Thursday, 28 November 2013

Chemoreceptor VfcA Mediates Amino Acid Chemotaxis in Vibrio fischeri

Flagellar motility is an important form of movement in bacteria. In this way microbes in suspension are mobilised and may travel to locations more beneficial for their sustained growth. To determine which direction to travel, microbes follow chemical gradients, this is chemotaxis. Chemotaxis is the recognition of gradients and the alteration of locomotion in response to this, with the intention of moving up or down a gradient. Methyl-accepting chemotaxis proteins (MCPs) are located on the cell's surface and act as receptors for specific attractants. If the chemical target attaches to the MCPs then the CheAY two-component system is initiated. This results in a change of tumbling frequency, which in turn leads to net movement towards the attractant.

The Euprymna scolopes-Vibrio fischeri symbiosis is transmitted horizontally i.e. from the environment not from the maternal line. For this to work there must be a method of attraction to the squid to allow rapid colonization. The V. fischeri genome has previously been sequenced and this lead to the identification of 43 predicted MCPs. To better understand these, we can use mutant strains and compare the behaviour to the wild type. In this study 12 MCP mutants were discovered and a further 7 plasmid integration mutants were produced and their characterization was attempted.

In a plate based chemotaxis assay the mutant strains as well as the wild type V. fischeri were tested for responses to glucose, serine, N-acetylglucosamine (GlcNAc), N,N'-diacetyl-chitoboise [(GlcNAc)2] (both are chitin derived sugars), thymidine and N-acetylneuraminic acid (NANA). Only one of these mutants, vfcA, displayed any change in behaviour. It did not display a chemotactic response to serine but otherwise functioned as the wild types. vfcA is regulated by the flagellar master regulator FlrA so was thought to be a likely candidate for chemoctactic regulation. The lack of response to serine strongly indicates the involvement of VfcA in serine chemotaxis. In a capillary assay, performed in suspension, the vfcA mutant, again, showed none of the chemotactic response to serine that the wild type strains did, this further confirmed serine attraction as one of its functions. Serine attraction was restored in the mutant with heterologous expression of vfcA meaning that it it is the product not the gene that is responsible for the attraction.

To discover if other amino acids also induced vfcA mediated chemotaxis, a capillary assay was performed for all 20. Serine, cysteine, threonine and alanine all displayed strong a chemotactic response in the wid type but not in the vfcA mutant. The mutant did however have an increased response to the hydrophobic aromatic amino acids. This could be due to an increase in expression of other MCPs or that these act as a chemorepellant in the presence of VcfA.

To test if this was the primary driver in colonization of squid, wild type strains, vfcA mutants and a combination of the two were allowed to colonise squid. Both mutant and wild type colonized to comparable levels so there must be other more important factors driving colonization. These results indicate that chemotaxis towards amino acids is not of great importance in the colonization of the squid, however it could be that attraction to the hydrophobic amino acids compensates for that of serine, alanine, threonine and cysteine. There are another 42 MCPs in V. fischeri that might yield the answer to what drives colonization and the other 3 MCPs regulated by the flagellar master regulator would be a good place to start. The most beneficial aspect of this work is the development of the capillary assay which allows chemotaxis to be quantified in suspension rather than on plates, This is much more similar to the natural environment so results will hopefully be more comparable.

Brennan, C. A., DeLoney-Marino, C. R., & Mandel, M. J. (2013). Chemoreceptor VfcA mediates amino acid chemotaxis in Vibrio fischeri. Applied and environmental microbiology, 79(6), 1889–96. doi:10.1128/AEM.03794-12

Wednesday, 27 November 2013

Are Marine Actinomycetes Just Terrestrial Visitors?

Actinomycetes are a clade of prokaryote that has produced some of the most economically and biotechnologically valuable secondary metabolites. Isolated strains have rendered unto us, various antibiotic agents, antitumor properties and, immuno-suppresive agents and enzymes. The number of novel strains isolated from the terrestrial environment has recently declined in conjunction with an increase in re-isolated known compounds. Due to this decline and high re-discovery rate it is essential that new Actinomycetes be discovered from lesser explored and unexploited habitats.
It has been estimated that the marine floor is more diverse in microbial life than that of a terrestrial rainforest. Whether or not this is the case, the marine floor provides a stunning array of extraordinarily different habitats ranging from high acidity, high pressures, anaerobic conditions and temperatures exceeding 100ºC; all of which have are known to have been colonized by actinomycetes. It is expected that the diversity of habitats populated by this microbe, will yield new and exciting metabolites that can be further examined for practicality. The purpose of this study is to define whether known marine actinomycetes are locally indigenous or simply swept in from nearby terrestrial habitats. The results could influence whether their conceived origins reduce or increase the novelty of their secondary metabolites.

Jensen et al collected 5 sediment samples from 15 different locations, ranging from ocean atolls, mangroves, sand spits and scrub vegetation. Each sample's dry weight was recorded and a serial dilution was conducted with sterilized seawater. The diluted sediment samples were innoculated onto two types of nutrient agar: Medium 1 for general hetrotrophic bacteria and Medium 2 designed to isolate actinomycetes. Each medium was inoculated with 2 x 10-2 and 2 x 10-4 dilutions. Separate samples were heated in favour of actinomycetes and were inoculated onto the two different agar media.
The plates were incubated for 14 and 21 days before bacterial and actinomycete counts were conducted. It is worth noting that due to the problems associated with actinomycete inoculations, some of the counts were not included. All actinomycetes were isolated and analyzed chromatographically for isomeric diaminopimelic acid configurations and whole-cell sugar analysis. This was conducted to compare results and identify each strain. The isolated actinomycetes were then grown in sterile, deionized water and total biomass was used to evaluate growth.

