Can viruses have a positive effect? The effect of Viral lysis on Synechococcus growth

A review of: Weinbauer, MG. Bonilla-Findji, O. Chan, AM. et al (2011) Synechococcus growth in the ocean may depend on the lysis of heterotrophic bacteria. Journal of Plankton Research, Vol. 33 (10) 1465-1476.

Carbon cycling is an important process which occurs in the oceans, Carbon is the basic unit of life and therefore all organisms rely on it for survival. Synechococcus and Prochlorococcus, two genera of unicellular Cyanobacteria are responsible for an astounding 50% of the total primary production in some parts of the ocean and understanding the fate of the carbon which they produce is important to understanding the complex nutrient and energy cycles which occur.

Synechococcus is very abundant and causes various blooms during the year, Heterotrophic nanoflagellates graze on Synechococcus, however the cyanobacteria is so abundant that this does not have much affect on the population. Mortality can also be due to viral lysis caused by Cyanophages; viral lysis results in the release of dissolved organic matter (DOM) such as nitrogenous and iron containing compounds, as well as the release of inorganic matter. However in their natural environment several studies have shown that viral lysis is only a moderate source of Synechococcus mortality compared to that of heterotrophic bacteria. This study looks into the effects of viral lysis in the ocean and concentrates on the lysis of heterotrophic bacteria and its effects on the growth and abundance of Synechococcus.

Two sampling sites were used throughout this study, one in the Gulf of Mexico and the second in the Mediterranean; both are areas where Synechococcus contributes >50% to primary production. Samples taken from these sites between September 2003 and April 2005 were filtered to remove viruses, heterotrophic bacteria and cyanobacteria (namely Synechococcus). Different methods were used with each sampling site but all followed a general consensus of filtrations, dilutions and in some heat treatments to inactivate viruses. A TEM (transmission electron microscope) was even used to make observational views on infected cells.

In both experiments Synechococcus abundance increased regardless of the treatment or dilution when viruses had contact with host cells; however when diluted with virus free seawater the growth rate of Synechococcus was much lower. It was also shown that Synechococcus growth and division was significantly higher with active viruses. The most likely explanation is that viral lysis of heterotrophic bacteria releases nutrients required for the growth of Synechococcus.

Since the abundance of viruses present in sea water has been recognised (10 to every bacterial cell), it has become important and fascinating to find out what role they play in the oceans. The idea that viruses could be beneficial to an organism is interesting as they are normally thought of in a negative light even though theories have already encompassed their use in the evolution of the eukaryotic cell. However there are many possible explanations for the above results and the idea that viruses could be removing competition is also another viable theory. I believe that it is more likely that there were various elements contributing to the abundance of cyanobacteria and that the combination of these is what is making Synechococcus growth so successful.

Giant Viruses and the Creation of Eukaryotes

Trying to understand how the complex eukaryotic cell evolved is not fully understood. How did a simple prokaryote cell with no membrane-enclosed organelles give rise to cells that contained mitochondria, chloroplasts and a nucleus? One possible answer for this is the viral eukaryogenesis (VE) hypothesis.

According to the VC hypothesis the mitochondria was believed to have originally been a free-living marine alpha-proteobacterium. Possibly 2 billion years ago, this alpha-proteobacterium found its way into a larger archaeon. Whether this was an infection of the archaeon or the archaeon cell consuming the alpha-proteobacterium via phagocytosis remains unclear. In either case, the cells then developed an evolutionary symbiosis. The same hypothesis explains the rise of chloroplasts having come from an engulfed cyanobacterium and eventually gave rise to algae and then plants. What remains less understood is the sudden development of a nucleus within a cell. The VC hypothesis tries to explain this as being the result of a virus that infected the cell but instead of killing the cell became an essential part of it.

In 2003, a new virus was described that infects the genus of amoebae called Acanthamoeba. What is special about this virus is its giant size, both physically (750nm) and the size of its double-stranded DNA genome (1.2Mb) (Raoult et al, 2004). This is actually bigger than some marine bacteria. These viruses are now called mimiviruses (Mimiviridae) and have been given the nickname ‘giruses’.

