Evidence for a persistent microbial seed bank throughout the global ocean

This paper attempts to answer the question, “Do bacterial taxa demonstrate clear endemism?”, are they restricted to specific geographical ranges to the extent that macroorganisms are, or can one sample site contain the total phylogenetic diversity of the world’s oceans.

The microbial community within an environment at any given time is determined by current environmental and biological factors, so it changes across space and time as niches open and close. Different species bloom and then decrease in certain conditions, but they must all be present at low levels for these blooms to occur, in what is known as the “microbial seed bank”.

For this study, across a 6 year period, around 10 million 16S rRNA sequences were recorded at a deep water sampling site in the West English Channel, known as the L4-DeepSeq dataset. Their first finding was that all operational taxanomic units (OTUs) identified throughout the 6 years were present at any time point within the 6 years. All taxa were present at all times, but their abundance varied dramatically. These results contrast the widely accepted model that it is presence/absence of taxa that controls community structure, suggesting instead that all taxa persist and it is their abundances that control the community composition.

From this they suggest that sufficient sequencing would allow the testing of the null hypothesis “all bacteria are found in any particular environment because of an immense and persistent microbial seed bank”, this would be falsified by evidence of clear endemism, the lack of certain taxa in some environments.

In order to begin investigating this, they compared the L4-DeepSeq dataset with the global International Census of Marine Microbes (ICoMM), which contains 356 datasets of bacterial 16S rRNA sequences ranging from pelagic and sediment samples to mangrove and sponge environments.
They found that the L4-DeepSeq dataset overlapped significantly with ICoMM datasets, containing 31.7-66.2% of taxa in any given ICoMM biome. They also discovered the relationship that the greater the sequencing depth in L4 samples, the greater their overlap with global ICoMM data. Sequencing depth here does not mean depth in the ocean but the number of sequences within a sample. By extrapolating the relationship, they suggested that 100% overlap between L4-DeepSeq data and ICoMM global data would be achieved when a 16S rRNA sequencing depth of 1.93x10¹¹ reads was reached. Achieving this would require over 200L of seawater to be filtered, rather than the 2L per sample used in this study, in order to collect enough cells to allow a sequencing depth that great. It would also cost an estimated $1.34million in sequencing costs. Regardless of the feasibility of such a study, it seems incredibly implausible that 100% overlap would ever truly be found, I think it far more likely that the relationship between sampling depth and phylgenetic overlap would at some point ‘level off’, where increased sequencing effort would cease to produce additional operational taxomic units. Though it seems the opinion of the authors that every taxa will be able to be found in every environment, if the sequencing depth is great enough.

The main value of this paper is not so much that it proves the existence of microbial endemism, as such a thing seems obvious given the existence of highly specialised extremophiles. It is valuable because it shows that an arbitrary sample of a marine microbial community overlaps on average 44% with other samples from varying marine environments around the world, with the suggestion that increased detection resolutions may show even greater overlap. So rather than 100% of taxa being present in all environments, I think they provide evidence for a global seedbank for a large number of cosmopolitan taxa.

One weakness of the study in my opinion is that comparing one sample point in Britain against many sample points from around the world, and generating the overlap percentages is not the best method for investigating global taxa overlap of communities. I think that comparing each sample point in the ICoMM to every other point in the ICoMM would give a higher average overlap, rather than using L4-DeepSeq as the central reference point. The average overlap between all samples is likely greater than the average overlap between L4-DeepSeq with each other sample.
The use of extreme habitats such as hydrothermal vents, cold seeps and estuaries likely pulled the average down due to their large number of specialised and endemic taxa. Excluding them from the dataset may produce stronger evidence for a global microbial seedbank for the general ocean.

Gibbons, S. M., Caporaso, J. G., Pirrung, M., Field, D., Knight, R., & Gilbert, J. A. (2013). Evidence for a persistent microbial seed bank throughout the global ocean. Proceedings of the National Academy of Sciences, 110(12), 4651-4655.

