Behind the Paper: Interspecific Diversity Reduces And Functionally Substitutes For Intraspecific Variation In Biofilm Communities
I caught up with Kelvin Lee and Scott A Rice from the Singapore Centre for Environmental Life Sciences Engineering (SCELSE) to discuss their paper, recently published in The ISME Journal.
What was the main aim of your research and why did you decide to investigate this?
Scott: The main focus of my laboratory has been biofilm development, with an interest in how different developmental stages are regulated. Increasingly, we are shifting towards mixed species biofilm systems as this reflects the natural environment more closely. As a consequence we are again asking similar questions about biofilm development, but it now takes on the added dimension of the role of interactions between organisms, which impact gene expression and metabolism, as well as adaptive responses, of the organisms around them. Some of those observations were presented in our previous publication using this mixed species model.
Kelvin: For some time, ecologists and evolutionists have been debating on the relationship between intraspecific and interspecific diversity in a community. Community genetics, a single discipline that unifies ecology and evolution was thus born, but still faces many challenges today. The long temporal and large spatial scales involved in the studies of macro organisms are some of the hurdles encountered for such studies. In contrast, it is possible to develop microbial communities in the laboratory, where their small size and fast generation times are amenable to experimentation. The main aim of the research, as described in the article, is to investigate the relative importance of intraspecific and interspecific diversity within a biofilm community.
“Once we had the basic observation that the mixed species community did not generate variants at the same frequency as the single species biofilms, we knew we were on to something very exciting.”
How did you go about designing your study?
Kelvin: We have previously established a biofilm community model consisting of P. aeruginosa, P. protegens and K. pneumoniae, which we believe can serve as a good model for this study. The three microbes were shown to interact with each other, giving rise to emergent properties such as tolerance to stresses and delayed biofilm development. An added advantage to this model is the genetic information (whole genomes) we have for all three microbes, thus enabling the study of evolutionary genetics. To investigate the relationship between intraspecific and interspecific diversity, we grew mono- as well as mixed-species biofilms, measured their intraspecific diversity by recording the frequency of self-generated morphotypic variants and tested their tolerance to SDS stress.
Scott: Once we had the basic observation that the mixed species community did not generate variants at the same frequency as the single species biofilms, we knew we were on to something very exciting. From there, we began to develop hypotheses about why the variants did not appear. We compared the relative fitness of the different variants, which did not explain the results (the variants were invariably more fit than their parents) and performed a range of mixing experiments to determine whether the variants were competitive. They were. As we refined our hypotheses, we modified the experimental design.
“Results wise, we are also surprised by our observations, as they are completely different from our initial hypothesis… The new idea is breathtaking and we had a hard time convincing our reviewers, who made some excellent suggestions to improve the manuscript.”
What challenges did you face?
Kelvin: On the experimental aspect, growing up biofilms and measuring the frequency of morphotypic variants are labour intensive. In addition, being able to identify and record the number of morphotypic variants accurately requires substantial training, thus limiting the manpower we can employ for the huge amount of work.
Results wise, we are also surprised by our observations, as they are completely different from our initial hypothesis. We did multiple repeats to confirm our results before we came out with the current explanation and conclusion. The new idea is breathtaking and we had a hard time convincing our reviewers, who made some excellent suggestions to improve the manuscript.
What were the key findings from your research?
Kelvin: We found that:
1. Both intraspecific and interspecific diversity can enhance biofilm’s tolerance to SDS stress;
2. Interspecific diversity reduces and functionally substitutes for intraspecific diversity in a biofilm community;
3. The reduction in intraspecific diversity can be achieved by extracellular factors that might reduce the accumulation of mutants.
What next? What further research is needed in this area?
Scott: Well, as highlighted above, the data suggest there is an extracellular factor that when added back to monospecies biofilms, results in decreased variant formation. So we really want to work out what is that compound or factor. If could turn out to be something really simple like scavenging reactive oxygen species which might otherwise lead to mutations. On the other hand, if it is a cue or signal that modifies DNA repair activity, it could be pretty spectacular.
Kelvin: We would like to extend the research to other microbial species and even eukaryotes (protozoa) to investigate the generality of the current conclusion.
The genetic responses of the community when grown together will also be particularly exciting to understand, especially if coupled with changes in metabolic pathways, as these are also likely to help us understand how such mixed species biofilms display these emergent properties.
Image: a confocal image of the mixed-species biofilm.