Blog entries closed for coursework

Many thanks for all your submissions. Students are welcome to continue adding posts and comments  but anything entered after 11 April  will not count towards the coursework mark (unless you are given referred coursework at the Assessment Panel).

Between October 2012 and April 2013 the class of 25 students wrote

231 posts
430 comments
177,108 words - that's about the same as JJR Tolkein's "Fellowship of the Ring"!

There were 13993 page views of the blog
59% of views were from the UK (mostly us?)
20% were from Europe, 16% from USA, and 6% from RoW

Here's a link to a Wordle, showing the most commonly used words in the blog.(apart from common words like and, the etc.)  Not surprisingly, "bacteria" and "bacterial" scored top. Not such a strong showing for "viruses" this year!

http://www.wordle.net/show/wrdl/6584057/MicrobesRuletheWaves2012

Are Gorgonian Corals a Source of Novel Antibiotics?

Quorum sensing (QS) is a method of cell-to-cell communication employed by a variety of bacteria and such signals are often used to facilitate colonisation of host organisms. Gorgonian corals are already known to possess compounds that mediate cellular interactions and may be used to regulate microbial colonisation. The researchers in this study used extracts of twelve species of gorgonian corals sampled from the Caribbean to search for compounds that exhibited antibacterial activity that could potentially be developed for bacterial control purposes.

The original coral samples were collected at depths of 5-10m from La Parguera, Puerto Rico and Looe Key Reef, Florida Keys, USA. The following species were sampled: Briareum sp., Eunicea laciniata, Eunicea tourneforti, Plexaura flexuosa, Plexaura homomalla, Pseudoplexaura porosa, Pseudopterogorgia americana, and Pseudopterogorgia acerosa, for Puerto Rico, and Gorgonia ventalina, Plexaurella sp., Muriceopsis flavida, and Eunicea mammosa for Florida Keys. In the Texas-based lab, ethanol extracts were prepared and antibacterial assays performed.

A variety of gram-negative and gram-positive bacteria of both marine and non marine origin were used for the assays, including known coral pathogens such as Serratia marcescens and Vibrio alginolyticus. Human pathogens were also used in the antibacterial assays. The assay itself was a bacteria turbidity assay which was conducted in a 96-well flat bottom plastic microplate, an assay chosen as it requires less of the coral sample than others, thus less damage to the reefs. The assays were simultaneously replicated three times with the controls of bacteria only, bacteria and antibiotic and bacteria and ethanol. Growth inhibition of bacteria was measured by drawing comparisons between the growth rates of the bacteria with the coral antibiotic extracts and the bacterial with ethanol as a control.

Bacteria chosen for the assays had to be isolated and identified, and shown to be culturable. Firstly, mucus samples were collected and cultured on glycerol agar. Bacteria that had grown was then subcultured in a marine broth an identified by species. PCR was used to amplify DNA and amplified sequences were compared with data from the GenBank database. Vibrio parahaemolyticus was the only bacteria identified and it was isolated from Pseudoplexaura porosa.

Bioassays to detect long chain N-acylhomoserine lactones (AHLs) in the ethanol extracts were carried out using Pseudomonas aeruginosa as a QS reporter strain. Inhibition or stimulation of quorum sensing was tested for, and P. aeruginosa is known to be sensitive to the long chain AHLs that result in these responses, making it an ideal reporter strain to reveal the compounds. Fluorescent signals that indicate transient gene expression were measured using spectrophotometry, this allowed for real-time QS detection.

Another biosensor strain, Chromobacterium violaceum, was used as an indicator organism in experiments to measure AHL presence by quantifying violacein synthesis. The synthesis of this pigment indicates a bacterium under QS control. The bioassay in this experiment was based again on spectrophotometry to compare absorbances at A₅₉₀ of the bacteria exposed to antibacterial compounds and the bacteria with ethanol as a control.

Their main findings were that the gorgonian corals possessed compounds that could inhibit and stimulate quorum sensing, suggesting selection on behalf of the corals of the bacteria that could colonize. P. americana, P. acerosa, and P. flexuosa displayed the highest QS inhibition and P. porosa displayed the highest QS stimulation. These differences in effects on QS hint at corals actively selecting the bacteria that can colonize the coral holobiont. More antimicrobial activity was noted against non-marine strains than marine strains of bacteria, suggesting that corals employ several mechanisms of bacterial control. Bacillus subtilis, vancomycin resistanct Enterococcus and methicillin-sensitive Staphylococcus aureus were most sensitive to compounds from the gorgonian extracts and can all be pathogenic to humans. Gram-negative strains of bacteria were less susceptible to gorgonian compounds than gram-positive strains from both marine and non-marine origins, suggesting that gorgonian antimicrobial activity is not broad-spectrum, this may be expected as they would have evolved mechanisms to deal with the bacteria present in the coral holobiont.

The researchers closed by calling for further work on the Caribbean gorgonians which exhibited strong antibacterial action against human pathogens, as these represent a potential for much needed novel antibiotics.

Reference

Hunt., L.R., Smith, S.M., Downum., K.R. and Mydlarz, L.D. (2012) Microbial regulation in gorgonian corals. Mar. Drugs 10: 1225-1243.

Degradation of dispersed crude oil

As we have found out in lectures dispersants can play an important role in aiding biodegradation of large oil spills. Many studies have demonstrated how effective the use of such dispersants but few studies have actually assessed the biodegradation of oil once it has been dispersed in environmentally relevant conditions. Once dispersants have been added oil droplets are very small and become diluted within the water column. This study looked at the degradation of oil at these low concentrations, close to those observed following a real spill when dispersants have been used in order to discover degradation is affected by the presence of a dispersant. 

