Bees aren't the only ones: swarming in Gram-negative bacteria

Summary
Swarming is a form of active surface motility that is widespread among flagellated. Gram–negative bacteria. In the laboratory, growth of the bacteria on certain agar surfaces leads to induction of the differentiated swarmer-cell state. Swarmer cells are generally long and multinucleate, always hyperflagellated, and can move rapidly over the agar surface in a coordinated manner. Some swarm colonies exude large amounts of 'slime', which could be essential for promoting intimate cell–cell contacts during swarming. There is evidence that the differentiated swarmer-cell stage facilitates pathogenic associations with host tissue. Almost nothing is known about the molecular signalling mechanism of surface sensing. Increased viscosity appears to be sensed by several bacteria, but other environmental cues, specific to each bacterium, are also important. In organisms in which swarming motility has been studied in some detail, the chemotaxis system has been shown to play an important rote. The recent discovery of swarming motility in two genetically well-characterized organisms – Escherichia coli and Salmonella typhimurium – should lead to rapid progress in understanding this process.     

Swarming of Pseudomonas aeruginosa is a complex adaptation leading to increased production of virulence factors and antibiotic resistance

In addition to exhibiting swimming and twitching motility, Pseudomonas aeruginosa is able to swarm on semisolid (viscous) surfaces. Recent studies have indicated that swarming is a more complex type of motility influenced by a large number of different genes. To investigate the adaptation process involved in swarming motility, gene expression profiles were analyzed by performing microarrays on bacteria from the leading edge of a swarm zone compared to bacteria growing in identical medium under swimming conditions. Major shifts in gene expression patterns were observed under swarming conditions, including, among others, the overexpression of a large number of virulence-related genes such as those encoding the type III secretion system and its effectors, those encoding extracellular proteases, and those associated with iron transport. In addition, swarming cells exhibited adaptive antibiotic resistance against polymyxin B, gentamicin, and ciprofloxacin compared to what was seen for their planktonic (swimming) counterparts. By analyzing a large subset of up-regulated genes, we were able to show that two virulence genes, lasB and pvdQ, were required for swarming motility. These results clearly favored the conclusion that swarming of P. aeruginosa is a complex adaptation process in response to a viscous environment resulting in a substantial change in virulence gene expression and antibiotic resistance.     

Mutations that impair swarming motility in Serratia marcescens 274 include but are not limited to those affecting chemotaxis or flagellar function.

Serratia marcescens exists in two cell forms and displays two kinds of motility depending on the type of growth surface encountered (L. Alberti and R. M. Harshey, J. Bacteriol. 172:4322-4328, 1990). In liquid medium, the bacteria are short rods with few flagella and show classical swimming behavior. Upon growth on a solid surface (0.7 to 0.85% agar), they differentiate into elongated, multinucleate, copiously flagellated forms that swarm over the agar surface. The flagella of swimmer and swarmer cells are composed of the same flagellin protein. We show in this study that disruption of hag, the gene encoding flagellin, abolishes both swimming and swarming motility. We have used transposon mini-Mu lac kan to isolate mutants of S. marcescens defective in both kinds of motility. Of the 155 mutants obtained, all Fla- mutants (lacking flagella) and Mot- mutants (paralyzed flagella) were defective for both swimming and swarming, as expected. All Che- mutants (chemotaxis defective) were also defective for swarming, suggesting that an intact chemotaxis system is essential for swarming. About one-third of the mutants were specifically affected only in swarming. Of this class, a large majority showed active "swarming motility" when viewed through the microscope (analogous to the active "swimming motility" of Che- mutants) but failed to show significant movement away from the site of initial inoculation on a macroscopic scale. These results suggest that bacteria swarming on a solid surface require many genes in addition to those required for chemotaxis and flagellar function, which extend the swarming movement outward. We also show in this study that nonflagellate S. marcescens is capable of spreading rapidly on low-agar media.     

