1 Section of Molecular Microbiology, Biocentrum-DTU, Building 301, Technical University of Denmark, 2800 Lyngby, Denmark
2 Section of Genetics and Microbiology, Department of Ecology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark
3 Marine Chemistry Section, The H. C. Ørsted Institute, University of Copenhagen, Copenhagen, Denmark
Correspondence
Michael Givskov
immg{at}pop.dtu.dk
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ABSTRACT |
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INTRODUCTION |
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Among the great variety of antifungal metabolites produced by fluorescent Pseudomonas spp., our attention has been focused on the cyclic lipopeptides (Kochi et al., 1951; Nielsen et al., 1998
, 1999
, 2000
, 2002
). In addition, there are several examples of a close correlation between the production of cyclic lipopeptides and the ability to carry out surface motility on low-agar nutrient plates (Nielsen et al., 2002
). One well-studied phenomenon regarding bacterial surface translocation and the involvement of cyclic lipopeptides, such as serrawettins and rubiwettins, is the swarming motility of Serratia spp. (Matsuyama et al., 1990
, 1992
; Lindum et al., 1998
). The swarming cells are found almost exclusively in a motile layer present at the perimeter of the expanding colony. This motile layer forms a bacterial biofilm, which precedes the bulk of bacterial mass. Serratia swarm cells are generally longer and more flagellated than their counterparts propagated in liquid media (the swimmer cells) and move within an encasement of a self-produced slime that surrounds the colony (see review by Eberl et al., 1999
). The morphology of swarm cells combined with the production of biosurfactants as well as the extraction of water from the underlying medium is believed to reduce the surface friction which in turn conditions the surface and as a consequence enables rapid expansion of the growing bacterial culture (Matsuyama et al., 1990
, 1992
; Bees et al., 2000
). The surfactant synthesized by Pseudomonas sp. DSS73 has been designated amphisin. The complete structural elucidation of amphisin has revealed that it is a cyclic lipopeptide consisting of an 11 amino acid cyclic peptide that is linked at the N-terminal end to ß-hydroxydecanoyl (Sørensen et al., 2001
).
In the rhizosphere, the potential benefits of bacterial motility include increased efficiency in nutrient acquisition, avoidance of toxic substances, ability to translocate to preferred hosts and access to optimal colonization sites within them (Turnbull et al., 2001). Since root-pathogenic microfungi appear to be highly motile in the rhizosphere, we speculated that the surface motility of root-associated biocontrol bacteria might play an important role in the effective protection of plant roots against microfungal attack. In this study, we characterized the surface motility phenomenon exhibited by the Pseudomonas biocontrol strain DSS73. Here, we present in vitro data implicating surface motility as an important factor for ensuring efficient impediment of the spreading mycelium of two plant-pathogenic microfungi that are known to induce damping-off disease in sugar beet seedlings.
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METHODS |
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Surface motility assays.
These were carried out on ABT minimal medium plates supplemented with 0·4 % (w/w) glucose and 0·4 % (w/w) Casamino acids (Difco) and solidified with 0·6 % Bacto Agar (Difco). In the following, these are referred to as the standard surface motility plates. When appropriate, the medium was supplemented with other carbon sources (see below) and different agar concentrations. The media were poured as 25 ml aliquots into 9 cm sterile Petri dishes and the resulting standard surface motility plates were dried for 2 h at 20 °C prior to inoculation with 1 µl culture droplets (harvested and washed twice with 1 ml of 0·9 % NaCl) of wild-type DSS73, DSS73-15C2 (AmsY-), DSS73-12H8 (GacS-) or DSS73-12H8 (GacS-) carrying the plasmid pEMH97 (this plasmid carries the gacS gene of Pseudomonas syringae pv. syringae). Following 20 h incubation at 20 °C, all surface motility plates were visually inspected and the surface motility phenotypes of the respective strains were recorded. All assays were done in triplicate and images of the representative surface motility phenotypes were captured using a Kodak DC240 digital camera. The resulting images were processed using Photoshop 5.5. To obtain bacterial inocula, strains were cultivated in liquid ABT medium containing 0·4 % glucose (w/w) and 0·4 % Casamino acids (w/w) at 20 °C for 24 h. Subsequently, 1 ml aliquots were harvested by centrifugation (2 min at 10 000 g) and washed twice with 1 ml of 0·9 % NaCl.
