Signalling-mediated cross-talk modulates swarming and biofilm formation in coral pathogen Serratia marcescens

Symbiotic relationships are formed between corals, dianoflagellates and associated bacteria. The stability and effectiveness of this symbiotic relationship determines the health of a whole reef ecosystem and its resistance to stress and diseases. Coral mucus is excreted onto a coral surface and mucus binds to bacterial receptors, this directly controls the composition of associated microbiota. The fate of mucus and its role in microbial communities are becoming clearer however less is known about microbe-microbe interactions within coral mucus. It has been suggested that microbe to microbe interactions within coral communities may have important functions relating to coral health and interactions with pathogens, some evidence has emerged showing that microbes associated with specific corals produce antimicrobials. One known mechanism in microbe-microbe interactions is exchange of small molecules. Cell to cell signalling and the resulting changes in the bacteria are known as quorum sensing. QS controls surface spreading, production of antibodies and exoenzymes, attachment to surfaces and timing of virulence gene expression. The aim of this study was to test whether bacteria associated with marine invertebrates and their endosymbiotic dinoflagellates, produce cell-to-cell signals capable of affecting behaviours in opportunistic pathogens by manipulating inversely regulated multicellular behaviours.

300 bacterial isolates from the mucus were screened with Chromeobacterium violaceum. Three luminescent reporters based on AHL receptors were used to analyse isolates which were capable of stimulating or inhibiting QS-mediated pigment. It was found that 4% of the tested bacteria were capable of affecting at least one reporter. In a similar study by Golberg et al. (2011), it was found that 30% of isolates affected reporters.  Thin-layer chromatography was used to further characterise 13 isolates. This was done to separate inhibitory from stimulatory QS activities produced by bacteria. Most of the tested strains produced one or two activities which co-migrated with AHLs of medium lengths. This shows a lack of complexity in the signals which is differs from other research.

The next few experiments focused on testing the behaviours of the isolates in the dual-species microbial consortia consisting of the coral pathogen S. marcescens PDL 100 and the isolate of interest. The hypothesis that marine isolates capable of affecting QS-reporters will also modulate behaviours from S. marcescens was tested. Ten out of the thirteen strains produced QS signals detectable with an Argobacterium tumefaciens reporter. This suggested that either QS activities detected with the Agrobacterium reporter are not AHLs or that in addition to QS signals; bacteria produce compounds which specifically disrupt swarming. The isolates were then inoculated with a wild-type Serratia strain and the PDL 100 strain. Five species inhibited swarming in the wild-type but not in PDL100. These results suggest that in addition to producing compounds that trigger responses in QS reporters, tested strains secrete other substances that inhibit swarming in S. marcescens. This is done by interfering with the regulation of the flagellar regulon or disrupting the synthesis of the surfactant.  It also suggests that mutant strains partially restore the ability to swarm and so will probably have similar signals; however the signals are usually strain specific.

The next hypothesis stated that in the absence of an interaction between a secreted compound from associated bacteria affecting global regulatory systems then there should be a measurable effect on biofilm formation by S. marcescens in the presence of the tested isolates. The results showed that a number of strains stimulated swarming and also inhibited biofilm formation by PDL100, even though growth of the pathogen in the suspension cultures was not affected. This suggested that the compound secreted by these organisms may target a global regulatory switch involved in the regulation of swarming and biofilm formation, without affecting growth of another organism. Not all strains showed the same results, some stimulated both swarming and biofilm formation, however some reduced both.

Finally, to test whether the isolates capable of affecting biofilm formation and swarming in the model opportunistic pathogen S. marcescens PDL 100, individual polyps were inoculated under laboratory conditions with the pathogen with or without the antagonistic marine isolates The results show that, PDL 100 can completely degrade the polyp within 3 -5 days. Pre-inoculation of the polyps with either a cocktail of isolates or a monoculture of  α-proteobacterium reduced the appearance of the disease symptoms in the polyps infected with the white pox pathogen. This indicates that the native microbiota associated with the invertebrates or their endosymbiotic dinoflagellates are capable of producing activities that reduce susceptibility of marine invertebrates to opportunistic pathogens.

Overall, there was no strong correlation between QS activities and the effect on swarming and/or biofilm formation by S. marcescens PDL100.

We found that was a significant paper as they used novel method, such as using the dialysis tubing (which isn’t mentioned here we had too much to talk about). The last experiment was the most significant as it showed some bacteria fully repressed the effect of pathogen damaging coral or anemones. However we found that the paper incorporated a number of methods, some of which are unnecessary. This made it confusing; also some methods are just mentioned in the results section and not the methods section. Sorry for such a long blog post, it was a very long paper!

This is a joint post by Georgia Smith and Sophie McKeeman relating to our seminar paper.

