The role of N-acetyl-D-glucosamine in facilitating colonisation of Euprymna scolopes by Vibrio fischeri

Vibrio fischeri is a bioluminescent bacterium that works in a symbiosis with the squid Euprymna scolopes. While the E.scolopes provides shelter and nutrients for the V.fischeri, the V.fischeri allows the E.scolopeses underside to mimic the moonlight during the night to protect itself from predators.

It has been found that one of the main nutrients that V.fischeri is provided with is chitin, which must be broken down by enzymes as it is not soluble. The chitin is broken down by exochitinases into N-acetyl-D-glucosamine (GlcNAc) and chitobiose (GlcNAc)₂. V.fischeri have a nag locus which regulates the uptake and metabolism of GlcNAc. The nag locus is made up of a number of genes including nagE and nagBAC. nagE encodes enzyme involved in transporting GlcNAc from outside the cell into the cytoplasm. During the transportation the GlcNAc is phosphorylated becoming N-acetylD-glucosamine-6-phosphate (GlcNAc-6P). nagA encodes for the deacetylase NagA which removes the acetyl group, giving glucosamine-6P. The deaminase Nag B, encoded by nagB, then removes the amino group. Nag C represses the nag locus by binding to operon sites. The Nag C is only released when GlcNAc-6P binds to the operon site.

One of the things that are not fully understood in the symbiosis of E.scolopes and V.fischeri is how E.scolopes is able to only allow V.fischeri to infect its light organ. We know that juvenile E.scolopeses have a complex ciliated epithelium around the pores of the light organ and that when the epithelium is stimulated by peptidoglycan from bacteria that colonise the squid produces mucus that stimulates V.fischeri, which then form aggregates. The V.fischeri somehow out-competes other, non-symbiotic bacteria and then migrates through the pores to colonise tissue within the light organ. It is thought that the expression of certain genes in the V.fischeri may act as an indicator to E.scolopes, informing the squid of which bacteria present would be suitable for the light organ.

This study tests the role of the gene nagC in colonisation. They tested the ability of both wild-type V.fischeri and a mutant strain that had a faulty NagC gene to colonise Euprymna scolopes and found that when both strains were present the wild-type did not facilitate the colonisation of the mutant strain but that the mutant strain did not hinder the wild-types ability to colonise either. This suggests that the squid is able to select symbionts that have a functional NagC , ensuring that the bacteria is able to regulate the expression of the nag locus and take advantage of the chitin present to the best of its ability. It would be interesting if a further experiment were conducted, looking into how the squid is able to be so selective about which bacteria colonise its light organ but such an experiment would be difficult to conduct.

Reference: Miyashiro, T et al (2011), The N-acetyl-d-glucosamine repressor NagC of Vibrio fischeri facilitates colonization of Euprymna scolopes. Molecular Microbiology, 82: 894–903

Veni, Vidi, Vici: A fitness trade-off in biofilms

A review of: Nadel CD and Bassler BL (2011) A fitness trade-off between local competition and dispersal in Vibrio cholerae biofilms. PNAS, 108 (34): 14181-14185.

Biofilms are critical for processes ranging from biogeochemical cycling, bacterial pathogenesis and industrial biofouling. Bacteria living in biofilms secrete extra-cellular polymeric substances (EPS) that form a matrix for bacteria to adhere to. Previous studies suggest that EPS secretion benefit the producer and neighbouring cells while enhancing multi-cellular development within the bacterial community, including cells which do not produce EPS. Consequently, non-EPS producing cells are able to invade, exploit and compromise the structural integrity of wild type biofilms because they do not pay the cost of EPS production.

Simulation models of biofilm growth indicate that bacteria that produce EPS can occupy locations with better access to nutrients compared to bacteria that do not produce EPS, suggesting that EPS secretion may be competitively advantageous in natural biofilms. This is contrary to the idea that such environments easily succumb to exploitation by non-EPS producing microbes.

To test the theoretical model, Vibrio cholerae was used for subsequent experiments, based on their ability to initiate biofilm growth upon surface adherence and their capacity to utilise quorum sensing to activate or suppress EPS production at low cell densities and high cell densities respectively. V. cholerae EPS⁺ and EPS^- mutants expressing different fluorescent proteins were used for microscopy analysis. Monoculture results reveal that EPS⁺ strains pay a substantial cost for diverting resources away from biomass production in favour of EPS synthesis when compared to EPS^- strains. In co-inoculated biofilm cultures, EPS⁺ strains proliferated whereas EPS^- strains decreased by more than 80%, indicating subjugation of EPS^- growth. The opposite result was observed in a mixed liquid environment where primary fitness is based on growth rate which is slower in EPS⁺ strains due to EPS production costs.

