Decomposing Predation: Testing for Parameters that Correlate with Predatory Performance by a Social Bacterium

Predator–prey interactions presumably play major roles in shaping the composition and dynamics of microbial communities. However, little is understood about the population biology of such interactions or how predation-related parameters vary or correlate across prey environments. Myxococcus xanthus is a motile soil bacterium that feeds on a broad range of other soil microbes that vary greatly in the degree to which they support M. xanthus growth. In order to decompose predator–prey interactions at the population level, we quantified five predation-related parameters during M. xanthus growth on nine phylogenetically diverse bacterial prey species. The horizontal expansion rate of swarming predator colonies fueled by prey lawns served as our measure of overall predatory performance, as it incorporates both the searching (motility) and handling (killing and consumption of prey) components of predation. Four other parameters—predator population growth rate, maximum predator yield, maximum prey kill, and overall rate of prey death—were measured from homogeneously mixed predator–prey lawns from which predator populations were not allowed to expand horizontally by swarming motility. All prey species fueled predator population growth. For some prey, predator-specific prey death was detected contemporaneously with predator population growth, whereas killing of other prey species was detected only after cessation of predator growth. All four of the alternative parameters were found to correlate significantly with predator swarm expansion rate to varying degrees, suggesting causal interrelationships among these diverse predation measures. More broadly, our results highlight the importance of examining multiple parameters for thoroughly understanding the population biology of microbial predation.     

The function of fimbriae in Myxococcus xanthus. II. The role of fimbriae in cell-cell interactions.

Anti-fimbriae antiserum specifically inhibited swarming but no gliding motility per se in Myxococcus xanthus. However, formation of motile aggregates on agar and clumps in liquid media correlated with the presence of fimbriae. Ethylenediaminetetraacetic acid which inhibited swarming also inhibited fimbriae formation. Direct electron-microscopic observations revealed that fimbriae establish contact with apposing cell surfaces. Intact but not depolymerized fimbriae exhibited hemagglutination activity against guinea pig erythrocytes. This activity was inhibited by mannose, N-acetyl-D-galactosamine, and to a lesser degree by fructose, raffinose, melibiose, and alpha-methyl-D-mannoside. It is concluded that fimbriae are organelles which function to establish and maintain intercellular contacts, perhaps by a lectin-like function, during the coordinated movement of cell aggregates' (swarming) in myxobacteria. This hypothesis is supported by the observations of other workers that genes determining movement of cells in groups also control fimbriation in M. xanthus.     

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.     

Resource Level Affects Relative Performance of the Two Motility Systems of Myxococcus xanthus

The adventurous (A) and social (S) motility systems of the microbial predator Myxococcus xanthus show differential swarming performance on distinct surface types. Under standard laboratory conditions, A-motility performs well on hard agar but poorly on soft agar, whereas the inverse pattern is shown by S-motility. These properties may allow M. xanthus to swarm effectively across a greater diversity of natural surfaces than would be possible with one motility system alone. Nonetheless, the range of ecological conditions under which dual motility enhances effective swarming across distinct surfaces and how ecological parameters affect the complementarity of A-motility and S-motility remain unclear. Here we have examined the role of nutrient concentration in determining swarming patterns driven by dual motility on distinct agar surfaces, as well as the relative contributions of A-motility and S-motility to these patterns. Swarm expansion rates of dually motile (A⁺S⁺), solely A-motile (A⁺S⁻), and solely S-motile (A⁻S⁺) strains were compared on hard and soft agar across a wide range of casitone concentrations. At low casitone concentrations (0–0.1%), swarming on soft agar driven by S-motility is very poor, and is significantly slower than swarming on hard agar driven by A-motility. This reverses at high casitone concentration (1–3.2%) such that swarming on soft agar is much faster than swarming on hard agar. This pattern greatly constrained the ability of M. xanthus to encounter patches of prey bacteria on a soft agar surface when nutrient levels between the patches were low. The swarming patterns of a strain that is unable to produce extracellular fibrils indicate that these appendages are responsible for the elevated swarming of S-motility at high resource levels. Together, these data suggest that large contributions by S-motility to predatory swarming in natural soils may be limited to soft, wet, high-nutrient conditions that may be uncommon. Several likely benefits of S-motility to the M. xanthus life cycle are discussed, including synergistic interactions with A-motility across a wide variety of conditions.     

