Coordinated regulation of two independent cell-cell signaling systems and swarmer differentiation in Salmonella enterica serovar Typhimurium

Almost all members of the genus Salmonella differentiate and migrate on semisolid surfaces in a coordinated population behavior known as swarming. Important virulence determinants are coupled to swarmer differentiation in several other pathogenic organisms, collectively suggesting that conditions that trigger swarming in the laboratory may fortuitously promote the cells to enter a robust physiological state relevant to the host environment. Here, we present evidence that expression of two independent cell-cell signaling systems are also coupled to swarmer differentiation in S. enterica serovar Typhimurium. Expression of both pfs and sdiA genes was up-regulated in the actively migrating swarmers compared to their vegetative counterparts propagated in broth or spread plated on the surface of swim, swarm, and solid media. Accordingly, swarmers produced elevated levels of a universally recognized signaling molecule, autoinducer-2, and exhibited increased sensitivity to N-acyl homoserine lactones (AHLs), signaling molecules that Salmonella does not produce. Expression of the rck operon was concomitantly up-regulated in the swarmers in an SdiA-dependent manner only in the presence of exogenous AHLs. In addition to the previously reported adaptive antibiotic resistance phenotype and global shift in metabolism, this work presents another component of the physiological changes that are specifically associated with swarmer differentiation in serovar Typhimurium and not simply due to growth on a surface.     

Swarming motility: a multicellular behaviour conferring antimicrobial resistance

Swarming is a type of social motility allowing the migration of highly differentiated bacterial cells. Swarming shares many similarities with biofilm communities, which are notable for their high resistance to antimicrobial agents. We investigate here if the swarming behaviour could also be associated with a widespread antimicrobial resistant phenotype. Challenged with 13 antibiotics from various classes, swarm cells of Pseudomonas aeruginosa , Escherichia coli , Serratia marcescens , Burkholderia thailandensis and Bacillus subtilis showed higher resistance than their planktonic counterparts to all the antibiotics tested, except for the antimicrobial peptides. Using P. aeruginosa as a model, this multiresistant phenotype was shown to be transient and intrinsically linked to the swarming state. Resistance of swarm cells towards other antimicrobial agents, such as triclosan and a heavy metal (arsenite), was also observed. Neither RND-type efflux pumps, including MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM, nor a biofilm-associated resistance mechanism involving periplasmic glucans, appear to account for the resistance of swarm cells. Together with the high resistance of biofilms, these results support the hypothesis that antimicrobial resistance is a general feature of bacterial multicellularity. Swarming motility might thus represent a form of social behaviour useful as a model to investigate biofilm antibiotic resistance.     

Importance ofeps genes fromBacillus subtilis in biofilm formation and swarming

Unicellular organisms naturally form multicellular communities, differentiate into specialized cells, and synchronize their behaviour under certain conditions. Swarming, defined as a movement of a large mass of bacteria on solid surfaces, is recognized as a preliminary step in the formation of biofilms. The main aim of this work was to study the role of a group of genes involved in exopolysaccharide biosynthesis during pellicle formation and swarming inBacillus subtilis strain 168. To assess the role of particular proteins encoded by the group ofepsI-epsO genes that form theeps operon, we constructed a series of insertional mutants. The results obtained showed that mutations inepsJ-epsN, but not in the last gene of theeps operon (epsO), have a severe effect on pellicle formation under all tested conditions. Moreover, the inactivation of 5 out of the 6 genes analysed caused total inhibition of swarming in strain 168 (that does not produce surfactin) on LB medium. Following restoration of thesfp gene (required for production of surfactin, which is essential for swarming of the wild-type bacteria), thesfp ⁺ strains defective ineps genes (exceptepsO) generated significantly different patterns during swarming on synthetic B medium, as compared to the parental strain 168sfp ⁺.     

MODE OF SWARMING IN RELATION TO REPRODUCTIVE ISOLATION IN MAYFLIES

Mayfly males swarm, that is they fly in a fixed pattern by a specific object, the swarm marker. Females orientate to the same markers. Leptophlebia marginata mayflies were observed to orientate to two kinds of objects in a single locality in central Finland: to trees and to horizontal pale objects on the ground; when dispersed or moved to the other type of marker, they returned to their former orientation. Tree swarming is by far the most common mode of swarming, but some horizontally orientating populations were found. Sympatric populations are genetically and morphologically distinct, whereas other populations appear to have some gene flow between the swarming types. The tree-swarming mode appears to be primitive and the horizontal mode derived; wind rather than predation is the factor favoring swarming close to the ground. Swarming constitutes an effective mechanism of premating isolation in mayflies.     