Alagely, A., Krediet, C. J., Ritchie, K. B., and Teplitski, M. (2011). Signaling-mediated cross-talk modulates swarming and biofilm formation in a coral pathogen Serratia marcescens. The ISME journal 5, 1609-1620.

http://www.ncbi.nlm.nih.gov/pubmed/21509042

The role of microorganisms in coral bleaching

The Role of Microorganisms in Coral Bleaching

This paper looks at how microbes, specifically Vibrio shiloi are the causative agents behind bleaching of the coral species Oculina Patagonica.  The secreted CaCO₃ exoskeleton, supporting the coral tissue layers, form a substrate not only for the photosynthetic zooxanthellae, but also for various bacteria, archaea and viruses. Environmental stressors are known to affect coral growth and survival and to be involved in bleaching of O. patagonica. for instance low salinity, sediment covering, exposure to excess heavy metals, species dependent poisons and changes in water temperatures have been shown to interfere with coral health. Recently, the influence of biotic factors, namely the infection by harmful bacteria, has been investigated in the context of coral bleaching..

In a related previous study, Kushmero et al. found a correlation between the presence of V. shiloi and coral bleaching.. In a further paper by Rosenberg and Falkovitz it is seen that the infection within the coral occurs by Vibrio after penetration of the epidermis, where the pathogen is thought to produce a peptide toxin which prevents zooxanthellae from photosynthesising.

Within the same study it was also noticed that the bleaching effects caused specifically by the Vibrio only occurred during the summer months when water temperatures are above the survival threshold of 25°C. With this in mind it is thought that the infection of O. patagonica by the Vibrio is temperature dependent, as during the winter months with water temperature <25°C these infections are not seen, but this does not mean that the vibrio is unable to survive as  V. Shiloi have a further trick up its sleeve in order to make sure it does not suffer mortality  in the inhospitable environment. The survival of  V. Shiloi is dependent on the bacterium’s ability to inhabit a particular  marine fireworm known as Hermodice carunculata (Sussman et al. 2003), and it is this that allows it to maintain life during the winter temperatures.

 After studying this particular phenomenon for over ten years, Rosenberg et al.  discovered that the corals had begun to build up a resistance to the V. shiloi which causes a problem within this line of research as the relationship between V. shiloi and the coral O.patagonica has been used as a model system demonstrating Koch’s postulates. This is a system designed by Robert Koch in 1984 based on 4 criteria that link a causative microbe to a disease. However, the development of resistance to V. shiloi is in contradiction to Koch’s first criterion, by this I mean that one of the criteria states  the microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms.

Further studies focus on two different approaches, one will look strictly at temperature and light, and how this affects the symbiotic algae by inhibiting photosynthesis and by favouring reactive oxygen species production and thus  promoting coral bleaching. Another direction will be to delve deeper into how high temperatures cause microbial shifts leading to the disruption of the crucial balance between symbionts and how this is linked to coral bleaching. Is it possible that resident microbial communities help the corals to build up  immunity to these pathogens as additional symbiotic function as in a study by Ritchie 2006  resident microbial communities most defiantly contribute to the corals resistance to harmful bacterial infection, so it is fair to assume that during the bleaching process the corals themselves lose their antibacterial qualities and have much greater risk to infection.    

Please see the paper below if you are interested further  in the paper this blog is based on.

Rosenberg E, Kushmaro A, Kramarsky-Winter E, Banin E, Yossi L (2009) The role of microorganisms in coral bleaching. International Society for Microbial Ecology (ISME) Journal 3: 139–146

From genome streamlining to symbiotic interaction: the curious case of UCYN-A

As discussed in previous posts and the seminar, symbiosis with a nitrogen-fixing bacterium is a smart strategy to deal with the low concentration of fixed nitrogen in oligotrophic waters. Thompson et al. (2012) focus on an uncultivated diazotrophic (“nitrogen-eating”) cyanobacterium (UCYN-A) and establish step by step its involvement in a symbiotic interaction by taking the reader through a series of hypotheses tested using state-of-the-art techniques.

First of all it is noted that UCYN-A has an unusual degree of genomic streamlining (the overall reduction in genome size via a number of mechanisms) and lacks photosystem II, RuBisCo and the TCA cycle. However, UCYN-A possesses all the necessary genes for nitrogen fixation, notably the nitrogenase gene (nifH), which in combination with the extremely reduced genome and the requirement for fixed organic carbon lead to the hypothesis that UCYN-A forms a symbiotic relationship to exchange fixed nitrogen for fixed carbon. 

To test this hypothesis, Thompson et al. first identified possible partners for a symbiotic relationship. Flow-cytometry was used to sort cells from raw seawater samples and UCYN-A-specific quantitative PCR revealed that 94% of the UCYN-A nifH genes were associated with photosynthetic picoeukaryotes (1-3 μm diameter cells). 