Identification of Novel Factors Involved in Modulating Motility of Salmonella enterica Serotype Typhimurium

Salmonella enterica serotype Typhimurium can move through liquid using swimming motility, and across a surface by swarming motility. We generated a library of targeted deletion mutants in Salmonella Typhimurium strain ATCC14028, primarily in genes specific to Salmonella, that we have previously described. In the work presented here, we screened each individual mutant from this library for the ability to move away from the site of inoculation on swimming and swarming motility agar. Mutants in genes previously described as important for motility, such as flgF, motA, cheY are do not move away from the site of inoculation on plates in our screens, validating our approach. Mutants in 130 genes, not previously known to be involved in motility, had altered movement of at least one type, 9 mutants were severely impaired for both types of motility, while 33 mutants appeared defective on swimming motility plates but not swarming motility plates, and 49 mutants had reduced ability to move on swarming agar but not swimming agar. Finally, 39 mutants were determined to be hypermotile in at least one of the types of motility tested. Both mutants that appeared non-motile and hypermotile on plates were assayed for expression levels of FliC and FljB on the bacterial surface and many of them had altered levels of these proteins. The phenotypes we report are the first phenotypes ever assigned to 74 of these open reading frames, as they are annotated as ‘hypothetical genes’ in the Typhimurium genome.     

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.     

Genetic population structure of the soil bacterium Myxococcus xanthus at the centimeter scale

Myxococcus xanthus is a gram-negative soil bacterium best known for its remarkable life history of social swarming, social predation, and multicellular fruiting body formation. Very little is known about genetic diversity within this species or how social strategies might vary among neighboring strains at small spatial scales. To investigate the small-scale population structure of M. xanthus, 78 clones were isolated from a patch of soil (16 by 16 cm) in Tübingen, Germany. Among these isolates, 21 genotypes could be distinguished from a concatemer of three gene fragments: csgA (developmental C signal), fibA (extracellular matrix-associated zinc metalloprotease), and pilA (the pilin subunit of type IV pili). Accumulation curves showed that most of the diversity present at this scale was sampled. The pilA gene contains both conserved and highly variable regions, and two frequency-distribution tests provide evidence for balancing selection on this gene. The functional domains in the csgA gene were found to be conserved. Three instances of lateral gene transfer could be inferred from a comparison of individual gene phylogenies, but no evidence was found for linkage equilibrium, supporting the view that M. xanthus evolution is largely clonal. This study shows that M. xanthus is surrounded by a variety of distinct conspecifics in its natural soil habitat at a spatial scale at which encounters among genotypes are likely.     

Social interactions in myxobacterial swarming

Swarming, a collective motion of many thousands of cells, produces colonies that rapidly spread over surfaces. In this paper, we introduce a cell-based model to study how interactions between neighboring cells facilitate swarming. We chose to study Myxococcus xanthus, a species of myxobacteria, because it swarms rapidly and has well-defined cell–cell interactions mediated by type IV pili and by slime trails. The aim of this paper is to test whether the cell contact interactions, which are inherent in pili-based S motility and slime-based A motility, are sufficient to explain the observed expansion of wild-type swarms. The simulations yield a constant rate of swarm expansion, which has been observed experimentally. Also, the model is able to quantify the contributions of S motility and A motility to swarming. Some pathogenic bacteria spread over infected tissue by swarming. The model described here may shed some light on their colonization process.     

Regulating pilin expression reveals a threshold for S motility in Myxococcus xanthus

An isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter was constructed in Myxococcus xanthus. The single-copy pilA gene encodes pilin, the monomer unit of M. xanthus type IV pili. To vary the level of pilA expression, we cloned its promoter in front of the lac operator, and a plasmid containing the construct was inserted into the chromosome of a DeltapilA strain. Induction of pilin expression increased smoothly as the dose of IPTG added to the culture was increased. IPTG-induced pilin rescued S motility of the DeltapilA strain to wild-type levels. The rate of S-motile swarming was found to be proportional to the number of pili (shear-sensitive pilin) produced rather than to the level of total pilin. In fact, S motility was not rescued until the total level of pilin was more than 50% of the wild-type level. This observation implies that a threshold concentration of pilin must be exceeded before the shear-sensitive material (pili) is polymerized in M. xanthus.     