From the samples collected, a total of 289 actinomycete colonies were observed. From the chromatographic analysis, 91 of the samples were shown to belong to the streptomycetes and 192 were shown to be actinoplanetes. Six colonies couldn't be identified as they were too few in number. In relation to depth it was shown that stretomycetes decreased in number whereas actinoplanetes population increased with depth. Actinoplanete numbers were variable with no particular habitat being a richer source; including the oceanic atoll of which had very little terrestrial influence. In all but 5 of the 32 streptomycetes tested, deionized water reduced total growth. With the exception of 1 actinoplanete out of 31, all reduced or ceased to grow in deionized water.

Jensen et al concluded that their work was not suitable to infer the population numbers of actinomycetes in marine sediments, as the mycelin of a colony could be fragmented during the vortexing of a sample – leading to an increase of colonies during plating. However, their work did not conform to previous theories of actinomycete origin. Previous works had speculated that actinomycetes were only found in the marine environment as terrestrial spores and did not play an ecological role or metabolise under such conditions. Their salt-tolerance was also put down to them being soil microbes that need to be adaptable to varying conditions.
The team believe that actinomycetes vary in their adaptation to the sea and have differing levels of function within the marine microbial community. From the decreasing population in relation to depth and the fact that they were not present at a marine atoll, streptomycetes were suggested to be of terrestrial origin and under appropriate conditions; able to grow in a marine environment. As actinoplanetes increased in number with depth, and that their growth was inhibited by the absence of saltwater, they were deemed to be of marine origin; inferring that these microbes metabolize in the marine environment. Through morphological analysis, it was found that most of the actinoplanetes were from the genus Micromonospora. This genus has previously been studied and found at depths ranging from 0 >2000m. The team concluded that the general theory of actinomycetes not being physically adapted for the marine environment and not active members of the marine microbial community is incorrect for some members of this microbial group.

Jensen P.R, Dwight R & Fenical W. 1991. Distribution of Actinomycetes in near-shore tropical marine sediments. Applied and Enviornmental Microbiology. 57 (40) : pp.1102-1108.

Tuesday, 26 November 2013

Evidence for possible syntrophic association of nitrifiying bacteria with Beggiatoa mats in hydrothermal vent sediments in the Guaymas Basin

Evidence for possible syntrophic association of nitrifiying bacteria with Beggiatoa mats in hydrothermal vent sediments in the Guaymas Basin

Deep sea hydrothermal sediments found in the Guaymas Basin (Gulf of California) are carpeted with thick microbial mats characterised by Beggiatoa - a filamentous species of nitrate-respiring, sulfide-oxidising bacteria.  These mats are fuelled by hydrothermal fluids, rich in ammonium, that mix below the surface and percolate up through the sediment where they cool before entering the water column.  The high ammonium concentration in the fluids suggests it is likely to be an important energy source for chemoautotrophic interactions, not only within the hydrothermal vent plumes but also in the surrounding sediments.

Beggiatoa is punctuated with vacuoles, known to accumulate and store nitrate up to 4,000 times the ambient concentration.  Due to the mats being at the interface of the oxic water column and cooled ammonium-rich vent fluids, mats such as these are hypothesised to be hotspots for nitrogen cycling.  Prior to this study, anaerobic ammonia oxidation (anammox) is the only process that has been identified in these deep sea sulphur mats.  The authors of this paper used both molecular and biochemical methods to assess whether nitrification was occurring.

O2, nitrogen oxide, nitrate & ammonium concentrations were measured both in situ, using microsensors and ship-board using washed and homogenised Beggiatoa mats, non-hydrothermal sediments free from ammonium and seawater taken 1m above the hydrothermal sediment surface.  Using 15N-labelled ammonium chloride, linear nitrate formation was measured to estimate nitrification rates at between 370 and 920 times higher than the ambient seawater sample and up to 2000 times greater than that associated with non-hydrothermal sediments.

Quantitative PCR (qPCR) techniques were used to ascertain the copy number of the amoA gene of bacterial and archaeal ammonia-oxidisers, which encodes for the ammonia mono-oxidase subunit A.  Presence of archaeal amoA gene copies were an order of magnitude higher within the
microbial mats than in the seawater sample and β-proteobacterial amoA was found in high concentrations in the mats but absent from the seawater.  Microbial diversity was assessed using a gene library for AmoA sequences and the composition for the mats was distinct from non-hydrothermal sediments, hydrothermal plumes and the seawater.

Thaumarchaeotes, which include the ammonia-oxidising archaea (AOA) were detected using catalysed reporter deposition, integrated with fluorescent in situ hybridisation (CARD-FISH), along with 16s rRNA pyrotag techniques. AOA were found to outnumber ammonia-oxidising bacteria (AOB) by 6-8:1 and were found attached to many of the Beggiatoa filaments.  These results suggest that when oxygen is depleted within the Beggiatoa mats, the close coupling, along with the high nitrification rates, is suggestive of inorganic nitrogen cycling between the ammonium and nitrate, triggering detoxification and oxidation of sulphide.  This process may reduce the loss of bio available nitrogen in the sediments and potentially contributes to a substantial proportion of chemoautotrophic processes occurring at hydrothermal sites.