The effect of shrimp chitin on the life cycle of Vibrio cholerae and the subsequent transmission of cholera

The bacterium Vibrio cholerae O1 causes epidemics of the disease cholera, and incidences have been well-documented in areas such as Bangladesh. This pattern is particularly clear at two main seasonal peaks each year, which coincides with plankton blooms in the spring and in the autumn. Chitin, a highly abundant substrate in the marine environment, is thought to affect the population dynamics of V. cholerae, and it is mainly found in the exoskeletons of crustaceans. These invertebrates feed on the highly abundant zooplankton during the blooms, which act as a reservoir for the bacterium. Chitin is heavily colonised by the chinolytic V. cholerae, which breaks it down into soluble constituents that can be further colonised by these bacteria.

                   

 There are three stages to the life cycle of V. cholerae; the non-culturable state, the reservoir, and the culturable, toxigenic stage which results in cholera transmission. However, the mechanisms and the capacity of the bacteria to regain their culturability are not well understood. Shrimp chitin from the estuarine of Bangladesh was used as the sole nutrient in two different types of microcosms (artificial ecosystems created for this experiment), the Mathbaria water (MW) and Mathbaria water supplemented with chitin chips (MW-CC), to determine how chitin influences both the natural cycle of V. cholerae and seasonal occurrence of cholera in this area of Bangladesh.

DFA Counts

In the initial counts for Direct Fluorescent Antibodies (DFA), there were similar numbers of V. cholerae in the MW and MW-CC microcosms. However, a gradual reduction in counts was found in both microcosms in the subsequent weekly intervals, with higher cell count for a longer duration in MW-CC (Cells in MW water were only counted for 49 days). The cells in the MW-CC microcosm remained culturable for longer than 174 days (when growing on LB agar), but the counts declined to <10 before the next interval at day 189.

Multiplex-PCR

Two particular toxigenic genes in V. cholerae, wbe and ctxA, could still be amplified using Multiplex-PCR (M-PCR) up to day 174 for MW-CC, but only for 49 days in the MW microcosm. However, the M-PCR only shows the presence of multiple genes, and do not mention whether the genes are expressed or not. It may be more appropriate to use reverse-transcriptase-PCR (RT-PCR) to give a more accurate length of time that the toxigenic genes are expressed for and hence how long V. cholerae remains in this life stage.

MW with Added Shrimp Chitin Chips

Chitin degradation was recorded for six months, and it was observed that chitin became degraded after the chips were initially colonised. During the experiment, it was noted that large numbers of cells formed clusters of biofilm, and after further degradation, the majority of the V. cholerae bacteria were embedded within the biofilm up to day 189, although there was still some further colonisation of the degraded residue. V. cholerae remained culturable up to 174 days, but no longer showed active growth by day 189.

Addition of HCl

The chitin biofilm formed by the bacterium was thought to act as a shelter from harmful environmental conditions, as it is suggested that V. cholerae can survive the stomach acid in humans when they consume drinking water from the Mathbaria waters containing chitinous material. When concentrated HCl was added, it was found that the homogenate of the chitin biofilm still had 10⁴ cfu/ml of toxigenic cells present. These results could be supported further by repeating the experiment in the control MW microcosm as a comparison.

The study gives support for the ability of V. cholerae O1 to persist in the plankton reservoir between epidemics using chitin in estuarine water, which shows that this biopolymer keeps the cells in the exponential active growth phase for a longer duration. The V. cholerae that are normally found in the environment are typically toxigenic, but the chitin acts as a food source to activate the toxigenic stage. However, this paper seems to assume that chitin degradation is the single cause for the activation of toxigenic V. cholerae, even though there could be a multitude of interacting factors involved, and the authors make many inferences biased on their experiments. 

Ellie Vaughan & Dave Watt

Nahar, S., Sultana, M., Naser, M.N., Nair, G.B., Watanabe, H., Ohnishi, M., Yamamoto, S., Endtz, H., Cravioto, A., Sack, R.B., Hasan, N.A., Sadique, A., Huq, A., Colwell, R.R., and Alam, M. (2012) Role of shrimp chitin in the ecology of toxigenic Vibrio cholerae and cholera transmission. Frontiers in Microbiology, 2(260). doi: 10.3389/fmicb.2011.00260

Stop releasing nutrient rich-wastewater directly into your coral reef if you want to save it from diseases and bleaching!

