Analysis of Proteus mirabilis Social Behaviors on Surfaces.

The opportunistic pathogen Proteus mirabilis engages in visually dramatic and dynamic social behaviors. Populations of P. mirabilis can rapidly occupy surfaces, such as high-percentage agar and latex, through a collective surface-based motility termed swarming. When in these surface-occupying swarm colonies, P. mirabilis can distinguish between clonal siblings (self) and foreign P. mirabilis strains (nonself). This ability can be assessed by at least two standard methods: boundary formation, aka a Dienes line, and territorial exclusion. Here we describe methods for quantitative analysis of swarm colony expansion, of boundary formation, and of territorial exclusion. These assays can be employed to assess several aspects of P. mirabilis sociality including collective swarm motility, competition, and self versus nonself recognition.

Cell Shape and Population Migration Are Distinct Steps of Proteus mirabilis Swarming That Are Decoupled on High-Percentage Agar.

Swarming on rigid surfaces requires movement of cells as individuals and as a group of cells. For the bacterium Proteus mirabilis, an individual cell can respond to a rigid surface by elongating and migrating over micrometer-scale distances. Cells can form groups of transiently aligned cells, and the collective population is capable of migrating over centimeter-scale distances. To address how P. mirabilis populations swarm on rigid surfaces, we asked whether cell elongation and single-cell motility are coupled to population migration. We first measured the relationship between agar concentration (a proxy for surface rigidity), single-cell phenotypes, and swarm colony phenotypes. We find that cell elongation and single-cell motility are coupled with population migration on low-percentage hard agar (1% to 2.5%) and become decoupled on high-percentage hard agar (>2.5%). Next, we evaluate how disruptions in lipopolysaccharide (LPS), specifically the O-antigen components, affect responses to hard agar. We find that LPS is not essential for elongation and motility of individual cells, as predicted, and instead functions to broaden the range of agar concentrations on which cell elongation and motility are coupled with population migration. These findings demonstrate that cell elongation and motility are coupled with population migration under a permissive range of surface conditions; increasing agar concentration is sufficient to decouple these behaviors. Since swarm colonies cover greater distances when these steps are coupled than when they are not, these findings suggest that collective interactions among P. mirabilis cells might be emerging as a colony expands outwards on rigid surfaces.IMPORTANCE How surfaces influence cell size, cell-cell interactions, and population migration for robust swarmers like P. mirabilis is not fully understood. Here, we have elucidated how cells change length along a spectrum of sizes that positively correlates with increases in agar concentration, regardless of population migration. Single-cell phenotypes can be decoupled from collective population migration simply by increasing agar concentration. A cell's lipopolysaccharides function to broaden the range of agar conditions under which cell elongation and single-cell motility remain coupled with population migration. In eukaryotes, the physical environment, such as a surface matrix, can impact cell development, shape, and migration. These findings support the idea that rigid surfaces similarly act on swarming bacteria to impact cell shape, single-cell motility, and collective population migration.

Swarmer Cell Development of the Bacterium Proteus mirabilis Requires the Conserved Enterobacterial Common Antigen Biosynthesis Gene rffG.

Individual cells of the bacterium Proteus mirabilis can elongate up to 40-fold on surfaces before engaging in a cooperative surface-based motility termed swarming. How cells regulate this dramatic morphological remodeling remains an open question. In this paper, we move forward the understanding of this regulation by demonstrating that P. mirabilis requires the gene rffG for swarmer cell elongation and subsequent swarm motility. The rffG gene encodes a protein homologous to the dTDP-glucose 4,6-dehydratase protein of Escherichia coli, which contributes to enterobacterial common antigen biosynthesis. Here, we characterize the rffG gene in P. mirabilis, demonstrating that it is required for the production of large lipopolysaccharide-linked moieties necessary for wild-type cell envelope integrity. We show that the absence of the rffG gene induces several stress response pathways, including those controlled by the transcriptional regulators RpoS, CaiF, and RcsB. We further show that in rffG-deficient cells, the suppression of the Rcs phosphorelay, via loss of RcsB, is sufficient to induce cell elongation and swarm motility. However, the loss of RcsB does not rescue cell envelope integrity defects and instead results in abnormally shaped cells, including cells producing more than two poles. We conclude that an RcsB-mediated response acts to suppress the emergence of shape defects in cell envelope-compromised cells, suggesting an additional role for RcsB in maintaining cell morphology under stress conditions. We further propose that the composition of the cell envelope acts as a checkpoint before cells initiate swarmer cell elongation and motility.IMPORTANCEProteus mirabilis swarm motility has been implicated in pathogenesis. We have found that cells deploy multiple uncharacterized strategies to handle cell envelope stress beyond the Rcs phosphorelay when attempting to engage in swarm motility. While RcsB is known to directly inhibit the master transcriptional regulator for swarming, we have shown an additional role for RcsB in protecting cell morphology. These data support a growing appreciation that the Rcs phosphorelay is a multifunctional regulator of cell morphology in addition to its role in microbial stress responses. These data also strengthen the paradigm that outer membrane composition is a crucial checkpoint for modulating entry into swarm motility. Furthermore, the rffG-dependent moieties provide a novel attractive target for potential antimicrobials.