This study is carried out in laboratory with sea water collected in April and January 2010 from the shore of New Jersey and stored in Carboys. No bacteria or nutrients were added so only indigenous bacteria and nutrients were present throughout the study in an attempt to mimic natural conditions for biodegradation. Oil was added to the samples, one lot of oil samples was lightly weathered in the laboratory by evaporation at room temperature until 20% of its weigh was lost (to mimic 24 hours exposure to sea water) and the other was fresh oil. To both types of the dispersant Corexit 9500 was added. A second set of experiments were carried out where no dispersant was added the sea water samples. The experiment was carried out in a cold room at 8 °C.

At designated times water was extracted from each Carboy 3 times and analysed using gas chromatography and mass spectrometry. Oil biodegradation was monitored with respect to 17α(H),21β(H)-hopane as a conserved internal marker within the oil. Oil was found to be degraded quickly and extensively (more than 80% after 60 days). The half times of the biodegradation were found to be similar with and without dispersant at 13.8 and 11 days respectively. The level of oil present was 2.5 ppm by volume which is clearly low and represents levels expected when dispersants have successfully been added. The authors note that this study cannot be used to assess the effectiveness of dispersants but the study shows that under natural conditions biodegradation is not affected by the presence of dispersants but is rapid and extensive.

I found this study interesting as it used natural nutrient levels and only indigenous bacteria. I think for future work it would be good to look at which bacteria are actually present in the sample and in what numbers to gain a more clear understanding of the whole process. It would also be good to compare the results from this study to with water taken from other areas as there would presumably be different naturally occurring bacteria and nutrient levels which would be likely to change the degradation rates.

Prince. R, McFarlin. K, Butler. J,  Febboe. E, Wang. F, Nedwed, T. 2013 The primary biodegradation of dispersed crude oil in the sea. Chemosphere. 90; 521-526. 

Inactivation rates in pathogens: lab vs in situ

Managed aquifer recharge (MAR) schemes involve water being recycled and stored underground in an aquifer for reuse and relieve water shortages. Microbial pathogens can be present and survive in the recharged water, which subsequently cause health hazards. The potential survival times of enteric pathogens can be assessed to alter the MAR scheme or determine the need for additional treatment. In this study the authors have evaluated the effectiveness of using in situ diffusion chambers for assessment of health risks associated with MAR, this involved comparing inactivation rates in both laboratory microcosms and in situ diffusion chambers.

Groundwater samples seeded with pathogens and indicators were tested in laboratory microcosms (50ml polypropylene centrifuge tubes) and in situ Teflon diffusion chambers. The chambers contained membranes with pore sizes 0.010µm and 0.025 µm and were suspended in a well for 50 days with the upper chambers 1 m below the water table to intercept flow of groundwater. The pH, temperature, redox potential, dissolved oxygen (DO) and EC were measured in situ while dissolved organic carbon (DOC) was analysed by a commercial laboratory. These parameters remained almost constant during the in situ study.

To tackle the issue of potential clogging in groundwater mentioned in previous studies, the chambers were recovered and Rhodamine WT (RWT) a stable fluorescent dye was used to measure diffusion of water across the constructed diffusion chambers and to determine whether clogging of membranes has reduced the flow of water across the chambers. No biological growth or biological clogging was observed on the membranes, so investigating the potential effect of clogging on water flow requires further studies that involve greater turbidity and nutrient concentrations.

All microorganisms tested in this study were observed to decay both in laboratory microcosms and in diffusion chambers. The bacteriophage MS2 was found to decay at much faster rate than adenovirus, which suggests that MS2 is not a suitable indicator for enteric virus inactivation in groundwater. The results are simplified in figure 3, showing that the inactivation times of seeded bacteria were lower when 0.010µm membranes were used compared to 0.025 µm membranes. The higher inactivation time found in the smaller pore size (0.010µm) suggests that reduced rate of water flow across the membranes does influence the rate of bacterial inactivation. The laboratory microcosm inactivation rates were similar to the 0.025 µm membranes, yet significantly different compared to the 0.010µm membranes.

Previous studies have used laboratory microcosms and in situ diffusion chambers however in situ studies are preferred for accurate assessment of pathogen inactivation. Reasons for this preference seem to be laboratory microcosms can cause an underestimation of inactivation rates. I agree with this preference because an in situ model is likely to simulate the amount of time the recharged water is contained  within the aquifer, as well as the conditions it is subjected to. The authors provided several possible factors (for example, low water flow or water temperature) that could have influenced the inactivation rates of the pathogens, but they were unable to explain several  mechanisms behind some of the variations in inactivation rates which suggest that further research into the factors associated with diffusion chambers is needed. 

Sidhu, J.P.S. and Toze, S. (2012), Assessment of pathogen survival potential during managed aquifer recharge with diffusion chambers. Journal of Applied Microbiology, Volume 113, pages 693–700. 

Potential Interactions of Particle-Associated Anammox Bacteria with Bacterial and Archaeal Partners in the Namibian Upwelling System

Nitrogen is often considered a factor that limits the growth rate of phytoplankton. A high proportion of nitrogen loss (30-50%) occurs in oxygen minimum zones (OMZ) such as the Nambian Shelf, partially due to micro-organisms that carry out anaerobic oxidation of ammonium (anamox species). Previous research has been carried out into anamox species that reside in the Nambian Shelf under conditions that are similar to that near sewage systems, which contain more ammonium than naturally occurs in these waters. Woebken et al (2007) investigated the distribution and particle association of anamoxx species to improve understanding of their occurrence in their natural habitat. This topic is worthy of investigation because of the importance of the phytoplankton species influenced by the activity of anamox species.