The next step was to further narrow down the identity of the photosynthetic picoeukaryote associated with UCYN-A. Firstly they needed to sort the cells from the picoeukaryote sample again in order to isolate only those cells that were positive for UCYN-A nifH. Amplification using PCR of the 18S rRNA gene of the positively sorted single cells revealed that the closest known relative was the calcareous nanoplankton Braarudosphaera bigelowii, which is a free-living photosynthetic prymnesiophyte (e.g. coccolithophorids). 

So a potential partner for symbiosis with the unicellular cyanobacterium had finally been identified. But do they really form a symbiosis and is there exchange of nutrients? Halogenated in situ hybridization nanometer-scale secondary ion mass spectrometry (HISH-SIMS) and nanoSIMS techniques were used to follow and quantify potential exchange of fixed compounds by introducing nitrogen and carbon isotopes. They observed that up to 95% of the fixed nitrogen were transferred from UCYN-A to the partner cell and UCYN-A received 1 to 17% of fixed carbon, which is consistent with the lower carbon requirements of a small slow-growing heterotrophic symbiont. 

Thompson et al. provide very convincing evidence for the symbiosis between a prymnesiophyte and a unicellular cyanobacterium. They hope this symbiosis can be used as model to study the absence of nitrogen fixing plastids, since this interaction is reminiscent of the endosymbiotic events leading to chloroplasts and mitochondria, and briefly mention the implications of their findings for nutrient fluxes.

This paper seems exceptional to me in the way that is unravels the whole story of the symbiosis between two organisms, from the hint in the unusual genome streamlining to quantification of exchanged compounds in the association with another cell. Moreover it reveals yet another element in the nitrogen cycle and further studies are required to fully understand the implications and the spatial and temporal scale of this symbiosis.

Thompson, A., Foster, R. & Krupke, A., 2012. Unicellular Cyanobacterium Symbiotic with a Single-Celled Eukaryotic Alga. Science, 337, pp.1546–1550.

Tidal Fluctuations: Exactly what you think, happens.

Fluctuations in marine population abundances can be attributed to a combination of intrinsic and extrinsic factors. For larger mammals or birds these factors include dynamics such as migration, birth and death rates and environmental influences.  However for microorganisms these factors are harder to determine. Factors such as nutrient availability and light intensity are commonly reported, however the specific effect of these dynamics are rarely studied. The research I am summarising aims to offer an insight into the effects of tidal fluctuations as an extrinsic effect on phytoplankton growth.

This study analysed fluctuations in coastal phytoplankton concentrations in the southern North Sea through the use of an automated mooring station. Chlorophyll fluorescence, suspended particle matter, salinity and temperature were measured at a resolution of 12-30 minutes over the course of nine years.

Water levels were found to fluctuate at 12hr and 15 day cycles, explicable by the semidiurnal tidal cycle and spring-neap tidal cycle respectively. In direct correlation to this chlorophyll concentration displayed daily and bi-weekly fluctuations, but also gradual longer term fluctuations over seasonal changes. Interestingly, Suspended Particle Matter (SPM) reached higher values in winter than summer, but gradually built up over the course of several years resulting in a spring bloom and sudden decline. 

The authors conclude that ‘high resolution monitoring programmes are essential to capture the natural variability of phytoplankton in coastal waters’ however I believe the results of this study are obvious common sense and the study has little significance.

Review: Blauw, A.N., Beninca, E., Laane, P.M., Greenwood, N., Huisman, J. (2012) Dancing with the Tides: fluctuations of Coastal Phytoplankton Orchestrated by Different Oscillatory Modes of the Tidal Cycle, PLoS ONE. Published Online.

Accessible from: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0049319

Coral microbiota: temperature and your inhibition

It has been found that there are bacteria which colonise the coral mucus layer and can act as a barrier to pathogenic colonisation. This is thought to be because of the bacteria-bacteria interactions between the pathogens and the microbiota associated with the corals. Previous studies have already found that bacteria which are attached to some form of surface are more likely to produce antibiotics than bacteria which are free living within the water column. The authors of this study have outlined that many of the bacteria associated with corals are closely related to bacteria which produce antibiotics.  

The authors of this study outline the need for study within the topic of antagonistic interactions between pathogens and the associated bacteria of the coral Montastrea annularis, and the relationships between rising sea temperatures and coral disease. This study found that antagonistic interactions were fairly common although it is noted that each individual isolate was only inhibited by a few other isolates. This suggests that each isolate has a specific other type or types of bacteria which it can inhibit and be inhibited by. The isolates themselves were predominantly α-proteobacteria and γ-proteobacteria, within the γ-proteobacteria the most common genera were Vibrionales and Alteromonadales. When subjected to temperatures of 25°C the data collected showed that most bacteria would show sensitivity to inhibition to at least on other bacterium. However, at temperatures of 31°C more bacteria were able to grow unchecked. Though there are indications that the bacteria which can be agonistic have the ability to inhibit the growth of other bacteria at both 25°C and 31°C.