Genome-wide screening of genes required for swarming motility in Escherichia coli K-12

Escherichia coli K-12 has the ability to migrate on semisolid media by means of swarming motility. A systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection) was used to identify the genes involved in the swarming motility of this bacterium. Of the 3,985 nonessential gene mutants, 294 were found to exhibit a strongly repressed-swarming phenotype. Further, 216 of the 294 mutants displayed no significant defects in swimming motility; therefore, the 216 genes were considered to be specifically associated with the swarming phenotype. The swarming-associated genes were classified into various functional categories, indicating that swarming is a specialized form of motility that requires a wide variety of cellular activities. These genes include genes for tricarboxylic acid cycle and glucose metabolism, iron acquisition, chaperones and protein-folding catalysts, signal transduction, and biosynthesis of cell surface components, such as lipopolysaccharide, the enterobacterial common antigen, and type 1 fimbriae. Lipopolysaccharide and the enterobacterial common antigen may be important surface-acting components that contribute to the reduction of surface tension, thereby facilitating the swarm migration in the E. coli K-12 strain.     

Lateral Flagella and Swarming Motility in Aeromonas Species

Swarming motility, a flagellum-dependent behavior that allows bacteria to move over solid surfaces, has been implicated in biofilm formation and bacterial virulence. In this study, light and electron microscopic analyses and genetic and functional investigations have shown that at least 50% of Aeromonas isolates from the species most commonly associated with diarrheal illness produce lateral flagella which mediate swarming motility. Aeromonas lateral flagella were optimally produced when bacteria were grown on solid medium for approximately 8 h. Transmission and thin-section electron microscopy confirmed that these flagella do not possess a sheath structure. Southern analysis of Aeromonas reference strains and strains of mesophilic species (n = 84, varied sources and geographic regions) with a probe designed to detect lateral flagellin genes (lafA1 and lafA2) showed there was no marked species association of laf distribution. Approximately 50% of these strains hybridized strongly with the probe, in good agreement with the expression studies. We established a reproducible swarming assay (0.5% Eiken agar in Difco broth, 30 degrees C) for Aeromonas spp. The laf-positive strains exhibited vigorous swarming motility, whereas laf-negative strains grew but showed no movement from the inoculation site. Light and scanning electron microscopic investigations revealed that lateral flagella formed bacterium-bacterium linkages on the agar surface. Strains of an Aeromonas caviae isolate in which lateral flagellum expression was abrogated by specific mutations in flagellar genes did not swarm, proving conclusively that lateral flagella are required for the surface movement. Whether lateral flagella and swarming motility contribute to Aeromonas intestinal colonization and virulence remains to be determined.     

Swarming motility by Photorhabdus temperata is influenced by environmental conditions and uses the same flagella as that used in swimming motility

Photorhabdus temperata, an insect pathogen and nematode symbiont, is motile in liquid medium by swimming. We found that P.?temperata was capable of surface movement, termed swarming behavior. Several lines of evidence indicate that P. temperata use the same flagella for both swimming and swarming motility. Both motility types required additional NaCl or KCl in the medium and had peritrichous flagella, which were composed of the same flagellin as detected by immunoblotting experiments. Mutants defective in flagellar structural proteins were nonmotile for both motility types. Unlike swimming, we observed swarming behavior to be a social form of movement in which the cells coordinately formed intricate channels covering a surface. The constituents of the swarm media affected motility. Swarming was optimal on low agar concentrations; as agar concentrations increased, swarm ring diameters decreased.     

Extracellular proteolytic activity plays a central role in swarming motility in Bacillus subtilis

Natural isolates of Bacillus subtilis exhibit a robust multicellular behavior known as swarming. A form of motility, swarming is characterized by a rapid, coordinated progression of a bacterial population across a surface. As a collective bacterial process, swarming is often associated with biofilm formation and has been linked to virulence factor expression in pathogenic bacteria. While the swarming phenotype has been well documented for Bacillus species, an understanding of the molecular mechanisms responsible remains largely isolated to gram-negative bacteria. To better understand how swarming is controlled in members of the genus Bacillus, we investigated the effect of a series of gene deletions on swarm motility. Our analysis revealed that a strain deficient for the production of surfactin and extracellular proteolytic activity did not swarm or form biofilm. While it is known that surfactin, a lipoprotein surfactant, functions in swarming motility by reducing surface tension, this is the first report demonstrating that general extracellular protease activity also has an important function. These results not only help to define the factors involved in eliciting swarm migration but support the idea that swarming and biofilm formation may have overlapping control mechanisms.     

Preparation,Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more “temperate swarmers” that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. “Wettability”, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.     

