    Via single-gene fluorescence in situ hybridisation (FISH), signals from hupL gene probes were observed to overlap 16S rRNA FISH signals from the same sulphur-oxidising symbiont, further implying that this symbiont uses hydrogen.  Finally, hydrogenase gene expression in the sulphur-reducing symbiont was confirmed via immunohistochemistry: anti-hydrogenase antibody signals were observed to overlap with sulphur-reducing symbiont 16S rRNA FISH signals in single Bathymodiolus gills cells; FISH signals from the methane-oxidising symbiont did not overlap.

The thorough nature of the authors’ investigation is persuasive as to the reality of hydrogen use in mussel biomass production at hydrothermal vents.  The environmental significance of H₂ use was also explored by Peterson et al. (2011):  temperature and hydrogen concentration of vent fluids that had crossed mussel beds were measured (via en situ mass spectrometry), as were vent fluids that had not been exposed to macrofauna; a significantly lower slope in the regression line of the former condition suggested that fluids in the mussel beds were hydrogen depleted.  The authors’ calculate that this level of hydrogen consumption may well make Bathymodiolus mussels a significant hydrogen sink; it is also proposed that hydrogen use may be widespread in chemoautotrophic symbiosis, such as those observed in tubeworms and shrimps, and that this may be especially important at hydrogen rich sites.  Further investigation of these theories appears warranted: a better comprehension of hydrogen as an energy source in hydrothermal vent symbioses may affect not only our understanding of how vent ecosystems function, but also of how the marine hydrogen cycle operates.

Petersen JM, Zielinski FU, Pape T, Seifert R, Moraru C, Amann R, Hourdez S, et al. (2011) Hydrogen Is an Energy Source for Hydrothermal Vent Symbioses. Nature 476: 176–80.

http://www.nature.com/nature/journal/v476/n7359/full/nature10325.html

Planctomycetes & Kelp, possible Symbiosis?

Species from the phylum “Planctomycetes” are not typical of most bacteria; they posses cell compartmentalisations with membrane bound organelles. Additionally their cell wall’s lack peptidoglycan, a trait solely shared with members within Chlamydiae, which are obligate intracellulars. Planctomycetes are also found to be prolific through a plethora of environments across the planet. It has also been found that planctomycetes favour a biofilm lifestyle, where they may adhere to surfaces such as those of seaweeds, sediments or particulates of marine snow.

Genome sequences of a well-studied planctomycete, Rhodopirellula baltica, have shown a considerable number of genes involved in the breakdown of sulfated polysaccharides. These are produced by a number of marine photoautotrophs, such as seaweeds, for instants Laminaria hyperborea. A hypothesis was developed expressing that heterotrophic planctomycetes are specialist degraders of sulphated polymeric carbon because of the overrepresentation of these genes in R. baltica and other species. 

Bacteria associated with the kelp Laminaria hyperborea are believed to be important in the carbon and nitrogen cycling of kelp forest. A recent study investigating biofilms of L. hyperborea found planctomycetes through out the year, however their abundace and phylogenies were not examined. Moreover this paper by Bengtsson and Øvreås, (2010) attempts to:

(1) Enumerate planctomycete abundance and distribution within the biofilms on the Laminaria hyperborea.

(2) Construct clone libraries to make clear phylogenetic relationships, community composition and diversity at different times of year. 

(3) And assess culturability of planctomycetes from kelp surfaces. 

(1) On the kelp surface cells were found in uneven distributions growing in strait lines, rings or clusters. Planctomycetes within these aggregations were cocci of small and medium sizes displaying typical ring shaped cell organization. These were evenly intermingled with cells of different morphologies, from rods to long filaments. 

(2) Samples of planctomycetes from all three months, (July 2007, February 2007 and September 2008) were found to have high abundance within the kelp biofilm community. In July there was high abundance but low diversity. In February there was the greatest diversity but abundance was lowest. September showed high abundance, similar to July, and intermediate diversity.

