Bacterial Vesicles in Marine Ecosystems

Vesicles are known to be released from many heterotrophic organisms, gram negative bacteria release vesicles containing lipids, DNA, RNA and proteins during growth. Though the mechanism for this are not yet understood, though it can be influenced by many external factors. Vesicles have been shown to influence model organisms in many ways, but research up to this point has not uncovered their functions and abundance in marine ecosystems.

Biller et al. (2014) discovered what they deem to be a vesicle produced from the photoautotroph Prochlorochoccus  (and a strain of Synechoccus), and confirmed this finding using Negative stain and thin section micrographs and independently with nanoparticle tracking analysis of unperturbed cultures.

Prochlorochoccus are dominant photoautotrophs world wide with a global population of ~1027 cells and are the most abundant photoautotroph in ogliotrophic oceans (Synechoccus is the second).

Biller et al.’s experiments with the vesicles found that vesicles were produced during light and dark periods, with production consistent with other vesicle producing microbes. And the initial data predicts Prochlorochoccus produces 10^27 – 10^28 vesicles per day, though this is likely an overestimation.

Similarly to other vesicles Prochlorochoccuss’ contain lipopolysaccharides as well as normal cyanonbacterial lipids. They have rigid membranes, many proteins (though not known if all are functional), and importantly DNA.

Bacterial membrane vesicle release may account in part, for the relative abundance of membrane proteins among all dissolved proteins in seawater.

Supporting their findings with former field and lab data, observations of vesicle structures from coastal and ogliotrophic (Sagrasso sea) sites were classified as similar vesicles, though the origin was not confirmed through further analysis.

In the Sargasso Sea such vesicles were also found in and below the euphotic zone. Mechanisms dictating their distribution are unknown, but as vesicles and bacterial abundance correlate, they lean to the likelihood of microbial origin.

Prochlorochoccus are thought to produce large fractions of dissolved organic carbon (DOC) for oligotrophic regions, and these vesicles may be a contributing mechanism. Lower estimations of the initial data from this study predict Prochlorochoccus  supplies 10^4 – 10^5 tonnes of fixed C exported into the oceans per day. Also vesicle lipids have structural similarities to, and may account for a proportion of lipid DOC.

Biller et al. (2014) examined whether the Prochlorochoccus vesicles could support heterotrophic bacteria in culture and found that Alteromonas and Halomonas could both grow in sea water supplemented by Prochlorochoccus vesicles as the only carbon source. They contain about 1/100^th of a cells carbon and likely influence carbon movements in the microbial food web. The presence of proteins and nucleic acids infer that they influence N & P cycles as well.

Prochlorochoccus has adapted to have very low phosphorus and nitrogen demands, though why an organism would supply vital materials in harsh oligotrophic environments is puzzling. Vesicle functions could be that the growth of Prochlorochoccus is positively influenced by the presence of heterotrophs,, so supporting them increases Prochlorochoccus fitness. Vesicles could be diversions for cyanophages – their use as such confirmed by Biller et al.s’ experiments.

As a purpose or side affect, they will also influence microbes through horizontal gene transfer. Vesicles have been shown to facilitate horizontal gene transfer before in Escherichia coli and Acinetobacter. Metagenomics of “wild vesicles found DNA pool of significant homology to 33 species phyla from the 3 domains. These sequences could reflect the export of prophage sequences within vesicles or DNA arising from phage infection of vesicles in the field. These vesicles could even assist microbes by providing a reactive surface!

It seems most likely that photoautotroph vesicles are important in many biotic and abiotic microbial processes in the sparse environment of ogliotrophic seas. I look forward to further research on this subject, and how this will fit in and help shape our knowledge of ocean processes.

Biller, S. J., Schubotz, F., Roggensack, S. E., Thompson, A. W., Summons, R. E., & Chisholm, S. W. (2014). Bacterial Vesicles in Marine Ecosystems. science, 343(6167), 183-186.

The EU Bathing Directive: More information required!

Although the current EU bathing directive has certainly had a positive effect on the condition of our beaches and waters there are still concerns, particularly when considering urban beaches, which are subjected to higher environmental pressure due to their location, but also experience seasonal increases in visitors increasing the risk of human exposure to possible infection.  One of the main concerns of the EU bathing directive has been to monitor and control pollution relating to sewage but when sampling methods have been designed, they have not taken into consideration possible small scale temporal variations.

Recent research has focused on this aspect in a study conducted in North West Portugal.  Four beaches were chosen, to be sampled hourly for a period of 11 hours, on several different occasions.  All samples were analysed to determine numbers of microbial indicators, Escherichia coli and intestinal enterococci. 