Knowledge of the distribution, diversity and activity of microbial associations at and around hydrothermal vents is likely to be spatially patchy due to the expense and inaccessibility associated with deep sea research.  In addition, bacterial/archaeal associations are likely to differ at each vent site due to the unique composition and concentrations of compounds found there, but this study contributes to the evidence bank piecing information together to identify important processes and understand local community structure.

Winkel, M., Beer, D., Lavik, G., Peplies, J., & Mußmann, M. (2013). Close association of active nitrifiers with Beggiatoa mats covering deep‐sea hydrothermal sediments. Environmental Microbiology. In Press

Monday, 25 November 2013


Alkanes are a class of saturated hydrocarbon (carbon and hydrogen containing) compounds.  The simplest, possible alkane, with which most of us are familiar, is methane (CH4). They are key energy source for various human activities as they are a major component of petroleum and natural gas. They are toxic to some organisms whereas source of energy and carbon for others. In nature, alkanes originate from various biological, especially microbial, as well as geochemical activities. Nevertheless, major source is through microbial activities. Thus, understanding microbial transformations of alkanes is very important. The bacteria that use hydrocarbons as their sole source of carbon and energy are called “Hydrocarbonoclastic bacteria”. Oxidation of alkanes involves breaking of a strong non-polar C-H bond, which is chemically very difficult. In marine bacteria, eight different families of enzymes employed in aerobic alkane oxidation, have been identified. These include monooxygenases, hydroxylases, cytochrome P450 (CYPs) and others.

High to moderate amounts of hydrocarbons, including n-alkanes are found in hydrothermal vent fluids. Hydrothermal vent fluids are usually highly reducing and anoxic, which are mixed with colder oxygenated waters of depths, forming thermal and redox gradients. Microbes exploit these gradients and support complex vent ecosystems. For example, there is experimental evidence for microbial oxidation of methane and other short-chain alkanes – playing critical role in supporting higher trophic levels in the vent ecosystem. Similarly, anaerobic oxidation of methane in vent sediments is critical in carbon cycling. Microbes that oxidize long chain alkanes have also been isolated from plume waters and vent sediments. Nevertheless, little is known about the diversity and ecological role of long chain-alkane oxidizers of hydrothermal vents. There is also industrial potential for finding powerful enzymes from vent microbes that can transform alkanes into other organics in more efficient ways.

This study explores reaction mechanisms of alkane metabolism in six different bacteria isolated from deep sea vents. Norcarane (a mid-chain alkane) was used to culture the tested strains of bacteria. It is called as “diagnostic substrate” because its enzymatic oxidation produces distinct profile depending upon the enzyme which has been used.

This study confirms presence of medium-chain alkane oxidizing mesophilic bacteria among hydrothermal vents. Alkane oxidation may be a key component of microbial metabolism in this extreme environment. The enzymes involved in this are AlkB like hydroxylase and CYPs, both of
which are thought to be involved in hydroxylation of medium-chain alkanes in surface waters.  Thus, mechanisms for medium-chain alkane oxidation are similar among hydrothermal vents, surface waters and other environments. 

Interestingly, there is a redundancy among different classes of alkane-oxidizing enzymes; rendered by having more than one enzyme for oxidizing same sort of alkane. Some bacteria express only one kind of enzyme but they still possess multiple genes for its isozymes whereas others have multiple types of enzymes (e.g. CYPs & AlkBs) employed in the same job and expressed simultaneously. Metal availability and subcellular enzyme localization could be the explanations for such redundancies.

This study found two different enzymes (CYPs & AlkBs) potentially doing same task, where only one strain expressed CYPs and others expressed AlkB-like hydroxylases. Both are iron-containing metalloenzymes and as the strains are from hydrothermal vent – an iron-rich environment; metal availability is unlikely to be a factor. Slightly different substrate ranges could be a cause for coexistence of these enzymes. 

In my opinion, in an evolutionary perspective, this redundancy or having more than one enzyme for doing the same task may characterize acclimatory abilities of an organism to different environmental conditions such as different temperature and salinity regimes, resource availability (e.g. metal availability). Gene-knock out experiments could resolve the question of redundancy where under certain conditions; all the isozymes of an enzyme may be knocked-out to see if desired reaction can be catalysed by that particular enzyme under those certain condition!

In summary, this is the first study to investigate mechanisms of medium- to long-chain alkane oxidation and metalloenzymes involved in it by aerobic vent bacteria. Although being from an extreme environment, vent bacteria do not seem to have novel alkane oxidising mechanisms; but express enzymes, functionally similar to bacteria from other environments. It is intriguing to see that extreme environment has not prompted its resident microbes to do things differently, at least alkane oxidation.

Bertrand, E. M., Keddis, R., Groves, J. T., Vetriani, C. & Austin, R. N. (2013). Identity and mechanisms of alkane-oxidizing metalloenzymes from deep-sea hydrothermal vents. Frontiers in Microbiology, 4 (109), 1-11.

Sunday, 24 November 2013

Bacterial Associations with Developing Coral Embryos

Many studies have focused on the bacteria associated with adult corals, but this paper investigates the initiation and  importance of these relationships to the survival of the coral in early development. Embryos of the coral Pocillopora meandrina were collected from Kaneohe Bay, Oahu. A sub sample of 4 hour old embryos were prepared and frozen for immediate analysis while the majority were put into individual petri dishes containing raw seawater, sterile seawater or individual bacterial strains. Once the coral planulae reached an age of 170 hours, terminal restriction fragment length polymorphism (T-RFLP) analysis was used to determine the associated bacterial communities. Fluorescence in situ hybridization (FISH) was used to demonstrate the density of bacteria associated with coral planulae while electron microscopy located the position within the planulae.