This may be linked to the age of the kelp tissue, as the kelp lamina is older in February compared to in July and September due to the seasonal growth cycle of the kelp. Aging of the kelp tissue could be associated with lowered antibacterial chemical defense by the kelp, as the old kelp lamina is to be shed soon after February, and does therefore not need to be defended against microbial colonization. Without the presence of chemical defenses, the planctomycetes could loose their competitive advantage over other bacterial groups. Specifically the lack of peptidoglycan in their cell walls may leave them exempt from the affects of antibiotics that inhibit other species. This may explain their lower abundance in February.

(3) One strain of planctomycetes, named “P1” was isoloated from the kelp biofilm. It was found be related to R. baltica and posses similar morphological characteristics, though it was not closely related to any of the clone libraries.

Final notes

This paper shows that this species of bacteria may be more closely associated with eukaryotes than previously thought. The findings discussed above lend support to the hypothesis of an algal holobiont. The genetic evidence for planctomycetes as degraders of sulphated polysaccharides, and their preference to a biofilm life style and the knowledge that kelps produce these polysaccharides can be viewed as fledgling evidence for symbiosis. 

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Bengtsson M & Øvre#as L (2010) Planctomycetes dominate biofilms on surfaces of the kelp Laminaria hyperborea. BMC Microbiol 10: 261–273. 

Available at: http://www.biomedcentral.com/1471-2180/10/261

Culturable halophilic archaea at the initial and crystallization stages of salt production in a natural solar saltern of Goa, India

Haloarchaea, a group of archaea that depend on high salt concentrations, have been found to inhabit salterns that produce salt that has a wide use domestically. There are possible implications for human health, dependant on the species present, if they survive to inhabit the salt that is harvested. This study investigates the diversity of culturable haloarchaea present in the salt, during two stages of salt production: pre-harvesting, and at peak harvesting time.

Sampling of water and sediments (surface and at a depth of 10cm) was carried out. Species were isolated, purified, and identified, then the lytic effect of distilled water and low-salt water was tested. Isolation was carried out using plating techniques on two types of medium: NaCl Tryptone Yeast Extract (NTYE) and NaCl Tri-Na-citrate (NT). Initial plates were created using 100ul aliquots of water and a loop full of sediment. Enrichments were created by transferring 1ml of water samples and 1g off sediment samples onto media. The plates were incubated at 37oC for up to 5 days. Appropriate isolates were purified by selecting 10ul aliquots from these and incubating at 30oC, and repeating until orange or red colonies were observed. This colour is a noted character for identifying haloarchaeal species. There were other colonies found in both phases that were white or cream, which were removed when plated onto agar containing ampicillin. The colonies that were investigated are therefore ampicillin resistant.  The morphology of these isolates was checked using Gram staining and electron microscopy, to determine if they were in fact haloarchaea. The lytic ability of water and low-salt concentrations (3.5%) were determined of those characterised as haloarchaea by subjecting cells to their presence, followed by incubation for 24 hours. Viable colony formation was observed on medium plates. Genomic sequencing using the 16S rRNA was done to identify strains of haloarchaea. The salinity of the sampling sites were determined at the point of sampling by testing conductivity.

The species present and their responses vary depending on the sampling time, and the type of agar. Organisms present in the pre-salt collecting phase were all found to be under the species Halococcus salifodinae, to not lyse in water or low salt concentrations, and all but one to be of bright orange-red colour. There were also highly similar genetically, with 98% minimum similarity to Halococcus. Whereas, of the haloarchaea found at the peak of harvesting, all but one lysed in water and low salt concentrations, 3 gave orange colonies and 7 gave bright red. Their morphology also varied, though they were all gram negative, confirming their haloarchaeal classification.

Colonies in the pre-harvesting phase appeared at 20-30 days, suggesting there are slow growers or in lower quantities initially. Both are likely theories as the environment varies greatly, with an extreme increase in salt concentrations allowing for organisms that are more adapted to these conditions to have a competitive advantage. Also, their ability to survive at the lower concentrations suggests they do not have a high demand for salt, suggesting a slow metabolism and slow growth. This could also account for low number. The strain found in both concentrations, Halococcus salifodinae AB588757, exhibited a colour change from red-orange to orange when in high salt. This could be a reflection of lower numbers.