Water samples containing bacterioplankton were collected from 4 stations around the Nambian Shelf, and water at 6 stations along a transect (23.0°S from 14.36°E to 12.0°E) were investigated. Profiles were created in terms of turbidity, oxygen levels and ammonium levels. Correlation was seen between anamox numbers and particulate organic carbon, and particulate organic nitrogen. Anamox species were present in depths of 30 m down to the sediment, across suboxic to anoxic conditions but not oxic, with the highest concentrations occurring near the coast.
            DNA from collected water samples was isolated, then replicated using PCR with universal bacterial and archaeal primers for the 16S rRNA sequences. The results were used to choose specific primers for a second PCR and specific probes from FISH and CARD-FISH (Catalyzed reporter deposition Fluorescence In Situ Hybridization).
            From the second PCR, they created several bacterial clonal libraries from the water samples at station 182 (119 m and 130 m deep) and one archaeal library based on samples from station 182 (130 m deep). The bacterial diversity and species numbers found were greater than that of Archaeal species: 235 bacterial species versus 23 archaeal species, belonging to 5 major groups as opposed to only 2 major groups for archaeal species. Several sequences were found to belong to uncultured bacteria. The species identified occur worldwide.
            FISH and CARD-FISH performed using group- and species-specific probes found a majority of bacteria (56.3%) occurred in clusters; 24.5% attached to particles; and 19.3% were individual cells.
            In situ observations were also carried out in the water column through a remotely operated camera, showing high densities of macroparticles.

This research shows the ability for anamox species to inhabit a wide range of habitats, limited by high oxygen levels. Their reliance on nitrogen suggests that natural cycles in terms of population numbers occur due to their removal of nitrogen as their numbers increase. It would be interesting to investigate the interactions between the cells considering they seem to flourish in group situations, such as quorum sensing, co-operative metabolism and gene transfer. Perhaps it is inter-bacterial interactions that allow them to inhabit regions with varying environmental conditions? If these species prove problematic for valuable species of phytoplankton it may be beneficial to consider research into breaking down these aggregates. Future methods of collection that did not disturb the aggregates may provide a more accurate representation of the frequency of clusters.

Woebken, D; Fuchs,B. M; Kuypers,M. M.M. and Amann, R. (2007) Potential Interactions of Particle-Associated Anammox Bacteria with Bacterial and Archaeal Partners in the Namibian Upwelling System Appl Environ Microbiol. 73(14): 4648–4657.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1932835/

Megan

Gradients in Microbial Methanol Uptake in Atlantic Waters

Methanol biogeochemistry is an emerging area of research with importance to understanding marine microbial carbon cycling. Methylotrophic bacteria have been known to exist in oceans for a number of years, but it is only recently that extensive research on them has been carried out. Researchers have begun to establish where methylotrophs are located, often in coastal waters, and understand the significant roles they, and bacteria of similar metabolism, play in carbon cycling, such as the heterotroph SAR11 Alphaproteobacteria’s oxidation of one-carbon compounds representing a source of CO₂ in the upper ocean. This and similar research shows how methanol turnover may be common and be integral in the marine environment.

The researchers (Dixon et al, 2013) in this study investigated the microbial demand for methanol along a gradient of metabolic productivity from highly productive coastal waters to comparatively arid oligotrophic gyres. They collected seawater samples along a transect in the Atlantic ocean during research cruises and started experiments within an hour of sampling.

Microbial methanol uptake was investigated by incubating the seawater samples with ¹⁴C labelled methanol for 5-10 hours at the temperature of their origin. Uptake of this labelled methanol into particulate cell biomass was assessed. Bacterial production rates were determined by measuring the incorporation of ³H-leucine into protein synthesis. ¹⁴C-bicarbonate incorporation was used as a measure of primary production and fluorometric analysis of acetone-extracted pigments was used to determine chlorophyll a concentrations. Flow cytometry on SYBR Green I DNA-stained bacterioplankton cells and unstained Prochlorococcus sp. and Synechococcus sp. cells was used to established community composition and plankton community composition was determined by inverted settlement microscopy. Inorganic phosphate and nitrite concentrations of the samples were also determined. Finally, DNA was extracted from the samples, amplified and analysed using PCR and sequences compared to databases to identify bacteria.

The researchers found that microbial methanol uptake varies between 0.1–24.8 nmol l^-1 d^-1 in coastal upwelling waters and decreased at depths below 20m, though overall methanol uptake was up to 10 times lower in samples from the gyres. Most of the assimilation of methanol in both upwelling waters and gyres was attributed to microbes of 0.2-2.0 mm in size. Leucine uptake rates varied between 190-2279 pmol Leu l^-1 d^-1 in the upwellings, and uptake was 3-7 lower in the gyres. Primary production rates of the upwelling waters decreased at depths below 20m and 99% of primary production in the top 20m of the waters was associated with larger cells such as flagellates and diatoms, the primary production rates of the gyre samples were again lower but were also associated with larger cells. Chlorophyll a concentrations ranged from 6.5 mg m^-3 in recently upwelled waters to 0.5mg m^-3 10 days later and seemed to correlate with methanol uptake. Prochlorococcus sp. were absent from the upwelling waters but Synechococcus was present as were pico-plankton and nano-phytoplankton. All microbes with the exception of pico-plankton (numbers of which were higher in the gyres than the upwellings) were less abundant in the gyres. Inorganic nitrate and phosphate concentrations in the upwelling samples initially ranged between 6.1–7.7 mM and 0.43–0.58 mM but lowered slightly after and were low in the gyre samples.

