Impact Of Respiration On Biofilm Formation And Its Molecular Basis In Shewanella Oneidensis

Bacteria adopt a variety of lifestyles in their natural habitats and can switch between different life styles in response to environmental changes. At high cell densities, bacteria can form extracellular matrix encased cell population on submerged tangible surfaces (biofilms), or at the air-liquid interface (pellicles). Compared to biofilm, pellicle lifestyle allows for better oxygen access, but is metabolically more costly to maintain. Bacterial biofilm/pellicle formation process is composed of a complex series of biochemical processes, which are regulated and influenced by environmental factors through a variety of regulators and the regulation of multiple genes.Shewanella species are a group of facultative Gram-negative microorganisms with remarkable respiratory abilities that allow for the use of a diverse array of terminal electron acceptors (EA). Previous studies showed that addition of metal-chelator EDTA in the culture medium could significantly reduce pellicle biomass of S. oneidensis, indicating a significant role played by divalent metal cations in pellicle maturation. Moreover, a similar study on S. oneidensis showed that biofilm formation was completely inhibited by EDTA at a concentration of 0.3 mM, while adding EDTA exogenously can lead to biofilm disintegration. All these observation indicated that divalent metal cations were required for proper cell aggregation. It was reported previously that divalent cations are essential in the initial stage of biofilm life cycle; for instance, phosphate and iron play an important role in microcolonies formation and maturation stage. Furthermore, divalent metal cations in culture medium can interact with polysaccharides, proteins and DNA in extracellular matrix by neutralizing negatively charged carboxy, hydroxyl or sulfhydryl electrostatically to maintain biofilm structure.In this study, S. oneidensis cells were cultured in 24-well plates with supplementation of various divalent cations, and pellicles formed under such conditions were evaluated. Mutants defective in respiration were used to further characterize and confirm unique impacts of iron. We present evidence that small amount of Fe2+was essential for pellicle formation, but presence of over-abundant iron (0.3 mM Fe2+or Fe3+) led to pellicle disassociation without impairing growth. Such impacts were found due to formation of insoluble alternative electron acceptors (i.e., Fe3O4, Fe2O3) under physiologically relevant conditions. Furthermore, we demonstrated that cells preferred to form biofilm and respire on such insoluble electron acceptors under conditions permitting pellicle formation. Our research explored the unique influence of iron on S. oneidensis pellicle in depth, indicating that its special respiration mechanisms determined respiration substrates selection and life style transformation. Since the anaerobic respiration of S. oneidensis has been characterized extensively, our findings described above promoted a subsequent study focusing on the terminal oxidases involved in aerobic/microaerobic respiration, as well as an investigation concerning the unique respiratory system and regulation mechanism of S. oneidensis.Like most bacteria, S. oneidensis possesses multiple terminal oxidases, including two heme-copper oxidases(caa3- and cbb3-type) and a bd-type quinol oxidase. As a facultative anaerobe, S. oneidensis thrives in redox-stratified environments, and the underlying mechanisms remain unexplored. In this work, we discovered that the cbb3-type oxidase is the predominant system for aerobic respiration, especially when O2 is abundant. Under microaerobic conditions, however, the bd-type quinol oxidase has a significant role. In contrast, multiple lines of evidence suggest that under tested conditions the caa3-type oxidase, an analog to the mitochondrial enzyme, has no physiological significance, likely because of its extremely low expression level. In addition, expression of both cbb3-and bd-type oxidases is under direct control of Crp (cAMP receptor protein). These data, collectively, suggest that adaptation of S. oneidensis to redox-stratified environments is likely due to functional loss of the caa3-type oxidase and switch of the regulatory system for respiration.From the above, we take the impact of divalent metal cations on S. oneidensis pellicle as a starting point to explore the effects of unique respiratory system on electron acceptor selection and life style transformation, and further unravel its unique terminal oxidase system and respiratory regulation mechanism.