Killing of Escherichia coli by Myxococcus xanthus in Aqueous Environments Requires Exopolysaccharide-dependent Physical Contact

Abstract

Nutrient or niche-based competition among bacteria is a widespread phenomenon in natural environment. Such inter-species interactions are often mediated by secreted soluble factors and/or direct cell–cell contact. As ubiquitous soil bacteria, Myxococcus species are able to produce a variety of bioactive secondary metabolites to inhibit the growth of other competing bacterial species. Meanwhile, Myxococcus sp. also exhibit sophisticated predatory behavior, an extreme form of competition that is often stimulated by close contact with prey cells and largely depends on the availability of solid surfaces. Myxococcus sp. can also be isolated from aquatic environments. However, studies focusing on the interaction between Myxococcus and other bacteria in such environments are still limited. In this study, using the well-studied M. xanthus DK1622 and E. coli as model interspecies interaction pair, we demonstrated that in a aqueous environment, Myxococcus xanthus was able to kill Escherichia coli in a cell contact-dependent manner, and that the observed contact dependent killing required the formation of co-aggregates between M. xanthus and E. coli cells. Further analysis revealed that exopolysaccharide (EPS), type IV pilus (TFP) and lipopolysaccharide (LPS) mutants of M. xanthus displayed various degrees of attenuation in E. coli killing, and it correlated well with the mutants' reduction in EPS production. In addition, M. xanthus showed differential binding ability to different bacteria, and bacterial strains unable to co-aggregate with M. xanthus can escape the killing, suggesting the specific nature of co-aggregation and the targeted killing of interacting bacteria. In conclusion, our results demonstrated EPS mediated, contact-dependent killing of E. coli by M. xanthus, a strategy that might facilitate the survival of this ubiquitous bacterium in aquatic environments.

Introduction

Cooperation and competition within and between bacterial species to enhance their survival in natural environments is a common theme in microbial ecology [1, 2]. For example, metabolic cooperation among different bacteria species is essential for obtaining nutrients and the maintenance of the bacterial consortia [3, 4]. Meanwhile, nutrient or niche-based competition is a more widespread phenomenon among different bacterial species within natural microbial communities [5]. During competition, bacteria often employ different strategies, such as producing antibiotics or toxic secondary metabolites to inhibit or kill the competing microbial species, thus gaining growth advantage [6, 7]. One of the extreme forms of competition is predation, where predatory bacterial species kill and lyse their targeted prey cells to obtain cellular components as nutrients needed to sustain their growth [8, 9].

M. xanthus, the model species of Myxococcus, is an ubiquitous soil bacterium that competes with other microbial species by producing a slew of secondary metabolites to inhibit their growth. More importantly, on solid surfaces, M. xanthus demonstrates several complex social behaviors, such as social motility, fruiting body formation, and particularly, predation behavior [10-13], a competition strategy that is often stimulated by close contact with prey cells and enables M. xanthus to prey upon other bacteria for better survival. The detection of prey cells, as well as the coordinated release of bacteriolytic enzymes, proteases, and antibiotics for prey killing require extensive and well-regulated intra- and interspecies interactions.

Although Myxococcus sp. are commonly found in various types of terrestrial habitats, many of them, including M. fulvus, M. visrescens, M. xanthus etc., can also be frequently isolated from many aquatic environments [14-16], presumably as a result of soil organism being regularly washed into the water and surviving there [13, 17, 18]. As flagella-less bacteria, they are non-motile and often lose solid surface dependent social behaviors under liquid conditions [19] and little is known regarding their strategies to compete with other bacteria to survive in aqueous environments. The most frequent places one can isolated myxobacteria is animal dung, which is full of E. coli cells. Thus the M. xanthus and E. coli is a well-known model system to study interspecies interaction in the filed of myxobacteria [17]. In this study, by using the well studied M. xanthus DK1622 and E. coli MG1655 as model microorganisms, we aimed to study the interspecies interaction between these two species in aquatic environments. The killing of E. coli by M. xanthus under aqueous condition was analyzed, and the cell components involved in interspecies interaction were further investigated.

