Environmental Microbiology Reports (2010)
doi:10.1111/j.1758-2229.2010.00209.x
Pseudomonas fluorescens BBc6R8 type III secretion mutants no longer promote ectomycorrhizal symbiosis 209
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Angela M. Cusano,1† Peter Burlinson,1,2† Aurélie Deveau,3 Patrice Vion,1 Stéphane Uroz,1 Gail M. Preston2 and Pascale Frey-Klett1* 1 INRA, UMR1136 INRA-Nancy Université, «Interactions Arbres/Micro-organismes», Centre de Nancy, IFR110, 54280 Champenoux, France. 2 Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, UK. 3 Department of Microbiology and Immunology, Dartmouth Medical School, 208 Vail Building, Hanover, NH 03755, USA. Summary The Mycorrhiza Helper Bacterium (MHB) Pseudomonas fluorescens BBc6R8 promotes the ectomycorrhizal symbiosis between Douglas fir roots and Laccaria bicolor. In this study, we identified a nonflagellar type III secretion system (T3SS) in the draft genome of BBc6R8 similar to that described in the biocontrol strain P. fluorescens SBW25. We examined whether this T3SS plays a role in the BBc6R8 mycorrhizal helper effect by creating a deletion in the rscRST genes encoding the central channel of the injectisome. The in vitro effect of BBc6R8 T3SS mutants on the radial growth rate of L. bicolor was unchanged compared with the parental strain. In contrast, T3SS mutants were unable to promote mycorrhization, suggesting that type III secretion plays an important role in the mycorrhizal helper effect of P. fluorescens BBc6R8 independent of the promotion of hyphal growth that BBc6R8 exhibits in vitro. Introduction The type III secretion system (T3SS) is one of six secretion pathways currently identified in Gram-negative bacteria. It is a complex molecular injection machinery used to translocate bacterial type III secreted effector (T3SE) proteins across the bacterial envelope and directly into the cytoplasm of a eukaryotic cell. T3SSs have been Received 28 May, 2010; accepted 12 July, 2010. *For correspondence. E-mail:
[email protected]; Tel. (+33) 383394149; Fax (+33) 383394069. †These authors contributed equally to this work.
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intensively studied because of their important roles in mediating bacterial pathogenic or symbiotic interactions with plant and animal hosts (McCann and Guttman, 2008). However, there is now increasing evidence to suggest that the ecological functions of T3SSs are more diverse than had previously been thought (Preston, 2007). T3SSs have been identified in a wide variety of environmental and commensal bacteria that have not been shown to have detrimental effect on plants or animals and which may even have beneficial applications in promoting plant health and nutrition (Preston et al., 2001; Mazurier et al., 2004; Rezzonico et al., 2004; 2005; Preston, 2007). This fuels speculation that bacteria naturally use T3SSs to interact with the diverse eukaryotes they encounter in the environment (Preston, 2007). Supporting this concept, several studies have shown that T3SSs can be directed against eukaryotes that inhabit soil systems; for example, T3SSs can target the amoebae Dictyostelium discoideum and Acanthamoeba castellanii, while the T3SEs of plant and animal pathogens are biologically active when expressed in yeast, indicating the potential of T3SEs to target fungal cells, although delivery across a fungal cell wall has yet to be demonstrated (Lesser and Miller, 2001; Pukatzki et al., 2002; Jamir et al., 2004; Valdivia, 2004; Matz et al., 2008). Recent reports have indicated that bacteria associated with fungi harbour T3SSs, raising the intriguing possibility that the T3SS may be involved in fungal–bacterial interactions (Mazurier et al., 2004; Warmink and van Elsas, 2008). One setting in which bacteria play an important role in fungal biology and ecology is the ectomycorrhizal complex, a mixed fungal–bacterial continuum at the interface between soil and tree roots (Frey-Klett and Garbaye, 2005). In this niche, ectomycorrhizal fungi form an intimate association with plant root systems in which their hyphae remain extracellular to plant cells and form a mantle that encloses short lateral roots (Frey-Klett et al., 2007; Fig. 1). The establishment of ectomycorrhizal symbiosis can be significantly improved by selected soil and mycorrhizosphere bacterial strains, called ‘Mycorrhiza Helper Bacteria’ (MHB; Frey-Klett et al., 2007). This system constitutes an exemplar of plant–fungal–bacterial interactions within forest communities, which are of particular interest since establishment
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A. M. Cusano et al. (Deveau et al., 2007) and also significantly promotes the establishment of ectomycorrhizal symbiosis between Douglas fir and L. bicolor S238N (Frey-Klett et al., 1997). The BBc6R8 MHB effect is related to the enhanced survival and growth of L. bicolor during its pre-symbiotic life in the soil, under unfavourable growth conditions (Brule et al., 2001). Here, we report that P. fluorescens BBc6R8 harbours a T3SS gene cluster, the operon organization of which is similar to the T3SS of the biocontrol strain P. fluorescens SBW25 and phytopathogenic Pseudomonas syringae. Since this strain has no known pathogenic potential towards plants or fungi, we hypothesized that the T3SS of BBc6R8 may fulfil an alternative role, modulating the functioning of the ectomycorrhizal complex and potentially contributing to the promotion of the ectomycorrhizal symbiosis. To test this hypothesis, the objective of the present work was to assess the effect of a targeted knockout in the T3SS cluster on the ability of BBc6R8 to stimulate growth of L. bicolor S238N in vitro, and on the mycorrhizal helper effect in a glasshouse microcosm with Douglas fir seedlings.