Swarming motility

Swarming involves differentiation of vegetative cells into hyperflagellated swarm cells that undergo rapid and coordinated population migration across solid surfaces. Cell density, surface contact, and physiological signals all provide critical stimuli, and close cell alignment and the production of secreted migration factors facilitate mass translocation. Flagella biogenesis is central to swarming, and the flhDC flagellar master operon is the focal point of a regulatory network governing differentiation and migration.     

Swarming and mating behaviour ofChironomus flaviplumus (Diptera: Chironomidae), compared with a sympatric congeneric species,C. yoshimatsui

The swarming and mating behaviour ofChironomus flaviplumus was observed and compared with a sympatric congeneric species,C. yoshimatsui. C. flaviplumus males swarmed around sunset near foliage or angles of buildings near the emergence site and copulated with females entering the swarm. Swarming and mating occurred under conditions of higher light intensity in cooler seasons than in warmer ones. Results suggested that temperature had an effect on the timing of flight to the swarming site in both sexes. TheC. flaviplumus swarm marker and swarming behaviour seemed very similar to that toC. yoshimatsui, and their respective daily swarming time zone greatly overlapped. No mixed swarm, however, was observed in the study area. This is probably due to the distance between the species' larval habitats. Possible premating isolation mechanisms between these 2 species are discussed.     

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 motility in undomesticated Bacillus subtilis

Swarming motility was identified and characterized in an undomesticated strain of Bacillus subtilis. Rapid surface migration was preceded by a cell density-dependent lag period, which could be eliminated if actively swarming cells were used as the inoculum. The leading edge of the swarm was characterized by multicellular rafts of highly flagellated cells. Flagellum biosynthesis and surfactant production were required for swarming. Swarming was not found in any of several standard laboratory strains. Laboratory strains are characteristically unable to produce surfactant, but such a strain remained unable to swarm even when surfactant was provided by extracellular complementation. We conclude that robust swarming is a feature of undomesticated B. subtilis and that this behaviour has been lost or attenuated in laboratory strains through the accumulation of multiple genetic defects.     

Assessment of efflux-mediated antibiotic-resistant Salmonella enterica serovar Typhimurium under simulated gastrointestinal conditions

The objective of this study was to evaluate the efflux-mediated antibiotic resistance and virulence potential in Salmonella enterica serovar Typhimurium exposed to bile salts. S. enterica serovar Typhimurium KCCM 40253, S. enterica serovar Typhimurium CCARM 8009, and plasmid-cured S. enterica serovar Typhimurium CCARM 8009 were used to evaluate the antimicrobial susceptibility, adherence ability, and gene expression in the presence of 0.3 % bile salts. The sensitivity of S. enterica serovar Typhimurium CCARM 8009 to tetracycline was significantly increased in the presence of phenylalanine-arginine β-naphthylamide (PAβN), showing the decrease in the minimum inhibitory concentration (MIC) values from 256 to 8 mg/ml. The relative ethidium bromide (EtBr) fluorescence intensity was rapidly decreased from 1 to 0.47 in S. enterica serovar Typhimurium CCARM 8009 after 20 min of exposure to bile salts. The highest adhesion ability was observed in S. enterica serovar Typhimurium CCARM 8009 exposed to both absence and presence of bile salts. The tolC and tetA genes were up-regulated in S. enterica serovar Typhimurium CCARM 8009 exposed bile salts. The results suggest that the antimicrobial resistance were positively correlated with efflux pump activity, and virulence potential in antibiotic-resistant S. enterica serovar Typhimurium when exposed to bile salts.     

细菌群集运动特性的研究进展

群集运动(swarming motility)是细菌以群体方式协调性地依靠鞭毛和Ⅳ型菌毛(type Ⅳ pili,TFP)在半固体表面共同运动,是一种典型的协同运动。群集运动因其与生物被膜、子实体的形成、病原体的侵入和微生物的扩散及共生等过程都有着密切的关系而备受人们的关注,是当前微生物领域的一个研究热点。人们对细菌群集运动开展了大量的研究,包括群集运动中关键蛋白表达的变化、细胞间化学交流的变化以及机械性变化等。鞭毛蛋白的表达以及胞内环二鸟苷酸(cyclic diguanosine monophosphate,c-di-GMP)的水平等会对群集运动产生一定的影响,在菌落中复杂地调控着细菌集体行为;群集运动细胞独特的物理性质表现有益于菌落整体的扩张;细菌周围生长环境中的营养和水分含量等因素也在不同程度上影响细菌群集运动的能力。未来,在解析群集运动分子机制的基础上,如何构建一个统一的群集运动模型成为该领域研究面临的一个挑战。     

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.     

