Co-culturing reveals shift in gene expression levels compared to solo cultures

Co-Cultures of Pseudomonas aeruginosa and Roseobacter dentrificans Reveal shifts in Gene Expression levels compared to solo Cultures

Interactions among diverse microbial species are dynamic and most likely the basis for many adaptations that allow the occupation of diverse niches. These interactions may be beneficial, forming a mutualistic relationship such as symbiosis (Rosenberg & Zilber-Rosenberg 2011) or antagonistic through competition for resources or space etc (Long & Azam 2001; Rypien et al. 2010). The molecular basis of some ecological interactions have been linked to the production of secondary metabolites noted by (Allen et al. 2010), who also discusses there different uses, such as intraspecies signalling or defence.  Many biologically active secondary metabolites have potential to be used for future medicines (Nunnery et al. 2010), and so the need for reliable biosynthesis of these metabolites is high. However, pure cultures are often unreliable in the yield, or consistent biosynthesis, of secondary metabolites (muscholl-Silberhorn et al. 2008).

This study attempted to induce, measure and track the expression of microbial genes while they grew in mixed cultures, in order to mimic antagonism and interaction in the natural environment. Two model bacteria were chosen Pseudomonas aeruginosa (P.a) PAO1 and marine Roseobacter denitrificans (R.d) Och114, due to the availability of their complete genomic sequence. All cultures and co-cultures (growth of >1 bacterial species within one flask), were sampled for standard RNA extraction at different time points, and levels of specific gene expression were tracked and quantified by using real-time quantitative PCR, using the SYBR green detection (Ginzinger 2002; Livak & Schmittgen 2001). Two genes from the two model bacterial genomes were chosen, and used to create the gene specific primer design (using PRIMER BLAST), these were; PhzA, RhdA, Betalact and DMSP. Gene expression in solo and co-cultures were compared using qPCR at intervals, with the solo cultures acting as controls.

Conway et al found that P.a, when co-cultured with R.d, had a much lower gene expression of both RdhA and PhzH when compared to the solo, control culture of P.a. However, when the gene expression of R.d co-cultured with P.a was measured a different pattern emerged.  Both Betalact and DMSP were lower than solo culture levels during the initial stages but after 30 minutes rose by a factor of 2 and then levelled off. After 2 hours both Betalact and DMSP decreased below solo culture levels. These results show that gene expression of certain target genes could be reproducibly induced, or affected, by systematic co-culturing in multistrain growth conditions.

Although not measured directly, Conway et al went on to suggest that quorum sensing (QS) may have played roles in the co-culture gene expression in this study. QS is the regulation of gene expression in response to changes in cell-population density, QS bacteria produce and release chemical signal molecules, called autoinducers, which increase in concentration with cell density (Miller & Bassler 2001). The detection of a minimal threshold stimulatory concentration of the autoinducer leads to an alteration in gene expression. It is possible that after the initial mixing of P.a and R.d, any autoinducers released by either species was diluted by at least a half and may have fallen below the minimum stimulatory concentration. On the other hand, it may be possible that interactions in mixed cultures involve the degredation, or modification, of autoinducers produced by other members of the community. Though they do state there are other other possible explanations for the patterns seen in these results, but it is clear that these patterns resulted from the mixed species co-culturing.

I originally came about this study during my project research on interactions between bacterial communities. I thought it was particularly interesting as it combined bacterial interactions with gene expression, instead of just stating whether one bacterium inhibited the other.

Here is a link to the study if anyone is interested; http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3330761/

Allen, H.K. et al., 2010. Call of the wild: antibiotic resistance genes in natural environments. Nature reviews. Microbiology, 8(4), pp.251–9. Available at: http://www.ncbi.nlm.nih.gov/pubmed/20190823 [Accessed March 3, 2012].

Ginzinger, D.G., 2002. Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Experimental hematology, 30(6), pp.503–12. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12063017.

Jaiswal, P., Singh, P.K. & Prasanna, R., 2008. ` SE REVIEW / SYNTHE Cyanobacterial bioactive molecules — an overview of their toxic properties. , 717, pp.701–717.