Fig. 1. Douglas fir seedling – L. bicolor microcosm (left) used in glasshouse assays to monitor mycorrhization. Enlarged (right) are examples of a non-mycorrhizal root (- myc) with root hairs and a mycorrhizal root (+ myc) with a fungal white/purplish mantle. Scale bar = 1 mm.
Results and discussion
of a good mycorrhizal symbiosis between tree roots and fungi is highly beneficial to tree nutrition and therefore to forest health. This has been demonstrated by the successful use of the controlled mycorrhization process for commercial reforestation in France. A thorough understanding of forest ecosystems functioning is of particular pertinence given the increased pressures on such systems in recent times caused by anthropogenic factors. However, while many physiological aspects of mycorrhizal interactions between plant root systems and fungi have been studied, the mechanisms by which MHB stimulate mycorrhiza formation are poorly understood. Despite this, direct effects of MHB on the pre-symbiotic survival and growth of ectomycorrhizal fungi in the soil have been well documented, highlighting the importance of fungal–bacterial interactions in the mycorrhiza helper process (Brule et al., 2001; Founoune et al., 2002; Schrey et al., 2005; Deveau et al., 2007). Little emphasis has been given so far to the significance of the T3SSs harboured by bacteria associated with ectomycorrhizal fungi, although they could potentially modulate the plant or the fungal partner in the symbiosis. The model MHB strain Pseudomonas fluorescens BBc6R8, a rifampicin-resistant spontaneous mutant of the BBc6 strain isolated from a sporocarp of the ectomycorrhizal fungus Laccaria bicolor S238N (Duponnois and Garbaye, 1991), promotes L. bicolor in vitro growth
We recently generated a draft genome of the MHB strain P. fluorescens BBc6R8 using 454 pyrosequencing (P. Frey-Klett, unpublished). During annotation of the data using the RAST server (Aziz et al., 2008) we found evidence of sequences corresponding to T3SS genes. We searched further for T3SS genes by performing TBLASTX (Altschul et al., 1990) comparisons with the T3SS sequences from the genomes of the biocontrol strain P. fluorescens SBW25 and the phytopathogen P. syringae pv. tomato DC3000. This revealed a total of 20 open reading frames (ORFs) that display a high percentage amino acid identity to corresponding sequences in one or both strains (typically > 60% amino acid identity, calculated using BLASTP) (Fig. 2 and Table 1). T3SS genes in BBc6R8 have a higher sequence similarity at both the amino acid and nucleotide level to those found in SBW25, and the BBc6R8 T3SS operon organization is broadly similar to that of SBW25 but also contains homologues of genes from the P. syringae hrpJ operon (designated rspJ, rscV, rspQ and rscN according to the nomenclature of Preston et al., 2001) which are missing (or in the case of rspJ truncated) in SBW25. Unlike SBW25, BBc6R8 lacks homologues of the T3SS structural proteins rspA, rspG and rspF, although other small putative ORFs with no sequence similarity to known T3SS components are present in the same genomic context. The contig containing the BBc6R8 T3SS (desposited in GenBank,
Identification and characterization of the BBc6R8 T3SS gene cluster
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MHB effect lost in T3SS mutants 3
Fig. 2. Alignment by TBLASTX of the P. fluorescens BBc6R8 and SBW25 T3SSs, generated using WebACT (Carver et al., 2005). BBc6R8 genes were predicted using FgenesB (Softberry). Regions of high amino acid identity are indicated by red bars between the two sequences, the colour intensity indicating the level of identity between the two. Predicted coding sequences are shown as coloured boxes: blue for T3SS genes, green for the ropE putative SBW25 effector and grey for genes not believed to be involved in type III secretion. The region deleted in the BBc6R8DrscRST mutants is indicated by the yellow triangle.
