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For bacteria to survive desiccating conditions they must make adaptations to facilitate water retention in all cellular compartments. In the cytosol, accumulating compatible solutes reduce the cytosolic water potential in a well characterized process. However, we do not know how the periplasm of gram negative bacteria is hydrated. Housing many critical functions, hydration of the periplasm is important. Osmoregulated periplasmic glucans accumulate under water-replete (hypo-osmotic) conditions to reduce the periplasmic water-potential relative to the solute rich cytosol, limiting swelling of the cytoplasmic compartment. Interestingly, compared to cells grown on solute stress or water-replete media, Pseudomonas aeruginosa biofilm cells accumulate more linear glucans under matric stress conditions. Furthermore, there are three populations of linear glucans with varying anionic properties, ranging from neutral to highly anionic. Under matric stress there is a shift towards a greater proportion of highly anionic linear glucans. A transient increase in the linear glucan biosynthesis operon expression suggests accumulation is regulated transcriptionally, though regulation of enzyme activity is also likely. While we were unable to purify cyclic glucans from P. aeruginosa biofilm cells, we show that cyclic glucan-deficient mutants exhibit lower matric stress tolerance than the wild type. Collectively, our data suggests that to facilitate periplasm hydration under desiccating conditions anionic linear glucans accumulate and cyclic glucans contribute to matric stress tolerance by a presently unknown mechanism. 24 Introduction For many terrestrial bacteria, their environment can be quite dry. Even under non-drought conditions bacterial communities living in soil, the phyllosphere or on the skin of animals often experience conditions of limited water availability. The availability of water can be determined by measuring the total water potential of an environment, which is primarily comprised of two components; the solute potential and matric potential. Dissolved solutes, such as NaCl, reduce the water potential by structuring water. As water moves to areas of lower potential, bacteria must regulate the water potential within the cell relative to their immediate surroundings (6). Porin channels in the outer membrane of gram negative bacteria allow solutes to enter periplasm, hydrating this compartment with water of reduced potential, while imposing a solute (osmotic) stress due to water potential differences across the inner (cytosolic) membrane. To hydrate the cytosol, compatible solutes including potassium ions, glutamate, glutamine, trehalose, proline and glycine betaine accumulate (8, 33, 39, 42), reducing the water potential to retain water. In water-replete (hypo-osmotic) conditions, the environmental water potential is greater than the cytosol, raising the possibility that over swelling of the cell will occur from an influx of water. In response, excess cytosolic solutes are secreted, increasing the water potential of the compartment (24). However, as the cytosol contains at least 300 mOsM of solutes required for normal metabolism (41) it is necessary for there to be an equilibrium between the cytosol and periplasm water activities to avoid overswelling of the cytosol. Consequently, many gram negative bacteria accumulate oligosaccharides called glucans in the periplasm, which can have anionic substitutions to improve hydrophilic interactions and to increase the osmolarity of the compartment, reducing the water potential differential across the inner membrane (2, 16, 27). 25 As a habitat dries the matric potential becomes the predominant factor contributing to the total water potential due to the interaction of water with matrix (e.g. soil, leaves). Under these low-water-content conditions, bacteria are desiccated due to the physical removal of water and experience “osmotic” pressure across the outer membrane, periplasm, and cytosol: this is referred to as a matric stress (32). We can simulate low-water-content in the laboratory by amending media with non-outer membrane permeable polyethylene glycol MW8000 (PEG), creating a matric stress, since it tightly sorbs water and reduces the water potential like dry soil (11). We know that similar to solute stress, the cytosolic compartment is hydrated during a matric stress by the accumulation of compatible solutes (6, 30). However, we do not know how the periplasm is hydrated under dry or low-water-content conditions. This compartment can comprise 20-40% of the cell volume and houses many important structural, metabolic, signaling and transport functions, necessitating proper hydration to function (33, 41). Accumulation of the small compatible solutes found in the cytosol would likely not effectively hydrate the periplasm as they could diffuse out of the periplasm into the environment through outer membrane porins. Intermediately sized anionic molecules, such periplasmic glucans, could fulfill this role. Unfortunately, no studies have directly investigated glucan abundance in response to low-water-content conditions. In a transcriptomic study, confirmed by qRT-PCR, Nielsen observed that expression of the periplasmic glucan biosynthesis gene opgG was elevated 3.9 fold in Pseudomonas putida after 15 minutes of growth under matric stress (29). Additionally, it was found that the extracellular matrix of P. putida contains a significant amount of low molecular weight, glucose-rich polysaccharide when grown under matric stress (3). This was observed even in a cellulose-deficient mutant. These findings suggest that these low molecular weight polysaccharides could be periplasmic glucans accumulating in 26 response to low-water-content conditions that may have been secreted or released from the periplasm during our extraction procedure. Found within diverse gram negative species (2, 16, 26, 27), there are two types of periplasmic glucans: linear and cyclic. Studied primarily in enteric bacteria, linear glucans are oligosaccharides composed of 5-16 glucose residues with a β1→2 linked backbone and β1→6 linked branches (2, 19, 37). They are produced by the membrane-bound glycosyl transferases encoded by the opgGH operon (formerly mdoGH) from UDP-glucose (7). In E .coli, they can be anionically decorated with both succinyl and phosphogycerol groups by OpgC and OpgB, respectively (15, 17). Cyclic glucans have been studied extensively in Rhizobia. Depending on the species, cyclic glucans are 10-40 glucose residues comprised of β1→2, β1→3 or β1→6 linkages synthesized by the glycosyl transferase NdvB and can have phosphoglycerol and succinyl substitutions (2, 19, 27, 36, 43). Interestingly, most Pseudomonads have the potential to produce both linear and cyclic glucans. As of January 2014, based on gene annotation we identified 43 opgGH and 36 ndvB orthologs in the 44 genomes available in the Pseudomonas Genome Database (pseudomonas.com; (Table S1)) (45). All genomes harbored at least one opgGH or ndvB ortholog. Separate BlastP searches confirmed the absence of opgGH or ndvB orthologs in genomes missing an ortholog. Pseudomonas aeruginosa linear and cyclic glucans have both been characterized when produced under water-replete conditions (19, 22, 36), making this genetically-tractable and commonly-used model organism an excellent subject for glucan

Hydrating the Pseudomonas aeruginosa periplasm under desiccating conditions