An observation noted by the authors was that there was a group of γ-proteobacteria were far more specific in their inhibition as they primarily targeted α-proteobacteria. This could mean that these microbes have co-evolved together to produce an antibiotic that can only target the α-proteobacteria or it could mean that other bacteria have evolved mechanisms which render the antibiotic ineffective against them leaving the α-proteobacteria the only bacteria still sensitive to the particular antibiotic produced.

This does not bode well for coral populations in the future if sea temperatures continue to rise as it means that the inhibition of bacteria is lessened and therefore increases the chances of the coral protracting diseases such as black band or white pox disease.

The authors have stated the need for techniques to investigate this in situ to be developed so that we can more accurately understand how the antagonistic interactions in the microbial communities associated with the corals work. Further research also needs to be done in regards to the interactions of the nutrient availability, predation and the competitive interactions of the community so that we can achieve a more full appreciation for the ecology of Scleractinian corals and their associated microbiota.

Rypien Kl., Ward JR., Azam F., 2010, Antagonistic interactions among coral-associated bacteria, Environmental Microbiology, 12, 28-39

Are we missing half the viruses in the ocean?

Are we missing half the viruses in the ocean?

It has been assumed most seawater viruses that infect bacteria contain DNA, however the quantity of RNA infecting bacteria has never been measured. Viruses influence the ecology, evolution and mortality of plankton and there is increasing indication that there are a diverse range of RNA viruses that infect important members of the marine plankton food web. Therefore, this study was important in gaining a better understanding of marine viral dynamics, especially as RNA viruses predominantly target eukaryotes.  

The reason for there being little knowledge about the abundance of RNA viruses in the ocean is due to difficulties in the technical methods required to achieve this. Studies investigating the abundance of DNA viruses have previously used fluorescence techniques, however it has been found that as the stains differ in sensitivity, some are unable to detect individual virions that have a very small genome. Therefore, this study instead measured the relative mass of DNA and RNA and coupled this with estimates of the nucleic acid mass for each in order to calculate an abundance of marine RNA viruses.

The seawater samples were taken from Kāne Bay in Hawaii and immediately taken to the laboratory where they were filtered at 0.22μm and purified. A fraction analysis was then conducted and the nucleic acid from each sample removed. The study used fluorometry, as this provided the sensitivity required to assay the nucleic acids, and measured the DNA and RNA contents from each fraction separately. In order to prevent overestimation of the abundance of viral RNA they also used spectrophotometry to measure the mass of RNA and compared the results obtained from the fluorometry. Metagenomic analysis was applied to verify the RNA composition. This gave rise to specific ranges of RNA density, which were used to estimate the quantity of RNA virus genomes.

Due to losses encountered during the study, the number of viruses in the final sample was a minimal estimate and it was assumed this loss was similar for both DNA and RNA viruses. One of the main limitations to the methods was the fact that the filter used was 0.2μm, meaning some viruses were lost at the beginning stages of the study, perhaps causing bias against DNA viruses, since the largest known viruses contain double stranded DNA. Furthermore, they stated that the viral concentration method used was only tested on DNA, and just assumed that it would be as efficient for RNA viruses. This, combined with the fact they were more hesitant to allocate RNA than DNA viruses suggests the results may be biased towards DNA viruses.

Nonetheless, the findings from this study demonstrated that RNA viruses were more abundant than DNA viruses. The most dominant species present, with 50-57% of the reads from the viral fractions, were RNA viruses that infect eukaryotes, most of which were of the order Picornavirales, which other studies have found to be very diverse in seawater. Although there is a smaller concentration of eukaryotic plankton than bacteria in the ocean, these results suggest that eukaryotic viruses are as abundant as bacteriophages. Therefore if these results are a correct representation, it could have a large impact on the understanding of marine viral ecology.

This study provides evidence that the current fluorescence based methods employed to measure virus abundance may need modification in order to allow the smallest of viruses to be included in these counts. The method used in this study provides an estimate of the contribution of RNA viruses in the ocean and could be utilised to validate whether this is also representative of other marine habitats.

Grieg F Steward^1,3, Alexander I Culley^1,3, Jaclyn A Mueller¹, Elisha M Wood-Charlson^1,4, Mahdi Belcaid² and Guylaine Poisson²

The ISME Journal advance online publication 15 November 2012; doi: 10.1038/ismej.2012.121

Trichodesmium and Fe-cycling the possibilities are endless…

Iron is a nutrient that is associated with several processes, for instance nitrogen fixation, photosynthesis, DNA biosynthesis and electron transfer. It is considered a limiting factor because it is scarce in surface waters. Iron can be present in the open waters as organic iron, colloidal and soluble inorganic iron or even as complexes. Organic ligands which are molecules that binds to a central metal (in this case dissolved Fe) and makes a complex. These iron-ligand complexes are believed to be an important source of iron. There have also been observations of dust particles migrating into Trichodesmium colonies, suggesting it is an iron source.