There were two months in between the sampling times. This is sufficient time for new bacterial species to be introduced to the saltern through natural means. It would be interesting to investigate if the species found in the pre-harvest phase are also present in the harvesting phase, since not all bacteria were represented in this study: those that did not produce visible colonies were not identified. They could be present in small quantities, therefore require a different method to that used here. The lysis test shows that this does not need to be done in the pre-harvest phase since these bacteria lyse in low salt concentrations, which the pre-harvesting phase environment consists of: 3-4% salt.

I was surprised that the species found at peak harvesting were so varied, since it makes sense for the most competitive organisms to outcompete the rest. Presumably this is not the case because of the frequency the environment changes, acting as a form of disturbance, which is said to increase diversity. The salt increase act as a disturbance, allowing a variety of species to colonise initially, before the salt concentrations become more stable, at which point the influence of competition is increased and the variation in species is reduced. This is seen by 10 species being found at peak salt and only 1 species, but several strains, are found pre-salt harvesting.

It was beneficial to use multiple agars as NT agar is capably in supporting more ‘choosey’ bacteria, hence more strains were found compared to NTYE. This of course does not enable bacteria that are not currently culturable to be identified. Another method would be to extract all the DNA from the samples, then PCR and run on a gel such as DGGE, and sequence significant bands. This would create a more representative view of the diversity present. 

The results of this study provide useful information about the diversity and change in species compositions as a result of altering the salt concentrations, with links to ecology and disturbance.

Mani et al. (2012) Culturable halophilic archaea at the initial and crystallization stages of salt production in a natural solar saltern of Goa, India, Aquatic Biosystems, 8:15

http://www.aquaticbiosystems.org/content/pdf/2046-9063-8-15.pdf

Rapid diversification of coevolving marine bacteria Synechococcus and a virus

Viral lyses of bacteria play a key role in the cycling of nutrients in the ocean. This said, our understanding of how phenotypic plasticity between bacteria and viruses influences the biogeochemical cycling of nutrients is limited. Marston et al (2012) investigated how the phenotype and genotype of Synechococcus sp. WH7803 and the Myoviridae virus RIM8 may change when incubated in a chemostat experiment for six months. Marston e al (2012) aimed to answer three questions during this investigation. Firstly, what is the potential for coevolution and diversification in Synechococcus and RIM8? Secondly, are there candidate genes that underlie the phenotypic diversification? Thirdly, do mutations that arise from pair wise coevolution exposures have consequences for interactions with other Synechococcus and and cyanophage strains?

Marston t al (2012) preformed four replicate chemostat experiments and one control. To test for the potential of antagonistic coevolution to lead to the diversification of Synechococcus and RIM8 single cells and viruses were isolated from each of the chemostats by colony isolation and plaque purification at six time points over the six month investigation. Infection assays were conducted to determine the ability of the viral isolates to infect various Synechococcus isolates from the same chemostat.

Over the six months up to 13 viral and 4-11 bacterial phenotypes were isolated from the chemostats indicating that antagonistic coevolution is possible between these two strains. In both cases the phenotypic change was directional. The viral infectivity increased over time, such that viruses from the later time points had a wider host range than earlier time points.  Similarly, Synechococcus increased its resistance to viral infection over the course of the experiment. The development of phenotypes over time differed between RIM8 and Synechococcus. Viral phenotypes appeared to replace one-another over time, whereas, Synechococcus showed multiple phenotypes at any one time point. Marston et al (2012) suggests that the actual number of Synechococcus phenotypes present in the investigation may have been greatly under estimated because the low number of isolates taken at each time point.  The identification of the genes controlling the changes phenotype is complex in this investigation. Although obvious changes were observed in amino acid sequence it did not always lead to the same phenotype. In one example, on day 84 the RIM8 virus was shown to have the same amino acid sequence but this represented five different phenotypes. Synechococcus also showed similar complexity. It appeared that different genes controlled the resistance response to the same virus.