As the first slide of our last lecture on coral diseases describes, disease epizootics do not occur just by pathogens infecting the host and producing the disease. However, it is the result of complex set of interactions between various factors such as immune status of the host, pollution, virulence of pathogens and many others. Nutrient enrichment and subsequent eutrophication of the ecosystem is one of the factors that may induce the occurrence and severity of diseases in a variety of organisms. Caribbean Sea has been witnessing frequently occurring epizootics of numerous coral diseases and mass bleaching events, both of which are reducing the coral cover of Caribbean. Many previous studies have shown significance of nutrient enrichment in coral health, diseases and its susceptibility to bleaching. Nevertheless, there is yet no experimental evidence that nutrient loading encourages frequency of coral bleaching and disease in the field.

            This in situ experiment investigated effects of chronic (long-term) exposure of nutrient loading on a coral reef in Florida Keys, USA, as an assessment of impacts of anthropogenic nutrient pollution on the reef. The experiment was conducted by enrichment of phosphorous and nitrogen for a period of three years.

The results revealed that nutrient loading caused disease outbreaks on the coral reef, with the dark spot syndrome (DSS) being the dominant disease. Nutrient enrichment caused more than hundred percent increase in the occurrence of DSS in a common Caribbean coral Siderastrea siderea. It increased not only occurrence of a disease but also its severity. This study confirms that nutrient enrichment is one of the etiological factors for this disease and probably other diseases as well. Though such a link between nutrient enrichment and coral diseases has previously been suggested; separating it from other stressors and experimentally prove it as one of the key etiological factors for diseases and bleaching is challenging and the authors have discussed these challenges.

This study reinforces this link and proposes “nutrient loading” as a critical etiological factor for disease progression in many corals, even in the absence of other etiological factors. Surprisingly, nutrients also increased the prevalence of bleaching in a coral Agaricia spp. under normal temperature conditions. Nutrients may lower temperature threshold at which bleaching usually occurs in this species of coral. Explanation of this nutrient enrichment-induced bleaching probably lies in the abrupt elevation of zooxanthellae density within the host, which is prone to oxidative stress by zooxanthellae; making the host more susceptible to bleaching. Interestingly, this effect of disease and bleaching prevalence was no longer visible after nutrient loading was stopped.  

At present, DSS in S. siderea is a mystery. It is caused by whether a pathogen/s or by physiological disorder, is still unknown. Hence, even though, knowing nutrient enrichment increases prevalence and severity of DSS, it is difficult to answer how does it do that; as the disease causing agent is unknown. The authors hypothesized few mechanisms of how nutrient loading might increase the severity of DSS, based on possible scenarios of origin of DSS. In my opinion, the explanation that excessive nutrients may alter coral holobiont’s homeostasis and at the same time may also impair its ability to resist the pathogen (whether a pathogen causes DSS is not known but it is possible that an opportunistic pathogen, already present in the holobiont may become virulent under such conditions) seems likely.  

In future, finding the DSS causing agent will be very important. The authors suggested comparative metagenomics amplicon sequencing of bacterial, fungal and archaeal markers and continuous analysis of microbiomes and physiology of healthy individuals under nutrient stress until healthy individuals show signs of DSS. I think DSS causing agent would probably be a pathogen or a consortium of pathogens; whatever it would be, it would certainly be unable to produce DSS in the coral host, unless coral holobiont’s physiological status is compromised. This physiological status would include health of the coral animal, its bleaching status, its associated microbiota and any taxonomic and/or functional alterations in the associated microbiota.  

Vega Thurber, R. L., Burkepile, D. E., Fuchs, C., Shantz, A. A., McMinds, R., & Zaneveld, J. R. (2013). Chronic nutrient enrichment increases prevalence and severity of coral disease and bleaching. Global change biology. doi: 10.1111/gcb.12450. 