Trichodesmium is an abundant cyanobacteria, found in open waters. They are responsible for a large majority of nitrogen cycling and have been related to the formation of new nitrogen pools. Trichodesmium lives in colonies that create a microbial consortium which is a mixture of different organisms with unique roles. The organisms commonly associated with Trichodesmium are bacteria, dinoflagellates, amoeba, ciliates and diatoms. Bacteria in particular are common and are known to be present in Trichodesmium colonies. Heterotrophic bacteria can produce siderophores (molecules released by bacteria to obtain iron) under iron limiting conditions, Trichodesmium have not been shown to produce any siderophores however it does up regulate an iron deficiency-induced protein an IdiA homologue under conditions of reduced iron availability.

In this study the rate of iron cycling was tested using laboratory cultures of Trichodesmium and two strains of Trichodesmium-associated bacteria. The two strains representative of Trichodesmium-associated bacteria used for the iron uptake experiments were an alpha proteobacteria from Roseobacter lineage Silicibacter TrichCH4B and a bacteriodetes Microscillia marina. The bacteria strains were supplied with various iron sources after acclimation to PC media where iron was the limiting nutrient. The cultures were non-axenic, which means they were not contaminated with any other living organism. The genomes of both M.marina and TrichCH4B have been sequenced through the Moore Microbial Genome Sequencing Project.

For determining Trichodesmium growth rates in Fe-low and Fe-High medium, aliquots of a single Fe-high Trichodesmium culture was gravity filtered onto a 3.0µm filter, rinsed twice and resuspended with either Fe-low or Fe-high R medium. The Fe-high and Fe-low cultures were then sampled over 3 weeks of incubation.

To insure that the Trichodesmium cells were healthy the authors measured the photosynthetic efficiency (Fv/Fm) after 2 weeks. A FRRFII sensor was used to measure the kinetics of chlorophyll fluorescence induction and decay in Trichodesmium.

The Fe uptake in Trichodesmium was measured using a labelled 55^Fe solution, which was added to the cultures and then incubated at 24ºc with a 12h light/dark cycle. To examine Trichodesmium Fe uptake during the diel cycle, uptake experiments were initiated by rinsing and resuspending all cultures at the beginning of the corresponding light or dark periods.

The Trichodesmium was gravity filtered onto a 3.0µm polycarbonate filter and rinsed. The amount of radioactivity on the filter was analysed. The Fe concentration was determined by converting 55^Fe activity to molar concentrations using a standard curve.

Cultures were maintained for a month but the first 16 days were used for the growth curves to show exponential growth. After day 6 the Fe-high and Fe-low cultures start to diverge and there was a Fe stress showed in Fe-low cultures. The exponential growth calculated for Fe-high was 0.12 and for Fe-Low was 0.08, which was clearly slower. The Fe-low culture was however able to survive longer (1 week) than the Fe-high culture. The decreases in Trichodesmium growth rates show that there is an indication of Fe stress in the Fe-low cultures. This was supported by the observations of morphological differences present in the Fe-low cultures compared to Fe-high cultures. The morphological features were longer trichomes containing more cells per trichome and individual cells that were longer and narrower.

To make up for bacterial Fe uptake in each sample, samples were size fractionated at each time point. In all the Trichodesmium experiments the filtration reduced the abundance of bacteria by 80% in the Trichodesmium fraction. Active Trichodesmium Fe uptake could then be calculated by subtracting the 3.0µm bacterial Fe uptake and the 3.0µm glutaraldehyde killed control.

The Trichodesmium associated bacteria were grown on PC medium which were grown in room temperature. They were under iron limiting conditions, created by transferring the culture 3 times through iron poor PC media. The iron limitation was confirmed by comparing the growth rate of subcultures of M. marina (36h) and TrichCH4B(20h).

The bacteria were rinsed and resuspended 3 times with chelexed artificial seawater . Bacterial 55^Fe uptake experiments were set up in triplicate with a 0.01% glutaraldehyde killed control and cultures were incubated at room temp in the dark.

Trichodesmium and the two bacteria strains were also analysed for carbon content.