Materials and methods

Bacterial Strains and Growth Conditions

Bacterial strains used in this study are listed in Table 1. E. coli strains from the Large-scale Chromosome Deletion and E. coli Transposon Insertion Mutation Libraries (NBRP, National Institute of Genetics, Japan), Klebsiella pneumoniae IA565 [20], Pseudomonas aeruginosa PA01 and Micrococcus luteus were cultured in liquid Luria-Bertani (LB) broth or on solid LB containing 1.5% agar. The M. xanthus strains were cultured in CYE liquid medium at 32 °C on a rotary shaker at 300 rpm or on CYE plates containing 1.5% agar (Difco) [21]. When needed, kanamycin (Km, 100 μg/ml) was added to the media.

Table 1. Bacterial strains used in this study.

Designation	Genotype or description	Reference
Myxococcus xanthus DK1622	Wild type strain	[26]
SW504	DK1622, ΔdifA	[27]
DK10410	DK1622, ΔpilA	[36]
SW810	DK1622, ΔepsA	[28]
ΔMXAN4619	DK1622, ΔMXAN4619	This study
Escherichia coli MG1655	Wild type strain	NBRP, National Institute of Genetics Japan
Klebsiella pneumoniae IA565	Clinical isolate	[20]
Pseudomonas aeruginosa PA01	Wild type strain	Laboratory strain
Micrococcus luteus	Collection from ATCC	ATCC272

Two Chamber Assay

The two-chamber assay was carried out as reported [22, 23] with some modifications. M. xanthus and E. coli strains were grown to the exponential phase, harvested, and re-suspended to OD₆₀₀ of 1 in fresh CYE medium. The M. xanthus cells alone or together with E. coli cells were inoculated into the bottom chamber of a 12-well plate containing a 0.4-μm PET membrane insert (Millipore, Billerica, MA, USA), while wild-type E. coli was added to the top chamber. Meanwhile, the mixed M. xanthus - E. coli cells as well as E. coli cells alone were inoculated into the 12-well plate without the top chamber, serving as controls. The culture condition for this assay was at 32 °C on a rotary shaker at 130 rpm. The M. xanthus-to-E. coli ratio for the two-chamber assay was 100:1. Three replicates were performed for each experiment.

Trypan Blue Binding Assay

To monitor EPS production of the selected M. xanthus mutants, the Trypan blue binding assay was performed as described [24]. In brief, cells growing in liquid CYE medium were collected, adjusted to 5 × 10⁸ cells/ml. 300 μg protein equivalent of cells was mixed with 1 ml trypan blue working solution (25 μg/ml trypan blue in cohesion buffer) and incubated in the dark at room temperature for 30 min. The cell suspensions were then pelleted at 16,000 g for 10 min and the absorbance of the supernatants was measured at 585 nm. The relative amounts of EPS were calculated relative to trypan blue bound to the wild-type DK1622 cells.

Agglutination Assay

The cohesion of M. xanthus cells was measured with an agglutination assay as reported by Shimkets [25]. Briefly, the exponentially growing cells were harvested, washed and re-suspended to an OD₆₀₀ of 1.0 in MCM buffer (10 mM MOPS, 1 mM CaCl₂, 1 mM MgCl₂). Agglutination or cellular cohesion was determined by measuring absorbance at OD₆₀₀ every 10 min. The experiment was repeated three times.

Co-aggregation Assay

The co-aggregation assay was performed to determine the binding ability of M. xanthus strains to E. coli. The assay was based on the agglutination assay with some modifications. M. xanthus and E. coli cells were harvested at the exponential growth phase, and re-suspended to OD₆₀₀ of 1 in MCM buffer. Equal volumes of M. xanthus and E. coli cell suspensions were thoroughly mixed by vortex for 10 sec and immediately transferred to a plastic disposable cuvette. Co-aggregation was determined spectrophotometrically by measuring absorbance of the supernatant in the cuvette at 600 nm every 10 min. M. xanthus cells suspended in the MCM buffer were used as positive control, while E. coli cells were used as negative control. Similarly, the binding ability of M. xanthus to other bacterial strains, including K. pneumoniae IA565, P. aeruginosa PA01, and M. luteus (Table 1), was also analyzed using the same assay.