Marine Microbe Drug Production? An overview of 2013.

Drugs developed from natural products (NP’s - most commonly secondary metabolites) to combat widespread illnesses are common place; almost half of all anticancer drugs originate from NP’s. Microbial targets have contributed heavily to this stock of NP’s but traditionally only terrestrial microbes have been exploited extensively. NP discovery rates have been falling since the mid 70’s and now investigations into marine microbe natural products (MMNP’s) are being stepped up as the increase in antibiotic resistant pathogens, or ‘superbugs’, is causing growing concerns for public health.

MMNP’s are already undergoing clinical/pre-clinical trials, many providing promising treatments for many types of cancers but there is still a global deficit of new drug development from NP’s. The largely untapped reservoir of candidate marine microbes presents a large scale source of potential MMNP’s to combat persistent drug-resistant pathogens such as the Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) to new emerging threats such as Gram-negative New Delhi metallo-beta-lactmase (NMD-1) bacteria.

Marine microbes are notoriously difficult to culture, so new and innovative culture methodologies are paving the way to larger scale exploitation and production of candidate MMNP’s. A recent review by Xiong et al., (2013) discusses this topic as it currently stands and outlines new pretreatment strategies for physical isolation of specific microbes along with refined culture mediums and alterations of ‘classical’ culture approaches. These advances have allowed the culture of previously uncultivated microbes e.g., the SAR11 marine bacterioplankton clade (Xiong et al. 2013: 702-704) but future investments in isolation and cultivation techniques will be vital to fully exploit the potential pool of microorganisms suitable for MMNP production. Xiong et al., (2013) also provides a valuable overview of innovative screening strategies being implemented to aid the discovery and development of MMNP’s. These valuable products have proven to be indispensable in past medical history so in order to generate and maintain an increasing NP discovery curve, efforts such as those outlined in the review by Xiong et al. (2013) will be critical to the future of NP’s and MMNP’s in industrial scale applications.

Xiong, Z.Q., Wang J.F., Hao Y.Y. and Wang Y. (2013). Recent Advances in the Discovery and Development of Marine Microbial Natural Products. Marine Drugs 11, 700-717.

The plasticity of nitrogen metabolism in chemoautotrophic symbionts

I found this paper while I was looking for more research on the vent tubeworms that are dependent on microbial symbionts in order to survive. This paper focuses on the chemoautotrophic symbionts of the vent tubeworm Ridgeia piscesae and aims to determine whether the nitrogen metabolism of these bacteria show some sort of plasticity depending on the environmental conditions since the tube worms display a variation in the phenotype in different conditions as well.

Nitrogen is important for the growth and maintenance of organisms and an exogenous nitrogen source is required; however, dissolved organic nitrogen is rare around hydrothermal vent habitats and inorganic nitrogen is rather abundant (e.g. NO3- or NH4+). Nitrogen metabolism of the tubeworm symbioses is not well studied in comparison to carbon and energy metabolism, especially to what extent they utilise of exogenous ammonium (NH4+) is unclear. As found in Riftia tubeworms (e.g. Robidart et al., discussed in the lecture), most symbionts have roles in dissimilatory/assimilatory nitrate reduction (DNR/ANR) or in dissmilatory nitrate reduction to NH4+ (DNRA; gene glnA) as a source for reduced nitrogen for biosynthesis. However, it is unclear if there are variations in nitrogen uptake and utilisation by the same species of tube worm living in different habitats where ammonium and nitrate concentrations fluctuate dramatically.

R. piscesae are ideal model organisms since they occur in a range of habitat with NH4+ concentrations varying between 10 and 950 µM, and for this study, the authors collected two phenotypes of R. piscesae, long-skinny (LS) and short-fat (SF) which exhibit very distinct variations in size, tube shape and colour, and plume shape from two different locations. The LS phenotype occurs around low temperature vent fluids with lower NH4+, and the SF phenotype at rather high temperature chimneys with higher NH4+. However, both phenotypes identified to provide a habitat for identical symbionts as confirmed by 16S rRNA analyses. Gene expression of key enzymes involved in symbionts DNR and ANR, as well as host and symbiont ammonium assimilation from the two sites were examined.