Local competition is not the only determining factor for long term evolutionary success as micro-organisms must disperse to new resource patches after old ones have been depleted. Whether EPS production affects dispersal ability was assessed using adjacent microfluidic chambers. Frequency of EPS⁺ and EPS^- strains in the biofilm, the liquid effluent and the adjacent chamber was measured at regular intervals. Results show the dominance of EPS⁺ strains in the biofilm while remaining a minority in the liquid, correlating to poor colonization of the adjacent resource patch.

These findings suggest a trade-off between competition and colonization governed by EPS production. Bacteria have evolved to find a balance between local competition and dispersal ability with long term competition dynamics depending on resource availability. Bacteria like V. cholerae have finely tuned regulatory mechanisms such as quorum sensing which modulates the timing and strength of EPS production relative to environmental conditions.

10 Reaons to exclude viruses from the tree of life...

A Review of: Moreira and Lopez-Garcia. Ten Reasons to exclude viruses from the tree of life. Nature. Vol 7. 2009.

This is an opinion article in which the authors argue quite passionately that to include viruses in the tree of life would mean that it would become a ‘tree of genes from multiple origins’ instead as, without cells, viruses are nothing more than ‘inanimate complex organic matter’.

The first point they argue is that viruses are not alive. Many scientists, including Aristotle, have proposed definitions of life but as of yet none of them can include viruses. The first definition postulates that an organism is alive if it can ‘self-replicate and self-maintain’. As viruses lack any kind of carbon metabolism, they are excluded from this definition. The second involves ‘self-replication and evolution’ of the organism. Again, viruses cannot be included as they require a cell to carry out these tasks. Under this second definition it could be argued that computer viruses are alive as they can be designed to produce copies of themselves with slight changes in their code which, by extension can be seen as mutation and therefore evolution.

The fact that viruses are polyphyletic and have no ancestral viral lineages is also argued as there is no single gene shared by all viruses or viral lineages and therefore viruses have various evolutionary origins. Common protein motifs in distinct viral lineages have been found leading to the theory of a common ancient origin that predates the last common ancestor of cellular organisms: the Cenancestor. The authors argue here that horizontal gene transfer and convergence are more likely explanations.

The existence of a genetic membrane provides strong evidence that all modern cells are derived from a single common ancestor as it can only be formed by splitting pre-existing membranes. Its absence in viruses is additional evidence for their polyphyletic origin because viral constituents are synthesized de novo at each viral infection cycle and shows that viral lineages lack structural continuity.

Some viruses have been found to contain metabolic and translation genes. The authors argue that these are the result of horizontal gene transfer from a cellular origin. They describe viruses as a reservoir of cellular genes that can be transferred between different hosts and could therefore play a part in cellular adaptation and evolution but in no way help in the modification of pre-existing genes or the creation of completely new genes, hypothesized by some scientists.

Many people believe that because viruses are so small they must be very old. Moreira and Lopez-Garcia argue that structural simplicity neither implies antiquity or primitiveness but is a consequence of parasitism. Viruses are subject to strong selective pressures to keep a minimal genome size in order to have faster reproduction rates, which is a major force preventing complexification.

They conclude that viruses are not alive and should not be included in the tree of life but that they had and continue to have a significant role in the evolution of life on earth. Being abundant and comprising a major selective pressure that exerts strong control on the populations of many cellular organisms’ means that they are an important source and means of maintaining biodiversity.

The passion and opinions of the authors comes across very clearly in this article. However they do appear to be a tad closed minded about certain topics at times, dismissing reasonable hypothesis and explanations rather rashly. This article does appear to have caused somewhat of a stir in the scientific world which at times is great in provoking people to get passionate about their ideas and research- (There have been a couple of reply articles). However, I wonder if Moreira and Lopez-Garcia would stand by their arguments in this article after doing a little reading about all the new discoveries of Megaviruses and the information surrounding them…?

Vibrio fischeri, a symbiotic bacterium with pathogenic congeners

There are a number of species found within the Vibrionacea family of marine proteobacteria; some of which live symbiotically and others which have pathogenic interactions with animal tissue.

