Genetics of swarming motility in Salmonella enterica serovar typhimurium: critical role for lipopolysaccharide

Salmonella enterica serovar Typhimurium can differentiate into hyperflagellated swarmer cells on agar of an appropriate consistency (0.5 to 0.8%), allowing efficient colonization of the growth surface. Flagella are essential for this form of motility. In order to identify genes involved in swarming, we carried out extensive transposon mutagenesis of serovar Typhimurium, screening for those that had functional flagella yet were unable to swarm. A majority of these mutants were defective in lipopolysaccharide (LPS) synthesis, a large number were defective in chemotaxis, and some had defects in putative two-component signaling components. While the latter two classes were defective in swarmer cell differentiation, representative LPS mutants were not and could be rescued for swarming by external addition of a biosurfactant. A mutation in waaG (LPS core modification) secreted copious amounts of slime and showed a precocious swarming phenotype. We suggest that the O antigen improves surface "wettability" required for swarm colony expansion, that the LPS core could play a role in slime generation, and that multiple two-component systems cooperate to promote swarmer cell differentiation. The failure to identify specific swarming signals such as amino acids, pH changes, oxygen, iron starvation, increased viscosity, flagellar rotation, or autoinducers leads us to consider a model in which the external slime is itself both the signal and the milieu for swarming motility. The model explains the cell density dependence of the swarming phenomenon.     

Swarming populations of<Emphasis Type="Italic">Salmonella</Emphasis> represent a unique physiological state coupled to multiple mechanisms of antibiotic resistance

Salmonella enterica serovar Typhimurium is capable of swarming over semi-solid surfaces. Although its swarming behavior shares many readily observable similarities with other swarming bacteria, the phenomenon remains somewhat of an enigma in this bacterium since some attributes skew away from the better characterized systems. Swarming is quite distinct from the classic swimming motility, as there is a prerequisite for cells to first undergo a morphological transformation into swarmer cells. In some organisms, swarming is controlled by quorum sensing, and in others, swarming has been shown to be coupled to increased expression of important virulence factors. Swarming in serovar Typhimurium is coupled to elevated resistance to a wide variety of structurally and functionally distinct classes of antimicrobial compounds. As serovar Typhimurium differentiates into swarm cells, thepmrHFIJKLM operon is up-regulated, resulting in a more positively charged LPS core. Furthermore, as swarm cells begin to de-differentiate, thepmr operon expression is down-regulated, rapidly reaching the levels observed in swim cells. This is one potential mechanism which confers swarm cells increased resistance to antibiotics such as the cationic antimicrobial peptides. However, additional mechanisms are likely associated with the cells in the swarm state that confer elevated resistance to such a broad spectrum of antimicrobial agents. Published: September 26, 2003     

Metabolic differentiation in actively swarming Salmonella

Most current paradigms of microbial metabolism have been derived from studying cells grown under a variety of nutrient compositions in aqueous environments. With recent advances in genomics and experimental techniques, alternative forms of bacterial growth are increasingly being explored. When propagated on nutrient-rich semi-solid media, several species of bacteria undergo a morphological differentiation into swarmers that are capable of migrating on surfaces. Recent studies indicate that swarmer differentiation represents much more than a motility phenotype, as several clinically important attributes are also co-regulated. We demonstrate that migrating swarmer cells of Salmonella are metabolically differentiated compared to the vegetative swimmer cells grown in the same nutrient environment. Furthermore, once the cells have differentiated, the swarmers remain in this physiological state under conditions that do not promote the initial differentiation. The bacterium's capacity to override some of the classic paradigms of metabolic regulation established in aqueous environments represents a unique physiological response by the pathogen that may be advantageous in polymicrobial environments such as the host.     

Swarmer cell differentiation in Proteus mirabilis

Under the appropriate environmental conditions, the gram-negative bacterium Proteus mirabilis undergoes a remarkable differentiation to form a distinct cell type called a swarmer cell. The swarmer cell is characterized by a 20- to 40-fold increase in both cell length and the number of flagella per cell. Environmental conditions required for swarmer cell differentiation include: surface contact, inhibition of flagellar rotation, a sufficient cell density and cell-to-cell signalling. The differentiated swarmer cell is then able to carry out a highly ordered population migration termed swarming. Genetic analysis of the swarming process has revealed that a large variety of distinct loci are required for this differentiation including: genes involved in regulation, lipopolysaccharide and peptidoglycan synthesis, cell division, ATP production, putrescine biosynthesis, proteolysis and cell shape determination. The process of swarming is important medically because the expression of virulence genes and the ability to invade cells are coupled to the differentiated swarmer cell. In this review, the genetic and environmental requirements for swarmer cell differentiation will be outlined. In addition, the role of the differentiated swarmer cell in virulence and its possible role in biofilm formation will be discussed.     