Livak, K.J. & Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif.), 25(4), pp.402–8. Available at: http://www.ncbi.nlm.nih.gov/pubmed/11846609 [Accessed October 3, 2012].

Long, R. & Azam, F., 2001. Antagonistic interactions among marine pelagic bacteria. Applied and Environmental Microbiology, 67(11), pp.4975–4983. Available at: http://aem.asm.org/content/67/11/4975.short [Accessed October 19, 2012].

Miller, M. & Bassler, B., 2001. Quorum sensing in bacteria. Annual Reviews in Microbiology. Available at: http://www.annualreviews.org/doi/pdf/10.1146/annurev.micro.55.1.165 [Accessed October 23, 2012].

Nunnery, J.K., Mevers, E. & Gerwick, W.H., 2010. Biologically active secondary metabolites from marine cyanobacteria. Current opinion in biotechnology, 21(6), pp.787–93. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3034308&tool=pmcentrez&rendertype=abstract [Accessed March 13, 2012].

Rosenberg, E. & Zilber-Rosenberg, I., 2011. Symbiosis and development: the hologenome concept. Birth defects research. Part C, Embryo today : reviews, 93(1), pp.56–66. Available at: http://www.ncbi.nlm.nih.gov/pubmed/21425442 [Accessed October 18, 2012].

Rypien, K.L., Ward, J.R. & Azam, F., 2010. Antagonistic interactions among coral-associated bacteria. Environmental microbiology, 12(1), pp.28–39. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19691500 [Accessed July 17, 2012].

Chemotactic response to extracellular products of Cyanobacteria

Chemotactic response to extracellular products of Cyanobacteria

Dissolved organic carbon (DOC) is released into the water column through extracellular exudation and cell lysis by cyanobacteria, such as the two important marine groups Synechocococcus and Prochlorococcus, which often constitute the bulk of photosynthetic biomass and are therefore responsible a significant proportion of primary production in oligotrophic waters. DOC is essential to the growth of heterotrophic bacteria and while they are expected to cluster around living phytoplankton cells, using chemotaxis to take advantage of the exuded DOC, yet they had not previously been examined quantitatively in association with prokaryotic phytoplankton. Seymour et al. (2010) tested whether the exuded chemical products of Synechococcus and Prochlorococcus are chemoattractants for three heterotrophic marine bacterium strains; Pseudoalteromonas haloplanktis, Silicibacter TM1040 and Vibrio alginolyticus. Global Ocean Sampling data from 15 open ocean sites showed that they occurred in 100%, 100% and 93% of samples, respectively, and are therefore likely to co-occur with Synechococcus and Prochlorococcus.

Synechococcus exudants were found to induce a strong chemotactic response from all three strains, with P. haloplanktis accumulating in concentrations up to 9-fold higher than the background levels. While all three strain exhibited a marked response, this was far stronger in P. haloplanktis than either of the other two strains, but was significantly different from only V. alginolyticus. Response time is driven by differences in the chemotactic velocity; typically a fraction of the maximum swimming speed but it also depends on the sensitivity of their chemoreceptor’s. Similar results were found for Prochlorococcus, with P. haloplanktis exhibiting the strongest response, but again this was not significantly different from Silicibacter TM1040. Although a slight chemotactic response was observed in V. alginolyticus, it was not significantly different from the control. Interestingly, V. alginolyticus was found to have a higher mean swimming speed than Silicibacter TM1040 (54 and 52µm s^-1 respectively), so perhaps it may much less sensitive chemoreceptor’s than Silicibacter TM1040. P. haloplanktis was found to have a much higher mean swimming speed than either of the other strains; 85µm s^-1.

The DOC values used here were 3-5 fold higher than commonly found in the oligotrophic ocean, however they were still within an environmentally relevant range as they are within concentrations that would be observed during a bloom event or large, localised aggregation of cells. Rapid chemotactic responses to the DOC released by these cyanobacteria are likely to provide a strong competitive advantage for heterotrophic bacteria, which will gain exposure to the nutrients before competitors. The rapid response of P. haloplanktis is likely to give it a strong competitive advantage in comparison to Silicibacter TM1040 and V. alginolyticus.