In this study, an ancestor of the mimivirus is believed to be the origin of the eukaryotic nucleus for several reasons. Just like a eukaryotic nucleus the mimivirus genome is double-stranded, there are repetitive base sequences at their telomeres and have DNA polymerases that are homologous to eukaryotes but not prokaryotes. Mimiviruses also contain enzymes that are used in the capping of mRNA to protect it from degradation inside the host’s cytoplasm. This is not seen in prokaryotic cells but is in eukaryotic nuclei. This also means that once the viral capsid is inside the host cell it is able to carry out DNA replication and transcription independently. Although it still relies on the cell for translation and energy production.

Bell (2009) suggests that like some other viruses, the mimivirus causes the cell membrane to pinch off internal vesicles that is then packaged with DNA to produce new virions. Possibly, as mimivirus are so big, the host genetic material started to get packaged into these vesicles too. The virus remained in the cell and took on the role of DNA storage instead.

The VC hypothesis was originally suggested back in 2001 but with the discovery of the mimivirus a couple of years later this hypothesis had even more evidence to back it up. It is also an expansion on the endosymbiotic theory which is an older and better known theory. It is still just a theory of coarse but I think one that has a lot of strong evidence to back it up. It will be very interesting to see if there are more breakthroughs in this field in the near future.

A Review of:
Bell, P. J. L. (2009) The Viral Eukaryogenesis Hypothesis: A Key Role for Viruses in the Emergence of Eukaryotes from a Prokaryotic World Environment. Ann. NY Acad. Sci, 1178, 91-105.

Additional References:
Raoult, D. Audic, S. Robert, C. Abergel, C. Renesto, P. Ogata, H. La Scola, B. Suzan, M and Claverie, J (2004) The 1.2-Megabase Genome Sequence of Mimivirus. Science, 306, 1344-1350.

Hydrogen is an energy source for hydrothermal vent symbioses

Deep sea hydrothermal vent ecosystems are dominated by organisms that are in a symbiotic relationship with chemosynthetic bacteria. Before this paper only two types of metabolism in the symbiotic bacteria had been established, these were the reduction of sulphur compounds and the reduction of methane. The aim of the investigation was to determine if symbiotic bacteria in hydrothermal vent organisms could utilise hydrogen as an energy source. The authors state that it is likely there are different types of metabolism in symbiotic bacteria because bacteria which can utilise hydrogen, ferrous iron, ammonium and manganese have been found in hydrothermal vents, however these metabolism pathways have not been found in bacteria which are in a symbiosis.

Hydrogen would be an ideal source of energy, as it is a favourable electron donor, using a thermodynamic model it was predicted that hydrogen oxidation could release as much as 7 times more energy per kilogram of hydrothermic vent fluid compared to methane oxidation while compared to sulphur oxidation as much as 14 times more energy could be released. Furthermore there are some hydrothermal vents in which the vent fluid has a high concentration of hydrogen, for example the Logatchev vent field is located in a ridge segment characterized by ultramafic outcrops, the fluids that vent from this site have the highest hydrogen concentration ever measured in a hydrothermal vent system, this would act as a rich energy source for chemosynthetic microbes.

The experiment began by sequencing genes from bacteria found in the gill tissue of mussels from the Logatchev vent field (high hydrogen concentration). The enzymes that are involved in hydrogen metabolism are hydrogenases, enzymes of the group 1 NiFe hydrogenases are membrane bound respiratory enzymes and provide a link from hydrogen oxidation with energy production by channelling hydrogen into the Quinone pool. The genes which encode for the membrane bound uptake hydrogenases is the hupL gene, the gene was amplified and sequenced from B. puteoserpentis symbiont containing mussel gill. The sequence then underwent phylogenetic analysis and was compared with a similar sequence from Oligotropha carboxidovorans which is known to utilise both CO or H₂ as electron donors. The enzyme from B. puteoserpentis was found to be placed in a cluster with other group 1 NiFe hydrogenases, showing that B. puteoserpentis has the genetic capability to utilise hydrogen as an energy source. As a comparison endosymbionts of mussels found in hydrogen poor vents were also analysed for the hupL gene, it was found that the gene could be amplified in hydrogen poor endosymbionts suggesting that the potential to use hydrogen as an energy source is not restricted to those microbes in high hydrogen concentrations.