The authors concluded that Trichodesmium was rationed to inorganic iron (FeCl₃) or iron in association with weak organic ligands (FeCit) in iron limiting conditions. However the bacteria strains were able to acquire iron from all of the Fe sources provided, although the uptake of iron by TrichCH4B was somewhat slower than M.marina. M.marina’s preference for iron complexes over FeCl₃ could be the result of siderophore receptors being upregulated in low Fe conditions, because it increases the efficiency of ferric siderophore uptake. Several theories were provided for the contrast between the bacteria and Trichodesmium, for instance because Trichodesmium is a diumal diazotroph its ability to acquire and transport iron might be expected to vary temporally. Other iron acquisition mechanisms were also considered such as iron reduction in trichodesmium or possibility of cell lysis to cycle within a colony.

The field colonies of Trichodesmium would contain bacteria; this environment could impact the nutrient and iron cycling. Diazotrophs need high concentrations of iron and combined with the limited iron accessibility of Trichodesmium it is possible a mutualistic relationship is present. This relationship would not have been represented in the laboratory experiments because the cultures were non-axenic. It is also possible that the Trichodesmium-associated bacteria which acquire iron by using siderophores may compete with Trichodesmium.

The authors created a model to understand the possible relationship between the Trichodesmium and the bacteria. It showed that bacteria directly competed with Trichodesmium during the 3 hour uptake incubations. Although the model shows competition, mutualism cannot be ruled out because even though Trichodesmium cannot access from strong siderophore sources they can acquire it elsewhere, for instance mineral dust. Trichodesmium and its colony-associated microbial community may differ substantially in terms of iron acquisition strategy.

I was interested in this paper because it focused on Trichodesmium, which in my last summary was used in a comparison with diatom-cyanobacteria complexes. It was interesting to look at its connection to nitrogen fixation through iron cycling. This paper was very detailed into the different methods of iron acquisition and the idea that trichodesmium-associated bacteria like TrichCH4B and M.marina are either competing or mutualistic would be very important in terms of colonial densities of Trichodesmium. If it is mostly competing, the possible knock on effect on the formation of new nitrogen pools in the open oceans, should be considered.

Roe, K.L, Barbeau K, Mann E.L, Haygood M.G, (2012) Acquisition of iron by Trichodesmium and associated bacteria in culture, Environmental Microbiology, 14(7), 1681-16951..

Prediction of Salmonella in seawater by total and faecal coliforms and enterococci

Microbial quality of marine bathing water in terms of public health has been concern for the general public for years. There as several directives for different countries to follow in order to monitor the microbial quality bathing water. The main directives include the European Union (EU) Framework Directive (2000) and the US Environmental Protection Agency (USEPA) Water Quality Guidelines. Indicator organisms are currently used to detect microorganism in bathing water rather than the direct detection of waterborne pathogens as this proved to be too expensive and time-consuming. The microorganisms used presently as faecal indicators include Enterococci and Escherichia coli. Enterococci have been found by major epidemiological studies to relate best to adverse health effects to bathers.

A large proportion of gastrointestinal symptoms occurring in the general population after contact with contaminated bathing water are due to Salmonella infection. However it has been found that faecal indicators in marine water have a low predictive capability for the presence of Enteroviruses, for example Salmonella.

This study looks at the ability of enterococci to predict the presence of Salmonella spp. better than total or faecal coliforms and that indicator bacteria limits set by the USEPA and the EU are satisfactory in excluding Salmonella from marine bathing water.

Samples were obtained, over a 24 month period, from 9 sites along the coast south of Athens, Greece. Three sampling sites were affected by sewage outlets and the other 6 were close to the mouth of a polluted small river. Total coliforms, faecal coliforms and Enterococci were enumerated by membrane. Total coliforms were then enumerated on Membrane Lauryl Sulphate Agar, mFC Broth Base plus agar was used for faecal coliforms. Finally enterococci were measured on Slanetz and Bartley medium. Salmonella detection was qualitative according to the World Health Organisation method (I can give you more details if you are interested).

The results show that 33 out of 240 samples were positive for Salmonella. Total coliforms were isolated from 209 samples, faecal coliforms from 192 and enterococci from 234. A statistical increase in frequency of Salmonella isolation was observed in an increase in densities of each of the 3 indicator bacteria. The percentage of Salmonella isolations depended on statistically significant increase on each indicator organism separately. The statistics used showed when TC or FC were used for Salmonella prediction in logistic regression, the addition of another indicator did not have a statistically significant effect. When enterococci were used for prediction, then the addition of one or two other indicators led to a statistically significant improvement.

Overall, this study shows that the presence of Salmonella can be predicted using total or faecal coliforms alone. Also Enterococci can be used to discriminate between the presence and absence of Salmonella. This can be statistically significantly improved by also looking at total or faecal coliforms.  The US EPA Water Quality Guidelines and the EU Framework Directive are suitable to find out if Salmonella is present in bathing water. If Salmonella is found to be present then total or faecal coliforms could be used to identify it more accurately. I think that this paper is significant as it backs up previous studies that have looked at the capability of faecal indicators to predict the presence of Salmonella. Salmonella can be a big health threat and so this study is important to the health of the general public.