Results showed that all Ridgeia hosts take up NO3- since it is the most readily available inorganic nitrogen source, and they use it via dissimilatory nitrate reduction pathways. It was also found that the LS phenotype directly uses NH4+ as major nitrogen source for the symbiosis, whereas the SF phenotype uses a certain DNR pathway for the synthesis of NH4+. A difference in genes expression between the two phenotypes has also been observed, for instance glnA expression was much higher in the LS phenotype and it can be assumed that this reflects an increase in enzyme production to enable the acquisition of NH4+ in colder conditions with less NH4+ around.

This study was the first of its kind to highlight the flexibility of the symbionts for nitrogen metabolism in changing availability of nitrogen sources and this could mean that the symbionts have great impact on the host’s fitness and survival which enables the symbioses to live in physiologically changing environments.

Liao, et al. (2013). Characterising the plasticity of nitrogen metabolism by the host and symbionts of the hydrothermal vent chemoautotrophic symbioses Ridgeia piscesae. Molecular Ecology.

Antimicrobial activity of bacteria isolated from haemolymph of bivalve holobionts

 The lens of the hologenome concept does not see the host and associated microbiota as separate entities, but rather as a single evolutionary unit, the holobiont. The principles underlying this idea lie in how all multicellular life has symbiotes, transmission of symbiotes, benefits of symbioses and how microbiota changes enhance the plasticity of the holobiont against environmental stress.

Bivalves are an ideal model for how microbiota protect marine invertebrate holobionts by competing with pathogens and producing antimicrobial compounds, because filter feeding exposes bivalves to many microbes. The microbiota of healthy bivalve haemolymph has been found to produce antimicrobials and could play an important role in protecting bivalve holobionts from infection by pathogens. This study investigated the antimicrobial activity of culturable bacteria isolated from the haemolymph of an oyster, clam, mussel and scallop species; bacteria with the strongest antimicrobial activity were also tested for cytotoxicity towards bivalve immune system phagocyte cells (hemocytes) and resistance to antibiotics commonly used in aquaculture.

On marine agar, haemolymph bacteria (HB) isolated from the more mobile bivalves Pecten maximus and Tapes rhomboides were almost below detection levels more frequently than those from the fixed bivalves Crassostrea gigas and Mytilus edulis. On average M. edulis had the highest HB concentrations and P. maximus the lowest. The paper posits that HB densities are individual and species dependent, possibly influenced by environmental factors. 843 HB strains in total were isolated from the bivalves and 26 created inhibition zones for at least one pathogen strain; most affected were Vibrio. This apparently differs from most antibacterial spectra, which are never as narrow as this; the specificity of HB antimicrobial activity suggests that the holobionts containing them have been selected because they inhibit common pathogens of bivalves. Other findings for potential further research include the lack of antimicrobial activity in any clam isolates and the rapid loss of HB viability and activity.

Anti Vibrio activity has potential applications in aquaculture, since this genus is a prolific pathogen of fish, molluscs and crustaceans. Oyster and mussel isolates had the most potent antimicrobial activity and were identified to be either the Vibrionales or Alteromonadales orders within the Gammaproteobacteria; nine strains were from Pseudoalteromonas, a genus known to produce bioactive secondary metabolites and increase survival in bivalves. However the strains isolated in this study are phylogenetically distinct from those already used in probiotics and have unique properties.

Hemocytes are short lived and die quickly when incubated in sterile seawater, but the presence of HB strains, their mortality actually decreased and some did in a concentration dependent manner. Reduction of hemocyte mortality by microbes has never been shown before and it demonstrates the role of HB in stimulating the immune system of marine invertebrates. Many HB were found to be resistant to tetracycline, the most common antibiotic in aquaculture, which could apparently be useful for picking out individual strains in future experiments.