 From the experiment, it was concluded that the composition of bacteria in the surrounding environment influences the density and variety of bacterial associations formed with coral planulae. The planulae formed prominent associations with bacteria within raw seawater, Roseobacter clade strain HIMB1 and Pseudoalteromonas strain HIMB1276 but didn't with other isolates. This suggests that the planula, bacteria or both play an active role in the relationship. Proteins within the planulae are thought to facilitate in the recognition of certain bacterial cells in order for them to cross the epithelium therefore bacteria which aren't recognised cannot form an association.

 Roseobacter clade strain HIMB1 formed the closest internal association, although this is not surprising as Roseobacters commonly form relationships with adult corals, performing a variety of beneficial functions for the coral. As bacteria was not externally or internally associated with 4 hour embryos, it is suggested that the bacteria form associations with the coral surface and are later incorporated into the ectodermal tissues, during development.

 The majority of the Pseudoalteromonas strain HIMB1276 was found on the very edge of the ectoderm, possibly playing a role in the settlement or adhesion of the coral planulae, as well as chemical signals produced by microbial biofilms. The other, less prominent, isolates used in the experiment were considered not suitable in a host environment.

 Viruses were also found to be associated with the coral planulae but further research is needed to understand the beneficial effects and threats to the coral and its holobiont. Further investigation is also required to establish the importance of bacterial communities for the successful development of coral embryos into adult colonies.

 Apprill, A., Marlow, H.Q., Martindale, M.Q. and Rappe, M.S. (2012) Specificity of Associations between Bacteria and the Coral Pocillopora meandrina during Early Development. Applied and Environmental Microbiology. 78: 7467-7475

A new probiotic for aquaculture, Phaeobacter gallaeciensis reduces abundance of a pathogenic Vibrio species.

Vibrio anguillarum is a marine pathogen that causes the disease ‘vibrosis’, a lethal haemorrhagic septicaemic disease that affects various marine and freshwater fish, bivalves and crustaceans. The pathogen is responsible for severe economic losses in aquaculture worldwide due to its high mortality rates.
In this study, Phaeobacter gallaeciensis of the Rhodobacteraceae was shown to antagonize V. anguillarum, ultimately reducing mortality levels significantly in cod larvae (Gadus morhua), highlighting the potential of P. gallaeciensis as a probiotic for marine fish larvae and their feed cultures.
The typical first feed for marine fish larvae are rotifers (Brachionus plicatilis), which are themselves fed or enriched with microalgae such as Tetraselmis suecica and Nannochloropsis oculata. These species can harbour high concentrations of the pathogens, which can be combated using antibiotic prophylactics. However, their use must be avoided as it can lead to the production of resistant strains. The use of a P.gallaeciensis as a probiotic bacteria to reduce Vibrio concentrations is a far more natural way to prevent the disease, capitalising on the competitive exclusion caused by this superior coloniser.
The antibiotic mechanism of P.gallaeciensis was suggested by a previous study to be the production of the compound tropodithietic acid (TDA). This study compared the effect of a TDA-negative mutant of P.gallaeciensis with the TDA-positive type strain to confirm if this is the mechanism. Axenic cultures of the rotifer and microalgal species were used in the different treatments to study the probiotic effect of P.gallaeciensis.
In Tetraselmis microalgae, wild-type P. gallaeciensis reduced the numbers of Vibrio cells a thousand-fold, and complete elimination of Vibrio was achieved in 3 out of 4 replicates. The presence of the TDA-negative mutant reduced Vibrio numbers ten-fold, though it was only significant for two of the four replicates, it still suggests however that there is another mechanism of antagonism.
In Nannochloropsis cultures, Vibrio was completely elmintated by wild-type P. gallaeciensis in 1-2 days in high algal density.
In the rotifer species Brachionus plicatilis, wild-type P. gallaeciensis reduced Vibrio numbers a thousand-fold, whereas the effect of the TDA-negative mutant was non-significant.
In cod larvae, those challenged with V. anguillarum in the absence of P. gallaeciensis saw 100% mortality, whilst larvae challenged in the presence of wild-type P. gallaeciensis saw only 12.5% mortality. Which, bizarrely, was less than the control larvae grown in the absence of both bacteria, which saw 37.5% mortality. The TDA-negative mutant reduced mortality to 68.8% mortality, which again suggests a second mechanism of antagonism. Larvae that were not challenged by V. anguillarum had a lower mortality in the presence of P. gallaeciensis, indicating that it has additional beneficial effects beyond its action against V. anguillarum.
P. gallaeciensis is therefore able to colonise two aquaculture-relevant algae species and a rotifer species without compromising their growth. It strongly reduces or eliminates V. anguillarum numbers within those species and reduces mortality significantly in cod larvae.
The findings from this study are likely the first stage in the process of manufacturing a new probiotic. Now that P. gallaeciensis has been shown experimentally to effectively reduce pathogenic Vibrio numbers in cod larvae, its plausibility as a probiotic will need to be tested.

D’Alvise, P. W., Lillebø, S., Prol-Garcia, M. J., Wergeland, H. I., Nielsen, K. F., Bergh, Ø., & Gram, L. (2012). Phaeobacter gallaeciensis reduces Vibrio anguillarum in cultures of microalgae and rotifers, and prevents vibriosis in cod larvae. PloS one, 7(8), e43996.

Thursday, 21 November 2013

Mussels chose where to keep their symbionts.

Shift from widespread symbiont infection of host tissues to specific colonization of gills in juvenile deep-sea mussels.