The results of this study were therefore mostly consistent with expectations, with higher nutrient uptake, primary production and assimilation rates noted in more biochemically active waters than in waters that were less so. Coastal waters showed the highest methanol carbon assimilation rates when compared to northern temperate and equatorial upwelling waters and and in contrast with oligotrophic gyres. Their results also suggested that all surface waters of upwellings may contain a population of methylotrophic microbes, or microbes that use methanol-derived carbon for growth. Preliminary characterization of bacteria in the equatorial waters revealed a variety of methylotrophs supporting this finding. Around 50-60% of the total methanol was assimilated into carbon biomass in upwelling waters, which was in contrast to 97% of methanol being used oligotrophic microbes in gyres as an energy source.

Lastly, the researchers suggested that correlations between methanol uptake and chlorophyll a concentrations in upwelling and coastal waters could be used by climate scientists to infer methanol biological loss rates by imaging chlorophyll a remotely. Leucine uptake was not as stongly correlate and therefore couldn’t offer a similar use. The researchers recognized that further work would be needed to refine a technique that could use chlorophyll a concentrations to effectively estimate methanol loss rates and that further research was needed to narrow down the microbial species which were utilizing methanol.

Reference

Dixon, J.L., Sargeant, S., Nightingale, P.D. and Murrell, J.C. (2013) Gradients in microbial methanol uptake: productive coastal upwelling waters to oligotrophic gyres in the Atlantic Ocean. J ISME 7: 568-580.

Furunculosis, Aeromonas salmonicida & Fish Farming

The disease furunculosis caused by the bacteria Aeromonas salmonicida, continues to be a major health problem for the growing salmonid aquaculture. Continuing to raise high concerns in European salmonid fishfarms due to high mortality rates leading to detrimental economic losses.

In spite of effective vaccination programs frequent outbreaks occur at the fish farms calling for repeated antibiotic treatment. However, side effects following oil-adjuvanted vaccination have raised a series of ethical and welfare questions connected to the use of vaccines. Additionally antimicrobial residues left from treating out breaks can persist in the environment and lead to resistance spreading to other microbes.

Lars Holten-Andersen et al (2012) hypothesized that a difference in natural susceptibility to this disease might exist between Baltic salmon and the widely used rainbow trout. The researchers tested the hypothesis using a cohabitation challenge model; this was used to investigate the relative susceptibility to infection with A. salmonicida in rainbow trout and Baltic salmon. They monitored the course of the infection on a daily basis over the course 30-day period post challenge summerising the results as mortality curves.

The cohabitation infection model proved to be effective in terms of disease

transmission. Bacteriological examinations provide confirmation that the mortalities occurred as a result of the infection. This verifies the transmission of the disease from East-Atlantic salmon to Baltic salmon and rainbow trout.  Lars Holten-Andersen and co found that survival at day 30 was 6.2% and 34.0% for rainbow trout and Baltic salmon, respectively.  The differences in susceptibility to A. salmonicida were significant between the two salmonids. The risk of dying from the infection was found to be 3.36 higher Baltic salmon compared to rainbow trout.

FINAL NOTES:

Studies such as this one provide valuable data and evidence that we can use to make better-informed decisions in the future. For example, which particular species of fish would one select for cultivation? If a fish already possessing naturally high resistance was selected this might enable us to reduce the amount of antibiotic residues left in the environment do to lower frequencies of outbreaks. 

Full article here :) 

Holten-Andersen L, Dalsgaard I, Buchmann K (2012) Baltic Salmon, Salmo salar, from Swedish River Lule A ̈ lv Is More Resistant to Furunculosis Compared to Rainbow Trout. PLoS ONE 7(1):

Vibrio cholerae Pathogenicity Islands Form Circular Intermediates

The acquisition of novel DNA via horizontal gene transfer is known to play a significant role in both the emergence and recurrence of pathogenic bacteria.  A number of mobile genetic elements have been identified in isolates of pathogenic Vibrio chloreae, the causative agent of cholera, a disease which has resulted in thousands of deaths worldwide in the last decade alone.  Such mobile genetic elements include pathogenic genomic islands (PAIs), such as Vibrio pathogenicity island-1 (VPI-1, which encodes toxin-coregulated pili), VPI-2, Vibrio seventh pandemic island-I (VSP-I), and VSP-II.  These PAIs have been associated with endemic and pathogenic isolates of V. cholerae.  The nucleotide sequences of genomic islands display certain features which differentiate them from the rest of the bacterial genome.  Such features are used for identification purposes, and include the presence of mobility genes (e.g. integrases and transposases), a G+C (base) content which differs significantly from the overall content of the host, flanking direct repeat sites (that mark where incoming DNA recombined with the genome), association with a tRNA gene, and large regions of the chromosome that are present in one set of isolates, but absent in closely related isolates. 