As the genetic potential to utilise hydrogen as an energy source was expressed, gills from the mussels were then incubated with hydrogen of 100ppm. The mussel gills from the Logatchev site (B. puteoserpentis) were found to take up hydrogen at a rate of 650+200nmol h^-1 (g wet weight)^-1 , whereas the symbiont free gill tissue did not take up hydrogen above the rates of the negative control. Furthermore mussels from the basalt hosted vent fields (low hydrogen) were found to take up hydrogen, however this was 20-30 times lower than the mussels from the Logatchev vent.

Furthermore to determine if hydrogen was in fact acting as an energy source, because hydrogen uptake is not necessarily coupled with carbon fixation, the mussel gills were incubated in water containing ¹⁴C-bicarbonate. The control gills used were those incubated with sulphur and those incubated without an electron donor; it was found that ¹⁴C uptake was stimulated by sulphur (which is known to be en energy source for the symbiont) and also hydrogen. As the rates of ¹⁴C uptake were similar in both hydrogen oxidation and sulphur oxidation the authors concluded that hydrogen was in fact acting as an energy source.

The effect of hydrogen concentrations on hydrogen uptake was also tested; some hydrogen oxidising microbes only express hydrogenases in the presence of hydrogen, while some express them at low levels and then increase them in the presence of hydrogen. The mussel gills were incubated with hydrogen at partial pressures up to 3,000 ppm, the hydrogen uptake increased by 135, 21 and 8 nmol h^-1 (g wet weight)^-1 respectively for each increase of 100 ppm in hydrogen partial pressures, therefore hydrogen uptake is clearly stimulated by increasing hydrogen concentrations.

The final experiment which was conducted was to test to see if sulphur oxidising symbionts can also utilise hydrogen as an energy source. There were two different conditions, those were the fluid from the vent had not been exposed to B. puteoserpentis mussels, and situations where they had. The slope of the regression line calculated for hydrogen concentration versus temperature was significantly lower in the mussel bed compared to the source fluid, therefore indicating that the fluids inside the mussel bed are depleted of hydrogen in comparison with the sources fluid. This result indicates that the hydrogen is being utilised by the bacteria and therefore showing that significant amounts of hydrogen are consumed at the Logatchev trench. This is corroborated through the findings that sulphur oxidising symbiont of Bathymodiolus was found to possess the genes needed for uptake and oxidation of hydrogen, these were found on the same genome fragment that contained the genes for sulphur oxidation and carbon fixation showing the hydrogen oxidising genes were from the symbiont.

The paper was extremely well written and easy to understand the results and the reasoning behind the experiments. However when it came to addressing the significance of finding that many bacteria in a symbiotic relationship use hydrogen as an energy source, I feel this was poorly addressed, only really stating that it may act as a major hydrogen sink, but not really explaining why this would be important.

Reference: Petersen, J.M., Zielinski, F.U., Pape, T., Seifert, R., Moraru, C., Amann, R., Hourdez, S., Girguis, P.R., Wankel, S.D., Barbe, V., Pelletier, E., Fink, D., Borowski, C., Back, W., Dubilier, N. 2011. Hydrogen is an energy source for hydrothermal vent symbioses. Nature., 476, pp. 176-180.

The importance of marine microbes and their relation to the productivity of the deep sea.

A review of: Das, S., Lyla, P and Khan, A. 2006. Marine microbial diversity and ecology: importance and future perspectives. Current science. Vol 90. No 10.

The largest habitat biospheres on earth provides the largest living space for organisms, especially for marine microbes. the term microorganism encompasses a wide variety of organisms including bacteria, viruses, protists and fungi. the deep sea is a unique and physically extreme environment. Barotolarent bacteria from depths of 500m were first discovered by certes, 1983. It thought that these microbes may exist in a state of suspended animation. deep see expeditions to work on deep sea microbiology undertook by Zobell 1952, stated that these heterotrophic bacteria utilize the abundant organic carbon or dissolved organic matter in a threshold concentration. These microbacterial split refractory polymeric substances, and further monomeric and oligomeric molecules to either the bacterial metabolism or the dissolved organic matter (DOM) pool. The deep sea bacteria are therefore efficiently adapted to extreme oligotrophy and the activity is mainly limited by substrate availability. Bacteria have adapted themselves for life in the extreme marine environment. the bacteria require high levels of Na+. This is vital for maintaining the osmotic environment for protection of cellular integrity.

