Marine bacteria Alteromonas spp. require UDP-glucose-4-epimerase for aggregation and production of sticky exopolymer.

The physiology and ecology of particle-associated marine bacteria are of growing interest, but our knowledge of their aggregation behavior and mechanisms controlling their association with particles remains limited. We have found that a particle-associated isolate, Alteromonas sp. ALT199 strain 4B03, and the related type-strain A. macleodii 27126 both form large (>500 µm) aggregates while growing in rich medium. A non-clumping variant (NCV) of 4B03 spontaneously arose in the lab, and whole-genome sequencing revealed a partial deletion in the gene encoding UDP-glucose-4-epimerase (galEΔ308-324). In 27126, a knock-out of galE (ΔgalE::kmr) resulted in a loss of aggregation, mimicking the NCV. Microscopic analysis shows that both 4B03 and 27126 rapidly form large aggregates, whereas their respective galE mutants remain primarily as single planktonic cells or clusters of a few cells. Strains 4B03 and 27126 also form aggregates with chitin particles, but their galE mutants do not. Alcian Blue staining shows that 4B03 and 27126 produce large transparent exopolymer particles (TEP), but their galE mutants are deficient in this regard. This study demonstrates the capabilities of cell-cell aggregation, aggregation of chitin particles, and production of TEP in strains of Alteromonas, a widespread particle-associated genus of heterotrophic marine bacteria. A genetic requirement for galE is evident for each of the above capabilities, expanding the known breadth of requirement for this gene in biofilm-related processes. IMPORTANCE: Heterotrophic marine bacteria have a central role in the global carbon cycle. Well-known for releasing CO2 by decomposition and respiration, they may also contribute to particulate organic matter (POM) aggregation, which can promote CO2 sequestration via the formation of marine snow. We find that two members of the prevalent particle-associated genus Alteromonas can form aggregates comprising cells alone or cells and chitin particles, indicating their ability to drive POM aggregation. In line with their multivalent aggregation capability, both strains produce TEP, an excreted polysaccharide central to POM aggregation in the ocean. We demonstrate a genetic requirement for galE in aggregation and large TEP formation, building our mechanistic understanding of these aggregative capabilities. These findings point toward a role for heterotrophic bacteria in POM aggregation in the ocean and support broader efforts to understand bacterial controls on the global carbon cycle based on microbial activities, community structure, and meta-omic profiling.

Cell aggregation is associated with enzyme secretion strategies in marine polysaccharide-degrading bacteria.

Polysaccharide breakdown by bacteria requires the activity of enzymes that degrade polymers either intra- or extra-cellularly. The latter mechanism generates a localized pool of breakdown products that are accessible to the enzyme producers themselves as well as to other organisms. Marine bacterial taxa often show marked differences in the production and secretion of degradative enzymes that break down polysaccharides. These differences can have profound effects on the pool of diffusible breakdown products and hence on the ecological dynamics. However, the consequences of differences in enzymatic secretions on cellular growth dynamics and interactions are unclear. Here we study growth dynamics of single cells within populations of marine Vibrionaceae strains that grow on the abundant marine polymer alginate, using microfluidics coupled to quantitative single-cell analysis and mathematical modelling. We find that strains that have low extracellular secretions of alginate lyases aggregate more strongly than strains that secrete high levels of enzymes. One plausible reason for this observation is that low secretors require a higher cellular density to achieve maximal growth rates in comparison with high secretors. Our findings indicate that increased aggregation increases intercellular synergy amongst cells of low-secreting strains. By mathematically modelling the impact of the level of degradative enzyme secretion on the rate of diffusive oligomer loss, we find that enzymatic secretion capability modulates the propensity of cells within clonal populations to cooperate or compete with each other. Our experiments and models demonstrate that enzymatic secretion capabilities can be linked with the propensity of cell aggregation in marine bacteria that extracellularly catabolize polysaccharides.

Multiscale modeling of innate immune receptors: Endotoxin recognition and regulation by host defense peptides.

The innate immune system provides a first line of defense against foreign microorganisms, and is typified by the Toll-like receptor (TLR) family. TLR4 is of particular interest, since over-stimulation of its pathway by excess lipopolysaccharide (LPS) molecules from the outer membranes of Gram-negative bacteria can result in sepsis, which causes millions of deaths each year. In this review, we outline our use of molecular simulation approaches to gain a better understanding of the determinants of LPS recognition, towards the search for novel immunotherapeutics. We first describe how atomic-resolution simulations have enabled us to elucidate the regulatory conformational changes in TLR4 associated with different LPS analogues, and hence a means to rationalize experimental structure-activity data. Furthermore, multiscale modelling strategies have provided a detailed description of the thermodynamics and intermediate structures associated with the entire TLR4 relay - which consists of a number of transient receptor/coreceptor complexes - allowing us trace the pathway of LPS transfer from bacterial membranes to the terminal receptor complex at the plasma membrane surface. Finally, we describe our efforts to leverage these computational models, in order to elucidate previously undisclosed anti-inflammatory mechanisms of endogenous host-defense peptides found in wounds. Collectively, this work represents a promising avenue for the development of novel anti-septic treatments, inspired by nature's innate defense strategies.