Microscopic Evaluation of Co-aggregation between M. xanthus and GFP-labeled E. coli For a more accurate microscopic assessment of co-aggregation between M. xanthus and E. coli, GFP-labeled E. coli cells were used as co-aggregation partner for M. xanthus. The co-aggregation assay was set up as described above. Cell-cell co-aggregation was observed after 30 min using by fluorescence microscopy (Nikon E400 equipped with a mercury lamp and a C17414 filter). GFP-labeled E. coli cells suspended in MCM buffer were used as negative control.

Killing Assay in Liquid

M. xanthus and E. coli strains were grown to the exponential phase, harvested and re-suspended in the fresh CYE medium to adjust the cell concentration to 5 ×10⁹ cells/ml. M. xanthus mutants and E. coli were co-cultured (in a 100:1 M. xanthus-to-E. coli ratio) in liquid CYE medium in a twelve-well plate on a rotary shaker operating at 130 rpm. After a 16 hr incubation at 32 °C, the cell mixture was collected, vortexed thoroughly, serial diluted and spread onto selective LB plates to monitor the viability of E. coli cells. Isolated EPS (1 mg/ml) and heat inactivated M. xanthus cells (5 ×10⁹ cells/ml) were also used as controls during the killing assay. The kinetics of killing of E. coli by M. xanthus was further analyzed by monitoring the viability of E. coli cells every two hours for twelve hours. Similarly, the killing ability of M. xanthus to other bacterial strains, including K. pneumoniae IA565, P. aeruginosa PA01, and M. luteus (Table 1), was also analyzed using the same assay.

Results

M. xanthus Killed E. coli in a Aqueous Environment

Using a model system comprised of M. xanthus DK1622 and E. coli MG1655 dual species, we aimed to investigate interspecies interaction between M. xanthus and E. coli cells under aqueous conditions. M. xanthus wild type strain DK1622 and E. coli strain MG1655 were co-cultured overnight, and the viability of E. coli cells was monitored by cfu counting on LB agar plates. As shown in Fig. 1A, the viability of E. coli cells suffered five orders of magnitude reduction, from initial 10⁷ cells/ml to 10² cells/ml after overnight co-cultivation with M. xanthus DK1622. Furthermore, the killing kinetics of M. xanthus against E. coli was analyzed. The most dramatic decline in the number of viable E. coli cells occurred in the first two hours, followed by a much slower decrease in the viability of E. coli cells with extended incubation time (Fig. 1D). Isolated EPS and heat inactivated M. xanthus cells had no E. coli-killing effect (data not shown here). The result indicated that, in a aqueous environment M. xanthus was still able to kill E. coli cells, despite being non-motile and absent of its characteristic solid surface dependent social behaviors [19].

Fig. 1.

Survival of E. coli under different conditions A: Wild-type E. coli MG1655 was co-cultivated with M. xanthus DK1622 in a 1:100 ratio. B: Two-chamber assay in which E. coli was inoculated into the upper chamber and physically separated from M. xanthus cells in the lower chamber. C: Two-chamber assay in which E. coli was added to the upper chamber, while M. xanthus cells and E. coli (in a 100:1 ratio) was inoculated into the lower chamber. Viability of E. coli was monitored after 16 hrs incubation in all conditions. Dark grey bars represent the initial inoculate cell numbers of E. coli and grey bars represent viable count of E. coli after 16 hrs. The viability measurement of group C is the E. coli in the upper chamber. D: Killing kinetics of M. xanthus against E. coli cells. The viability of E. coli cells was monitored every two hours for twelve hours. The experiments were repeated three times. Star indicates significant difference between the two values (P<0.05).