In the third part of the experiment the authors wanted to see how the evolved Synechococcus would react to 31 different strains of viruses isolated from seawater in an attempt to make the results more realistic. The results showed that the resistance increased with time, but also, that the increased resistance to viruses was pleiotropic- resistance to one virus lead to resistance to other similar viruses. The opposite is also possible, with resistance to one increasing the susceptibility to other viruses. In this study increasing resistance to RIM8 lead to increased sensitivity to RIM26.

I chose this investigation because I believe it highlights some of the complexities in how marine bacterial/virus interaction evolves and how dynamic it is. I also thought it was interesting how the same amino acid sequence can produce differing phenotypes in such simple organisms, leading to differing levels of resistance, either positive or negative.   

 

 Marston.M. F., Peirciery. F. J., Sherperd. A., (2012). Rapid diversification of coevolving marine Synechococcus and a virus. PNAS. 109. 12. 4544-4549.

http://www.pnas.org/content/109/12/4544.full

Activity and abundance in varying bacterial communities.

Campbell and Kirchman (2013) attempt to determine growth properties of the bacterial community in the Delaware bay to help gage the contribution of bacteria to bio-chemical cycling. Using 16S ribosomal RNA as an indication of , and the presence of rRNA genes (rDNA) as an indication of abundance. The ratio between the rRNA and the rDNA can then be used as an indication of relative contribution of bacterial communities to biochemical processes. Campbell and Kirchman (2013) examine the potential growth rates and activity of individual bacterial taxa, and how certain environmental factors may impact this.

Campbell and Kirchman (2013) took samples along a transect spanning Delaware Bay and at a nearby coastal ocean observatory. Samples were either collected directly onto 0.22mm membranes, the whole water community, or filtered through a 0.8mm filter before collection on a 0.22mm membrane, the free living community. DNA and RNA was isolated, quantified and sequenced. After “cleaning” sequences were clustered using the average neighbour algorithm at 0.03 distance which resulted in 2446 operational taxonomic units (OTU), and phylogenetic distances were calculated.

It was found that there were distinct differences in richness and diversity between different salinities. Richness was observed to be highest in the whole water communities between 1.2 PSU and 6.4 PSU, with a steep drop to less than half the number of OTUs present by 10 PSU. There was no significant difference in richness observed (Chao1 index) between the low and high salinities in the free-living communities. The diversity (inverse Simpson) of the samples on the other hand, showed a distinct U curve in both the whole and free living communities with both the lowest and highest salinities having a significantly higher overall diversity than the mid salinity samples. 

Community structure changed drastically over the salinity gradient. In low salinities and fresh water the community was dominated by Actinobacteria, Verrucomicrobiota and Betaproteobacteria, and shifted to a community dominated by the SAR11 taxa, Rhodobacterales, Gammaproteobacteria and Bacteroidetes. 

Ratios or rRNA to rDNA were also examined. Using rRNA as a model for activity and rDNA as a model for abundance, relationships between abundance and activity can be determined. This relationship was examined for each OTU. It was found that overall the contribution to activity did not follow the relative abundance of phylotypes. Though when the whole-water and free-living communities were examined separately, it was found that for the whole water communities the ratios did differ whereas the ratios between phylotypes in the free-living communities did not. Also noted was the difference in ratio at different salinities. Results suggest that the ratios of some taxa were higher at certain salinity ranges compared to others. Though other factors may also be involved, as nitrate levels and light attenuation are mentioned to be correlated with the rRNA : rDNA ratio.

This study attempts to help understand how constantly fluctuating environmental conditions may impact bacterial communities and how these communities respond. Campbell and Kirchman (2013) suggest that rRNA:rDNA ratios may be more informative than abundance alone, to understand how environmental factors can influence bacterial communities. Especially if these ratios can be converted into estimates of individual bacterial growth. 