Murphy & Boyd (2008) examined the genomic structures of VPI-2, VSP-I, and VSP-II, in the fully sequenced genomes of 12 different strains of V. cholerae.  Comparative analyses showed that the VPI-2 regions were highly conserved at the 5’ and 3’ insertion sites.  This prompted the authors to examine whether VPI-2 had the potential to excise from the genome (as previously shown in VPI-1), with subsequent formation of an extra-chromosomal, circular intermediate (CI).  The presence of both Phage-4-like integrase sequences (an enzyme that facilitates integration of transferred genetic material into host DNA), and two direct repeat sequences (one at the 5’ and one at the 3’ end, detected by comparative sequence analysis of VPI-2 positive and negative strains), suggested that an excision mechanism was present in the integrated VPI-2 region.  Therefore, the authors attempted to detect circular excision products of VPI-2 via inverse PCR and nested PCR (note: the primers used in both cases were designed to form products only if the VPI-2 region could excise and form a circular product).  No PCR product was detected in the initial, inverse PCR assays, but nested PCR (of inverse PCR products) resulted in amplified products from six strains, indicating that VPI-2 did indeed excise from its chromosomal insertion site, forming a circular excision product (although at low levels in the conditions applied).  The excision potential was likewise ascertained for the PAIs VSP-I and VSP-II, and the circular products were detected initially via inverse PCR, and subsequently confirmed via nested PCR.  All PCR products were sequenced to confirm the excision events.

The PAIs of several bacterial species have previously been shown to similarly excise from their chromosomal insertion sites.  Although the fate of circular excised islands is generally unknown, it has been shown that in some V. cholerae isolates, post-excision islands have the capability of reinserting into the genome at new locations.  Murphy & Boyd (2008) conclude that the excision and formation of CIs are probably the initial steps of PAI horizontal gene transfer (by transduction, transformation or conjugation), and go on to discuss the probable importance of chitin to the mechanisms underpinning naked DNA transfer in V. cholerae.  The authors also include analyses and investigations of specific PAI genes (the description of which is not possible in this short blog post).   In summary, Murphy & Boyd (2008) appear to have undertaken a thorough investigation which elucidates an important part of the process by which PAIs are transferred between V. cholerae strains.

Murphy R. A. & Boyd E. F. (2008) Three pathogenicity islands of Vibrio cholerae can excise from the chromosome and form circular intermediates. Journal of bacteriology 190: 636–47.

Where are the viruses? (Flaws of virus count methods)

Marine viruses represent a major driving force in plankton ecology and, due to perhaps false assumptions that the majority of marine bacteriophages were DNA-containing viruses, most study in the field over the last 20 years has been on such viruses. Steward et al set out to quantify eukaryote infecting RNA viruses by comparing the total mass of viral RNA and DNA from seawater samples and estimating the abundances of each virus type. They found that RNA virus abundance was comparable to, and possibly exceeding, that of DNA viruses. Picorna-like viruses were found to dominate the RNA viral fraction of their samples, which was in agreement with previous metagenomic surveys of the marine RNA viral fraction that suggested that positive-sense, single-strand picornavirads were dominant. However, as these picornavirads posses small genomes that are often below the detection threshold of most fluorescence-based counting methods, the authors concluded that virus counts may be vastly underestimating true numbers.

Direct quantification of RNA virus abundance is difficult and unreliable due to a number of limiting factors such as stain sensitivity when using stains to differentiate DNA and RNA viruses for counts and small genome size preventing detection by such stains. The researchers took a novel approach and measured the relative masses of RNA and DNA viruses from tropical seawater samples collected from Kane’ohe Bay, O’ahu, Hawaii, and paired their data with estimates of the mass of nucleic acid per RNA and DNA virion to estimate marine RNA virus abundance.

After the seawater samples were collected, they were filtered and processed into subsamples, from which the viruses were concentrated and purified into fractions of 0.5ml each. The RNA and DNA content of these fractions were measured via fluorometric assays in parallel tests and nucleic acid masses were predicted from the assay results to a ±95% confidence interval. The RNA concentration for each sample was plotted as a function of the given RNA and DNA concentrations and statistics including regression and a two-tailed t-test performed to test for differences.

The researchers created and analysed metagenomes of one subsample of each fraction. RNA treated with DNase from these subsamples was pooled and amplified. They used pyrosequencing to generate sequence libraries from the amplified samples (now DNA). The community composition of each sample was analysed and taxonomic assignments applied. Sequences were compared with the SILVA database to rule out contamination from cellular RNA and the initial RNA mass estimates were adjusted accordingly. Abundances of RNA viruses were estimated by calculating the number of genomes from nucleic acid masses, done by using the relative representation of different taxa in the metagenomic library to calculate an average RNA mass per virion. DNA virus abundances were calculated from the DNA content of the fractions. It should be noted that they based their assumption of DNA content per virion on an average of data from a wide range of environments in their calculations and admitted overestimation may have occurred.

Their results showed that 50-57% of the RNA viral genomes likely originated from eukaryote-infecting RNA viruses, mostly positive-sense ssRNA viruses belonging to Picornavirales. An implication of this is that protists, while of low abundance, possibly contribute more to marine viral dynamics than expected via RNA virus infection. Around 1% of the sequences matched known double-strand RNA viruses when compared with the metagenomic library. Around 42% of the sequences had no match in the library and remain unidentified, although they exhibited virus-like qualities. They estimated the contribution of RNA viruses to the total number of viruses at 38-63%, taking cellular RNA into consideration. These numbers were minimum estimates to account for processing losses. DNA virus abundances varied throughout the year, from 17-33% in June to 5-9% in August. This variability suggests uncertainty which may relate to laboratory practices and to unreliability of the counting methods. For example, some physically larger viruses (above 0.2mm) may have been lost during filtration when preparing the samples for analysis. Since all larger viruses are double stranded DNA types, the results are biased against DNA viruses. However the magnitude of this bias is unknown and may only be slight if the contribution of larger viruses to the fraction mirrors electron microscopy data suggesting that these virus are low in number.

