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].