It was found that waters with lower salinity due to the beaches being closer to river run off had significantly higher numbers of both indicators.  In addition during the summer months, reduced rainfall lead to a higher concentration of microbes in the run off.  Wind direction was also found to have a significant influence on readings, driving contaminated surface waters from river mouths towards the monitored beaches at times.  As wind direction may change regularly, it is important that this be taken into consideration when samples are taken as this could have significant effects on the readings acquired.

Although water quality varied at all sample sites throughout the day, the highest readings for indicator species were generally found in the morning.  Readings could be as high as 5100 cfu 100 ml^-1, and as low as 21 cfu 100 ml^-1 with in one 11-hour period.  These readings could mean the difference between the beach being closed or being given a rating of excellent. 

The current directive requires a minimum of four samples to be taken from each bathing area during any season however there appears to be no specification regarding times that samples should be taken.  Previous studies have shown that if samples are taken five times per week they highlight 80% of non-conformity events however only 5% of these events were detected when sampling was monthly.

I believe that this study along with previous evidence highlights the need for not only an increase in sampling efforts to ensure that those beaches considered more at risk from pollution are maintained at a safe level but also a requirement to have a greater understanding of the local area.  If these studies were carried out on a more local basis rather than by a national organizations it would allow for a greater understanding of how daily variations and seasonal patterns may affect the cleanliness of bathing waters.  This could allow local authorities to better inform visitors highlighting the safest times for swimming or watersports.

Amorim, E., Ramos, S., & Bordalo, A. A. (2014). Relevance of temporal and spatial variability for monitoring the microbiological water quality in an urban bathing area. Ocean & Coastal Management, 91, 41-49.

Antifouling Bacteria in Sponges (Aplysina gerardogreeni)

Antifouling in marine environments as big business, but to date few microbes have been screened for this purpose, with fewer yielding extractable compounds. Marine microbes are exposed to myriad of environmental conditions and so are likely to hold many novel compounds, as well as being able too produce differing compounds in different culture conditions, and having a higher output of these compounds than could be gained from other sources (invertebrates, algae). Aguila-Ramirez et al. (2014) examined bacteria of the sponge Aplysina gerardogreeni (from a family well known for harbouring high numbers of associated bacteria) from the Gulf of California to assess its antifouling qualities.

Samples of 5g tissue were collected bimonthly from April for one year. Though the study does not specify from which region the samples were taken, and whether this was consistent across the study. Bacteria were isolated and cultured in soy broth, and tested for antifouling, antimicrobial, antimicroalgael properties. Bacteria that showed the greatest activity were also identified by partial sequencing of 16S rRNA gene fragments, and matched or compared to their closest recorded relative. This latter method is quite exclusionary, as some bacteria may in a  viable non culturable (VNBC) state, or just less efficient, in the soy cultures used – however as this study seems to ultimately be for commercial purposes, perhaps ease of access in regular mediums is part of the attraction.

The results showed Bacillus, Micrococcus, Paracoccus, Pseudobacter, Pseudovibrio, Psychrobacter, Staphylocuccus and Terribacillus to be present in A. gerardogreeni. These bacteria are reflective of it’s the varied habitats it is distributed in, including chemo-heterotrophs, aerobes or facultative anaerobes, with a fermentative or respiratory metabolism. Preforming functions from bioactive metabolites to protect A. geradogreeni from harmful microbes The latter being an antifouling function, which 50% of the bacteria were found to posses.

The most active isolates were closely related to the Bacillus species, which as Auigla-Ramirez pointed out falls in line with other such research involving this family found in different sources (nudibranch, microalage). Bacillus are widely studied for antimicrobial properties and produce a secondary metabolites synthesised n the ribosome which are especially effective against other members of the Bacillus family.

The temporal variation in activity ranged from the highest levels in February to the lowest in October, with Bacillus by far taking up the largest chunk of time. Other antifouling strains likely compensate for times of inactivity. The lack of activity in winter months indicate that the study has missed out cultures, likely due to VNBC, or/and that biofouling organisms associated with A. gerardogreeni are less prevalent at this time in the Gulf of California.

This study demonstrates some the rich treasure trove of tools waiting to be harvested from the ocean, and that those from sponges will hopefully help along antifouling technology. If there was a greater understanding of the likely symbiotic relationship between A. gerardogreeni and the bacteria which inhabit it, and the reasons fir such temporal variation (ecological or physiological), would most definitely help spur this research along. Use of metagenomics in this study would likely show many more antifoulant bacteria that that may no be able to be cultured, and perhaps change the temporal data gathered in this study. Especially following Deans latest post the A. gerardogreeni and the rest of the Aplysina family are likely more diverse than this study suggests.