In their closing paragraph, they suggest a review of RNA virus and single-strand DNA virus count methods to improve reliability and account for smaller and undetectable genomes that may skew data, and push for the development of new infallible methods.

Reference

Steward, G.F., Culley, A.I., Mueller, J.A., Wood-Charlson, E.M., Belcaid, M. and Poisson, G. (2013) Are we missing half the viruses in the ocean? J ISME 7: 672-679.

Seeing spots: Decapod crustaceans and their susceptibility to WSD

White Spot Disease (WSD), caused by White Spot Syndrome Virus (WSSV), is largely responsible for the loss of productivity in penaeid shrimp farms. WD results in high mortalities which affects the entirety (100%) of exposed stock. Turnbull et al. (2005) have described the practice of ‘emergency harvesting’ as a way of preventing complete productivity loss following the discovery of infected individuals within the stock. Even though WD was discovered nearly two decades ago it is still the most significant pathogen in regards to the impact it has upon the continued growth and sustainability of penaeid shrimp farming. This has important implications for humans as emergency harvesting may not be able to produce the crop that would otherwise be produced by a full harvest. This inevitably costs the farmers money in terms of profit gained and may, ultimately, lead to a loss of livelihood.

Guidelines which are currently in place for the methodological approaches to diagnosing WSD are primarily based around detecting viral nucleic acids within infected individuals. This study assessed the susceptibility of non-model species to WSSV infection using the criteria set by the European Food Safety Authority (EFSA). The criteria are; replication of the organism during infection, viability of the organism, any changes to/ presence of the pathogen and the location of the pathogen within the host. The use of non-model species within this study is key in helping to further our knowledge of just how strongly the definition of susceptibility can be applied to non-model species.

The authors found that there is a universal susceptibility to WSSV infection within a range of decapod crustaceans native to European waters when exposed to WSSV via feeding. It was also found that WSSV was able to replicate and retain its ability to cause disease in the ambient water temperatures found in European waters. It was also found that the virus was still viable and able to be passed onto other susceptible hosts, spreading the infection. Even though the decapod species tested within this study were found to be universally susceptible to WSSV, the way in which the disease itself manifested after infection by WSSV varied between species. This is to be expected as different species will usually have different tolerance levels to infections.

The results from this study helped to define three categories of susceptibility for the non-model decapods species used, high, low and moderate susceptibility. These three categories were defined using a variety of different factors. The categories also have application for measuring susceptibility at both the species and individual level. The authors conclude by suggesting that, in order to assess the true risk that WSSV poses to wild decapod populations, experiments investigating transmission from asymptomatic carrier hosts to other hosts of either the same species or disease susceptibility are required.  Overall WSSV and, consequently, WSD are not understood enough for us to be able to start bringing preventative measures to bear to prevent crop loss in farmed penaeid shrimp or other farmed decapods species and further study is needed.  

Bateman KS., Tew I., French C., Hicks RJ., Martin P., Munro J., Stentiford GD., 2012, Susceptibility to infection and pathogenicity of White Spot Disease (WSD) in non-model crustacean host taxa from temperate regions, Journal of invertebrate pathology, 110, 340-351

Turnbull JF., Corsin F., Mohan CV, Padiyar PC., Thakur M., Madhusudan NV., Hao NV., Morgan KL., 2005, Optimising emergency harvest strategy for White Spot Disease in a semi-intensive Penaeus monodon culture system in Karnataka, India, Walker P., Lester R., Bondad-Reantaso MG (Eds), Diseases in Asian aquaculture V, Fish health section, Asian fisheries society, Manila, 405-414

SAR11 viruses in the ocean- could these affect ecology?

Abundant SAR11 viruses in the ocean

SAR11 bacteria thrive in the oceans despite the high abundance of viruses. These bacteria are the most abundant microbes in all of the world's oceans, and as such are an important factor in carbon cycling.  Several reports have proposed that the dominance of the SAR11 clade in the oceans is due to resistance to Bacteriophages. But alternatively, the evolution of high surface-to-volume ratios coupled with minimal genomes with high-affinity transporters enables an unusually efficient metabolism for oxidizing dissolved organic matter in the world’s oceans that could support vast population sizes despite being vulnerable to phages.

The ‘Kill-the-Winner’ hypothesis is a theory for understanding the impact of top-down control in microbial communities. The basis is that rising population densities and cell metabolic activity expose cells to more viral predation by increasing host–phage encounter rates. Evidence supporting this hypothesis has already been demonstrated. The global dominance of SAR11 in marine communities has led to speculative theories that SAR11 avoids phage predation either by having a small cell size or by growing so slowly as to make infection inefficient. But the surface area and volume of SAR11 cells are comparable to those of Prochlorococcus spp, where multiple phages have been isolated. The contribution of SAR11 to bacterial heterotrophic production in the open ocean is greater than their relative abundance which suggests that SAR11 does not avoid viral predation by growing slowly.