Using strain ES114 (the model-light organ symbiont of the squid Euprymna scolopes) of the proteobacteria V. fischeri the aim was to begin identifying features that were common to both the beneficial and the pathogenic bacteria in order to understand the symbiotic activities by which the bacteria adjust to the environment of host tissues.

Within this family, pathogenic species such as Vibrio cholera, Vibrio parahaemolyticus and Vibrio vulnificus have had their genome sequenced, but a symbiotic species of the Vibrio genus had never been sequenced.

The genomic DNA of V. fischeri was mechanically sheared and 2-3kb (kilo-base pair) fragments were isolated. Ends of the fragments were filled using Klenow fragment and ligated into SmaI-digested pGEM3 to produce a high-copy-number, small-insert library. Greater molecular mass genomic DNA was partially digested with Sau3A to construct a cosmid library. Undigested, unsheared DNA was used in PCR as a template for amplification of chromosomal regions which were not represented in the plasmid or cosmid libraries.

Whole genome shotgun sequencing was performed on around 35,000 plasmids and 400 cosmids, as well as gap-spanning PCR products.

They found that automated contig-assembly algorithms were ineffective in determining the number and orientations of the highly homologous rRNA operons; therefore those regions where assembled manually from sequenced PCR products. Potential open reading frames (ORF’s) were identified.

The finished genome underwent initial automated annotation followed by a manual gene-by-gene curation. (Results of their analysis are found at www.ergo-light.com)

The genome sequencing revealed many points that both show differences between and also links the pathogenic species to the beneficial symbiotic species of Vibrionacea.

Firstly, a plasmid known as pES100 was found within ES114; homologous plasmids are common among other strains of V. fischeri, but it is not required for host association and similar plasmids are found in pathogenic species.

Secondly, in V. fischeri G+C content is lowest of all 27 species. Despite this low content V. fischeri is more closely related to the higher G+C containing pathogenic Vibrio than to any other sequenced bacterium.

Thirdly, chromosomal density of apparent ORFs is almost 10% greater in V. cholera compared to V. fischeri.

Fourthly, a 4 fold greater percentage of unique genes are present on Chr II of this species when compared to others on a completed database. Thus, the smaller chromosomes characteristic of this genus may turn out to be a rich source of genes that define the unique potential of individual Vibrio species, and perhaps their specific lifestyles.

Fifthly, V. fischeri contains mobile genetic elements which share sequence similarity with V. cholera.

Sixthly, there is a presence of multiple pilus gene clusters in V. fischeri which suggest that different pili may be expressed to aid this bacterium either in the diverse environments it inhabits or during the multiple stages of its development as a symbiont.

Finally, Vibrio genes encode a putative toxin called RTX (which seems to affect regulators of host cell actin polymerisation). RTX has not yet been investigated, but it is known to have both beneficial and pathogenic effects on host cells.

This study begins to shed light on the unifying themes underlying contrasting bacteria-host interactions, using comparative genomic approaches of two closely related beneficial and pathogenic species from the Vibrionacea family; studies similar to this must continue to use genomic techniques to help us reveal the mechanisms by which pathogens associate with marine invertebrates as benign or even beneficial symbionts.

A review of:

Ruby, E. G., Urbanowski, M., Campbell, J., Dunn, A., Faini, M. et al. (2004). Complete genome sequence of Vibrio fischeri: A symbiotic bacterium with pathogenic congeners. Proceedings of the National Academy of Sciences. Vol. 102 (8). pp. 3004-3009

Mixotrophic Mussels

Bathymodiolus azoricus (Ba) is a dominant species of mussel which inhabits many hydrothermal vents of the mid-Atlantic Ridge (MAR). They contain two metabolically distinct endosymbionts (methanotrophic (Mb) and thiotrophic (Sb)) however have also been shown to capture particulate organic matter. The gill filaments contain three different zones consisting of a ciliated frontal zone, followed by a transitory zone, followed by the bacteriocyte zone. The objective of this study was to see the effects of starving the mussels of methane and dissolved sulphur on their gill filaments using microscopy and PCR based methods. The study also gives insight into how to maintain this deep-sea species in laboratory conditions for extended periods of time.