The ability of Proteus mirabilis to sense surfaces and regulate virulence gene expression involves FliL, a flagellar basal body protein

Proteus mirabilis is a urinary tract pathogen that differentiates from a short swimmer cell to an elongated, highly flagellated swarmer cell. Swarmer cell differentiation parallels an increased expression of several virulence factors, suggesting that both processes are controlled by the same signal. The molecular nature of this signal is not known but is hypothesized to involve the inhibition of flagellar rotation. In this study, data are presented supporting the idea that conditions inhibiting flagellar rotation induce swarmer cell differentiation and implicating a rotating flagellar filament as critical to the sensing mechanism. Mutations in three genes, fliL, fliF, and fliG, encoding components of the flagellar basal body, result in the inappropriate development of swarmer cells in noninducing liquid media or hyperelongated swarmer cells on agar media. The fliL mutation was studied in detail. FliL- mutants are nonmotile and fail to synthesize flagellin, while complementation of fliL restores wild-type cell elongation but not motility. Overexpression of fliL+ in wild-type cells prevents swarmer cell differentiation and motility, a result also observed when P. mirabilis fliL+ was expressed in Escherichia coli. These results suggest that FliL plays a role in swarmer cell differentiation and implicate FliL as critical to transduction of the signal inducing swarmer cell differentiation and virulence gene expression. In concert with this idea, defects in fliL up-regulate the expression of two virulence genes, zapA and hpmB. These results support the hypothesis that P. mirabilis ascertains its location in the environment or host by assessing the status of its flagellar motors, which in turn control swarmer cell gene expression.     

Surface-associated flagellum formation and swarming differentiation in Bacillus subtilis are controlled by the ifm locus

Knowledge of the highly regulated processes governing the production of flagella in Bacillus subtilis is the result of several observations obtained from growing this microorganism in liquid cultures. No information is available regarding the regulation of flagellar formation in B. subtilis in response to contact with a solid surface. One of the best-characterized responses of flagellated eubacteria to surfaces is swarming motility, a coordinate cell differentiation process that allows collective movement of bacteria over solid substrates. This study describes the swarming ability of a B. subtilis hypermotile mutant harboring a mutation in the ifm locus that has long been known to affect the degree of flagellation and motility in liquid media. On solid media, the mutant produces elongated and hyperflagellated cells displaying a 10-fold increase in extracellular flagellin. In contrast to the mutant, the parental strain, as well as other laboratory strains carrying a wild-type ifm locus, fails to activate a swarm response. Furthermore, it stops to produce flagella when transferred from liquid to solid medium. Evidence is provided that the absence of flagella is due to the lack of flagellin gene expression. However, restoration of flagellin synthesis in cells overexpressing sigma(D) or carrying a deletion of flgM does not recover the ability to assemble flagella. Thus, the ifm gene plays a determinantal role in the ability of B. subtilis to contact with solid surfaces.     

Bacterial lateral flagella: an inducible flagella system

Flagella are complex surface organelles that allow bacteria to move towards favourable environments and that contribute to the virulence of pathogenic bacteria through adhesion and biofilm formation on host surfaces. There are a few bacteria that possess functional dual flagella systems, such as Vibrio parahaemolyticus, some mesophilic Aeromonas spp., Rhodospirillum centenum and Azospirillum brasilense. These bacteria are able to express both a constitutive polar flagellum required for swimming motility and a separate lateral flagella system that is induced in viscous media or on surfaces and is essential for swarming motility. As flagella synthesis and motility have a high metabolic cost for the bacterium, the expression of the inducible lateral flagella system is highly regulated by a number of environmental factors and regulators.     

Initiation of Swarming Motility by Proteus mirabilis Occurs in Response to Specific Cues Present in Urine and Requires Excess l-Glutamine

Proteus mirabilis, a leading cause of catheter-associated urinary tract infection (CaUTI), differentiates into swarm cells that migrate across catheter surfaces and medium solidified with 1.5% agar. While many genes and nutrient requirements involved in the swarming process have been identified, few studies have addressed the signals that promote initiation of swarming following initial contact with a surface. In this study, we show that P. mirabilis CaUTI isolates initiate swarming in response to specific nutrients and environmental cues. Thirty-three compounds, including amino acids, polyamines, fatty acids, and tricarboxylic acid (TCA) cycle intermediates, were tested for the ability to promote swarming when added to normally nonpermissive media. l-Arginine, l-glutamine, dl-histidine, malate, and dl-ornithine promoted swarming on several types of media without enhancing swimming motility or growth rate. Testing of isogenic mutants revealed that swarming in response to the cues required putrescine biosynthesis and pathways involved in amino acid metabolism. Furthermore, excess glutamine was found to be a strict requirement for swarming on normal swarm agar in addition to being a swarming cue under normally nonpermissive conditions. We thus conclude that initiation of swarming occurs in response to specific cues and that manipulating concentrations of key nutrient cues can signal whether or not a particular environment is permissive for swarming.     

