Bacterial aggregation by depletion attraction: sinorhizobium meliloti and its extracellular polysaccharide succinoglycan
In their natural environments microorganisms exist predominantly in aggregates and biofilms. The ability of bacteria to form aggregates is associated with the biosynthesis of polymers such as polysaccharides. In this study the physical mechanisms underlying bacterial aggregation by extracellular polysaccharides are investigated by utilising the bacterium Sinorhizobium meliloti. S. meliloti biosynthesises an extracellular polysaccharide called succinoglycan, which is well characterised in terms of its structure and biosynthesis. A range of previously constructed succinoglycan biosynthesis mutants were screened for altered aggregation. An S. meliloti exoS mutant (a gain of function mutation that results in a constitutively active two component regulator called ExoS) overproduces succinoglycan and has enhanced aggregation compared to the parent strain, Rm1021. The aggregates settle to the bottom of the culture vessel resulting in loss of turbidity of the cultures and phase separation. Microscopic observation showed that succinoglycan did not appear to be attached to the aggregates, which formed ordered structures of laterally aligned cells. By addition of purified succinoglycan it was found that the critical concentration of polymer required to induce aggregation and phase separation of the cultures decreased with increasing cell concentration. These observations suggest that aggregation of S. meliloti cultures in the presence of succinoglycan is driven by macromolecular crowding, otherwise known as depletion attraction. Depletion attraction can drive the ordered arrangement and aggregation of colloidal particles in the presence of polymers. Aggregation of the particles increases the volume available to the polymers, maximising their entropy and the entropy of the system. Addition of succinoglycan to stationary phase Escherichia coli cultures and polystyrene colloids also resulted in aggregation consistent with depletion attraction. Furthermore alternative polymers such as the bacterial extracellular polysaccharide xanthan produced by Xanthomonas campestris can result in aggregation of bacteria by depletion attraction. Depletion attraction may therefore be a ubiquitous force driving aggregation of crowded dispersions of bacteria and polymers. The second part of the thesis focuses on how depletion driven aggregation can lead to surface-associated biofilm formation. Imaging of the sediment formed by the exoS mutant showed that the structure formed at the base of the culture vessel leads to development of an ordered structure composed of interlinked aggregates. The role of succinoglycan in surface attachment is complex and varies with culture conditions. Depletion attraction may facilitate interaction with a surface but alternative factors may then play a role in anchoring the cells to the surface. Under certain conditions the cells may produce factors which allow binding of the cells to a surface independently of succinoglycan. This study has demonstrated for the first time that an extracellular polysaccharide produced by bacteria can result in aggregation via depletion attraction which may be an under explored mechanism by which aggregation of bacteria can occur.