Zhao et al (2013) reported the isolation of diverse SAR11 viruses belonging to two virus families in culture. They proposed the name ‘pelagiphage’. The pelagiphage genomes were highly represented in marine viral metagenomes, which indicated their importance in nature. One of the new phages, “HTVC010P”, represents a new virus subfamily more abundant than any seen previously and may be the most abundant virus subfamily in the whole biosphere. This discovery disproves the theory that SAR11 cells are immune to viral predation and is consistent with the alternative explanation that the success of this highly abundant microbial clade is the result of successfully evolved adaptation to resource competition.

Zhao et al (2013) propose that the dominance of SAR11 in marine bacterial communities is a result of their superior competitiveness for nutrient uptake. High host abundance can provide protection against population decimation by viral predation.  For example high population densities increase encounter rates with infective phage particles, but they also increase recombination. Recombination enables genomic elements to pass in populations more rapidly than is possible by a sexual reproduction and thus offers an advantage to the spread of immunity. Furthermore, recombination rates in SAR11 populations were reported previously to be among the highest on record. This is what led Zhao and co to propose that high recombination rates in SAR11 populations allow for rapid adaptation to novel phage phenotypes. This mechanism for co-evolution and co-existence of pelagiphage and SAR11 is archetypal of the Red Queen Hypothesis,  an evolutionary theory which where organisms must constantly adapt, not just to gain reproductive advantage, but also simply to survive while pitted against ever-evolving opposing organisms in an ever-changing environment

I think this study is important for understanding plankton ecology because the authors emphasise the potentially important role of top-down mechanisms in predation, thus determining the size of SAR11 populations and their concomitant role in biogeochemical cycling. In summary this study describes the isolation and subsequent culture of several viruses that infect SAR11 from water samples taken on the Oregon coast and off Bermuda. Metagenomic analysis reveals that these 'pelagiphages' are abundant in the Pacific Ocean. These findings argue against a recent hypothesis developed to explain the success of SAR11 — that they might be immune to viral predation. Rather, the authors suggest, SAR11's dominance may reflect successfully evolved adaptation to resource competition.

http://www.nature.com/nature/journal/v494/n7437/abs/nature11921.html

Survival of Faecal Microorganisms in Marine and Freshwater Sediments.

Survival of Faecal Microorganisms in Marine and Freshwater Sediments.

 

Marine and freshwaters can contain an assortment of pathogenic and non-pathogenic faecal microbes. Faecal contamination can come from a number of sources such as sewage effluents from farming livestock (Sheep, cattle, pigs, etc.), human populations and wildlife. These microbes can pose a significant health risk to bathers who use these waters for recreation. Illness and disease can occur when an infective dose of the pathogen is able to enter the body and colonize. Monitoring and maintaining a safe level of water quality is therefore important.

 

The study by Davies et al (1995) looked at the survival of faecal microorganisms in marine and freshwater sediments in the vicinity of Sydney’s three deep-water ocean outfalls (see paper for exact location of sampling sites). The study looked at the survival of culturable faecal coliforms, faecal streptococci and Clostridium perfringens spores from sites near sewage outfalls. Three different studies were carried out to analyse survival rates: a predation study in the presence and absence of predators, an in situ microcosm survival study and a VNC (Viable but non-culturable) survival study. For the full details of the experiment please read the materials and method section of the paper.

 

The study’s key findings showed that in the predation studies, inhibition of protozoan predators with cycloheximide allowed the faecal coliforms to grow in the sediment whereas in the presence of predators a net die-off occurred. The study also showed that C. perfringens spores did not appear either to be affected by predators or to die off throughout the duration of the experiments (28 days). The situ survival experiments showed that all the test organism except C. perfringens, die-off to 10% of their initial numbers. This die-off occurred in both the marine and freshwater sediments within the 85 days of testing. The usual exponential decay model could not be applied to the survival data except for the faecal streptococci data. This was thought to be due to the complex interrelationship between predation and growth. In the NVC survival study the proportion of culturable E.coli remained the same for the entire duration of the experiment (68 days). This suggests that the sediment does provide a favourable, nonstarvation environment for the faecal bacteria.

 

Overall the study shows that in the absence of predators faecal coliforms may be capable of growth in marine and freshwater sediments however in natural conditions where predators are present a net die-off does occur. The study also suggests that C. perfringens where not preyed upon as much resulting in no net die-off. The NVC experiment suggests a survival strategy to overcome unfavourable conditions while still maintain the ability to form colonies. These results suggest that the bacteria can survive for extended periods of time in the marine sediment however as marine sediment is more often favourable for growth this strategy is unlikely and growth and predation more probable. To conclude, marine and freshwater sediment does provide favourable conditions for faecal bacterial to grow and it can be possible for extended survival of bacteria in the sediment. However the most likely outcome is for predation to result in a net die-off for the bacteria.

 

Cheryl M. Davies, Julian A. H. Long, Margaret Donald and Nicholas J. AShbolt (1995) Survival of Faecal Microorganisms in Marine and Freshwater Sediments, Applied and Environmental Microbiology. Vol 61, No 5 Pg 1888-1896

 

 

 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC167451/pdf/611888.pdf

 

-->

A light-driven sodium ion pump in marine bacteria

Light-driven proton-pumping rhodopsins are widely distributed in many microorganisms. For example, proteorhodopsin is a membrane protein that contains retinal, during light absorption conformational changes cause protons to be transported across a membrane. This results in an electrochemical gradient across the membrane, which is utilized in ATP synthesis serving as an energy source of the cell.

Keiichi Inoue et al (2013) describe a new functional class of a microbial rhodopsin, a light-driven sodium ion pump. They found that flavobacterium Krokinobacter eikastus possesses two rhodopsins, KR1 and KR2.

