Bacterial aggregation by depletion attraction: sinorhizobium meliloti and its extracellular polysaccharide succinoglycan
Abstract
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.