The chemotactic behaviour exhibited here provides a potential mechanism for the development of associations, recently found to commonly occur in the open ocean (in a separate study), between Synechococcus and heterotrophic bacteria. These associations may have major implications for nutrient cycling rates and microbial competition.

Seymour, J., Ahmed, T., Durham, W. & Stocker, R. (2010) Chemotactic response of marine bacteria to the extracellular products of Synechococcus and Prochlorococcus. Aquatic Microbial Ecology. 59, 161-168
http://web.mit.edu/romanstocker/publications/SeymourEtAl_AME_2010.pdf

Separate study mentioned is Malfatti, F. & Azam, F. (2009) Atomic force microscopy reveals Microscale networks and possible symbioses among pelagic marine bacteria. Aquatic Microbial Ecology. 58, 1-14http://www.int-res.com/articles/feature/a058p001.pdf

Marine Snow: Whole versus Fragmented

Marine Snow can be described as sinking particles of organic matter or macro-aggregates, which are aggregates greater than 5mm in size. The marine snow collects at the surface water, where there is an abundance of bacteria and organic matter creating aggregates. The aggregates increase in density and eventually sink to the sea bed where due to the lack of light in the deep sea the aggregates are the primary source of carbon as many organisms are unable to photosynthesise. As the particles sink to the seafloor it is exposed to the currents of the ocean, the feeding and swimming of passing organisms; they are preyed upon by phytoplankton, zooplankton, bacteria, protists. These processes produce not only a plume but smaller particles (daughter particles) which still have the same composition, the division or fragmentation of the particles creates a greater surface area for bacteria to colonise. Daughter particles have a reduced density, so they sink at a slower rate. This means that smaller particles are more likely to remain in the surface area and according to Goldthwait et al (2005) remineralization is likely to occur.

Goldthwait et al (2005) tested two consequences of first time fragmentation on macroaggregates. 

•  The immediate release of dissolved organic carbon (DOC) and interstitial nutrients into surrounding seawater,

• The elevated solubilisation and remineralization of daughter-particle carbon due to the increased available surface area for bacterial colonization.

Approximately 200-300 aggregates of marine snow were collected by scuba divers at surface waters of Santa Barbara Channel in California during the summer months of 2002 -2003. Two types of experiments were carried out; 1 fragmentation experiment and 2 aggregate remineralization experiments. Each experiment was tested on the two treatments of aggregates (whole and fragmented) as well as a seawater control.

The seawater control was used to simulate the 10 aggregates which were added to 11 glass stopper BOD bottles filled with 300ml of unfiltered seawater. 4 replicates held the whole aggregates, 4 replicates held the fragmented and 3 held the control.

The fragmentation experiment analysed filtered particles for POC (particulate organic carbon) and PON (particulate organic nitrogen). Then Nutrient samples were collected from the filtrate, and analysed for their concentration of phosphate, nitrate/nitrite and ammonia.

3 replicates of 40ml DOC samples from the filtrate were stored at -20 then analysed using high temperature combustion, to determine the amount of the carbon dioxide present.

The Aggregate remineralization experiments consisted of 2 experiments, which were performed on 11 bottles kept at surface temperature and rotated end to end to keep aggregates in suspension

        Experiment 1: the bottles were re-suspended in unfiltered seawater, incubated for 3 days

        Experiment 2: the bottles were re-suspended in 0.2 µm filtered seawater and incubated for 5 days.

The amounts of solubilisation and remineralisation in whole and fragmented aggregates were compared, by assessing the pools of carbon from each. POC and DOC were measured and as a result TOC (total organic carbon) was determined (POC+DOC=TOC).

The authors concluded that fragmentation results in the immediate release of interstitial DOC and macronutrients to surrounding seawater. Solubilision rates were the same for whole and fragmented aggregates suggesting the surface of aggregates do not regulate bacterial colonization. Fragmentation causes a decrease in the changes of aggregate-associated carbon that come from the DOC release and slower sinking rate of daughter particles. Fragmentation also does not seem to accelerate POC degradation.