This is only a preliminary in vivo study, so it seems to me that the extrapolating HB abundance to be species or fixed/mobile dependent is flawed, given that only four, very phylogenetically distinct species were used, leaving the results open to confounded by evolutionary differences. Future work should also look at how bivalves acquire their symbionts, as it would be interesting if they were acquiring them by filter feeding, the same activity that exposes them to pathogens. I am not aware of how they administer probiotics to bivalves in aquaculture, but it seems questionable that HB will necessarily be able to survive in the guts of other aquaculture species which obtain probiotics orally. The signalling pathways occurring between the HB and hemocytes should be investigated further, because it could represent a good model for how microbes and immune systems communicate with each other; for example, how do hemocytes recognise HB from pathogens? Are there pathogens which imitate beneficial bacteria to avoid phagocytosis?

Desriac, F., Le Chevalier, P., Brillet, B., Leguerinel, I., Thuillier, B., Paillard, C., & Fleury, Y. (2013). Exploring the hologenome concept in marine bivalvia: haemolymph microbiota as a pertinent source of probiotics for aquaculture. FEMS Microbiology Letters.

Bacterial DMSP Metabolism Inside Zooplankton

     Osmosis regulator, precursor to the climate altering gas dimethyl sulfide, cryoprotectant and occasional chess player; behold the famous dimethylsulfoniopropionate, or DMSP, a ubiquitous component of the water column and algal cells. The organic carbon in some phytoplanktonic species can be as much as 20 percent DMSP and once outside of a cell, DMSP can undergo a myriad of biological or chemical transformations. There are many free living DMSP-consuming bacteria (DCB) which cleave and demethylate DMSP for their carbon needs; 5 percent of microbial carbon demand is estimated to be fulfilled by DMSP alone. Most DMSP research has been aimed at its metabolism, but little has been done on the DCB associated with zooplankton; phytoplankton grazing is known to accumlate DMSP in copepod guts and thriving DCB communities have been identified in the copepod Acartia tonsa. This study predicted that since copepod associated DCB abundances and composition will vary with changes in which phytoplankton species are being grazed, then they should also change in relation to DMSP content of copepod diets. DCB abundances associated with A. tonsa were tested across five phytoplankton diets varying in DMSP content. The DMSP metabolising abilities of these bacteria were tested against 4 other carbon sources by measuring their growth rates, allowing a clearer picture of how DMSP is used by microbes and what marine adventures it undertakes in the environment.

     To ensure grazing behaviour was not a confounding factor, A. tonsa diets were standardised in carbon content and consisted of 5 different phytoplankton species, all of which had similar cell sizes. Despite prior knowledge that copepod consumption of DMSP rich phytoplankton can increase DCB abundances, there was no clear correlation between dietary DMSP content and DCB abundances. For example, abundances of DCB in copepods fed with DMSP rich Alexandrium tamarense algae were similar in copepods fed DMSP deficient Dunaliella tertiolecta algae. This could be because the DCB associated with copepods might be using other carbon sources; this is supported by previous work showing that bacteria associated with copepod faeces, bodies and habitats are usually from the same metabolically functional groups. DCB in this study were also shown to be able to grow on all the other substrates tested, with varying successes at assimilating either methyl or carboxyl groups from each carbon source treatment.

     Overall it appeared that these DCB associated with copepods were very metabolically flexible and whist not dependent on DMSP, they would still make opportunistic use of it when it became available, so this bacteria can maintain their populations even when DMSP is scarce. This link between DCB and copepods highlights a potentially large sink for DMSP spanning across all oceans, given how both copepods and DMSP have cosmopolitan distributions. Further studies should give attention to larger scale temporal and spatial variations in phytoplankton communities and how they influence the communities of DMSP metabolising bacteria which are a crucial part of how DMSP moves and changes through ecosystems. These findings are especially interesting because they establish that there is direct link in the DMSP cycle between the zooplankton and marine bacteria; they also show that DMSP has more to offer than just being a chemotactic signal.

Yuan Dong , Gui-Peng Yang & Kam W. Tang (2013) Dietary effects on abundance and carbon utilization ability of DMSP-consuming bacteria associated with the copepod Acartia tonsa Dana. Marine Biology Research, 9:8, 809-814.