Bacterial Swarming: A Biochemical Time-Resolved FTIR-ATR Study of Proteus mirabilis Swarm-Cell Differentiation Michae ¨l Gue ´, Virginie Dupont, Alain Dufour, and Olivier Sire* Laboratoire de Biologie et Chimie Mole ´ culaires, UniVersite ´ de Bretagne Sud, Campus de Tohannic, B.P. 573, 56017 Vannes Cedex, France ReceiVed March 5, 2001; ReVised Manuscript ReceiVed July 23, 2001 ABSTRACT: Fourier transform infrared spectroscopy was applied to the study of the differentiation process undergone by Proteus mirabilis. This bacterium exhibits a remarkable dimorphism, allowing the cells to migrate on a solid substratum in a concerted manner yielding characteristic ring patterns. We performed an in situ noninvasive analysis of biochemical events occurring as vegetative cells differentiate into elongated, multinucleate, nonseptate, and hyperflagellated swarm cells. The major findings arising from this study are (i) the real-time monitoring of flagellar filament assembly, (ii) the evidence for de novo synthesis of qualitatively different lipopolysaccharides (LPS) and/or exopolysaccharides (EPS) constituting the slime into which bacteria swarm, and (iii) the alteration in the membrane fatty acid composition with a concomitant 10 °C decrease in the gel/liquid crystal phase transition resulting in an elevated membrane fluidity in swarm cells at the growth temperature. The time course of events shows that the EPS-LPS syntheses are synchronous with membrane fatty acid alterations and occur about 1 h before massive flagellar filament assembly is detected. This study not only provided a time sketch of biochemical events involved in the differentiation process but also led to the identification of the major spectral markers of both vegetative and swarm cells. This identification will allow to resolve the time-space structure of P. mirabilis colonies by using infrared microscopy. Every living process is embedded in a unique temporal grid. Disregarding the entity considered, molecule, cell, or organism, its characteristic properties rely on elementary phenomena exhibiting particular frequencies: protein fluc- tuations, oscillating enzymatic reactions, cellular divisions, etc. This temporal axis is too often disregarded in our attempts to describe the mechanisms of life. A bacterial colony behaves as an entity in which biochemical modifica- tions occur in a concerted manner. Hence, the growth and behavior of individual cells become correlated in time and space to those of the whole population, which can be con- sidered as a multicellular organism (1, 2). Bacterial colonies are experimental systems of choice for studying fundamental problems of self-organization and pattern formation by com- plex biological systems. Such coordinated multicellular be- haviors are particularly well exemplified by bacterial swarm- ing, which is a form of active surface motility widespread among flagellated, Gram-negative bacteria (3-5). We used the uropathogenic Proteus mirabilis as a model since it is one of the bacteria exhibiting the strongest swarming abilities. From a central inoculum, a P. mirabilis colony is able to spread over the entire surface of a Petri plate within a few hours, by alternating periods of active mass migration and of cell division without colony expansion (consolidation) (5-8). As a result of this periodicity and of the cell coordina- tion, the final colony displays a concentric ring pattern. The colony is encapsulated into an extracellular slime, which is a complex mixture including polysaccharides, surfactants, proteins, peptides, etc. The slime acts as a surface lubricant, provides an aqueous environment allowing flagella rotation, and is likely involved in cell-cell communications (9). Three Proteus capsular polysaccharides (CPS) 1 have been described (10), including a colony migration factor (Cmf), which is part of the slime and has been shown to facilitate P. mirabilis mass migration (11). This striking swarming behavior has driven microbiologists to study the genetic bases of the morphogenesis during which short (2 μm) vegetative swim- ming bacteria differentiate into elongated (40 μm or more), multinucleate, nonseptate, and hyperflagellated swarm cells (6, 12, 13). As the differentiated cells display enhanced properties of invasion of human urothelial cells and of production of a number of virulence factors (14-17), it is likely that the swarming behavior does not constitute a laboratory artifact but is essential to P. mirabilis pathogenic- ity and warrants for its ability to colonize urinary tracts (17) or to block catheters with crystalline biofilms (18). To undertake their differentiation, the bacteria must meet a number of requirements, such as the contact with a solid substratum, the presence of glutamine (19), the ability to assemble flagella (20, 21), or a sufficient cell density (22). * Corresponding author: e-mail, osire@univ-ubs.fr; tel, 33 297 683 172; fax, 33 297 681 639. 1 Abbreviations: ATR, attenuated total reflection; Cmf, colony migration factor; CPS, capsular polysaccharide; DAPI, 4,6-diamidino- 2-phenylindole; EPS, exopolysaccharides; FTIR, Fourier transform infrared; GC, gas chromatography; IR, infrared; LB, Luria-Bertani culture medium; LPS, lipopolysaccharides; PBS, phosphate-buffered saline (pH 7.2) (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2PO4, 8.1 mM Na2HPO4); PMSF, phenylmethanesulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TCA, trichloroacetic acid; TEN, Tris-EDTA-NaCl buffer [10 mM Tris- HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl]. 11938 Biochemistry 2001, 40, 11938-11945 10.1021/bi010434m CCC: $20.00 © 2001 American Chemical Society Published on Web 09/06/2001