Surfactant complementation assays
Culture droplets (1 µl) were inoculated onto standard surface motility plates (see above) supplemented with 0, 1·25, 2·5, 5 or 10 µg ml-1 of amphisin (a cyclic lipopeptide isolated from Pseudomonas sp. DSS73) (Sørensen et al., 2001; Nielsen et al., 2002
), tensin (a cyclic lipopeptide isolated from Pseudomonas fluorescens 96.578) (Nielsen et al., 2000
), viscosinamid (a cyclic lipopeptide isolated from Pseudomonas fluorescens DR54) (Nielsen et al., 1999
), serrawettin W2 (a cyclic lipopeptide isolated from S. liquefaciens MG1 (Lindum et al., 1998
), NP40 (Nonidet P40, an artificial surfactant/non-ionic detergent; Sigma) or Triton X-100 (a detergent; Sigma). Following 24 h incubation at 20 °C, all plates were visually inspected and the surface motility phenotypes of the respective strains were recorded. All complementation assays were repeated three times and digital images of the representative surface motility phenotypes of the respective strains were captured and processed as described in the Surface motility assays' section of Methods.
Nutritional requirements for surface motility.
Aliquots (25 ml) of ABT minimal medium containing 0·6 % Bacto agar and either 0·4 % (w/w) glucose and 0·4 % (w/w) Casamino acids, 0·4 % (w/w) Casamino acids, 0·4 % (w/w) glucose, 0·4 % (w/w) D-xylose or 50 % sugar beet extract were poured into sterile Petri dishes. The plates were dried for 2 h at 20 °C and then inoculated with 1 µl culture droplets. Following 20 h incubation at 20 °C, all plates were visually inspected for surface motility. Since all of the strains employed in the experiment grew slowly on plates containing D-xylose as carbon source, their ability to perform surface motility on D-xylose plates was re-evaluated following 7 days incubation at 20 °C. All strains were tested three times for their ability to perform surface motility on the plates described above; based on these triplicates a rough estimate of the maximal colony expansion velocity was obtained by measuring the increase in colony radius as a function of time.
Biocontrol assays.
To compare the abilities of strains DSS73, DSS73-15C2 (AmsY-), DSS73-12H8 (GacS-) and DSS73-12H8(GacS-) carrying pEMH97 to limit the growth of the root-pathogenic microfungi R. solani and Pythium ultimum, the relevant bacterial and fungal strains were co-cultivated on standard surface motility plates and on nutrient agar plates composed of ABT minimal medium containing 0·4 % (w/w) glucose, 0·4 % (w/w) Casamino acids and 2 % Bacto agar (Difco). A 1 µl pre-culture of each bacterial strain was inoculated 2·5 cm away from the centre of a small agar plug containing mycelium of either R. solani or Pythium ultimum, and the plates were incubated at 20 °C. As a control, agar plugs containing mycelium of R. solani and Pythium ultimum were also inoculated alone onto the same types of nutrient plates. Following 24 and 48 h co-cultivation, all plates were visually inspected and the ability of each bacterial strain to inhibit mycelium spreading was evaluated. All biocontrol assays and appropriate controls were done in triplicate and digital images of the representative biocontrol phenotypes were captured and processed as described in the Surface motility assays' section of Methods. To address the viability of the microfungi contained by strain DSS73 on standard surface motility plates (see Fig. 4A as an example) agar plugs were withdrawn from the outer edge of the mycelium and from the area close to the original fungal inoculation site. These plugs were transferred to nutrient plates composed of ABT minimal medium containing 0·4 % (w/w) glucose, 0·4 % (w/w) Casamino acids, 2 % Bacto agar and 10 µg tetracycline ml-1 (tetracycline was added to prevent the growth of biocontrol strain DSS73). As positive controls, agar plugs containing non-challenged mycelium of R. solani and Pythium ultimum were inoculated onto the same types of nutrient plates. Following 3 days incubation at 20 °C, the plates were visually inspected for growth of the respective fungus. All viability assays were repeated three times.