A cell length‐dependent transition in MinD‐dynamics promotes a switch in division‐site placement and preservation of proliferating elongated Vibrio parahaemolyticus swarmer cells

Vibrio parahaemolyticus exists as swimmer and swarmer cells, specialized for growth in liquid and on solid environments respectively. Swarmer cells are characteristically highly elongated due to an inhibition of cell division, but still need to divide in order to proliferate and expand the colony. It is unknown how long swarmer cells divide without diminishing the population of long cells required for swarming behavior. Here we show that swarmer cells divide but the placement of the division site is cell length‐dependent; short swarmers divide at mid‐cell, while long swarmers switch to a specific non‐mid‐cell placement of the division site. Transition to non‐mid‐cell positioning of the Z‐ring is promoted by a cell length‐dependent switch in the localization‐dynamics of the division regulator MinD from a pole‐to‐pole oscillation in short swarmers to a multi‐node standing‐wave oscillation in long swarmers. Regulation of FtsZ levels restricts the number of divisions to one and SlmA ensures sufficient free FtsZ to sustain Z‐ring formation by preventing sequestration of FtsZ into division deficient clusters. By limiting the number of division‐events to one per cell at a specific non‐mid‐cell position, V. parahaemolyticus promotes the preservation of long swarmer cells and permits swarmer cell division without the need for dedifferentiation.     

Uncovering a large set of genes that affect surface motility in Salmonella enterica serovar Typhimurium

We describe a large set of genes affecting motility in Salmonella enterica serovar Typhimurium. Identified in microarray experiments as displaying flagellar gene expression patterns or controlled by known flagellar regulators, we show that null mutations in these genes primarily affect swarming motility. Three genes function in chemotaxis.     

A mechanical role for the chemotaxis system in swarming motility

The chemotaxis system, but not chemotaxis, is essential for swarming motility in Salmonella enterica serovar Typhimurium. Mutants in the chemotaxis pathway exhibit fewer and shorter flagella, downregulate class 3 or 'late' motility genes, and appear to be less hydrated when propagated on a surface. We show here that the output of the chemotaxis system, CheY approximately P, modulates motor bias during swarming as it does during chemotaxis, but for a distinctly different end. A constitutively active form of CheY was found to promote swarming in the absence of several upstream chemotaxis components. Two point mutations that suppressed the swarming defect of a cheY null mutation mapped to FliM, a protein in the motor switch complex with which CheY approximately P interacts. A common property of these suppressors was their increased frequency of motor reversal. These and other data suggest that the ability to switch motor direction is important for promoting optimal surface wetness. If the surface is sufficiently wet, exclusively clockwise or counterclockwise directions of motor rotation will support swarming, suggesting also that the bacteria can move on a surface with flagellar bundles of either handedness.     

Imaging and analysis of Pseudomonas aeruginosa swarming and rhamnolipid production

Many bacteria spread over surfaces by "swarming" in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacterium Pseudomonas aeruginosa. First, we quantify the temporal distribution of P. aeruginosa cells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming of P. aeruginosa and Salmonella enterica serovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of several P. aeruginosa strains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production.     

The cytokine balance in the maintenance of a persistent infection with Salmonella enterica serovar Typhimurium in mice

ClpXP, serine protease-disrupted mutant of Salmonella enterica serovar Typhimurium chi3306 exhibits attenuated but persistent infection in mice. During infection with S. enterica serovar Typhimurium ClpXP-disrupted mutant, gamma interferon (IFN-gamma) produced by CD4+ cells was up-regulated on day 10 and tumor necrosis factor-alpha (TNF-alpha) produced by CD8+ cells was up-regulated on day 30 after infection. Treatment of monoclonal antibodies against cytokines showed that IFN-gamma and interleukin 10 (IL-10) were involved in maintenance of growth of S. Typhimurium mutant on day 10 after infection, and IFN-gamma, TNF-alpha and transforming growth factor-beta (TGF-beta) were involved in maintenance of growth of this bacterium on day 30 after infection. During persistent infection of S. Typhimurium mutant, IFN-gamma, TNF-alpha, IL-10 and TGF-beta may play different roles to maintain the persistent infection. The cytokine balance might be important in persistent infection with ClpXP-disrupted S. enterica serovar Typhimurium.     

Salmonella enterica serovar typhimurium swarming mutants with altered biofilm-forming abilities: surfactin inhibits biofilm formation

Swarming motility plays an important role in surface colonization by several flagellated bacteria. Swarmer cells are specially adapted to rapidly translocate over agar surfaces by virtue of their more numerous flagella, longer cell length, and encasement of slime. The external slime provides the milieu for motility and likely harbors swarming signals. We recently reported the isolation of swarming-defective transposon mutants of Salmonella enterica serovar Typhimurium, a large majority of which were defective in lipopolysaccharide (LPS) synthesis. Here, we have examined the biofilm-forming abilities of the swarming mutants using a microtiter plate assay. A whole spectrum of efficiencies were observed, with LPS mutants being generally more proficient than wild-type organisms in biofilm formation. Since we have postulated that O-antigen may serve a surfactant function during swarming, we tested the effect of the biosurfactant surfactin on biofilm formation. We report that surfactin inhibits biofilm formation of wild-type S. enterica grown either in polyvinyl chloride microtiter wells or in urethral catheters. Other bio- and chemical surfactants tested had similar effects.     