Aguila-Ramírez, R. N., Hernández-Guerrero, C. J., González-Acosta, B., Id-Daoud, G., Hewitt, S., Pope, J., & Hellio, C. (2014). Antifouling activity of symbiotic bacteria from sponge< i> Aplysina gerardogreeni</i>. International Biodeterioration & Biodegradation, 90, 64-70.

 

Could global warming increase disease in shellfish farms?

Although we currently have a good understanding of how climate change will affect both physical and chemical processes within our oceans, we do not have the same level of knowledge when considering the effects of global warming on microbial agents responsible for disease.  The areas in and around the Irish Sea in particular are an important commercial source for a number of edible shellfish species such as lobster, edible crab and langoustines, and the farming industries in these areas are economically important.  These species are known to be susceptible to disease caused by marine microbes but it is not known to what extent these diseases affect sustainability in many cases, or how future environmental changes could alter these effects.

For diseases like haplosporidiosis, caused by bacteria of the Haplosporidium genus, which effects haemocytes, connective tissue and digestive gland epithelia in crabs and molluscs there is currently very little data, so possible future effects are unknown.  This disease causes high levels of mortality in infected species and this review highlights a definite requirement for research in this area to understand what conditions are required for this bacteria to thrive, or more importantly, not.

Hematodinium spp. are dinoflagellates that are known to be internal parasites affecting the hemolymph of crabs and lobster species, the disease is commonly known as pink crab disease due to the colour displayed in infected individuals.  It was recently discovered that the larvae of the hosts are also prone to infection and that it is not as previously thought, only the adults that can be infected.  This is of particular concern if global warming is to strengthen ocean currents possibly aiding the distribution of larval forms and therefore the parasites they may contain. 

A little more information is available for Vibrio spp. which include both human and shellfish pathogens.  We know that they generally prefer waters with temperatures of 15°C or above and salinities of less than 25ppt, meaning that costal areas and the species that reside there are at particular risk of infection.  The predicted increases in temperature of costal waters in addition to a decrease in salinity caused by an increase in rainfall due to a less stable climate will provide new areas for natural outbreaks.  It is also known that copepods in particular act as a reservoir for species such as V. cholerae and that they could also be expected to increase in numbers due to warming of costal waters.  There have already been cases of increasing sea temperatures being linked to Vibrio outbreaks in areas such as Chile, Peru and the Pacific Northwest of the United States, but again there are huge gaps in our knowledge of how widespread these effects are. 

It is unclear in many of these cases whether changes in environment will cause an overall increase in such diseases or if an increase in one area may be evened out by a decrease somewhere else.  There are certainly opportunities for research into not only the effects of temperature but also salinity and circulation / current patterns.  We must also consider the speed at which marine microbes may be able to adapt and evolve to allow them to survive in these changing conditions, this only increases the requirement to understand what part viruses may or may not play in gene transfer and control of outbreaks.  These problems could keep research facilities busy for many years, but can we get ahead of the game?

Rowley, A. F., Cross, M. E., Culloty, S. C., Lynch, S. A., Mackenzie, C. L., Morgan, E., ... & Malham, S. K. (2014). The potential impact of climate change on the infectious diseases of commercially important shellfish populations in the Irish Sea—a review. ICES Journal of Marine Science: Journal du Conseil, fst234.

Are globetrotting bacteria just hitching a ride or are they helpful?

The skin of any marine animal provides a unique interface between it and the environment.  Microbial communities found on the skin are known to cause disease in many fish and marine mammals, but could they also be an indicator of good health?

A team from Woods Hole has analysed skin samples from 56 Humpback whales, Megaptera novaeangliae.  This species is of particular interest as they are found in all of the worlds oceans, they also have huge annual migrations which will undoubtedly lead to changes in their environment, with temperatures varying between 6-25°C.  Using using 454 pyrosequencing of SSU rRNA genes, 23 major taxonomic groups were found to be associated with the skin of the humpback whales.  The majority of the bacteria found belonged to the Bacteroidetes and Gammaproteobacteria classes, and many skin samples also contained bacteria belonging to the Firmicutes and Alphaproteobacteria classes.  Two genera, Tenacibaculum (Bacteroidetes) and Psychrobacter (Gammaproteobacteria) were found to be abundant within the majority (97%) of whale skin samples, and collectively made up a large portion, 55–75%, of the skin-bacterial community.  Further analysis showed that differences in abundance of the Tenacibaculum and Psychrobacter spp. were linked to the geographic areas from which the samples were taken.   The same two species also significantly differed in abundance between samples taken from whales that were not actively feeding, in the Pacific Ocean and those who were feeding, in the Bering sea and Atlantic ocean. 