KR1 is a ‘classic’ proton pump, and likely plays a role similar to that of proteorhodopsin, (as described above). However the work conducted by Keiichi Inoue and friends (2013) describes KR2 as being able to pump sodium ions ‘outward’. In addition to functioning as a sodium pump KR2 has the ability to act as a pump for lithium ions as well. However when presented with potassium chloride or salts from larger cations it converts to a proton pump.

The data presented in this study is indicative that KR2 is a compatible sodium ion-proton pump. Additionally spectroscopic analysis revealed that binds sodium ions in its extracellular domain. The authors put forward that these findings are suggestive of light-driven sodium pumps being equally important in situ as their proton-pumping counterparts.

This research conducted by Keiichi Inoue et al (2013) is not only interesting, but additionally valuable as it builds on previous work done on other flavobacteria making the discoveries found here comparable. Again it’s interesting how far research in this area has come sine the work conducted by Beja et al (2001).

 http://www.nature.com/ncomms/journal/v4/n4/full/ncomms2689.html

Oil symbiosis in cold seeps!

Cold seeps arise in waters rich in methane and/or sulphides which come from the geology of the ocean floor. Life exists in these deep sea hot-spots due to the activity of two types of chemoautotrophic bacteria; Sulphur oxidisers and Methane oxidisers. These bacteria are symbionts to tube worms and mussels and can be found densely packed within specialised tissues. Many cold seeps have been studied in the north of the Gulf of Mexico but not many in the south. A new type of cold seep was discovered recently which is covered with solid asphalt. Asphalt flows are formed by the seepage of hydrocarbons which settle and remain on the sea floor. This study aimed to characterise symbiotic fauna of this new site (Chapopote) using molecular methods and stable carbon isotopes analysis and comparing the results to those found in cold seeps in the north of the Gulf of Mexico.

They found that the symbiotic relationships are very similar (if not identical) to those found in the north. An Escarpia sp. tube worm was identified with two species of Bathymodiolus mussels, all of which had intracellular sulphur-oxidising symbionts that are found in association with the same species in the northern cold seeps. Both mussel species contained methane oxidising symbionts identical to those found in the north. Until now only methane and reduced sulphur compounds have been shown to power cold seep chemosynthetic symbioses. However, novel hydrocarbon-degrading bacteria belonging in the Cycloclasticus genus were found in close relation to the mussel Bathymodiolus hecherae which also had genes for the metabolism of aromatic compounds, suggesting the utilisation of heavy aromatic hydrocarbons in addition to sulphate oxidation.

This study was very interesting and links Grahams and Colins lecturers. It reinforces the opportunistic nature of ‘nature’ and provides the first evidence of hydrocarbon symbiosis in a cold seep environment. 

Raggi et al. (2012) Bacterial symbionts of Bathymodiolus mussels and Escarpia tubeworms from Chapopote, an asphalt seep in the southern Gulf of Mexico

Bacteria helping corals survive oil pollution

Oil pollution as a result of anthropogenic activities is a major cause of damage to corals and is becoming a worldwide problem. The presence of crude oil causes sections of the exoskeleton to weaken and break off of the coral surface, the remaining tissue of which is then degraded. Oil pollution has also been observed to have detrimental effects on the zooxanthellae that are endosymbiotic to corals, thus resulting in coral bleaching. However some corals in the Arabian Gulf, which are constantly exposed to crude oil released from cracks in the seabed, surprisingly appear to be in a perfectly healthy state despite these conditions. Al-Dahsah and Mahmoud (2012) investigated the hypothesis of oil degrading bacteria being present on coral as an adaptive mechanism to enable the survival of oil in the surrounding environment.

The sample sites included two locations in the North Arabian Gulf. Qaro Island is an offshore reef system polluted by oil seeping from cracks in the seabed and Um Al-Maradim Island in South Kuwait is an unpolluted site that was used as the control.  Coral and mucus samples were collected from five colonies of the corals Acropora clathrata and Porites compressa at each site. Oil degrading bacteria was isolated both from fresh samples and from Porites compressa that had been enriched using a microcosm. These bacteria were tested for growth in a range of aliphatic and aromatic hydrocarbons and their ability to attenuate crude oil. DGGE was also used to determine the variation and richness of the bacterial populations.

They found the mucus and tissue from both sites to contain bacteria capable of degrading a wide range of aliphatic and aromatic hydrocarbons and crude oil. Although the control site appears to be clean by eye, it is in fact exposed to crude oil as a result of being transported by water currents, wind and waves from polluted regions and therefore appear to have obtained oil degrading bacteria to enable them to remain in a healthy state. The predominant bacteria were found to be associated with Gammaproteobacteria, Actinobacteria and Firmicutes. Increases in the concentration of crude oil caused a shift in the populations to include more oil degrading bacteria, suggesting that stressful conditions such as oil exposure may cause corals to change their residential bacterial communities or even incorporate exogenous bacterial communities obtained from the surrounding environment. This evidence suggests corals do have adaptations of oil degrading bacteria that enable them to survive oil pollution. Additional research investigating the viruses transferring the genes that enable the degradation of hydrocarbons would be of interest to gain further knowledge of these adaptations of corals.

Al-Dahash, L.M. and Mahmoud, H.M. (2012). Harboring oil-degrading bacteria: a potential mechanism of adaptation and survival in corals inhabiting oil-contaminated reefs. Marine Pollution Bulletin. Available online at: http://www.sciencedirect.com/science/article/pii/S0025326X12004298