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.     

Differentiation of Serratia liquefaciens into swarm cells is controlled by the expression of the flhD master operon.

The velocity with which a swarming colony of Serratia liquefaciens colonizes the surface of a suitable solid substratum was controlled by modulating the expression of the flhD master operon. In liquid medium, the stimulation of flhD expression resulted in filamentous, multinucleate, and hyperflagellated cells that were indistinguishable from swarm cells isolated from the edge of a swarm colony. Thus, expression of the flhD master operon appears to play a central role in the process of swarm cell differentiation.     

Inactivation of ompR promotes precocious swarming and flhDC expression in Xenorhabdus nematophila

The response regulator OmpR is involved in numerous adaptive responses to environmental challenges. The role that OmpR plays in swarming behavior and swarm-cell differentiation in the symbiotic-pathogenic bacterium Xenorhabdus nematophila was examined in this study. Swarming began 4 h sooner in an ompR mutant strain than in wild-type cells. Precocious swarming was correlated with elevated expression of fliC, early flagellation, and cell elongation. The level of flhDC mRNA was elevated during the early period of swarming in the ompR strain relative to the level in the wild type. These findings show that OmpR is involved in the temporal regulation of flhDC expression and flagellum production and demonstrate that this response regulator plays a role in the swarming behavior of X. nematophila.     

Cell differentiation of Proteus mirabilis is initiated by glutamine, a specific chemoattractant for swarming cells

Swarming by Proteus mirabilis involves differentiation of typical short vegetative rods into filamentous hyper-flagellated swarm cells which undergo cycles of rapid and co-ordinated population migration across surfaces and exhibit high levels of virulence gene expression. By supplementing a minimal growth medium (MGM) unable to support swarming migration we identified a single amino acid, glutamine, as sufficient to signal initiation of cell differentiation and migration. Bacteria isolated from the migrating edge of colonies grown for 8h with glutamine as the only amino acid were filamentous and synthesized the characteristic high levels of flagellin and haemolysin. In contrast, addition of the other 19 common amino acids (excluding glutamine) individually or in combination did not initiate differentiation even after 24 h, cells remaining typical vegetative rods with basal levels of haemolysin and flagellin. The glutamine analogue γ-glutamyl hydroxamate (GH) inhibited swarming but not growth of P. mirabilis on glutamine MGM and transposon mutants defective in glutamine uptake retained their response to glutamine signalling and its inhibition by GH, suggesting that differentiation signalling by glutamine may be transduced independently of the cellular glutamine transport system. Levels of mRNA transcribed from the haemolysin (hpmA) and flagellin (fliC) genes were low in vegetative cells grown on MGM without glutamine or with glutamine and GH, but were specifically increased c. 40-fold during glutamine-dependent differentiation. In liquid glutamine—MGM cultures, differentiation to filamentous hyper-flagellated hyper-haemorytic swarm cells occurred early in the exponential phase of growth, and increased concomitantly with the concentration of glutamine from a 0.1 mM threshold up to 10 mM. Differentiation in liquid culture was completely inhibited by GH but was further stimulated c. 30% in the absence of GH by the viscosity agent polyvinylpy-rollidone (PVP). Chemotaxis assays of bacterial cells in which the viscosity of liquid media was varied by PVP to allow either swimming or swarming motility demonstrated that glutamine was chemoattractive specifically to differentiated swarming cells.     

Scanning electron microscope observation of the swarming phenomenon of Vibrio parahaemolyticus.

Scanning electron microscopy was used to study the production of lateral flagella and the swarming phenomenon in Vibrio parahaemolyticus. Differences in the size and diameter of the sheathed, polar flagellum and lateral flagella were apparent in these preparations. Swarming of V. parahaemolyticus was found to be similar to the swarming of Proteus spp. in that swarm cells which are heavily flagellated and elongated are formed.