Mussels were collected from Menez Gwen as it is the shallowest location which removes the need to keep them under pressure. For the main experiment mussels were kept in tanks which were sulphide free for thirty days followed by fifteen days of exposure to normal sulphide conditions. However many other tanks were set up in different ways to provide controls. Light and Electron Microscopes were used at different time periods to see the effects on the gill filaments. They also did DNA extractions from gills using an old school method (not kits) followed by precipitation to clean the samples for PCR. In the PCR two sets of primers were used to target the endosymbiont 16s rRNA and ATP Sulphuryase genes to see whether the endosymbionts were present at different time periods. This was analysed using agarose gels. The method seemed to focus on Sb as no methane was introduced to any of the tanks.

The authors present the results extremely clearly with very nicely described images. There were very dramatic changes in mussel gills from tanks without dissolved sulphide. The bacteriocyte layer was highly reduced and all brown pigment was lost. The PCR and images clearly showed that all Sb and Mb were lost from the mussel gills very quickly. There was also an increase in the numbers of amoebocytes and lysosomes in gill filaments. The authors do not mention any mortality figures so I guess that there were no significant mortalities over the thirty days. Once they were re-acclimatised with dissolved sulphide the gill filaments seem to produce pit-like structures which had Sb associated with them and can be clearly seem in the images. The gill filaments seem to return to the form shown in the control group however do not seem to have fully “recovered” after fifteen days. It is very important to note that only mussels which were re-acclimatised by being placed in the control tanks showed this “recovery.” In all mussel gills Mb were shown to dissapear after twenty-four hours.

The study definitely seems to provide evidence for lateral transmission (possibly from other mussels or free living bacteria) of these endosymbiont bacteria. It also shows that the mussels can survive without these bacteria for at least thirty days. The paper provides evidence that the mussels gain bacteria through pit-like structures present on the gill filaments. Another key point which the paper makes which I have not really focused on is their method for keeping stable dissolved sulphide concentrations, which they proved did work. They also highlight other experimental procedures which they found to be highly important in keeping the mussels alive. Overall I thought the study was pretty impressive and the paper itself very nicely written. I thought it was a bit of a shame that they did not also try to see how long the mussels can survive without their endosymbionts to see how dependant the relationship is. I thought the mixotrophic nature of the mussels was quite cool and they also seem to show some level of plasticity.

A Review of Kadar, E. et al. (2005) Experimentally induced endosymbiont loss and re-acquirement in the hydrothermal vent bivalve Bathymodiolus azoricus, Journal of Experimental Marine Biology and Ecology 318, 99– 110.

Supply and Demand

A review of: Foster, R.A. et al. (2011) Nitrogen fixation and transfer in open ocean diatom-cyanobacterial symbioses, The ISME Journal, 5, 1484-1493.

Marine diatoms contribute significantly to primary production. This paper focuses on the diatoms found within low-nutrient waters which often exist symbiotically with nitrogen fixing cyanobacteria. Without actual evidence, it was assumed that the diatoms receive fixed nitrogen from the cyanobacterial symbionts, but little was known about the rates of fixation and supply within these symbiotic populations, and previous studies have included measurements from other unicellular populations. The paper took advantage of newer techniques which enabled the authors to measure stable nitrogen isotopes within individual cells and so gain a more accurate picture of rates of nitrogen fixation.

The seawater sampled in the experiment was collected from the Gulf of California and the subtropical North Pacific. A number of experimental designs were used, but all samples were amended with ¹⁵N_2. A combination of epi-fluorescence microscopy and nano-SIMS analysis (nanometer scale secondary ion mass spectrometry) were used to identify symbionts and measure ratios of ¹⁵N/¹⁴N. Cell dimensions were measured and numerous calculations performed to assess nitrogen fixation rates and transfer rates.

The results focused on three pairings; the diatoms Hemiaulus, Chaetoceros and Climacodium which are associated with the cyanobacterial symbionts Richelia, Calothrix, and Crocosphaera respectively. In all cases it was shown that the cyanobacteria were supplying fixed nitrogen to their diatom partners, and also fulfilling the diatoms requirements, as demonstrated by the equal or higher nitrogen enrichment observed within the diatoms, compared to within the vegetative cells of the cyanobacteria. Nitrogen transfer was very rapid, often within 30 minutes.

Growth and nitrogen fixation rates of the symbiotic Richelia and Calothrix cells were compared to those of free-living Richelia and Calothrix cells, and it was found that the symbiotic cells had accelerated rates. The authors suggest that the diatoms may co-ordinate changes to the symbionts growth and metabolism in order to ensure sufficient nitrogen is being fixed for both organisms. Further evidence for this was the discovery that Richelia fix much more nitrogen than needed for their own growth, with up to 97.3% being transferred to the diatom.

