COS 78-5 - Uncovering the Secrets of Rhodanobacter Biofilm Formation Under Environmental Challenges

Abstract: Biofilms enable bacteria to adhere to surfaces, and to each other, through a matrix of self-produced extracellular polymeric substances. It is well known that biofilm-formation is wide-spread and that such matrix-encased communities have better adaptability to survive under environmental stressors. While biofilms have been studied in model microorganisms for decades, biofilm formation by non-model environmental microorganisms is understudied. Much is unknown about the conditions under which biofilm develops or the molecular basis underpinning temporal progression of biofilm formation under fluctuating environmental conditions.

In this project, we report on the physiology of biofilm formation in subsurface microbial strains recovered from a mixed-waste field site (Oak Ridge Field Research Center-ORRFRC). ORRFRC encompasses a gradient of nitrate, pH, and heavy metals, making it ideal for studying biofilm variability among microorganisms under multiple environmental stresses. We performed high-throughput biofilm assays on strains belonging to genus Rhodanobacter that is prevalent in contaminated sites and highly correlated with stress gradients. Assays were performed under different incubation conditions that included different media (commercially available low nutrient media as well as media designed to mimic in situ conditions) and different times during the growth cycle of the strains. In general, different strains display distinct biofilm dynamics (i.e., biofilm formation and maintenance) over different growth stages and media. Moreover, different Rhodanobacter isolates from the same wells in the field show distinct biofilm dynamics under nitrate (0 to 1250 ppm) and pH (4 to 7) stresses, suggesting that both phylogeny and environmental stresses can impact the biofilm forming potentials. Transmission electron microscopy (TEM) images also show conspicuous flagella under certain stress conditions which can aid biofilm formation through surface recognition and attachment.
We further selected two Rhodanobacter isolates (FW104-10B01 and FW510-R12) for a deeper examination into molecular underpinnings of biofilm formation due to their distinct biofilm dynamics and their resilience to metal stressors. Metal tests (Mn2+, Al3+, Fe3+, Zn2+, Co2+, Ni2+, Cu2+) results showed that certain concentrations of Mn and Al can substantially promote biofilm formation. We then used barcoded transposon mutant (RB-Tnseq) libraries to investigate the fitness of strains under these conditions, and the gene determinants of these differences of these two Rhodanobacter isolates. Our results provide new insight into the molecular mechanisms behind biofilm formation in non-model, abundant subsurface metal-resistant bacteria and their distribution of microbial biomass from attached to planktonic in a shallow, subsurface environment.