Among endosymbioses it is unusual for the symbiotic bacteria to colonize much more of the animal then just a specialized organ, with the exception of hosts that pass on symbionts vertically through specialized gonadal cells. Within Bathymodilus mussels, a genus that is found throughout the ocean at both hydrothermal vents and cold seeps, symbionts are thought to be acquired horizontally, although it is unclear at which stage this occurs as mussels as small as 0.12mm have been shown to harbour symbionts.
            In adult and late juvenile Bathymodilus methane and sulfur oxidizing bacteria are constrained to specialized bacteriocytes in the gill tissue. This is the site where all the required nutrients are available and the large surface area to volume ratio and exposure to seawater ensure maximum efficiency of nutrient transfer both to symbiont and host (adjacent to haemolymph lacuna). Unusually though, early juvenile stages have been shown to harbour symbionts within epithelial cells of other tissues. To discover which tissues and whether or not these were exclusively symbiotic bacteria Wentrup, et al.,(2013) used fluorescence in situ hybridization (FISH) with probes specific to the symbiont groups as well as a general eubacterial probe on mussels at different stages of development (4-21mm).
            In all mussels they found both types of symbiont within bacteriocytes in the gill tissue. In the smaller individuals (4-7mm) there was evidence of both symbiotic bacteria in epithelial cells of the mantle, foot and retractor mussel (see fig. 1). In all individuals looked at there were no other bacteria found within host cells, indicating that there must be a mechanism for selecting only the beneficial bacteria. In all individuals larger then 7mm endosymbionts were exclusively in the gills.
            It is interesting to think that these bivalves ‘allow’ indiscriminate colonization of epithelia at early stages in development. It might be that the earlier stages require more energy from the symbiosis so allow colonization of other cells although it has been postulated that filtration feeding, that does still occur, would make up for any small amount of nutrients provided by these non gill symbionts. However the gill in many bivalves generally develops later then the mantle and foot so this theory does seem plausible. There may be some genetic mechanism, turned on at a specific stage in ontogeny that has a microbicidal effect in all tissues apart from the gill, perhaps a digestive enzyme. It may also be that the bacteria just cannot survive once the tissues have outgrown them, with diffusion distances increasing they may dwindle due to nutrient deprivation.
            Assuming that transmission is horizontal (same study could be performed on spawning individuals to determine if symbionts occur in the gonads) there must be a recognition mechanism that distinguishes the two symbiont groups from other potentially pathogenic bacteria and possibly initiates phagocytosis into specialized bacteriocytes though little is known about how this takes place. Bivalve immunity is carried out by haemocytes that engulf bacteria as well as producing microbicidal agents. At some point in the history of this symbiosis the haemocytes lost their ability to destroy these symbiotic bacteria and allowed them to enter the tissue of the animal. Perhaps the early (in ontogeny) immune system of these mussels is not developed enough to prevent bacterial contamination of other organs, though I am inclined to think that it has benefit to the developing mollusk. The problem with studying these kind of symbioses is the difficulty of sampling. in this study only 13 mussels were actually used so extrapolation of data to whole populations is risky.

Figure 1. FISH signals for sulfur oxidizing symbionts (green) and methane oxidizing symbionts (red) in the mantle and gill of juvenile mussels. Adapted from (Wentrup, et al., 2013).

Wentrup, C., Wendeberg, A., Huang, J. Y., Borowski, C., & Dubilier, N. (2013). Shift from widespread symbiont infection of host tissues to specific colonization of gills in juvenile deep-sea mussels. The ISME journal7(6), 1244–7.

Microbial chemoattraction to DMSP: the shaping of planktonic food webs and influencing global climate change

Dimethylsulfoniopropionate (DMSP) is solute produced by phytoplankton, and released into the surrounding water by grazing, exudation and cell lysis. The nature of these point source events results in spatiotemporal DMSP pulses. Such pulses have proved to be important underwater foraging cues for many species yet previous suggestions of the ecological function of DMSP amongst marine microorganisms have been contradictory to one another, and to date are largely unresolved. Though produced by microalgal species, many autotrophs that don’t produce DMSP have the capacity to uptake and assimilate it suggesting differing physiological needs for DMSP. 

The behaviour of seven marine microbial species were studied in response in to submillimeter diffusing pulses of DMSP, glycine betaine (GBT) and DMSP related degradation products: dimethylsulfide (DMS) and dimethylsulfoxide (DMSO). Though differences were found between chemicals, DMSP and its related products proved to the powerful chemoattractants with 74% of tested cases, across multiple trophic levels, resulting in positive chemotactic behaviour.  Five organisms displayed strong chemotactic responses to both DMSP and GBT (likely due to analogous physiological function and chemical structure between both chemicals). Weaker attraction was also shown to DMS and DMSO though that is congruent with the lower biological liability of these products. Relative response was quantified and stronger responses showed a 65% enhancement in chemical exposure.

Unlike most motile phytoplankton and despite possessing the ability to uptake DMSP cyanobacterium Synechoccus exhibited no chemotaxis. On the other hand Chlorophyte, Dunaliella tertiolecta, showed a positive response to DSMP despite not uptaking or assimilating it. Its strong attraction to DMS, supported by trials showing its capacity for extracellular transformation of DMSP to DMS could perhaps infer an ecophysiological requirement for DMS .

30-90% of oceanic DMSP is metabolised by heterotrophic bacteria. Two species, of which are known to demethylate DMSP, Silicibacter sp. and Pseudoalteromonas haloplanktis, exhibited strong chemotaxis to DMSP. Highly directional swimming and high chemotactic migration rates produced a rapid response to DMSP patches and resulted in a 66% exposure increase providing a substantial advantage over non-motile competitors.