Autonomous and in trans functions for the two halves of Srv2/CAP in promoting actin turnover.

Recent evidence has suggested that Srv2/CAP (cyclase-associated protein) has two distinct functional roles in regulating actin turnover, with its N-terminus enhancing cofilin-mediated severing of actin filaments and its C-terminus catalyzing actin monomer recycling. However, it has remained unclear to what degree these two activities are coordinated by being linked in one molecule, or whether they can function autonomously. To address this, we physically divided the protein into two separate halves, N-Srv2 and C-Srv2, and asked whether they are able to function in trans both in living cells and in reconstituted assays for F-actin turnover and actin-based motility. Remarkably, in F-actin turnover assays the stimulatory effects of N-Srv2 and C-Srv2 functioning in trans were quantitatively similar to those of intact full-length Srv2. Further, in bead motility assays and in vivo, the fragments again functioned in trans, although not with the full effectiveness of intact Srv2. From these data, we conclude that the functions of the two halves of Srv2/CAP are largely autonomous, although their linkage improves coordination of the two functions in specific settings, possibly explaining why the linkage is conserved across distant plant, animal, and fungal species.

Srv2/cyclase-associated protein forms hexameric shurikens that directly catalyze actin filament severing by cofilin.

Actin filament severing is critical for the dynamic turnover of cellular actin networks. Cofilin severs filaments, but additional factors may be required to increase severing efficiency in vivo. Srv2/cyclase-associated protein (CAP) is a widely expressed protein with a role in binding and recycling actin monomers ascribed to domains in its C-terminus (C-Srv2). In this paper, we report a new biochemical and cellular function for Srv2/CAP in directly catalyzing cofilin-mediated severing of filaments. This function is mediated by its N-terminal half (N-Srv2), and is physically and genetically separable from C-Srv2 activities. Using dual-color total internal reflection fluorescence microscopy, we determined that N-Srv2 stimulates filament disassembly by increasing the frequency of cofilin-mediated severing without affecting cofilin binding to filaments. Structural analysis shows that N-Srv2 forms novel hexameric star-shaped structures, and disrupting oligomerization impairs N-Srv2 activities and in vivo function. Further, genetic analysis shows that the combined activities of N-Srv2 and Aip1 are essential in vivo. These observations define a novel mechanism by which the combined activities of cofilin and Srv2/CAP lead to enhanced filament severing and support an emerging view that actin disassembly is controlled not by cofilin alone, but by a more complex set of factors working in concert.

A central role for the WH2 domain of Srv2/CAP in recharging actin monomers to drive actin turnover in vitro and in vivo.

Cellular processes propelled by actin polymerization require rapid disassembly of filaments, and then efficient recycling of ADF/cofilin-bound ADP-actin monomers back to an assembly-competent ATP-bound state. How monomer recharging is regulated in vivo is still not well understood, but recent work suggests the involvement of the ubiquitous actin-monomer binding protein Srv2/CAP. To better understand Srv2/CAP mechanism, we explored the contribution of its WH2 domain, the function of which has remained highly elusive. We found that the WH2 domain binds to actin monomers and, unlike most other WH2 domains, exhibits similar binding affinity for ATP-actin and ADP-actin (K(d) approximately 1.5 microM). Mutations in the WH2 domain that impair actin binding disrupt the ability of purified full-length Srv2/CAP to catalyze nucleotide exchange on ADF/cofilin-bound actin monomers and accelerate actin turnover in vitro. The same mutations impair Srv2/CAP function in vivo in regulating actin organization, cell growth, and cell morphogenesis. Thus, normal cell growth and organization depend on the ability of Srv2/CAP to recharge actin monomers, and the WH2 domain plays a central role in this process. Our data also reveal that while most isolated WH2 domains inhibit nucleotide exchange on actin, WH2 domains in the context of intact proteins can help promote nucleotide exchange.