Diatoms_,require dissolved fixed inorganic nitrogen pools (nitrate and ammonium) as they cannot obtain nitrogen from N₂, and this paper gives an important insight into how organisms can gain a significant advantage by exploiting other organisms in a low-nutrient environment. It would be interesting to further investigate the authors’ theory that the diatoms influence growth and metabolism of the symbionts. The authors were unable to determine how much nitrogen is released by the symbioses, but conclude by suggesting that the symbioses should be included in global N models.

oil indicators in Mangrove sediment is revealed by 18s rDNA sequencing.

A review of: Santos, H. Cury, J. Carmo, F. Rosado, S. Peixoto, R. 2010. 18s rDNA sequences from microeukaryotes reveal oil indicators in Mangrove sediment. Plos one 5 (8): e12437

The microeukaryotes are of vital importance to marine ecosystems, as they represent the base of the pelagic food web in the ocean, and changes in the composition and structure of this web can lead to profound changes at all trophic levels. Mangrove sediment is a well known habitat that provides a unique ecological niche to a number of endemic organisms- which includes many microeukaryotic species.

microeukayotes are used as an effective measure of a contaminant, as they demonstate the key features of a bioindicator, including their abundance, genetuc diversity and reduced generation time. This allows for rapid responces to environmental changes to occur.

in the past decade, 18s rDNA clone libraries have been crucial in the development of molecular surveys for the studying marine microbial diversity. recent studies have focused on the small subunits 18s ribosomal RNA gene fragments have revealed a great diversity of microeukaryotes. Mangroves are usually exposed to pollutants such as those released by oil spills.

This study, aimed to evaluate the impacts of oil on major microeukaryotes groups in mangrove sediment by PCR/DGGE and using clone libraries to search for potential candidates for use as bioindicators of oil, or in further studies of mangroves bioremediation and biomonitoring using microeukaryotes.

Methods:This study was conducted using opaque tube microcosms. Each microcosm then recieved 350g/L dry weight of sediment from the Restingua da inarambaia, rio de janerio, Brazil. after a setting period of 3 hours, oil contamination of the microcosm then occured. (2% v/w contamination)

T0 = Before oil contamination.

T23= 0% 23 days without oil.

T23= 2% 23 days with oil.

T66= 0% 66 days without oil

T66= 2% 66 days with oil.

to replace evaporated water 100ml of distilled water was added every 2 days. For each microcosm a 200g sediment a liquot was extracted so a total petroleum hydrocarbon analysis could be conducted. 0.5g from each microcosm sample was then used to extract the DNA and the clone library complete.

Results:

Library NS9 OTUs OUTs, Richness Shannons ESCd

0 125 46 97 69 319 080

23 91 20 47 37 205 087

66 87 32 51 42 289 083

The study found that the clone library analysis revealed a decrease in both diversity and species richness after contamination. The phylogenetic group that showed the greatest sensitivity to oil was the nematode. After contamination a large increase in the abundance of the groups Bacillariophyta and Biosoecida was detected. The oil contaminated samples were almost entirly dominated by organisms related to Bacillariophyta sp and cafetenia minima, which indicates that these groups are possible targets for biomonitoring oil in Mangroves. The DGGE fingerprints also indicated shifts in Microeukaryote profiles, specific band sequencing indicated the apperance of Bacillariophyta sp only in contaminated samples and nematoda only in non contaminated sediment. The richness estimators and the Shannon diversity index showed a decrease in microeukaryotic diversity and species richness after oil contamination occured.

The study was able to consider the application of molecular tools to evaluate the effects of oil on microeukaryotes in mangrove sediments.The clone library indicated a predominance of the fungi/ metazoa (70%), stramenoplies (25%), and alveolata (9%). The specifity of the groups suggests this diversity pattern is directly related to mangrove sediment. A number of Trichosporon sp, which was found in high numbers of the samples are extremely common in marine sediments and polluted waters. They are also able to assimilate phenolic compounds. The species that showed the greatest sensitivity to the oil contamination was nematodes. This species have been used in biomonitoring surveys as they are excellent indicators of pollution in marine ecosystem. in contaminated samples a large abundance of Bacillariophyta was detected. In conclusion the use of molecular techniques for the monitoring of oil contaminated mangroves is a quick and effective tool to indicate anthropogenic effects. With the presence of Bacillaiophyta sp and Cafeteria minima as targets for the presence of petroleum hydrocarbon in Mangrove sediments.