This paper originally interested me because it explained more about the presence and importance of marine snow in deep oceans. The fact that an aggregate of marine snow can divide into two smaller daughter particles and keep the same composition is incredible. Aggregate fragmentation is one of the primary removal mechanisms for sinking particles (Goldthwait et al 2005), this paper looks at its biochemical outcome and impact on the carbon cycle and this information and results could be useful in situations of large scale pollution and attempts at removing pollution.

Goldthwait S. A, Carlson C. A, Henderson G. K, Alldredge A. L,(2005) Effects of physical fragmentation on remineralization of marine snow, Marine Ecology Progress Series, Vol. 305: 59–65, Published December 23

http://www.int-res.com/articles/meps2005/305/m305p059.pdf

Potentially Human Pathogenic Vibrios in Marine and Fresh Bathing Waters Related to Environmental Conditions and Disease Outcome

Vibrios are more common in water temperatures over 17-20°C. They can survive in a range of adverse environmental conditions, in order to do this they enter a viable but non-culturable stage in which metabolic activities are minimal but they still retain pathogenicity. Several vibrio species are human pathogens and have been associated with wound infections (V. vulnificus) and ear infections (V. alginolyticus) after exposure to contaminated water, and gastroenteritis (V. parahaemolyticus and V. cholera) after consumption of contaminated food.

During the summer of 2006, four people developed V. alginolyticus infections after swimming in a large inlet on the North Sea, at separate but nearby locations. The water was then tested and both V. alginolyticus and V. parahaemolytis were found in the water samples. This study is follow up to previous finding of vibrio species in North Sea inlets. The quantification and typing of potentially human pathogenic vibrio species were looked at, as these areas had not been studied before. Vibrio numbers in bathing water were related to environmental conditions, for example water and salinity, and to European legislative requirements for bathing water quality.

In this study, Schet et al monitored four bathing sites in the Netherlands; samples at the bathing sites were taken biweekly or four-weekly from April to October. It was found that vibrio species were detected at all sites. There were 447 isolated vibrios from water samples found. 50.6% of these were V. alginolyticus and 8.5% were V. parahaemolyticus. The water temperatures ranged from approximately 10 to 21°C, no vibrio were detected in samples when the water was below 11°c. In the majority of the samples, faecal indicator levels were below the mandatory values for good water quality according to the European Bathing Water Directive. There was a correlation found between vibrio and E. coli concentration, however it was not a strong correlation. An even weaker correlation was found between intestinal enterococci and vibrio. Throughout the period of the study, only one case of a bathing water related vibrio infection was reported, swabs were taken and 2 V. clorae isolates were cultured from the wound.

Overall, potentially pathogenic vibrio species were detected, however lower numbers of vibrio were found than expected. This is due to colder bathing water temperatures than normal compared to other data looked at. This may be something to look at in the future and do the same experiments during another summer. The results underlie the need for further molecular studies on a larger number of clinical isolates and isolates from water samples to understand clonal distribution with seawater V. chloerae isolates in relation to bacterial pathogenicity, water salinity or environmental parameters. This study highlighted the need for education of public health workers, bathing water managers and the general public in order to prevent vibrio infections from bathing water exposure.

I chose to review this paper as I am interested in what actually is in bathing water; I was surprised to find these results especially as I have been swimming where some of the samples were taken from! I think that more studies need to address water quality in terms of vibrio content, especially as water temperatures could raise due to global warming and therefore have more favourable growth conditions for vibrio species.

http://www.sciencedirect.com/science/article/pii/S1438463911000526

F.M. Schets, H.H.J.L. van den Berg, A. Marchese, S. Garbom, A.M. de Roda Husman (2011). Potentially Human Pathogenic Vibrios in Marine and Fresh Bathing Waters Related to Environmental Conditions and Disease Outcome. International Journal of Hygiene and Environmental Health. Volume 214, Issue 5, September 2011, Pages 399–406.