Characterization of swarming motility in Citrobacter freundii

Bacterial swarming motility is a flagella-dependent translocation on the surface environment. It has received extensive attention as a population behavior involving numerous genes. Here, we report that Citrobacter freundii, an opportunistic pathogen, exhibits swarming movement on a solid medium surface with appropriate agar concentration. The swarming behavior of C. freundii was described in detail. Insertional mutagenesis with transposon Mini-Tn5 was carried out to discover genetic determinants related to the swarming of C. freundii. A number of swarming genes were identified, among which flhD, motA, motB, wzx, rfaL, rfaJ, rfbX, rfaG, rcsD, rcsC, gshB, fabF, dam, pgi, and rssB have been characterized previously in other species. In mutants related to lipopolysaccharide synthesis and RcsCDB signal system, a propensity to form poorly motile bacterial aggregates on the agar surface was observed. The aggregates hampered bacterial surface migration. In several mutants, the insertion sites were identified to be in the ORF of yqhC, yeeZ, CKO_03941, glgC, and ttrA, which have never been shown to be involved in swarming. Our results revealed several novel characteristics of swarming motility in C. freundii which are worthy of further study.     

Genetic circuitry controlling motility behaviors of Myxococcus xanthus

M. xanthus has a complex multicellular lifestyle including swarming, predation and development. These behaviors depend on the ability of the cells to achieve directed motility across solid surfaces. M. xanthus cells have evolved two motility systems including Type-IV pili that act as grappling hooks and a controversial engine involving mucus secretion and fixed focal adhesion sites. The necessity for cells to coordinate the motility systems and to respond rapidly to environmental cues is reflected by a complex genetic network involving at least three complete sets of chemosensory systems and eukaryotic-like signaling proteins. In this review, we discuss recent advances suggesting that motor synchronization results from spatial oscillations of motility proteins. We further propose that these dynamics are modulated by the action of multiple upstream complementary signaling systems. M. xanthus is thus an exciting emerging model system to study the intricate processes of directed cell migration.     

Differential effects of chemoreceptor methylation-domain mutations on swarming and development in the social bacterium Myxococcus xanthus

The soil bacterium Myxococcus xanthus is a model organism for the study of multicellular behaviour and development in bacteria. M. xanthus cells move on solid surfaces by gliding motility, periodically reversing their direction of movement. Motility is co-ordinated to allow cells to effectively feed on macromolecules or prey bacteria when nutrients are plentiful and to form developmental fruiting bodies when nutrients are limiting. The Frz signal transduction pathway regulates cellular movements by modulating cell reversal frequency. Input to the Frz pathway is controlled by the cytoplasmic receptor, FrzCD, a methyl-accepting chemotaxis protein (MCP). FrzCD lacks the transmembrane and periplasmic domains common to MCPs but contains a unique N-terminal domain, the predicted ligand-binding domain. As deletion of the N-terminal domain of FrzCD only results in minor defects in motility, we investigated the possibility that the methylation of the conserved C-terminal domain of FrzCD plays a central role in regulating the pathway. For this study, each of the potential methylation sites of FrzCD were systematically modified by site-directed mutagenesis, substituting glutamine/glutamate pairs for alanines. Four of the seven mutations produced dramatic phenotypes; two of the mutations had a stimulatory effect on the pathway, as evidenced by cells hyper-reversing, whereas another two had an inhibitory effect, causing these cells to rarely reverse. These four mutants displayed defects in vegetative swarming and developmental aggregation. These results suggests a model in which the methylation domain can both activate and inhibit the Frz pathway depending on which residues are methylated. The diversity of phenotypes suggests that specific modifications of FrzCD act to differentially regulate motility and developmental aggregation in M. xanthus.     

The RssAB two-component signal transduction system in Serratia marcescens regulates swarming motility and cell envelope architecture in response to exogenous saturated fatty acids

Serratia marcescens swarms at 30 degrees C but not at 37 degrees C on a nutrient-rich (LB) agar surface. Mini-Tn5 mutagenesis of S. marcescens CH-1 yielded a mutant (WC100) that swarms not only vigorously at 37 degrees C but also earlier and faster than the parent strain swarms at 30 degrees C. Analysis of this mutant revealed that the transposon was inserted into a gene (rssA) predicted to encode a bacterial two-component signal transduction sensor kinase, upstream of which a potential response regulator gene (rssB) was located. rssA and rssB insertion-deletion mutants were constructed through homologous recombination, and the two mutants exhibited similar swarming phenotypes on LB swarming agar, in which swarming not only occurred at 37 degrees C but also initiated at a lower cell density, on a surface with a higher agar concentration, and more rapidly than the swarming of the parent strain at 30 degrees C. Both mutants also exhibited increased hemolysin activity and altered cell surface topologies compared with the parent CH-1 strain. Temperature and certain saturated fatty acids (SFAs) were found to negatively regulate S. marcescens swarming via the action of RssA-RssB. Analysis of the fatty acid profiles of the parent and the rssA and rssB mutants grown at 30 degrees C or 37 degrees C and under different nutrition conditions revealed a relationship between cellular fatty acid composition and swarming phenotypes. The cellular fatty acid profile was also observed to be affected by RssA and RssB. SFA-dependent inhibition of swarming was also observed in Proteus mirabilis, suggesting that either SFAs per se or the modulation of cellular fatty acid composition and hence homeostasis of membrane fluidity may be a conserved mechanism for regulating swarming motility in gram-negative bacteria.     