The role of bacteria within the marine environment.
heterotrophic bacterial functions promote organic degradation, mineralisation and decomposition, and adds to the release of dissolved organic and inorganic substances. This organic matter which is mainly derived from primary producers, is recycled so these are again available for primary producers. These heterotrophic bacteria make up the bulk of the microbial populations inhabiting the water column. They are also responsible for the production of organic matter and carbon dioxide.
the study concludes that marine micro-organisms are able to breakdown all natural organic compounds into the compounds they originated from. The breakdown of protein takes place by proteoytic bacteria e.g. pseudomonas. cellulose within the ocean is mainly broken down by cellolytic bacteria e.g. cytophaga, chitin. This is then synthesised by some marine organisms e.g. cell wall of chlorophytes, extracellular material from algae and exoskeleton and molts from copepods.
The study also highlights the role that microbes play in the formation of sediment. The total amount of organic matter gradually decrease over time within the sediment, the structure of which therefore changes. It highlights the point that the bacteria will colonize suspended particles, changing their size, shape and therefore the sedimentation rate.
secondary metabolites. These are produced during the idiophase of microbial growth. One excellent example of a secondary metabolite only found in the marine environment is the production of antibiotics by marine bacteria.

Pairing Up Pays Off: Symbiosis confers tolerance to environmental stresses.

A review of: Richier S et al (2004) Symbiosis-induced adaptation to oxidative stress. Journal of Experimental Biology, 208: 277-285. France.

O₂ has low toxicity but can be converted to more toxic reactive oxygen species (ROS) such as superoxide during hyperoxia. Diurnal variations in O₂ levels in tissues are well documented in photosynthetic organisms such as plants and symbiotic cnidarians.

Superoxide dismutase (SOD) is the first line of anti-oxidant defence from ROS in aerobic organisms. This enzyme is highly conserved across kingdoms and is observed even when other enzymes (i.e. catalase, which works in conjunction with SOD) are not present. Plants are known to contain between 4-8 isoforms of SOD while animals have 2-3. The authors have previously described (Richier et al 2003) a large diversity of SOD isoforms from 3 well known SOD classes (Mn, CuZn and FeSOD) in symbiotic Anemonia viridis and Stylophora pistillata. A MnSOD (typically found in the mitochondria) in A. viridis tissues was also observed outside the mitochondria, along with an FeSOD isoform typical in CO₂ fixing organisms but not in animals.

The aim of the study was to determine the role of symbiosis in oxidative stress and thermal tolerance, as well as to assess the role of the symbiont in expression of specific SOD isoforms in animal cells. Richier et al (2004) compared effects of hyperoxia between two sea anemones, symbiotic A. viridis and non-symbiotic Actinia schmidti. Biomarkers such as protein carbonylation as a measure of protein oxidation and malondialdehyde as a measure of lipid peroxidation were used to gauge cellular damage. SOD activity was used as a biomarker for anti-oxidant defence.

Native gel electrophoresis identified five activity bands from Mn, CuZn and FeSOD in A. viridis and three bands from two isoforms of CuZnSOD, with one MnSOD in A. schmidti controls. Moreover, quantitative analysis revealed 1.8x higher SOD activity in A. viridis (10.5 U mg^-1) when compared to A. schmidti (5.88 U mg^-1). After 10 hours of exposure to hyperoxia, there was no change in SOD isoform expression in A. viridis but a new isoform of CuZnSOD and a slight decrease in SOD activity was observed in A. schmidti. In addition, there was no significant variation in damage biomarkers in the symbiotic anemone, in contrast to a six-fold increase in protein carbonylation seen in the non-symbiotic anemone. After 5 days of elevated water temperature a three-fold increase in protein oxidation was observed in A. viridis, but a ten-fold increase in protein oxidation and a three-fold increase in lipid peroxidation was detected in A. schmidti.

The effects of elevated temperature on symbiotic and aposymbiotic (bleached) specimens were described using anemones A. viridis and Aiptasia pulchella. Bleached A. viridis and A. pulchella exhibited an overall decrease in SOD activity in both endoderm and ectoderm, with the loss of some activity bands from Mn and FeSOD in A. viridis and the loss of 3 MnSOD isoforms in A. pulchella.