Prey ingestion and osmotrophic uptake are two ways in which microzooplankton achieve their reduced sulphur supply. Strong positive responses to DMSP and related products were observed in both Oxyrrhis marina and Neobodo designis, herbivorous and bacterivorous flagellates respectively. A pronounced shift in swimming behaviour and swim velocities, consistent with that exhibited by bacteria, suggest that for both zooplankton and bacteria DMSP can be used as a resource and an infochemical.  However, with predators being attracted to DMSP patches the ecological benefits of DMSP chemotactic responses of phytoplankton must outweigh the increased risk of grazing; perhaps explaining why Synechoccus exhibited no chemotaxis.

DMS is the principal natural source of sulphur gas, and is a significant factor affecting the climate system. Microbial exposure to DMSP and grazing on DMSP-producing prey are key processes involved in regulating ocean-atmosphere DMS flux. These demonstrated chemotactic responses could in fact enhance DMS production by increasing such processes, mediating a potentially large influence on global sulphur biogeochemistry.

Seymour J. R., Simó R., Ahmed T. and Stocker R. (2010) Chemoattraction to Dimethysulfoniopropionate Throughout the Marine Microbial Food Web. Science, 329, 342-345

Wednesday, 20 November 2013

The Microbes Living 3 Kilometres Beneath Antarctica

Welcome to Lake Vostok; beneath 3,700 metres of Antarctic glacier you can sit back, relax and enjoy unrelenting extremes of cold and heat. If you love crushing pressures, starvation and perpetual darkness, then Lake Vostok is the place for you.
Turns out Lake Vostok is the place for much microbial life. As the glacier creeps along at 3 metres per year, lake water freezes to the bottom, providing us with a historical record of its contents. Parts of this lake have been found to be rich in organic carbon, minerals and, of course, organisms. Average cell concentrations in this ice range from one to hundreds per millimetre, with most viable cells found in the ice formed by glacial movement. Previously, rDNA sequences have yielded the discovery of 18 unique Bacteria and 31 Fungi isolates. Phylogenetically, they resembled known species from deep-sea, sediment, polar and cold environments. This study used ice core samples from Lake Vostok ice formed by glacial movement to provide a more detailed view of Lake Vostok's life using metagenomics and metatranscriptomics.
Ice core sample quartering, melting in sterile conditions and ultracentrifugation provided RNA samples which were then copied in cDNA and followed by subsequent PCR amplification, ligation, chromatography and re-amplification with primer sequences. Finally, sequence analysis provided over 36 million base pairs of data.
3,169 sequences were identified as bacterial, 89% of which matched known database sequences by 97-99%; the taxa identified included the phyla Firmicutes, Proteobacteria, Cyanobacteria, Actinobacteria and Bacteroides. Only 2 sequences were from Archaea and they were most similar to deep ocean sediments Archaea.
6% of sequences were of eukaryotic origin; dominated by fungal groups, including one rRNA sequence which had 99% similarity to a known thermal-vent fungus. Other sequences found included those of Daphnia, springtails, rotifers, tardigrades, a deepsea bivalve and a anemone species. Many sequences were highly similar to those from uncultured bacteria from intimate parasitic or symbiotic associations with a range of eukaryotes including lobsters, annelids, salmon disease, fish intestines, bivalve larvae, sea squirts, tubeworms, sponges and Antarctic seaweeds.
The presence of certain metabolic capabilities was determined based on the rRNA sequences found; this diagram summarises the pathways they identified;
Figure 3. Summary of steps in nitrogen metabolism (above) indicated from the metagenomic/metatranscriptomic sequence
identities, as well as types of carbon fixation (lower left) and other functions (lower right) indicated by the sequence data.

Each unique sequence found is likely to represent a novel species or strain; especially of the Cyanobacteria and Proteobacteria, the most abundant groups. Metagenomics and transcriptomics have strongly suggested that Lake Vostok has a diverse community based on nitrogen cycling, symbiosis and high salinity toleration. Surprisingly few sequences originated from psychrophilic microbes, but this could be because there are relatively few psychrophile sequences for comparison in GenBank databases. The higher presence of thermophilic sequences supports the suggestions of other studies that Lake Vostok has hydrothermal activity. Sequences indicating arsenic oxidising bacteria also suggest hydrothermal activity, since arsenic is often present in volcanic activities.
The large representation of nitrogen metabolism in the sequences is probably due to frequent nitrogen gas introduction into the lake by glacial melting and movement. Carbon dioxide fixation was mostly represented by the reductive pentose phosphate cycle, with the TCA cycle in second place.
Lake Vostok was a normal lake 35 million years ago and part of a forest ecosystem and since then the lake has been partially exposed multiple times before it became completely frozen over. This means that life has had a number of opportunities to occupy the lake and this is reflected in the diversity hinted at by metagenomic techniques. Given the recent revelations from the discovery of hydrothermal vent ecosystems and the unlikelihood of contamination with the sequences found, Lake Vostok probably does have novel ecosystem dependent on the metabolism of unusual microbial groups; it will be interesting to see what innovations will have be developed to directly observe Lake Vostok's communities and even more so to know what lessons it will provide; ones which may even be extrapolated to how life elsewhere in the universe might exist, particularly on Europa, one of Jupiter's moons, where parallel conditions to Lake Vostok exist. This study also seems to imply that symbioses seem to become much more fundamental to life in extreme environments, for both host and microbe; this may have implications on our understandings on life's origins and the evolution of microbes into organelles.