How prey bacteria shape the community structure of their predators:

Predator and prey interactions have been noticed and studied since as far back as anyone can remember, unfortunately for microbiologists there havent been many studies as far down as microbes. This is due to numerous reasons; the most obvious being that it just isnt as easy due to the sheer range in sizes of them and that most microbes are minute, so compared to studying fish the difficulties are easy to spot.

Thankfully Huan Chen et al, decided to look at Bacteriovorax and how its prey; Vibrio vulnificus & Vibrio parahaemolyticus affect its population structure and number. The method that was used was to have flasks full of seawater and a specific concentration of the Bacteriovorax and either the V.vulnificus or the V.parahaemolyticus within the same flask as the predator and to monitor the abundance of the prey at specific time periods after the two were added together.

The results shown by this study showed that in the presence of a ready supply of prey a significant multiplication of abundance was demonstrated and this increase was extremely quick, the exact speed was a thousand fold increase within 24hours, from the 24 hour time mark to the 48 hour mark.  This increase led to a 2-4 fold log of prey reduction. Along with this it was shown that the Bacteriovorax had a faster predation on the V.p compared to the V.v. There were two clusters studied to see if there was a preferential prey for the predator and what was found was that V.v was preferential in one and V.p in the other, so although the rate of predation may have been quicker, given a choice of one or the other there isnt a favourite. Aswell as this as expected the paper showed that with an increase in prey this led to the standard predator-prey fluctuation where then predator levels increased and prey abundance decreased.

The reason why this paper is important and interesting is due to the fact that it is the start of a hopefully increasing area of study and it provides a backbone for the future studies. So it's important to the ever growing field of marine microbiology.

I hope more studies are done like this as it's always interesting to study predator and prey interactions and as there hasnt been much in this field it will be interesting to see just how far down the trophic scale predation plays a major part.

Here is the webpage if you want to read this paper:

http://www.nature.com/ismej/journal/v5/n8/pdf/ismej20114a.pdf

Huan Chen, Rana Athar, Guili Zheng and Henry N Williams

ISME J 5: 1314-1322; advance online publication, February 17, 2011; doi:10.1038/ismej.2011.4

Marine microalgae attack and feed on metazoans

The marine food web has been shown to be severely affected by mixotrophic dinoflagellates, with microalgal blooms causing the death of fish, copepods and other metazoans. Karlodinium is an example of a genus of toxic microalgae that form such blooms in eutrophic coastal waters. They work by releasing neurotoxins that stun their prey before feeding via myzocytosis. This investigation focused on the species Karlodinium armiger and Karlodinium veneficum and their effects on the motility and mortality of the copepod Acartia tonsa and other metazoans. Karlodinium veneficum is a well known ichtyotoxic bloom, which produces Karlotoxins to stun prey before ingestion. Karlodinium armiger is also believed to work in a similar manner, however has not been studied as extensively, so the specific neurotoxin used is currently unknown.

The interactions between the microalgae and copepods were observed using an inverted microscope. Immobilisation was characterised by copepods lying on the bottom of the microwell displaying erratic movements but with gut movements still occurring, and death was identified when gut movements stopped. Shortly after adding the copepods to the culture, Karlodinium armiger was attracted to them and first began to attach to the antennae and telson. These are important in sensing hydrodynamical disturbances in the surrounding area, which copepods then use to determine between prey and predator by size. As Karlodinium armiger is of a much smaller size in comparison, this suggests it is disguised as prey, allowing the attack of the copepod to occur whilst remaining unnoticed.

After 135 minutes Karlodinium armiger was shown to immobilise nearly all the copepods. Within 24 hours all copepods were dead and surrounded by swarms of Karlodinium armiger with feeding tubes attached. After 24 hours the copepods with the Karlodinium veneficum strains were alive and healthy, therefore further experiments used only Karlodinium armiger. The experiment was repeated with adult nematode, trochophore and late stage polychaete larvae, all of which experienced immobilisation and mortality, suggesting Karlodinium armiger affects a wide range of metazoans. Further experiments concerning cell density demonstrated that when Karlodinium armiger was below 1100 cells ml^-1 copepods were unaffected, however at 3500 cells ml^-1 they were immobilised and killed. As field studies have reported densities of Karlodinium armiger and Karlodinium veneficum between 10,000 and 100,000 cells ml^-1 in coastal areas, this suggests microalgal feeding is likely to have detrimental effects on metazoans.