Maria A. Efstratiou, Athena Mavridou, Clive Richardson. Marine Pollution Bulletin. Volume 58, Issue 2, February 2009, Pages 201–205.

http://www.sciencedirect.com/science/article/pii/S0025326X08004906

Is the ocean just one big “seed bank” of microbial diversity?

Many microbiology studies focus on differences in community “diversity” over time. The use of the word diversity and its real biological meaning is often not fully explained. Many recent studies which claim to have observed changes in community diversity have used some sort of diversity index to arrive at this conclusion, without discussing the limitations of using such statistical methods.

Caporaso et al. were specifically interested in the seasonal species diversity and richness changes at one sampling location in the Western English Channel. Previous studies have reported strong seasonal patterns in community dynamics at this location and the paper itself wanted to understand what was driving the patterns. They went about this by testing two opposing hypothesises; either the observed changes were driven by absolute changes in community composition (as the previous studies would suggest), or, that the changes were driven by differences in relative abundance of the same community members.

The paper used an entirely genomic method. They compared one very detailed sequenced sample (called the deep sequence) to many less detailed samples taken every month from January 2003 – December 2008 (called shallow sequences); the specific genome zone they looked at was V6 hypervariable region of our good friend the 16S rRNA gene. They found that when all the shallow samples were added together over the six year period, 95.47% of the operational taxonomic units were present in the single deep sequence. Furthermore when each shallow sample was compared individually with the single deep sample, an average of 99.75% operational taxonomic units present, were the same. They also used rarefaction (a statistical method to predict the maximum number of species actually present) and, despite analysing over 10 million 16s rRNA fragments, they did not reach the assumptotal predicted by rarefaction. This indicates that there is still some incredible rare species not yet discovered in this ecosystem.

Importantly this study provides evidence that traditionally observed seasonal changes diversity are actually driven by the relative abundance of the same taxonomic groups, rather than absolute changes in community as was previously thought.  Essentially this was revealed by very in depth sequencing which accounted for small branch length changes between classifiable and unclassifiable taxonomic units which traditional methods cannot detect. The findings suggest that the ecosystem at the sampled site has a huge core microbiome, which potentially changes the way we think about selection pressures and evolution. Is selection acting on the level of an individual microbe? Or is selection acting on the whole microbiome? Inside which we know genes are easily transferable via horizontal gene transfer. This study doesn’t provide any evidence for or against this, but I just thought I’d highlight it as a possibility…

Caporaso, J. G., Paszkiewicz, K., Field, D., Knight, R., & Gilbert, J. A. (2012). The Western English Channel contains a persistent microbial seed bank. The ISME journal, 6(6), 1089–93.

http://www.nature.com/ismej/journal/v6/n6/full/ismej2011162a.html

Symbiont Garden: Epibionts on Kiwa hirsuta

Most symbiotic relationships in deep sea hydrothermal vent systems are between an organism and endosymbionts which reside within bacteriocytes in the epithelium of the organism, however in decapods crustaceans the symbionts are epibiotic, living on the outside of their host. These usually occur on the setae of the decapods. Epibionts are particularly useful to their host as they can provide all stages of the life cycle with protection against pathogenic organisms and predators; this is done through the epibiont secreting compounds which facilitate the protection. In this article the authors investigate the trends between epibionts and decapods interactions and also investigate the possibility of epibionts that may be indigenous to invertebrates. They do this by taking samples of the epibionts from the setae of Kiwa hirsuta, a decapods crab found in the cracks along the Pacific Antarctic ridge where the temperature is relatively lower and also found within the mussel beds around black smokers where the temperature is tolerable to them, and using a comparative 16SrRNA analysis coupled with Fluorescent in situ Hybridisation analysed the phylogenies of the bacteria which were thought to be sulphur oxidising bacteria. The use of electron microscopy in order to assess the structure of the setae seemed to prove very insightful as it was then found that the bacteria only attached themselves to the chitinous outer layer of the setae and mostly at the distal end. The main morphologies of the bacteria included thick and thin filamentous communities, though it was noted that there was a distinct absence of structures which would allow for attachment of the filamentous communities to attach to the setae. This paper uses a variety of genes which are involved in the reductive Tricarboxylic Acid (rTCA) as well as the genes involved in sulphite oxidation and sulphate reduction to identify the potential function that the epibionts might perform for K. hirsuta. This does, I feel grant the papers results more power in determining the phylogeny of these epibionts and how they interact with the host organisms. As decapods crustaceans grow most need to undergo ecdysis to shed their rigid cuticle and grow a cuticle more appropriate to the new size of the organism, this raises the issue of how does the organism end up with new bacteria as the old onew would still reside on the cast off cuticle. The authors mentioned that it is possible that after ecdysis free living bacteria could colonise the newly moulted organism. They address the issue of recognition between the bacteria and the organism with the theory that the host could exude an exopolysaccharide which would facilitate the binding of epibiont to the host. They do admit that many questions remain about this process which should prompt the study of this mechanism more closely. The paper found that many of the bacteria residing on the setae of K. hirsuta are a species of Proteobacteria that possesses the correct enzymes to fix CO2 for the rTCA cycle and has the capacity to cycle sulphur species through the oxidative and reductive pathways, perhaps these bacteria are merely hitching a ride for the nutrition from the vents and in return the compounds that they create offer protection to their host? 