肠杆菌共同抗原的研究进展

肠杆菌共同抗原(Enterobacterial common antigen,ECA)是由多糖重复单元组成的多聚糖,几乎表达于所有肠杆菌细菌外膜,具有生物学功能。ECA由多基因协同作用而合成,这些基因在肠杆菌细菌基因组上成簇存在,形成ECA抗原基因簇。ECA是重要的毒力因子,在肠杆菌细菌入侵宿主、体内存活等过程中有一定作用。同时,ECA在维持细菌外膜渗透屏障、鞭毛表达、群集运动及抗胆酸胆盐等方面也有重要作用。此外,锚定在细菌脂多糖核心区的ECALPS还是细菌重要的表面抗原,能激发宿主产生高水平抗体,可以作为疫苗研究的靶点。结合笔者的研究,文中对ECA纯化、基因结构和合成、免疫特性、生物学功能及应用等方面进行了综述。     

Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili

We describe swarming in Pseudomonas aeruginosa as a third mode of surface translocation in addition to the previously described swimming and twitching motilities. Swarming in P. aeruginosa is induced on semisolid surfaces (0.5 to 0.7% agar) under conditions of nitrogen limitation and in response to certain amino acids. Glutamate, aspartate, histidine, or proline, when provided as the sole source of nitrogen, induced swarming, while arginine, asparagine, and glutamine, among other amino acids, did not sustain swarming. Cells from the edge of the swarm were about twice as long as cells from the swarm center. In both instances, bacteria possessing two polar flagella were observed by light and electron microscopy. While a fliC mutant of P. aeruginosa displayed slightly diminished swarming, a pilR and a pilA mutant, both deficient in type IV pili, were unable to swarm. Furthermore, cells with mutations in the las cell-to-cell signaling system showed diminished swarming behavior, while rhl mutants were completely unable to swarm. Evidence is presented for rhamnolipids being the actual surfactant involved in swarming motility, which explains the involvement of the cell-to-cell signaling circuitry of P. aeruginosa in this type of surface motility.     

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.     

Genetic Determinants of Swarming in Rhizobium etli

Swarming motility is considered to be a social phenomenon that enables groups of bacteria to move coordinately atop solid surfaces. The differentiated swarmer cell population is embedded in an extracellular slime layer, and the phenomenon has previously been linked with biofilm formation and virulence. The gram-negative nitrogen-fixing soil bacterium Rhizobium etli CNPAF512 was previously shown to display swarming behavior on soft agar plates. In a search for novel genetic determinants of swarming, a detailed analysis of the swarming behavior of 700 miniTn5 mutants of R. etli was performed. Twenty-four mutants defective in swarming or displaying abnormal swarming patterns were identified and could be divided into three groups based on their swarming pattern. Fourteen mutants were completely swarming deficient, five mutants showed an atypical swarming pattern with no completely smooth edge and local extrusions, and five mutants displayed an intermediate swarming phenotype. Sequence analysis of the targeted genes indicated that the mutants were likely affected in quorum-sensing, polysaccharide composition or export, motility, and amino acid and polyamines metabolism. Several of the identified mutants displayed a reduced symbiotic nitrogen fixation activity.     

The mechanism of swarming of Vibrio alginolyticus

Factors leading to swarming of Vibrio alginolyticus cells on solid media were studied. Polar flagellated rods from liquid medium develop into small colonies on solid medium. Byproducts, accumulating in the colony area, induce at certain critical concentrations, the formation of peritrichous flagella and development of long heavily flagellated filaments which swarm away form the high by-product concentrations. Several types of nonswarming mutants were isolated, among them, mutants which lack the capacity to form swarming-inducing pyproducts, but can be induced to swarm by byproducts of other mutants incapable of swarming. Different compounds could replace the natural metabolic byproducts; at very low concentration these compounds induce peritrichous flagella and swarming in some of the nonswarming mutants mentioned above. The natural metabolic byproducts accumulating in yeast-extract-peptone medium are suggested to be volatile acids belonging to the valine and isoleucine pathway. Wild-type V. alginolyticus cells cannot swarm on certain substrates but its mutants, able to swarm on many substrates in minimal media, are easily selected.