Role of cell-cell signaling, differentiation and fitness in multi-species biofilms
Lee, Kelvin Kai Wei
Date of Issue2014
School of Biological Sciences
Bacteria in the environment exist mostly as sessile communities known as biofilms. Biofilms are widely found on various biological surfaces such as on the leaves and roots of plants as well as in the lungs of humans, where they are associated with diseases. Biofilms can also grow on abiotic surfaces of rocks, water pipelines, contact lenses and surgical implants. Some biofilms can form independent of a surface, called suspended biofilms, and are represented by either floes or granules that are important for the proper functioning of wastewater treatment plants. Biofilms, which are difficult to eradicate, have many negative impacts. For example, they are reservoirs for many pathogens and their activities also lead to biocorrosion of different surfaces. Biofilms are also essential, contributing to the biogeochemical cycling in the environment and are key players in processes such as sewage treatment and bioremediation. As a result, intensive research has been carried out with the aim to manipulate biofilms to our advantage. However, a main limitation of such research to date is the focus on mono species biofilms. In reality, biofilms in the environment consist of multiple interacting species, where interspecies interactions play an important role in determining the development, structure and function of the biofilms. Hence, a mixed species biofilm model that can be explored experimentally for research and development projects, with relevance to specific environmental, industrial and medical settings, is much needed. Here, a reproducible mixed species biofilm consisting of Pseudomonas aeruginosa PAOl, Pseudomonas protegens Pf-5 and Klebsiella pneumoniae KP-1 was studied and compared to the respective mono species biofilms. The three bacterial species were stably labeled with three different fluorescent proteins using a Tn7 transposon expression system. The development of the mixed species biofilms was delayed relative to all mono species biofilms. While mushroom and tower like microcolonies were observed for mature PAOl and Pf-5 mono species biofilms respectively, these structures were not seen for the mixed species biofilms. In contrast, KP-1 in the mixed species biofilms formed mound-like microcolonies, which were not observed when it was grown alone. The proportions and structures of the three bacterial species within the mixed species biofilms grown at the inlet end and outlet end of the continuous-culture flow cell also differed, most likely as a result of decreasing glucose concentration and associated changes in the specific growth rate for KP-1 along the channel. Most importantly, the mixed species biofilms were more resilient to stresses such as tobramycin and sodium dodecyl sulfate than the mono species biofilms. Intriguingly, such community level resilience was found to be contributed by the resistant species to the whole community rather than selection for the resistant species. In addition, community level resilience was not observed for mixed species biofilms with loosely associated members and for mixed species planktonic cultures, suggesting that community level resistance was unique to a structured biofilm community with closely associated members. The formation of morphotypic variants, which is typical of almost all biofilm forming bacteria to date, was also observed and quantified in this study. One morphotypic variant each was identified for PAO 1 and KP-1 while four morphotypic variants were observed for Pf-5. The formation of these morphotypic variants was specific to biofilms as planktonic cultures produced either no variants or a significantly reduced number of variants. The morphotypic variants were also stable after daily passaging, while sequencing of their genomes showed that they possessed insertions/deletions and single nucleotide polymorphisms that might lead to changes in their phenotypes and morphotypes. In fact, each morphotypic variant was shown to differ from its parent strain in various phenotypes such as swimming and swarming motilities, attachment, biofilm formation as well as pyoverdine and pyocyanin productions. In addition, the morphotypic variants also formed biofilms with distinct structures and outcompeted their parent strains in co-cultured biofilms. The morphotypic variants also outperformed their respective parent strains when grown with the other two species in a mixed species biofilm. Despite their fitness, the frequency and diversity of these morphotypic variants decreased in the mixed species biofilms. Since each strain sacrificed the production of individual variants in the presence of other species, the results suggest that interspecies diversity may be more important than intraspecies diversity in microbial biofilms. Notably, the addition of PA01 and KP-1 cell-free biofilm effluents to Pf-5 biofilms also decreased the frequency and diversity ofmorphotypic variants formed by Pf-5. Hence, molecules present in the biofilm effluents ofPA01 and KP-1 may be responsible for an interspecies cell-cell signaling that regulates self-generated genetic diversity. Subsequently, metatranscriptomic analyses were performed to determine the genes that might be involved in biofilm response to sodium dodecyl sulfate. The results showed that extracellular polymeric substances production and export as well as stress response proteins were found to be involved in the resistance ofPA01 and KP-1 biofilms to SDS. Particularly, the up regulation of siaA has been associated with the autoaggregation of PA01 cells during SDS treatment, thereby protecting them from lysis by SDS. Similarly, the induction of Cpx envelope stress response proteins in SDS treated KP-1 biofilms may be responsible for protecting KP-1 cells from lysis by SDS through ensuring proper folding of the envelope proteins and maintaining the integrity of the cells. The induction of extracellular polymeric substances and relevant stress response proteins was not observed for SDS sensitive Pf-5 biofi lms. In conclusion, results from this study have shown that mixed species biofilms differ from their mono species counterparts in terms of development, structure, resilience and intraspecies diversity. As a result, studies on mono species biofilms cannot predict the behaviours of mixed species biofilm communities in nature. The mixed species biofilm model used in this study is reproducible and is one of the few emperical studies that provided evidence for the proposition that interspecies diversity could substitute intraspecies diversity. Furthermore, all three bacterial species are genetically tractable and amenable to molecular techniques such as mutagenesis and 'omics' based approaches, thus making this mixed species model an excellent system to investigate interspecies interactions using both the top down and bottom up approaches.