The sequences of the Tenacibaculum and Psychrobacter spp. appear to be specific to Humpback whales although they are closely related to sequences from bacteria taken from dolphins.  Although Tenacibaculum spp. have been associated with disease in fish, it is unlikely that this is the case with this particular species due to the abundance in which it is found on the whale skin.  It has been shown in other studies that Tenacibaculum spp. could actually act as a predator species and could have an antifouling effect, actually helping to keep the whales skin clear and healthy.

Of the two bacterial species, Psychrobacter spp. is globally more widespread and is known to be tolerant of a number of different environmental conditions including temperature and salinity.  Other Psychrobacter spp. are known to be able to produce cold shock proteins and it is therefore likely that the species associated with these whales can also manufacture such proteins to protect themselves against the extreme cold conditions experienced during migration. 

Further investigation is required to determine if the feeding state of the whales which cause them to change to catabolic metabolism while in oligotrophic tropical waters can affect the composition of the associated microbial communities.  While in this state there is a decrease in repair of skin cells, and immune response, which could be linked to decrease in wound healing leaving the animals prone to infection.  So could any of the associated bacteria be producing antibiotics that could aid any healing, or could they be predatory and therefore preventing harmful bacteria from taking hold?  It is important to understand how these bacterial species may be beneficial for humpback whales and also to determine if they are could these properties be exploited for human health or anti fouling products?

Apprill, A., Robbins, J., Eren, A. M., Pack, A. A., Reveillaud, J., Mattila, D., ... & Mincer, T. J. (2014). Humpback whale populations share a core skin bacterial community: towards a health index for marine mammals?. PloS one, 9(3), e90785.

Marine Macroalgae: Defence against bacterial pathogens

Macroalgae supports a wide range of organisms, most importantly in temperate marine ecosystems. They act as a source of food; provide substrata to settle on and protective environments for early life history stages for many invertebrates. As well as being habitat formers and primary producers. Recently, there has been a massive decline in overall biodiversity and loss of algal species in coastal marine environments. Evidence has suggested that macroalgae are under threat from diseases caused by bacteria, viruses and fungi, could be a major cause of the decline. An increase in disease is attributed to opportunistic pathogens that can take advantage of an already weakened host. Therefore, scientists have said that with the impacts of global climate change could result in a greater increase in the rate and severity of bacterial disease of marine macroalgae.

Disease phenotypes often include rotting, abnormal tissue development or changes in pigmentation that appear as blotches, spots, rusts or bleaching. Over the past decade, there has been a gradual increase in the reporting of these symptoms. As there is increasing demand for macroalage as food sources and potential biofuels, it is important that they are well studied.

Macroalgae lack a cell-based, adaptive immune response but do have defence capabilities. Bioactive secondary metabolites are used to regulate colonization of bacteria and other epibionts. These algal metabolites can alter the bacterial community by selecting for beneficial bacterial populations. They can also interfere with bacterial communication networks and gene regulation, in particular quorum sensing systems. Additionally, macroalgal associated bacteria shows signs of inhibitory activities against other surface colonizers, allowing bacteria to outcompete and displace other bacteria.

Pathogen-induced defences include specific recognition of bacteria, followed by a series of immune signalling pathways involving oxidised polyunsaturated fatty acids or oxylipins. However, contrasting data suggests that the signalling pathways are different depending on the species. Oxylipin signalling in red algae increases the expression of stress-related genes and triggers oxidative burst activity in kelp.

Virulence traits in bacteria are highly varied which include toxins, adhesion factors, and mechanisms for nutrition acquisition from the host and to evade host immune responses. Another important virulence determinant of bacteria is the ability to detoxify reactive oxygen species released during oxidative burst of algal cells. Marine bacteria commonly harbour antioxidants and are essential for the progressive virulence, especially in Vibrio species.

Host-bacterial interactions are often highly complex and depend on multiple factors including the state of the host, the pathogenic potential of the bacterium and whether there are any environmental stressors affecting the macroalgae. Tolerances of environmental parameters will affect the overall performance of the algae, making it potentially susceptible to microbial pathogens. Therefore, with environmental changes happening as a result of global climate change, the roles in which bacteria play in structuring the future oceans ecosystems needs to be extensively investigated.

This review included many examples of these processes, however mostly done on terrestrial plants which has then been applied to marine algae, which suggests that more research is needed on this topic. As they contribute to primary production in the oceans and therefore are at the bottom of the food web, all organisms will be affected by the decline in macroalgae populations and this is why I think this is an important area of research.

Egan, S., Fernandes, N.D., Kumar, V., Gardiner, M. and Thomas, T. (2014) Bacterial pathogens, virulence mechanism and host defence in marine macroalgae. Environmental Microbiology. 16: 925-938