Shtarkman YM, Koc¸er ZA, Edgar R, Veerapaneni RS, D’Elia T, et al. (2013) Subglacial Lake Vostok (Antarctica) Accretion Ice Contains a Diverse Set of

Sequences from Aquatic, Marine and Sediment-Inhabiting Bacteria and Eukarya. PLoS ONE 8(7): e67221. doi:10.1371/journal.pone.0067221

Vibrio coralliilyticus Search Patterns across an Oxygen Gradient

Bacteria have to use chemotaxis in mobility search patterns for locating nutrient sources, arranging themselves in chemical gradients and to initiate pathogenesis. There are 4 known search patterns: run and reverse, run and tumble, straight swimming and the recently described 3-step run, reverse and flick technique. Previous studies show V. alginolyticus uses “3-step flick” to enter nutrient rich zones.

 Majority evidence suggests that bacteria utilise one search strategy per species, with marine bacteria most commonly utilising run and reverse.  However, in this paper V. coralliilyticus is shown to demonstrate the three search strategies of run and reverse, straight swimming and 3 step flick.

The 3 step flick is like a combination of run and tumble, and run and reverse tactics, allowing the re-examination of nutrient areas with reverse movement, but paired with a beneficial 90 degree flick, altering orientation of the microbe, increasing the probability of entering desirable environments.

V. coralliilyticus is a pathogen reliant on chemotaxis to invade organisms such as corals. This 3 flick technique is thought to allow V. coralliilyticus to navigate the differing mediums of corals (water, mucus and tissue cells). Turbulent waters in the marine environments cause constant flux of variables affecting V. coralliilyticus. Nutrient levels change diurnally and nocturnally while oxygen saturation in coral tissues flips to extreme highs (250%) and lows (<2%). Water around the coral stays at a constant, only layers of 1mm on the surface of the coral tissue changing with light and dark conditions.

The authors investigate how V. coralliilyticus’ search patterns change relative to fluctuating oxygen gradients.

An observation chamber was set up, where the centre of the slide was highly anoxic and the edges oxic. V. coralliilyticus was shown to exhibit more flick-search patterns in the oxic conditions, and more run and reverse techniques in anoxic conditions.
The chemotaxic search pattern behaviour of this bacterium changed over the oxic-anoxic interface, identified over a microscope transect.
When entering the anoxic environment there was a significant decrease in 3 step flick behaviour, though over all no preferences were found for search patterns in oxic and anoxic conditions.
This lead to the conclusion that V. Coralliitycus can use both search patterns in both anoxic and anoxic conditions while maintaining search pattern velocities over the oxic-anoxic intersect.

The relationship between oxygen saturation and a bacterium flick or non-flick response is still unknown. The authors discuss that the 3-flick mechanism could be either triggered oxygen saturation (as seen in Escherichia coli) – or perhaps due to bacteria monitoring changes in the electron transfer rate via redox sensors through sensory pathways (the sensory system can convert environmental signals directly into rotational changes of the flagella motor). Further research should investigate the actual mechanisms behind these actions.

One criticism of this paper is the short discussion that does not segregate the results to infer meaning to the reader. Leading to statements that could be construed as misleading such as the “3-step flick”. Is present in oxygenated environments and not in deoxygenated” – though this may infer to statistical differences in data there are still trends that should not be ignored. Having said this, it is still useful paper as it is one of the first to investigate a relationship between oxygen and 3-flick search patterns.

Further research could be focused on how temperature affects the “3-step flick” due to the temperature dependence of this micro-organism. Secondly it would be interesting to repeat this experiment with more extreme oxygen saturation levels/gradients, with perhaps more sophisticated methods in place. It may also be useful to investigate V. coralliiticus response to oxygen saturation in light and dark settings, to see whether any search pattern behaviours are influenced by this factor – perhaps shedding light on whether infection of coral is nocturnal or diurnal specific.  

Winn, K. M., Bourne, D. G., & Mitchell, J. G. (2013). Vibrio coralliilyticus Search Patterns across an Oxygen Gradient. PloS one, 8(7), e67975.
Adam Battishill & Caroline White

Monday, 18 November 2013

The breakdown of the oil spill from the 2010 Gulf of Mexico disaster by bacterial EPS

Dissolved Organic Matter (DOM) is one of the largest and least understood pools of carbon in the ocean, and ~10-25% of the total oceanic DOM is composed of biopolymers. A large proportion of DOM is produced from the synthesis and release of exopolysaccharides (EPS) from bacteria and eukaryotic phytoplankton.  This has led to research into the potentially significant role of EPS, particularly in bacteria, in the degradation of hydrocarbon pollutants in the ocean. There has been much interest in the fate of the oil released following the explosion and sinking of the Deepwater Horizon oil rig in the Gulf of Mexico in 2010, the worst accidental marine oil spill in the history of the oil and gas industry. The fate of the oil from the ecological disaster is poorly known, but Gutierrez et al. stress the importance of understanding the capacity of EPS produced by marine bacteria to affect the dissolution of aromatic hydrocarbons to enhance the bioavailability of these compounds for indigenous microbial communities and hence the rate of degradation.