The Observed Killing of E. coli by M. xanthus Required Direct Cell-cell Contact

To determine if the observed killing of E. coli by M. xanthus in aqueous condition required direct cell-cell contact between the two species, we employed the two-chamber assay, in which the two different bacterial species shared the same culture medium but were physically separated by a 0.4-μm membrane. As illustrated in Fig. 1B, when M. xanthus cells were inoculated in the lower chamber and E. coli cells were incubated in the upper chamber, no significant killing was observed for E. coli. Similar growth was monitored for the E. coli cells in the upper chamber when they were physically separated from a co-culture of M. xanthus and E. coli that were incubated in the lower chamber (Fig. 1C). The result suggested that cell-cell contact was essential for the observed killing of E. coli by M. xanthus under aqueous condition, and that the killing factor(s) involved is unlikely to be freely diffusible.

M. xanthus Cells Co-aggregated with E. coli Cells

The ability of M. xanthus to bind to E. coli cells under aqueous condition was analyzed using the modified co-aggregation assay described above to further demonstrate the contact-dependent interaction of these two species. While E. coli cells alone remained dispersed, a drastic reduction in the absorbance at 600 nm was observed for the suspension containing M. xanthus and E coli cells (Fig. 2), indicating that the two species formed co-aggregates and precipitated out of the suspension, which resulted in a reduction in the optical density reading. Furthermore, when GFP-labeled E. coli was used, the fluorescence microscopy evaluation revealed the formation of co-aggregates containing both M. xanthus and E. coli cells (Fig. 3).

Fig. 2.

Co-aggregation analysis of M. xanthus and E. coli in MCM buffer A: M. xanthus and E. coli were analyzed by measuring relative absorbance at 600 nm every ten minutes. Relative absorbance at 600 nm was calculated against the initial OD reading every ten minutes. B: Observed co-aggregation between M. xanthus and E. coli in static liquid suspensions.

Fig. 3.

Direct observation of the co-aggregation between M. xanthus mutants (including dif mutant SW504, EPS mutant SW810, TFP mutant DK10410, and LPS mutant ΔMXAN4619) and GFP-labeled E. coli using the fluorescence microscope (Nikon E400 equipped with a mercury lamp and a C17414 filter). The upper panel is micrographs without fluorescence, and the lower panel is fluorescent micrographs. The scale bar equals to 50 μm.

The EPS, TFP and LPS of M. xanthus Are Involved in the Cell-cell Aggregation

Co-aggregation is often mediated by cell surface components. In an initial effort to identify the cell components of M. xanthus that are involved in co-aggregation with E. coli, mutants that were deficient in the production of cell surface components, including EPS (EPS^- mutant SW810, dif^- mutant SW504 (dif locus is a chemotaxis-like operon, which participates in the regulation of EPS production), TFP (TFP^- mutant DK10410), and LPS (LPS^- mutant ΔMXAN4619) [26-29], were selected for further investigation. As shown in Fig. 4, a mixture of E. coli cells with M. xanthus mutants lacking either EPS or TFP maintained a steady absorbance reading during the 2 hrs incubation period, suggesting they were defective in co-aggregating with E. coli. Meanwhile, mixtures containing the LPS mutant also displayed a reduced drop in optical density reading, although not as drastic as observed for the other tested mutant strains. Furthermore, when mixed with GFP-labeled E. coli, the M. xanthus LPS mutant formed smaller co-aggregates with E. coli compared to wild type DK1622; while no obvious aggregates were observed when E. coli was mixed with M. xanthus EPS^- or TFP^- mutants (Fig. 3).

Fig. 4.

Co-aggregation analysis of M. xanthus mutants (including dif mutant SW504, EPS mutant SW810, TFP mutant DK10410, and LPS mutant ΔMXAN4619) and E. coli in MCM buffer. Relative absorbance at 600 nm was calculated against the initial OD reading every ten minutes.