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RESULTS |
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Amphisin production is required for surface motility of strain DSS73
The amphisin synthesized by strain DSS73 might promote or enable surface translocation. In a recent study by Koch et al. (2002), two surfactant-negative mutants, DSS73-15C2(AmsY-) and DSS73-12H8 (GacS-), carrying mutations in amsY (encoding the putative amphisin synthetase) and gacS, respectively, were isolated. These two mutants were subsequently demonstrated to be defective in the synthesis of amphisin. When stab inoculated on nutrient plates solidified with 0·3 % agar, the swimming motility phenotypes of DSS73-15C2 (AmsY-) and DSS73-12H8 (GacS-) were indistinguishable from the swimming motility phenotype exhibited by the parent wild-type strain, DSS73 (data not shown). This indicates the presence of functional flagella and a fully operational chemotaxis apparatus in both of the amphisin-deficient mutants. DSS73-15C2 (AmsY-) and DSS73-12H8 (GacS-) failed to carry out surface motility on 0·6 % agar plates unless the medium was supplemented with 2·5 µg amphisin ml-1 (Fig. 1B, C, E and F
). This clearly demonstrated that amphisin is the surface-tension-reducing compound that enables strain DSS73 to move over the agar surface (Fig. 1A
). In addition, surface motility could be restored in the gacS mutant upon introduction of pEMH97 encoding the heterologous wild-type gacS gene from Pseudomonas syringae pv. syringae (Hrabak & Willis, 1992
), supporting the finding of Koch et al. (2002)
that amphisin synthesis is regulated by gacS (compare Fig.1C and D).
Surface motility of the two mutants, DSS73-15C2 (AmsY-) and DSS73-12H8 (GacS-), was also restored by exogenous tensin (2·5 µg ml-1), viscosinamid (2·5 µg ml-1) or serrawettin W (2·5 µg ml-1) whereas the addition of synthetic surfactants such as NP40 and Triton X-100 failed to promote surface motility (Table 2). NP40 and Triton X-100 restored swarming motility in the surfactant-deficient S. liquefaciens strain PL10 (Table 2
).
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Antifungal properties of strain DSS73 are regulated by GacS
The ability of strain DSS73 to inhibit the growth of the root-pathogenic microfungi Pythium ultimum and R. solani is believed to originate from the production and excretion of amphisin, chitinases, proteases and hydrogen cyanide by the bacterium (Nielsen et al., 2002; Koch et al., 2002
). Interestingly, the production of these antifungal metabolites requires a functional gacS gene whereas only the production of amphisin seems to be impaired in the amsY mutant (Koch et al., 2002
). To characterize the antifungal properties of DSS73 in further detail, we compared the abilities of strains DSS73, DSS73-15C2 (AmsY-), DSS73-12H8 (GacS-) and DSS73-12H8 (GacS-) carrying pEMH97 to inhibit the growth of R. solani and Pythium ultimum. Following co-cultivation of strain DSS73 and R. solani on 2 % agar plates, a mycelium-deficient zone developed around the bacterial colony, confirming the antifungal properties of DSS73 (Fig. 3A
). A similar result was obtained when the mycelium of R. solani approached the bacterial colony of the amsY mutant, DSS73-15C2 (AmsY-) (Fig. 3B
). In striking contrast, R. solani appeared unaffected by the presence of the gacS mutant DSS73-12H8 (GacS-) when co-cultivated under identical conditions. Indeed, the mycelium of the fungus was eventually seen to cover the colony formed by DSS73-12H8 (GacS-), signifying a complete absence of antifungal metabolites in this strain (Fig. 3C
). However, upon provision of the wild-type gacS gene product from Pseudomonas syringae pv. syringae, present on pEMH97, to DSS73-12H8 (GacS-) a mycelium-deficient zone was maintained around the bacterial colony, demonstrating that the antifungal phenotype had been restored (Fig. 3D
). As similar observations were made when Pythium ultimum was challenged with strains DSS73 (AmsY-), DSS73-15C2(GacS-), DSS73-12H8 and DSS73-12H8 (GacS-) carrying pEMH97 (data not shown), these results clearly show that synthesis of antifungal metabolites in DSS73 requires the presence of a functional gacS gene. This strongly suggests that expression of the antifungal properties of strain DSS73 is controlled by a gacS/gacA-encoded two-component system similar to other Pseudomonas spp. (Chancey et al., 1999
; Laville et al., 1992
; Gaffney et al., 1994
; Pfender et al., 1994
; Corbell & Loper, 1995
; Schmidli-Sacherer et al., 1997
).
