Proteorhodopsin act 2- What is the ecological role it plays?

As discussed in my previous blog post, proteorhodopsin (PR) is an integral membrane protein, which may be used to ‘power’ marine bacteria when respiration is inhibited. This allows them to switch from normal respiration and become light powered, using the energy from the light to generate ATP. The previous study looked at was Walter et al., 2006, who modified E.coli to possess PR, to show how bacteria possessing it can survive in environments where respiration is inhibited. Studies such as this demonstrate PR’s function as proton pumps with energy-yielding potential, however the actual ecological role of them and how they contribute to the success of the bacteria containing them is still relatively unknown.

Furthering the work done by Walter et al., (2006), a study was conducted by Gomez-Consamau et al., (2010). They aimed to uncover the biological function of PR and how they contribute to the success of PR containing marine bacteria. They used a strain of the widespread genus Vibrio, a strain known to contain PR. The strain AND4 was isolated from ocean surface water. The whole-genome sequence of AND4 was carried out and a phylogenetic tree of 16s RNA genes constructed. Measures of AND4 PR absorption maximum and photolysis rates were also taken (Gomez-Consamau et al., 2010).

They investigated the survival of the Vibrio bacteria when incubated in light and dark conditions. They found that after 10 days of incubation in each condition, numbers decreased in all cultures, but remained 2.5 times higher in light conditions. This shows that bacteria growing in light conditions can respond more rapidly to improved growth conditions than those in dark conditions (Gomez-Consamau et al., 2010).

Therefore, this study provides evidence linking the PR gene to its biological function in marine bacterium, suggesting that it confers a fitness advantage, allowing marine bacteria to endure periods of resource deprivation at the ocean’s surface (Gomez-Consamau et al., 2010). Their function as proton pumps has been covered in previous studies, however this piece of research takes it step further in actually explaining the ecological role of PR.

I believe a key aspect of this study by Gomez-Consamau et al., is the fact that they generated a strain of Vibrio that was deficient in PR. The PR gene was removed by an in frame deletion of the near complete PR gene. This means that they were able to establish that the PR gene is the direct conveyer of the light-enhanced survival during starvation. This is of great importance as it shows that increased rates of survival and the ability to actively respond to increased growth conditions are as a direct consequence of having the energy harvesting potential of PR.

Gómez-Consarnau, L., Akram, N., Lindell, K., Pedersen, A., Neutze, R., Milton, D., González, J., Pinhassi, J.. (2010). Proteorhodopsin Phototrophy Promotes Survival of Marine Bacteria during Starvation. PLOS Biology Biol 8(4): e1000358. doi:10.1371/journal.pbio.1000358

Walter, J.M., Greenfield, D., Bustamante, C., Liphardt, J. . (2006). Light-powering Escherichia coli with proteorhodopsin. PNAS. Vol. 104 pp.2408-2412.

Small but Not Safe. Phages Fall Prey to Protists

Viruses are the most abundant biological entities on the planet. They play a key role in regulating biogeochemical cycles and are thought to constitute large reserves of carbon in the marine environment. However viruses do not persist for ever and maybe removed by biotic and abiotic mechanisms. For example, adsorption onto surfaces and the action of bacterial extracellular nucleases and proteases. Protistan grazing also removes viruses, but this area is poorly studied. Deng et al. (2014) set out to investigate if protists with different feeding mechanisms differed in their ability to remove viruses.

Three different heterotrophic flagellates, isolated from ground water, were used in the study. The Choanoflagellate Salpingoeca sp., a filter feeder, Thaumatomonas coloniensis a Thaumatomonad, which uses pseudopodia to graze sedimentary particles and Goniomonas truncata, a raptorial feeder which actively searches for prey. The model RNA phage MS2 was used to measure grazing. Flagellates were incubated in the dark at 12^oC with local bacteria and viruses along with the phage MS2. The numbers of MS2 were enumerated regularly using the standard double-agar overlay technique. When incubated with T. coloniensis and Salpingoeca sp. the numbers of MS2 declined steadily from day 2 to undetectable levels by the end of the experiment, day 92. This affect was not seen for G. truncata, indicating raptorial feeders are unable to uptake or digest phages.

Ingestion was then examined in detail using T. coloniensis. Random absorption of the phages was ruled out by performing qPCR on subsamples of nucleic acid which confirmed the decline in MS2 was real. Ingestion of the phage was then examined by labelling with a protein dye and feeding them to the flagellates. Confocal microscopy showed the labelled phages were taken into the cells (see picture). The use of phages as a carbon source was then investigated by incubating T. coloniensis with a low concentration of bacteria and either a high or low concentration of MS2 for 28 days. When incubated with a low concentration of phages, flagellate numbers only increased up to day 14 and then dropped off. But for the high phage concentration, numbers continued to increase until day 28. This suggests phages can be used as a supplementary carbon source.