EPS^-, TFP^- and LPS^- Mutants of M. xanthus Were Defective in Killing E. coli under Aqueous Condition

Our data showed that killing of E. coli by M. xanthus was cell-cell contact-dependent and requires the formation of co-aggregation between the two species. To further test if M. xanthus mutants defective in co-aggregating with E. coli were also deficient in the killing, E. coli cells were co-cultivated with these mutants, and their viability was monitored after 16 hrs co-cultivation. Results showed that, compared to the M. xanthus wild type strain, all mutants tested displayed various degrees of attenuated E. coli killing (Fig. 5A). Interestingly, the killing efficiency of each mutant towards E. coli was correlated with its EPS production. Mutants with less EPS displayed more drastic reduction in their ability to kill E. coli (Fig. 5).

Fig. 5.

EPS production and E. coli killing ability of M. xanthus dif (SW504), EPS (SW810), TFP (DK10410), and LPS (ΔMXAN4619) mutants in aqueous condition A: Survival of E. coli after co-cultivation with M. xanthus mutants for 16 hrs. Star indicates significant difference between the two values (P<0.05). B: Relative EPS production of M. xanthus mutants determined by Trypan blue assay.

M. xanthus Demonstrated Different Binding to Different Bacterial Species

Our data suggested an important role of M. xanthus EPS in co-aggregating with E. coli and mediating the observed contact-dependent killing. To test if EPS-mediated co-aggregation also occurs between M. xanthus and other bacterial species, we analyzed the binding ability of M. xanthus DK1622 to a panel of different bacterial species, including K. pneumoniae, P. aeruginosa and M. luteus. As shown in Fig. 6A, among all the tested strains, M. xanthus DK1622 was able to co-aggregate with E. coli and M. luteus, while it was unable to co-aggregate with K. pneumoniae and P. aeruginosa. The more accurate microscopic assessment was employed to demonstrate co-aggregation between M. xanthus and M. luteus, as M. luteus exhibited auto-aggregation in the aqueous condition we tested (Fig. 6B). Our results illustrated the differential co-aggregation ability between M. xanthus and other microbial species. More interestingly, M. xanthus DK1622 was not able to kill K. pneumoniae and P. aeruginosa (Fig. 7), the two bacterial species to which M. xanthus showed no significant binding under aqueous condition (Fig. 6A).

Fig. 6.

Co-aggregation analysis of different bacteria and M. xanthus A: Observed co-aggregation between M. xanthus and E. coli, M. luteus, K. pneumoniae (K. p) or P. aeruginosa (P. a) in static liquid suspensions. B: Direct observation of the co-aggregation between M. xanthus and M. luteus using the microscope (Nikon E400). The scale bar equals to 50 μm.

Fig. 7.

Survival of E. coli, M. luteus, K. pneumonia and P. aeruginosa after overnight co-cultivation with M. xanthus DK1622. Dark grey bars represent the initial inoculate cell numbers and grey bars represent viable count of E. coli, M. luteus, K. pneumoniae and P. aeruginosa after overnight co-cultivation with M. xanthus DK1622. The experiments were repeated three times. Star indicates significant difference between the two values (P<0.05).

Discussion

As a model soil bacterium, the surface dependent social behaviors of Myxococcus, including its predatory activity against other bacteria, such as E. coli, have been extensively investigated [8]. However, little study has been done to investigate the behaviors of Myxococcus sp. isolated from aqueous environments. In this study, we revealed for the first time that in an aqueous environment killing of E. coli by M. xanthus is cell-cell contact-dependent and requires the formation of co-aggregates between the two species (Fig. 1 and Fig. 2). Microbes have evolved many sophisticated strategies to interact and compete with other bacteria to ensure their successful survival in natural environments. Many bacteria can sense the presence of target cells and initiate a series of events in a cell-cell contact dependent manner, which eventually leads to the inhibition or killing of the competing bacteria. Often, the inhibition effect exerted by bacteria can be achieved by physically interacting with target cells and delivering specific effectors via one of the four main classes of bacterial cell surface structures, including type III, IV, V or VI secretion systems [9]. Recently, a novel contact-dependent growth inhibition (CDI) mechanism was revealed, in which E. coli strain EC93 can inhibit the growth of other closely related E. coli strains via a two-partner secretion family, including two novel gene products CdiA and CdiB [22]. The contact dependent killing has also been documented in the microbial community-based inhibition of bacteria of foreign origin. He et al demonstrated that, an in vitro oral microbial community was able to sense the presence of invading E. coli of gut origin in a cell-cell contact dependent manner, and responded by producing hydrogen to kill E coli and prevent its colonization [23].