Campbell, B., A.; Kirchman, D., L,. (2013). Bacterial diversity, community structure and potential growth rates along an estuarine salinity gradient. International Society for Microbial Ecology. 7 (1), 210-220.

Need to get somewhere? Call a fish!

 Bacterial bioluminescence, a mechanism for dispersion/propogation in free-living bacteria 

Bioluminescence has obvious advantages in symbiotic bacteria but its role in free-living bacteria is less certain, some theories such as anti-oxidative activity, enhanced DNA repair and UV resistance have been put forward as explanations but none are conclusive. The ecological function in propagation and dispersal was put forward 30 years ago (known as the bait hypothesis), the bacterium, by glowing, visually mark the presence of a food particle for fish in order to get into their nutritious guts but has received little attention.

The Objectives of this study was to test the following key points of the bait hypothesis;

1. Visual attraction of zooplankton to bacterial bioluminescence
2.  Promotion of luminescence in zooplankton by contacting or ingesting luminous bacteria
3. Attraction of zooplanktivorous fish to glowing prey
4. Survival of bacteria gut passage in both zooplankton and fish

The attraction of zooplankton to bacterial bioluminescence was tested by adding two dialysis bags of bacteria, P. leiognathi, in to a large tank of seawater. One bag containing luminous bacteria was placed at one corner of the tank, while on the other side there was a mutant strain (incapable of luminescence). Significant changes in zooplankton distribution were noticeable within 15 minutes, decapods and mysides were found almost exclusively over the corner with luminous bacteria, however copepods showed no significant attraction to either corner. Similarly, no difference was noted between non-motile organisms (which served as an internal control).


Fig 1  - Luminescent Artemia



The brine shrimp Artemia salina were used to show luminescence. Artemia became luminescent after swimming in a liquid culture of P. leiognathi after just 10 seconds, this was noticed from the guts (due to ingested bacteria) and the exoskeleton, where bacteria had attached. Artemia was used because they are readily eaten by zooplankton, easy to handle and lack evasive behaviour. The promoted glow in Artemia dramatically affected its risk of being preyed on by the nocturnal fish Apogon annularis in a recirculating laboratory flume in the dark. (Fig 1&2).

Fig 2 - Non-luminescent Artemia

            Fecal pellets of Artemia which had ingested luminous bacteria showed luminescence; indicating that P. leiognathi had survived the passage through the guts. High concentrations of viable P. leiognathi were found also in the feces of fish A. Annularis that had fed on luminious Artemia, in which the abundance of luminescence cells in there fecal pellets was fiver orders of magnitured high than that of fish fed on non-luminous Artemia.

This study shows experimental evidence for some key steps of the bait hypothesis, showing benefits of bioluminescence in non-symbiotic bacteria. Though the authors hint that this benefit of bioluminescence is most applicable in food-deprived environments, such as the deep sea, where food availability decreases with depth. However, this experiment did not differentiate between the luminescence generated when zooplankton ingested the bacteria or whether they attached externally via the exoskeleton. Also, the use of a non-marine zooplankton, Artemia, may have influenced bias. As well, the authors also make the assumption that the mechanism by which organisms becomes luminescent is the same, Artemia may not be applicable for all zooplankton.

I chose to review this study after beginning to read up on the symbiotic bacteria lectures, bioluminescence has obvious advantages when in a mutualistic relationships, such as in Vibryo fischeri and Euprymna scolopes. I however struggled to think of ways in which it may benefit free-living marine bacteria and so chose to look in to this, as there must be a reason some free-living bacteria are selecting for bioluminescence.

If anyone is interested in reading the paper, heres a link:
http://www.pnas.org/content/109/3/853

Zarubin, M., Belkin, S., Ionescu, M., & Genin, A. (2012). Bacterial bioluminescence as a lure for marine zooplankton and fish. Proceedings of the National Academy of Sciences of the United States of America, 109(3), 853–7. doi:10.1073/pnas.1116683109