Goffredi SK., Jones WJ., Erhlich H., Springer A., Vrijenhoeck RC., 2008, Epibiotic bacteria associated with the recently discovered Yeti crab, Kiwa hirsuta, Environmental Microbiology, 10, 2623-2634

Proteorhodopsin in Flavobacterium

Light-induced transcriptional responses associated with proteorhodopsin-enhanced growth in a marine flavobacterium

Ten years ago, Beja et al isolated a bacterial rhodopsin. Proteorhodopsin (PR) was discovered through metagenomic analyses of marine bacterioplankton genome fragments.  PRs were subsequently detected in many other marine bacteria. Since then additional studies have found PR’s in a wide range of bacteria. PR-containing marine bacteria have been recently cultured from a variety of marine environments. These isolates include members of Proteobacteria and Vibrionaceae. Laboratory experiments examining light-stimulated growth in some of these bacteria have been uncertain. Some studies have detected no significant light enhancement of either growth rates in PR-containing bacteria whilst light-enhanced growth rates have been  reported in one PR containing marine flavobacterium, Dokdonia sp. MED134 (Gomez-Consarnau et al., 2007). Additionally studies have suggested that some of marine flavobacteria populations have exhibited increased expression of the PR gene in the presence of light. Gomez- Consarnau et al (2010) demonstrated the enhanced long-term survival of PR-containing Vibrio cells in light, but not in darkness. The specific metabolic processes that cause PR enhanced growth or survival are not yet well understood due to inconsistent results reported from studies.

To increase our understanding of the photo physiology of PR containing flavo bacteria, Kimura et al exposed MED134 to variety of nutrient conditions in light and dark. The effect of light on growth in low nutrient concentrations and the effect of retinal biosynthesis inhibitors on light-enhanced growth were also tested in this study. In addition, the effects of sodium inhibitors on light-stimulated growth were also examined.

This study saw a significant influence of PR on growth rate at low carbon concentrations, and its lesser influence at higher carbon concentrations in light. These findings are concurrent with previous work of Gomez-Consarnau et al. (2007), which showed that light, has an impact on the growth of bacteria grown in low carbon conditions.

Gomez-Consarnau et al. (2007) demonstrated that MED134 had a higher expression of PR gene in the light than in the dark. Kimura’s results extend this and indicate that transcription of the entire PR photosystem is upregulated in the presence of light in this bacterium with the use of Transcriptomic studies.

To support the results that PR is important in light-stimulated growth in MED134, Cultures were set up with MPTA, a inhibitor in the retinal biosynthetic pathway. Kimura et al saw that MPTA prevented b-carotene generation, the precursor for retinal. Next, MED134 was grown in new cultures amended with MPTA. Cells incubated with MPTA, grew moderately in the presence of light. In contrast, MED134 incubated without MPTA in light, produced significantly higher yields, there were no differences found in the dark. The findings suggest that PR bound to retinal has a critical role in light-stimulated growth.

Transcriptomic analyses revealed that several genes, encoding for membrane transporters, exhibited significant upregulation in the presence of light, in particular Na-translocating NQR in light. This indicates the importance of the sodium ion gradient for transport functions in light. To test the importance of sodium ion exchange, Kimura et al performed growth experiments with HQNO, the inhibitor of NQR. Cells were incubated in cultures with and without HQNO. In cultures without HQNO, there was significant growth of MED134 in the light. Cell yields in cultures incubated with HQNO in the light were about three times less than those grown in the absence of inhibitor. These findings indicate that the Na-translocating NQR has a critical role in sodium pumping in light stimulated growth.

This study’s findings are concurrent with results previously reported by Gomez  but this study also expands these results to give us a more complete picture. I think this study provides a great example of just how far metagenomic analysis has come in the last 10 years.

 Hiroyuki Kimura,Curtis R Young,Asuncion Martinez,and Edward F DeLong. (2011). Light-induced transcriptional responses associated with proteorhodopsin-enhanced growth in a marine flavobacterium. Available: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3176510/. Last accessed 10/11/2012