In our study, the observed killing of E. coli by M. xanthus under aqueous conditions required formation of co-aggregates. Unlike aforementioned contact dependent inhibition, it appeared that E. coli cells were surrounded by M. xanthus and were exclusively in the middle in the aggregates (Fig. 3), which may facilitate the killing of E. coli by M. xanthus. Although we cannot rule out the involvement of other surface structures, such as TFP and LPS in the physical interaction with E. coli (Fig. 3 and Fig. 4), our data strongly suggested that EPS was likely the major cell surface component responsible for mediating the formation of co-aggregates between M. xanthus and E. coli, since the ability of M. xanthus to bind to E. coli was positively correlated with their EPS production (Fig. 5) and both TFP and dif chemotaxis system were reported to participate in the regulation of EPS production [17] . Furthermore, formation of co-aggregates was essential for the killing of E. coli by M. xanthus, and M. xanthus strains defect in co-aggregating with E. coli cells were unable to kill E. coli in a aqueous environment (Fig. 4 and Fig. 5A). Based on these findings, we propose that the EPS of M. xanthus plays an essential role in mediating cell-cell contact dependent E. coli killing by M. xanthus in a aqueous environment. EPS is a major cell surface component of M. xanthus [30] and involved in a variety of cellular functions. EPS is not only required for S-motility and fruiting body formation of Myxococcus on solid surface [28], but has also been implicated in its vegetative growth, stress survival and stationary phase recovery in aquatic condition [31]. Furthermore, EPS has been reported to be required for multicellular clumping of Myxococcus in aqueous condition [30]. In addition to the involvement of EPS in intraspecies interaction between M. xanthus [32, 33], our results further demonstrated its roles in mediating interspecies interaction in aqueous condition.

M. xanthus produces a variety of bacteriolytic/proteolytic enzymes and secondary metabolites [8, 34], which have been suspected to play important roles in killing and competing with other bacteria to enhance their survival and growth at the expense of the target cells. A critical population density was required for effectively hydrolyzing insoluble macromolecules released by target cells [35]. Thus, co-aggregating with E. coli under aqueous condition not only allowed for maximum physical interspecies interaction, but also ensured the optimal concentration of myxobacterial bacteriolytic/proteolytic enzymes and secondary metabolites for sufficient killing and feeding of E. coli cells. Our data was corroborated by the observation that many newly isolated Myxococcus strains from soil failed to grow as dispersed cells in normal liquid broth culture, but formed thin films or clumps instead of living as free cells [17], further suggesting that EPS mediated, contact-dependent killing of prey cells could facilitate the survival of M. xanthus in aquatic environments.

It is worth noting that myxobacteria only co-aggregated with certain bacterial species and for those that failed to co-aggregate with M. xanthus, they could escape the killing (Fig. 6 and Fig. 7). This result suggested the specific nature of the co-aggregation mediated killing of E. coli by M. xanthus, which might also require the participation of specific components on the E. coli cell surface. Meanwhile, the killing factor(s) involved remained to be determined. Although the myxobacterial antibiotic TA has been reported to be important in killing E. coli during predation on solid surfaces [7], TA mutants did not show drastically attenuated killing towards E. coli under aqueous condition (data not shown), which suggested that other killing factors, such as bacteriolytic/proteolytic enzymes were likely involved. Studies are underway to further identify cellular components of E. coli involved in co-aggregation as well as the exact killing factor(s) produced by M. xanthus during co-aggregation mediated killing under aqueous environment.