EPS-producing bacteria enriched during the oil spill were thought to have contributed to the formation of abundant oil aggregates on the surface and within deep water oil plumes, and the authors demonstrated this enrichment both in laboratory experiments and in the field. This study used one type of EPS-producing bacteria, the Halomonas species strain TG39, as a model to predict the potential influence of EPS on the oil, which was collected from surface waters during the active phase of the spill. This strain produces large amounts of EPS which display amphiphilic, or biosurfactant-like, properties, and can lead to the emulsification of aromatic hydrocarbons, crude oils or refined petroleum products by interacting with hydrophobic substrates on their surfaces and solubilising them to enhance their biodegradation.

An additional analysis of pyrosequence data from surface water samples collected during the spill revealed other distinct Halomonas strains which could produce EPS. Some of these strains had high (97%-100%) 16S rRNA sequence identity to strain TG39. Clusters of other EPS-producing bacterial communities, such as Alteromonas, Colwellia and Pseudoalteromonas, were also enriched in surface and deep waters, and could also emulsify the oil and form aggregates, which may have supplemented the bioavailability of EPS in the Gulf. They also used a roller bottle incubation experimental design with one of the other Halomonas isolates, TGOS-10, which exhibited an independent ability to emulsify the oil, as the aggregates directly formed on the extracellular cell surface of the strain.

Evidence from the results showed that the EPS produced by Halomonas species strain TG39 can facilitate the dissolution of poorly-soluble aromatic hydrocarbons, which enhanced the bioavailability to indigenous populations of microorganisms present during the active phase of the spill, particularly in the surface waters. The enrichment of EPS-producing bacteria, together with their capacity to produce amphiphilic EPS, is likely to have contributed to the eventual removal of the oil and to the formation of oil aggregates.

The authors focus on the enrichment and dominance of EPS in the Halomonas strain to influence the degradation of the oil in the surface waters, as well as other EPS-producing bacteria in the deep water oil plumes, but it would be interesting to see whether the rate of degradation differs in the surface waters compared to the deep water plumes and whether it is based on the level of enrichment alone. In addition, as this experiment was carried out during the active phase of the oil spill, further research should look at the after-effects to see how the abundance of the DOM pool changed once the oil had been degraded, and the effect it may have had on the carbon cycle in this region of the Gulf. 

Gutierrez, T., Berry, D., Yang, T., Mishamandani, S., McKay, L., Teske, A., and Aitken, M.D. (2013) Role of Bacterial Exopolysaccharides (EPS) in the Fate of the Oil Released during the Deepwater Horizon Oil Spill. PLoS one, 8 (6), e67717

Nephromyces, a beneficial apicomplexan symbiont in marine animals.

Nephromyces, a beneficial apicomplexan symbiont in marine animals. (SAFFO et al. 2010)

Nephromyces is an very unusual marine endosymbiotic protist. Its peculiar habitat, paired with unusual structural and developmental features has caused a great deal of debate over where exactly it belongs in the phylogenetic tree of life. For a while it was classified as a chytrid fungus due to several shared features with the fungi - such as chitinous walls and the absence of chloroplasts, however several features of this organism like its posteriorly biflagellate cell stage are atypical for fungi.

In this paper, SSU rDNA (short-sub unit ribosomal DNA) sequenced from Nephromyces cells isolated from four species of Molgula species: M.occidentalis, M.citrina, M. manhattensis and M. retortiformis, have given the evidence required to finally place this bacterium into the phylum Apicomplexa where it belongs. This analysis was confirmed via FISH (fluorescence in siti hybridization) which tested for both apicomplexan-specific and Nephromyces-species SSU rRNA oligonucleotide probes. Toxoplasma gondii was used a non-Nephromyces apicomplexan control.

Phenotypic features of the Nephromycesis also support this phylogenetic conclusion. For example characteristics such as  the non-flagellate, motile infective stage found in the blood and similarities between the only stage of Nephromyces known to cross epithelial boundaries strongly resembling the infective stages (sporozoits) of apicomplexans such as Toxoplasma. In addition, Nephromyces spores (the host-transfer stages, which give rise to the infective cells) contain inclusions reminiscent of rhoptries of the apical complex, a key structure in host-cell invasion among apicomplexa, and the ultrastructural hallmark of the apicomplexa and sister lineages

The most exciting point that arises from putting Nephromyces into the apicomplexan genus is the fact that this protist is currently (at the time of this paper) the only known beneficial apicomplexan – a stark constant from the other phylum members, which are either parasites or pathogens!

Nephromycesis has been documented in every adult individual of Molgula species (a highly derived group of ascidians)  surveyed, regardless of population, geographic location, environmental conditions, season or year of collection. This obligate symbiont has (in return) only even been discovered in molgulids, with most of its life history constrained to the renal sac lumen. The renal sac lumen is a “storage tissue” rich in calcium, oxalate, nitrogen and urate, and these characteristics provide a specific physiological niche for Nephromycesis. The high urate oxidase activity of Nephromyces, along with its urate-rich host habitat, suggests that Nephromyces may share, alongside other apicomplexans, a metabolic dependence on host purines

Nephtomycesis  also contains hereditarily transmitted intra-cellular bacteria. These bacteria are a symbiont to our original symbiont! It is hypothesised that they have important metabolic effects with regards to Nephromycesis, including the possibility that these bacteria are the source of urate oxidase activity found in their peroxisome-free apicomplexan hosts. 

There have only been two other descried apicomplexans with bacterial endosymbionts, and the sequence analysis reveals that at least one (Cardiosporidum cionae) is a close relative of Nephromyces.

Saffo, M. B., McCoy, A. M., Rieken, C., & Slamovits, C. H. (2010). Nephromyces, a beneficial apicomplexan symbiont in marine animals.Proceedings of the National Academy of Sciences107(37), 16190-16195.