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Cultivation of R. solani on standard surface motility plates in which the growth medium had been supplemented with varying amounts of amphisin revealed that fungal growth was not significantly affected until a concentration of approximately 10 µg amphisin (ml medium)-1 was reached (data not shown). In contrast, 2·5 µg amphisin (ml medium)-1 was the minimum concentration required to fully restore surface motility of the amsY and gacS mutants (see Table 3). Therefore, it was possible to clarify whether spatial limitations alone or in combination with synthesis of antifungal agents were responsible for the observed efficient containment of the fungus. To address this question, R. solani was challenged with DSS73-12H8 (GacS-) and DSS73-15C2 (AmsY-), respectively, on standard surface motility plates supplemented with 2·5 µg amphisin ml-1. Under these conditions, the ability of the amsY mutant to block mycelium spreading was restored (compare Fig. 4B and E
). Once surrounded by the bacterial colony, R. solani was never observed to resume growth, not even following prolonged incubation. A somewhat different scenario emerged following co-cultivation of R. solani with the gacS mutant on standard surface motility plates supplemented with 2·5 µg amphisin ml-1. Although fully capable of moving over the surface of the agar, strain DSS73-12H8 (GacS-) completely failed to stop the spreading mycelium. Even when completely surrounded by the bacterial colony, R. solani was still observed to maintain mycelium propagation into the agar beneath the DSS73-12H8 (GacS-) culture. Similar results were obtained by replacing R. solani with Pythium ultimum in all of the experiments described in this section (data not shown). This strongly suggests that the fungus had been effectively contained by strain DSS73 as a result of a combination of spatial limitations and a cocktail of antifungal agents.
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DISCUSSION |
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Similar to Serratia spp., the ability of Pseudomonas sp. DSS73 to move over the agar surface was clearly attributed to its production of an extracellular biosurfactant, since the presence of purified amphisin in the growth medium restored this phenotype to the amsY and gacS mutants. In contrast to S. liquefaciens, which is highly promiscuous with respect to accepting surfactants for swarming motility (Lindum et al., 1998; Eberl et al., 1999
), amphisin and other closely related cyclic lipopeptides are prerequisites for the surface motility of strain DSS73. This suggests that not only surface tension reduction but also additional physical/chemical properties of these closely related cyclic biosurfactants are crucial for the surface motility of DSS73.
Fungal growth was inhibited on agar plates supplemented with purified amphisin at a minimum concentration of 10 µg ml-1, which is four times the concentration required to efficiently promote bacterial surface motility. Therefore, amphisin can be considered a dual-functioning compound. For fungal antagonism, however, DSS73 relies not on amphisin alone. It produces a battery of antagonistic agents such as proteases, chitinases and hydrogen cyanide, all of which are controlled by the GacS/GacA system (Koch et al., 2002). Our results clearly demonstrate that the efficient elimination of competing fungi is due to the synergistic effect of surface motility and synthesis of antifungal agents. Without these factors the bacteria were unable to completely contain the fungi.
Our data also suggest that surfactant activity is a more important property than the antimicrobial action of amphisin. However, the artificial nature of the experimental set-up should be kept in mind. If the hardness of the surface in vivo prevents the bacterial culture moving over the surface, the antimicrobial activity of amphisin may be of greater value to the producer. With a MIC of 10 µg ml-1, amphisin clearly retains its potency as an antimicrobial compound.
Expression of the total antimicrobial cocktail (including motility) produced by fluorescent Pseudomonas spp. is controlled by the GacS/GacA system. Therefore, only a single signal is required to mount the entire protective arsenal of the bacteria. In a recent study, it was demonstrated that synthesis of amphisin was increased approximately sixfold when strain DSS73 was cultivated in the presence of a sugar beet seed extract (Koch et al., 2002). Since the growth rate of the organism was unaffected, this result indicates the presence of a signal compound in the seed extract. The structure of this putative signal compound has not yet been elucidated; however, experiments have revealed that signal transduction leading to increased transcription of amsY clearly requires a functional gacS/gacA system (Koch et al., 2002
).
The apparent interplay between strain DSS73 and the sugar beet seed might be considered as being mutually beneficial. Initially, the sugar beet seed supplies the bacteria with nutrients and releases signals that induce production of amphisin. We speculate that this enables a culture of DSS73 to efficiently expand on the seed surface, but in addition it also recruits the entire protective arsenal of the bacterium. In the environment, multicellular behaviour is considered an advantage for bacteria. Previous work by Eberl et al. (1997) and Mallory et al. (1983)
strongly suggests that the formation of flocks offers bacteria protection against grazing protozoa. Similarly, in the cause of infection, Pseudomonas aeruginosa grows in biofilms that protect it from the action of leukocytes (Høiby et al., 2001
; Costerton et al., 1999
). In this latter case, control over surface motility, biofilm architecture and the formation of a cocktail of virulence factors is integrated by quorum sensing. It is tempting to speculate that biocontrol bacteria, which coordinate their effort and work in a flock, are more successful in their interactions with other microbes and higher eukaryotes than their planktonic counterparts.
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ACKNOWLEDGEMENTS |
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Received 8 July 2002;
revised 17 September 2002;
accepted 25 September 2002.