Pseudomonad swarming motility is restricted to a narrow range of high matric water potentials

Using a novel experimental system that allows control of the matric potential of an agar slab, we explored the hydration conditions under which swarming motility is possible. If there is recognition that this physical parameter is a key determinant of swarming, it is usually neither controlled nor measured rigorously but only manipulated through proxies, namely, the agar concentration and the drying time of "soft" agar plates (swarming plates). We contend that this not only obscures the biophysical mechanisms underlying swarming but also impedes a full assessment of its clinical and environmental significances. Our results indicate that swarming motility is restricted to a narrow range of high matric water potentials in the three pseudomonads tested (Pseudomonas sp. DSS73, Pseudomonas syringae B728a, and Pseudomonas aeruginosa PA14). The threshold below which no swarming was observed was about -0.45 kPa for the first and about -0.1 kPa for the latter two. Above the threshold, the expansion rate of DSS73 swarms increased exponentially with the matric potential. Mutants deficient in surfactant production were totally or partially unable to expand rapidly on the surface of the agar slab. Our results thus suggest that swarming motility in pseudomonads is restricted to (micro)sites where ambient humidity is very high (relative humidity of >99.99%). The spatiotemporal occurrence of such sites is limited in many types of terrestrial environments.     

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.     

MotPS is the stator-force generator for motility of alkaliphilic Bacillus, and its homologue is a second functional Mot in Bacillus subtilis

The stator-force generator that drives Na+-dependent motility in alkaliphilic Bacillus pseudofirmus OF4 is identified here as MotPS, MotAB-like proteins with genes that are downstream of the ccpA gene, which encodes a major regulator of carbon metabolism. B. pseudofirmus OF4 was only motile at pH values above 8. Disruption of motPS resulted in a non-motile phenotype, and motility was restored by transformation with a multicopy plasmid containing the motPS genes. Purified and reconstituted MotPS from B. pseudofirmus OF4 catalysed amiloride analogue-sensitive Na+ translocation. In contrast to B. pseudofirmus, Bacillus subtilis contains both MotAB and MotPS systems. The role of the motPS genes from B. subtilis in several motility-based behaviours was tested in isogenic strains with intact motAB and motPS loci, only one of the two mot systems or neither mot system. B. subtilis MotPS (BsMotPS) supported Na+-stimulated motility, chemotaxis on soft agar surfaces and biofilm formation, especially after selection of an up-motile variant. BsMotPS also supported motility in agar soft plugs immersed in liquid; motility was completely inhibited by an amiloride analogue. BsMotPS did not support surfactin-dependent swarming on higher concentration agar surfaces. These results indicate that BsMotPS contributes to biofilm formation and motility on soft agar, but not to swarming, in laboratory strains of B. subtilis in which MotAB is the dominant stator-force generator. BsMotPS could potentially be dominant for motility in B. subtilis variants that arise in particular niches.     

Enhanced swarming of bacteria on agar plates containing the surfactant Tween 80

A widely used method for quantifying swarming motility is the swarm plate assay. A significant increase in the motility halo size formed by Escherichia coli or Azospirillum brasilense was measured on Tween 80-containing agar relative to untreated agar. This improvement could benefit the identification of mutants in swarming motility.     

Swarming by Pseudomonas syringae B728a requires gacS (lemA) and gacA but not the acyl-homoserine lactone biosynthetic gene ahlI.

Pseudomonas syringae pv. syringae B728a, a causal agent of bacterial brown spot on snap beans, swarms with a characteristic dendritic pattern on semisolid (0.4%) agar plates. Filamentation of swarming cells of B728a was not observed. Mutations in either the gacS (formerly lemA) or gacA gene of B728a eliminate the ability of this P. syringae isolate to swarm without obvious effects on bacterial motility. Three field isolates showed a similar dependence on gacS for swarming. Since gacS and gacA mutants are known to be deficient in N-acyl-L-homoserine lactone (acyl-HSL) production, a mutant was constructed by disruption of the ahlI gene of B728a. This mutant did not make any acyl-HSL detectable by the Agrobacterium traG::lacZ reporter system, yet was unaffected in its ability to swarm. Other phenotypes of gacS and gacA mutations were similarly unaffected in the ahlI mutant.     

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.