Although the species examined were from groundwater both Choanoflagellates and Thaumatomonads are represented in the marine environment (Munn 2011 & Ota et al. 2012). Considering a wide range of protists use similar feeding mechanisms it is likely that marine phages are also consumed in this manner. This has deep implications for marine ecology and biogeochemistry. It may be that viruses form another link in the microbial loop, as the carbon taken up via flagellates will feed through into higher trophic levels. Therefore the pool of viral carbon could be more accessible to the wider community than previously thought. Interestingly sponge choanocytes have a similar structure and feeding mechanism to Choanoflagellates. Is it possible that sponges feed directly on phages? In conclusion, the study highlights that such interactions must be investigated in the marine environment to fully understand the role that viruses play in marine food webs. 

Florescently labeled MS2 phage (green) after being ingested by T. coloniensis (Deng et al. 2014).

Deng, L., Krauss, S., Feichtmayer, J., Hofmann, R., Arndt, H. & Griebler C. (2014). Grazing of heterotrophic flagellates on viruses is driven by feeding behaviour. Environmental Microbiology Reports, 6(4), 325-330. 

Munn, C.B. (2011). Marine Microbiology: Ecology and Applications, 2^nd ed. New York: Garland Science. 

Ota, S., Eikem, W. & Edvardsen, B (2012). Ultrastructure and Molecular Phylogeny of Thaumatomonads (Cercozoa) with Emphasis on Thaumatomastix salina from Oslofjorden Norway. Protist, 163(4), 560-573.

The faster you are, the more you get to eat

The ocean is a turbulent environment and dissolved organic carbon (DOC) is quickly dissipated through diffusion and advection. The nutrient concentration is heterogeneous with patches of DOC appearing via sloppy eating, faecal pellets and cell lysis. Approximately 30% of oceanic bacteria have evolved the ability to use motility (Fenchel, 2002) and use chemotaxis to seek out patches of nutrients. But to what extent can they respond?

Previously, computer models have been used to predict bacterial response to nutrient patches, using known values for diffusion and observed bacterial motile behaviour, but are limited by being purely theoretical.

Here, Stocker et al (2008) has created new methods using microfluidics to analyse bacterial response to nutrient pulses in a micro-scale 1D environment.

Stocker et al (2008) visually examines how a known motile chemotactic bacterium (Pseudoaltermonas haloplanktis) responded to two types of nutrient pulses. He uses a small microfluidic chamber with a micro channel, which allowed flow when necessary.

A nutrient pulse mimicking a point source from a sloppy eater/cell lysis.

This was created, by adding the nutrient and bacteria simultaneously to the chamber with no flow, so that the nutrient dispersed laterally. To monitor nutrient diffusion dynamics, Stocker (2008) used a fluorescein as a proxy for a low molecular weight compound in DOM. The fluorescein is beneficial as it is not a chemoattractant to P. halopanktis and is a visible colour, being a dye. To visualise the bacterial response, Stocker et al (2008) used phase contrasting microscopy, recording numerous frames per second, and image analysis software to create trajectories of the bacteria.

Within tens of seconds the bacteria created a distinct band exactly where the nutrient was dispersing, and stayed in this band for 15minutes. When this response was compared to Escherichia coli (in the presence of two of the most chemoattractant compounds for this bacteria), P. halopanktis is ten times faster at chemotaxis.

Stocker et al (2008) mathematically simulated a 3D environment, and found that response time and swimming speeds could be even faster in 3D, and bacteria could potentially respond to patches of nutrient a fraction of the size simulated in the chamber.

A nutrient plume mimicking a sinking marine snow particle.

In a similar format to the previous, except Stocker et al (2008) used a stationary silicone particle in the chamber, and switched on the flow to create a plume projecting out behind the particle. He measured the response of P. haloplanktis to three ‘sinking speeds’, by increasing the flow from the micro channel.

The slowest sinking speed (66um/s) created the widest plume, and the bacteria aggregated in the plume right behind the particle. The concentration of bacterial cells was four-fold higher within the plume than outside. The intermediate speed (220um/s) was also rapidly aggregated, but the concentration of bacteria was slightly further back from the particle than the previous. The fastest sinking speed (660um/s) seemed to be too fast for the bacteria to aggregate, although they did swim into the nutrients, but couldn’t form an aggregation. However, this would be expected, as the swimming speed of these bacteria is 68um/s.

Next, Stocker et al (2008) used these results and calculated the nutrient exposure experienced by motile chemotactic bacteria compared to non-motile bacteria. The conclusion being that chemotaxis creates a considerable advantage when compared to non-motile bacteria. Motile bacteria utilising particles sinking at 66um/s will be exposed to nutrients by 4-fold compared to non-motile bacteria.

This paper was able to provide evidence to support theoretical models of how quickly motile bacteria respond to sudden pulses of nutrients in the ocean. It was achieved using novel methods - microfluidics. The results could then be mathematically simulated to give figures for 3D environment, which has improved the knowledge of bacteria’s ability to seek out nutrients in the ocean.

Primary reference:

Stocker, R., Seymour, J. R., Samadani, A., Hunt, D. E., & Polz, M. F. (2008). Rapid chemotactic response enables marine bacteria to exploit ephemeral microscale nutrient patches. Proceedings of the National Academy of Sciences, 105(11), 4209-4214.

Link: http://www.pnas.org/content/105/11/4209.full

Other reference:

 Fenchel, T. (2002). Microbial behavior in a heterogeneous world. Science,296(5570), 1068-1071.