Mucosal lymphatic-derived gammadelta T cells respond early to experimental Salmonella enterocolitis by increasing expression of IL-2R alpha

To better understand the roles of gammadelta T cells in mucosal infection, we utilized Salmonella enterica serovar Typhimurium (Salmonella serovar Typhimurium) infection in cattle as it closely approximates Salmonella serovar Typhimurium-induced enterocolitis in humans. Protein and gene expression in alphabeta and gammadelta T cells derived from lymphatic ducts draining the gut mucosa in Salmonella serovar Typhimurium-infected calves were analyzed. In calves with enterocolitis, general gene expression trends in gammadelta T cells suggested subtle activation and innate response, whereas alphabeta T cells were relatively quiescent following Salmonella serovar Typhimurium infection. An increase in IL-2R alpha expression on gammadelta T cells from infected calves and results from in vitro assays suggested that gammadelta T cells were primed by Salmonella serovar Typhimurium LPS to better respond to IL-2 and IL-15. Together with gene expression trends in vivo, these data support early priming activation of target tissue gammadelta T cells during Salmonella serovar Typhimurium infection.     

Proteus mirabilis mutants defective in swarmer cell differentiation and multicellular behavior

Proteus mirabilis is a dimorphic bacterium which exists in liquid cultures as a 1.5- to 2.0-microns motile swimmer cell possessing 6 to 10 peritrichous flagella. When swimmer cells are placed on a surface, they differentiate by a combination of events that ultimately produce a swarmer cell. Unlike the swimmer cell, the polyploid swarmer cell is 60 to 80 microns long and possesses hundreds to thousands of surface-induced flagella. These features, combined with multicellular behavior, allow the swarmer cells to move over a surface in a process called swarming. Transposon Tn5 was used to produce P. mirabilis mutants defective in wild-type swarming motility. Two general classes of mutants were found to be defective in swarming. The first class was composed of null mutants that were completely devoid of swarming motility. The majority of nonswarming mutations were the result of defects in the synthesis of flagella or in the ability to rotate the flagella. The remaining nonswarming mutants produced flagella but were defective in surface-induced elongation. Strains in the second general class of mutants, which made up more than 65% of all defects in swarming were motile but were defective in the control and coordination of multicellular swarming. Analysis of consolidation zones produced by such crippled mutants suggested that this pleiotropic phenotype was caused by a defect in the regulation of multicellular behavior. A possible mechanism controlling the cyclic process of differentiation and dediferentiation involved in the swarming behavior of P. mirabilis is discussed.     

Surface-induced swarmer cell differentiation of Vibrio parahaemoiyticus

Vibrio parahaemolyticus distinguishes between life in a liquid environment and life on a surface. Growth on a surface induces differentiation from a swimmer cell to a swarmer cell type. Each cell type is adapted for locomotion under different circumstances. Swimmer cells synthesize a single polar flagellum (Fla) for movement in a liquid medium, and swarmer cells produce an additional distinct flagellar system, the lateral flagella (Laf), for movement across a solid substratum, called swarming. Recognition of surfaces is necessary for swarmer cell differentiation and involves detection of physical signals peculiar to that circumstance and subsequent transduction of information to affect expression of swarmer cell genes (laf). The polar flagellum functions as a tactile sensor controlling swarmer cell differentiation by sensing forces that restrict its movement. Surface recognition also involves a second signal, i.e. nutritional limitation for iron. Studying surface-induced differentiation could reveal a novel mechanism of gene control and lead to an understanding of the processes of surface colonization by pathogens and other bacteria.     

Quorum sensing and swarming migration in bacteria

Bacterial cells can produce and sense signal molecules, allowing the whole population to initiate a concerted action once a critical concentration (corresponding to a particular population density) of the signal has been reached, a phenomenon known as quorum sensing. One of the possible quorum sensing-regulated phenotypes is swarming, a flagella-driven movement of differentiated swarmer cells (hyperflagellated, elongated, multinucleated) by which bacteria can spread as a biofilm over a surface. The glycolipid or lipopeptide biosurfactants thereby produced function as wetting agent by reducing the surface tension. Quorum sensing systems are almost always integrated into other regulatory circuits. This effectively expands the range of environmental signals that influence target gene expression beyond population density. In this review, we first discuss the regulation of AHL-mediated surface migration and the involvement of other low-molecular-mass signal molecules (such as the furanosyl borate diester AI-2) in biosurfactant production of different bacteria. In addition, population density-dependent regulation of swarmer cell differentiation is reviewed. Also, several examples of interspecies signalling are reported. Different signal molecules either produced by bacteria (such as other AHLs and diketopiperazines) or excreted by plants (such as furanones, plant signal mimics) might influence the quorum sensing-regulated swarming behaviour in bacteria different from the producer. On the other hand, specific bacteria can reduce the local available concentration of signal molecules produced by others. In the last part, the role and regulation of a surface-associated movement in biofilm formation is discussed. Here we also describe how quorum sensing may disperse existing biofilms and control the interaction between bacteria and higher organisms (such as the Rhizobium-bean symbiosis).