SummaryThe role of quorum sensing in Pseudomonas aeruginosa biofilm formation is unclear. Some researchers have shown that quorum sensing is important for biofilm development, while others have indicated it has little or no role. In this study, the contribution of quorum sensing to biofilm development was found to depend upon the nutritional environment. Depending upon the carbon source, quorum-sensing mutant strains (lasIrhlI and lasRrhlR) either exhibited a pronounced defect early in biofilm formation or formed biofilms identical to the wild-type strain. Quorum sensing was then shown to exert its nutritionally conditional control of biofilm development through regulation of swarming motility. Examination of pilA and fliM mutant strains further supported the role of swarming motility in biofilm formation. These data led to a model proposing that the prevailing nutritional conditions dictate the contributions of quorum sensing and swarming motility at a key juncture early in biofilm development.

Bacterial biofilms are structured multicellular communities that are responsible for a broad range of infections. Knowing how free-swimming bacteria adapt their motility mechanisms near a surface is crucial for understanding the transition from the planktonic to the biofilm phenotype. By translating microscopy movies into searchable databases of bacterial behavior and developing image-based search engines, we were able to identify fundamental appendage-specific mechanisms for the surface motility of Pseudomonas aeruginosa. Type IV pili mediate two surface motility mechanisms: horizontally oriented crawling, by which the bacterium moves lengthwise with high directional persistence, and vertically oriented walking, by which the bacterium moves with low directional persistence and high instantaneous velocity, allowing it to rapidly explore microenvironments. The flagellum mediates two additional motility mechanisms: near-surface swimming and surface-anchored spinning, which often precedes detachment from a surface. Flagella and pili interact cooperatively in a launch sequence whereby bacteria change orientation from horizontal to vertical and then detach. Vertical orientation facilitates detachment from surfaces and thereby influences biofilm morphology.

A searchable database of images allows detailed analysis of bacterial motility.

Mass spectrometry imaging (MSI) is a versatile tool for visualizing molecular distributions in complex biological specimens, but locating microscopic chemical features of interest can be challenging in samples that lack a well-defined anatomy. To address this issue, we developed a correlated imaging approach that begins with performing matrix-assisted laser desorption/ionization (MALDI) MSI to obtain low-resolution molecular maps of a sample. The resulting maps are then used to direct subsequent microscopic secondary ion mass spectrometry (SIMS) imaging and tandem mass spectrometry (MS/MS) experiments to examine selected chemical regions of interest. By employing MALDI undersampling, the sample surface is left mostly unperturbed and available for the SIMS analysis, while also generating an ablation array that can be used for navigation in SIMS. We validated this MALDI-guided SIMS approach using cultured biofilms of the opportunistic pathogen Pseudomonas aeruginosa; bioactive secondary metabolites, including rhamnolipids and quinolones, were detected and visualized on both macro- and microscopic size scales. MSI mass assignments were confirmed with in situ MALDI MS/MS and capillary electrophoresis–electrospray ionization MS/MS analysis of biofilm extracts. Two strains of P. aeruginosa were compared, wild type and a quorum sensing mutant, and differences in metabolite abundance and distribution were observed.

DNA is a common extracellular matrix component of bacterial biofilms. We find that bacteria can spontaneously order in a matrix of aligned concentrated DNA, in which rod-shaped cells of Pseudomonas aeruginosa follow the orientation of extended DNA chains. The alignment of bacteria is ensured by elasticity and liquid crystalline properties of the DNA matrix. These findings show how behavior of planktonic bacteria may be modified in extracellular polymeric substances of biofilms and illustrate the potential of using complex fluids to manipulate embedded nanosized and microsized active particles.