Diversity of SOD isoforms in animal cells living with photosynthetic dinoflagellates is associated with increase in overall SOD activity, as a consequence of host adaptation to symbiont photosynthesis and elevated ROS production during exposure to light. The sensitivity of non-symbiotic A. schmidti to elevated oxygen and temperature suggests symbiosis is crucial in conferring host cell adaptations to environmental stresses, such as enhanced antioxidant defences, to limit and/or avoid cellular damage.

The decrease in SOD activity in aposymbiotic specimen tissues in both endoderm and ectoderm (which doesn’t have zooxanthellae) suggests a decrease in oxidative stress in response to a decrease in photosynthetic activity, and possibly as a result of disruption of the anthozoans’ normal cellular processes.

The possibly cytosolic MnSOD and FeSOD described by Richier et al (2003) in animal tissues is rather unusual and could be an evolutionary adaptation to hypoxia. It is also possible that genes were exchanged between the two closely associated organisms. What is evident is hyperoxia induced by symbiont photosynthesis necessitates higher antioxidant defences in the host. Moreover, the hyperoxic adaptation of the symbiotic species may also be a preconditioning step to limit cellular damage by various environmental factors, such as elevated water temperature. There is clearly a complex interplay between host cells and symbionts which is to be the subject for further investigation.

Additional Reference:

Richier S et al (2003) Characterization of superoxide dismutases in anoxia- and hyperoxia-tolerant symbiotic cnidarians. Biochimica et Biophysica Acta, 1621: 84-91. France.

Photoheterotrophy in marine prokaryotes

A review of: Zubkov, M. V. (2009) Photoheterotrophy in marine prokaryotes. J. Plankton Res. 31: 933-938

Knowledge of light- utilizing prokaryotes has increased rapidly driven by the growing interest of the scientific community to understand the microbial use of solar energy. The intention of the author is to propose new avenues of enquiry. This is achieved by examining how solar radiation is exploited in the photic zone by investigating the different metabolic strategies used by marine microbes.

Over half of the prokaryotes found in our oceans populate the thin surface layer known as the ‘photic’ layer. These organisms have adapted to develop mechanisms which absorb and exploit solar energy as a way to increase their metabolism. Recently, the attention of the scientific community has largely been on aspects of oxygenic photosynthesis because of its global biological and geological significance in the generation of oxygen and carbon reduction. However, as evidence of alternative metabolic methods which contribute to the survival of microbial communities emerges, the author advocates that a shift in the attention of the scientific community may be of interest.

Photoheterotrophy is a common strategy adopted by marine prokaryotes. Photoheterotrophic acquisition of organic molecules containing nitrogen by cyanobacteria is suggested to be energetically beneficial to an individual when compared with de novo synthesis of the same molecules. Photoheterotrophic organisms are suggested to be better adapted to variable light conditions and dark periods by storing some light energy as chemical bonds or by switching to heterotrophy.

The author suggests that the role of photosynthesis in phototrophic organisms assumed in previous research is too narrow because other aspects of utilising light have been overlooked. Both principal types of light harvesting (rhodopsin-based and chlorophyll-based) produce energy via a photon induced electrochemical potential gradient. This can be used for photophosphorylation which is preserved as ATP. This ATP can be utilised in various metabolic processes, one of which is photosynthetic carbon reduction. Bacteriorhodopsin/proteorhodopsin based light absorption is discussed as an alternative to chlorophyll-based light harvesting. This method of utilising light is noted to be a relatively small evolutionary step and once acquired; proteorhodopsins could have ecological benefits for the organisms that possess them. These advantages are still to be experimentally shown in marine prokaryotes but can be identified in eukaryotes such as the alga, Acetabularia.

Oxygenic phototrophs rely on photophosphorylation to maintain photosynthetic carbon reduction, regulation of dark photoreactions, and elementary cell metabolism when nutrients are limited. However innovative photoheterotrophic strategies have been identified where ATP synthesis can be avoided and a proton –motive force is generated which can directly drive cell energetics, cell motility and importation of molecules in to a cell without producing oxygen. Photoheterotrophic strategies such as this could potentially give an organism an ecological advantage. The author implies that the heterotrophic strategies of marine prokaryotes, like cyanobacteria, could be as refined. Nevertheless, further investigation is needed.
The author advocates that photoheterotrophic prokaryotes may be able to regulate their uptake and use of solar energy. This is determined by the energetic demands of the metabolic processes of a cell. However further experimental evidence is needed.