Although Karlodinium armiger contains chloroplasts, when relying on only phototrophic growth, growth rates are very slow. However, feeding on prey such as copepods provides important growth factors and also stimulates photosynthesis, meaning consuming even a small amount of prey can result in a significant increase in growth rate. This was demonstrated in the 3500 cell ml^-1 cultures of Karlodinium armiger that contained copepods by an increase in 85% of the population growth rate in comparison to those without copepods.

This investigation has provided valuable insight into the flexibility of feeding exhibited by Karlodinium armiger. The fact that this species can consume a wide range of metazoan suggests competition for a variety of food sources may occur across many trophic levels, which could result in the disruption of the structure and function of the marine food web.  However, further studies are needed to establish the specific toxin produced by Karlodinium armiger and also into the behaviour of other such harmful species in order to reveal which organisms are most vulnerable.

Terje Berge, Louise K Poulsen, Morten Moldrup, Niels Daugbjerg and Per Juel Hansen

The ISME Journal 6, 1926-1936 (October 2012) | doi:10.1038/ismej.2012.29

http://www.nature.com/ismej/journal/v6/n10/full/ismej201229a.html

The “Cheshire Cat” escape strategy of the coccolihophore E. Huxleyi in response to viral infection. Frada et al (2008)

The theory of an ongoing evolutionary arms race that is taking place in biological systems has long been established. This is the idea that organisms develop defence mechanisms to out run, outwit or out perform their predators, and subsequently predators evolving strategies to overcome this. A similar dynamic can be observed in the relationship between organism and pathogen in cases of infection. This association has been described as the Red Queen dynamic due to the similarity with the Alice in Wonderland novel in which Alice is constantly running, yet remaining in the same place. However in some species this trend is questionable, for example in Emiliania Huxleyi there are no geographical subpopulations of host or virus; if local viruses only infected local hosts, geographical speciation would soon occur. This trend is not seen in E. huxleyi and its relationship with a giant phycodnaviruses called Emiliania huxleyi Virus (EhVs), as a virus strain isolated from the North Atlantic has been shown to be capable of easily infecting host cells originating from the Mediterranean Sea.

The coccolithophore E. huxleyi is one of the most successful eukaryotes in modern oceans and its blooms can be seen from space. This success can be attributed to a stage in its life-cycle which has exceptionally high phosphate uptake and very low photoinhibition of photosynthesis. At the end of its life-cycle these blooms are eradicated by a virus specific to E. huxleyi. This begs the question of why has little to no ‘evolutionary investment’ gone into a defence mechanism against this virus?

This paper shows that E. huxleyi occurs as a haplodiploid organism in which its lifecycle alternates between the two ploidies. The massive blooms occur as a calcified, coccolith-bearing diploid phase which can profoundly impact global biochemical equilibria, and a non-calcified flagellated haploid phase. This sexual cycling is temporarily separated by an as of yet unknown cause, however this paper hypothesises that viral infection may trigger meiosis and induces a shift from diploid to haploid. This viral escape mechanism has been described as a “Cheshire Cat” method of survival, due to a continuation of the Alice in Wonderland theme in which the Cheshire cat makes its body invisible in order to escape beheading. The susceptibility of diploid-stage and haploid-stage cells to EhVs was tested over a 50 day period and it was found that none of the haploid strains were sensitive, compared to a 100% infection rate of the diploid cells. From this it has been hypothesised that even if a diploid stage bloom is virally eradicated, the motile haploid stage is invisible to the virus and can therefore enable the continuity of the species.

As eukaryotic marine protists are responsible for nearly half of global primary productivity and carbonate production, further research in this area is essential to understand the genomic control and method of action of this escape strategy as this significantly advance assessment of marine eukaryotes on biogeochemical cycles and further reveal details of the control of the flux of matter between the atmosphere and the lithosphere.