The unknown complexity of oceanic photoheterotrophic prokaryotes could potentially influence carbon flow, and thus the application of current biogeochemical models. Energy could be channelled directly into cell metabolism, growth and nutrient acquisition by avoiding CO2 fixation because of a large, but unquantified, amount of solar energy being harvested by photoheterotrophic organisms. Due to this, element-based models may not be adequate. Models surrounding energy and protein flow are suggested as an alternative.

Photorhodopsin Phototrophy Promotes Survival of Marine Bacteria?

A review of: Gómez-Consarnau L, Akram N, Lindell K, Pedersen A, Neutze R, et al. (2010) Proteorhodopsin Phototrophy Promotes Survival of Marine Bacteria during Starvation. PLoS Biol 8(4)

Proteorhodopsins (PR’s) are light driven, membrane bound proton pumps that are found in some marine microbes at the photic level of the ocean. They allow a chemiosmotic gradient to be built up across their membranes resulting in the production of adenosine triphosphate (ATP) and therefore energy, without the need for chlorophyll. The ecological role of PR’s is largely unexplored and so they are being investigated as a contributor to energy flux and carbon cycling within our seas. Their role in bacterial reproduction success has also been investigated with little success. This journal, therefore, focuses on a member of the Vibrio species and investigates how PR’s allow the long term survival and growth of the AND4 strain when starved in seawater exposed to light compared to darkness.

Strain AND4 was isolated from the Andaman Sea and a PR encoding gene was identified from the whole genome sequence. After analysing its 16s rRNA it was found this gene shares a similarity of 87% with the PR encoded genome sequence of the V. haryeyi strain BAA – 1116, putting both within the Gammaproteobacteria group. However, their PR’s were found to cluster with Alphaproteobacteria when it came to similarity. This suggests that the PR genes and its chromophore have been acquired through lateral gene transfer from other, maybe distantly related marine bacteria. Does this mean that the genes do confer a competitive advantage to those which have obtained them? The PR gene has not being found in any other species of vibrio (to the authors knowledge). It was also discovered, from isolates of both Vibrio strains, that the amino acid Leu is found in a position that allows the PR light absorption peak to be concentrated within green light frequencies (absorption frequencies 535nm). This allows Phototrophy to be successful as this is the dominant light condition within sea waters.

To explore whether the PR’s do increase the success of the Vibrios that carry them, their effects on growth and survival was investigated. The AND4 growth experiments on a rich medium showed no difference in cell yields in light (133µmol photons m−2 s−1) or dark conditions. Transfer of these cells to a sterile and particle free natural sea water with low concentrations of nutrients also showed an increase in cells, however biomass did not increase. This means the cells were increasing in number but were smaller in size; this is called reductive division and is a technique shown in many Vibrios when nutrients become scarce. After 10 d of incubation however, although all bacterial numbers decreased, the PR cultures left in light were 2.5 times higher than those in darkness. This strongly suggests that PG phototrophy can improve marine bacteria survival when nutrients are not readily available in seawater.

Experiments completed at different light intensities also back up PR phototrophy as a plausible back – up energy source when there are limited nutrients. The cultures kept at high light intensity, rather than in 16:8h light:dark cycles, had optical densities that were higher by 40 – 60%. Differences between dark cultures and low light cultures were less pronounced but still showed PR’s would help during phases of starvation. Bacterial numbers peaked at day 3 of the experiments but decreased thereafter. However the cultures kept in high light remained nearly twice as high in numbers as all other experiments.

A photorhodopsin deficient Vibrio strain was also generated in this experiment and its growth recovery was compared to the wildtype. After 5 days the wildtype cultures kept at high light during starvation were at 3 -6 fold higher densities than those kept in darkness, there was no difference in density between the PR deficient strains kept in light or dark conditions during starvation.

All experiments completed in this paper suggest strongly that the presence of photorhodopsins and therefore photorhodopsin phototrophy can substantially help to increase life longevity in microbes when other resources are scarce. This is interesting as energy is created with light without the need for chlorophyll, a process that I have not come across before. It would be interesting to see which other microbes contain photorhodopsins and to study further how their presence will affect microbe communities in the future and what affect this could have on our seas.