accession: HM752578) does not contain large amounts of sequence flanking the T3SS region. The region adjacent to rspL in SBW25 contains the avrE effector homologue ropE, which is absent in BBc6R8; in its place a putative fosmidomycin resistance protein is encoded that is found
elsewhere in the SBW25 genome. However, the ORFs immediately adjacent to the rspR end of the SBW25 T3SS are conserved in BBc6R8, indicating that the genomic location of the T3SS in BBc6R8 and SBW25 is partially conserved.
Table 1. In silico genomic analysis of the T3SS gene cluster from P. fluorescens BBc6R8.
Encoded protein Operon L RspL Operon J RspJa RscV RspQ RscN RspO Operon U RscP RscQabb RscR RscS RscT RscU Operon C RspV RspT RscC RspG RspF Operon Z RspE RspD RscJ RspB RspZ RspA Operon R RspS RspR
Annotation (Psy DC3000 genome)
Present in Pfl BBc6R8?
Pfl SBW25 % AA identity (% positives)
Psy DC3000 % AA identity (% positives)
RNA polymerase sigma factor
Yes
79 (90)
47 (66)
T3S T3S T3S T3S T3S
protein IM channel protein protein cytoplasmic ATP synthase protein
Yes Yes Yes Yes Yes
58 (70) Absent Absent Absent 67 (83)
31 62 36 65 28
T3S T3S T3S T3S T3S T3S
protein protein protein inner membrane protein inner membrane protein inner membrane protein
Yes Yes Yes Yes Yes Yes
64 73 85 93 81 81
24 (43) 35/44 (50/60) 68 (84) 76 (89) 54 (73) 52 (71)
Negative regulator of hrp expression T3S protein T3S OM pore forming protein T3S protein T3S protein
Yes Yes Yes No No
58 (66) 81 (91) 81 (90) – –
36 (49) 37 (51) 45 (63) – –
T3S T3S T3S T3S T3S T3S
apparatus protein bridge lipoprotein protein protein protein
Yes Yes Yes Yes No No
42 (63) 44 (55) 74 (84) 38 (47) Absent –
30 30 61 30 – –
T3 transcriptional regulator T3 transcriptional regulator
No Yes
Absent 76 (84)
– 61 (76)
(75) (83) (93) (97) (89) (90)
(53) (75) (52) (81) (48)
(50) (46) (79) (67)
a. The rspJ gene is truncated in SBW25. b. HrcQab is present as two separate putative proteins in DC3000 (scores for both are indicated). The % identity is calculated based on BLASTP alignment with P. fluorescens SBW25 and P. syringae pv. tomato DC3000 predicted protein sequences.
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4 A. M. Cusano et al. Pseudomonas fluorescens BBc6R8 and SBW25 both lack hrpZ (encoding a pore-forming harpin protein) homologues and possess only a single gene corresponding to the positive regulatory hrpR/S enhancer-binding proteins found in P. syringae pv. tomato DC3000. Pseudomonas fluorescens BBc6R8 carrying the avrB effector gene on a plasmid (pAvrB1; Tamaki et al., 1988) was unable to elicit a hypersensitive reaction when inoculated into Arabidopsis thaliana Col-0 leaves at 108 cfu ml-1, although at higher concentrations (~5 ¥ 109 cfu ml-1) the wild-type strain produced some necrosis independent of the presence of the effector (data not shown). Failure to elicit a hypersensitive response in Arabidopsis may be the result of poor competence for P. fluorescens BBc6R8 type III secretion in the phyllosphere, as was found for SBW25, which could be due to regulatory or functional constraints on the BBc6R8 T3SS in an environment which is very different to its natural ecological niche. Generation and in vitro characterization of BBc6R8 T3SS mutants The T3SS is a multi-protein complex encoded by a number of genes in several operons. To avoid problems associated with polarity, we constructed an unmarked non-polar deletion mutant in a section of the BBc6R8 T3SS corresponding to rscS and sections of the rscR and rscT genes (Fig. 2), which form the central channel of the injectisome. This region was targeted as it has no known regulatory function and is also highly conserved in T3SShabouring bacterial strains, and therefore likely to be essential for the functioning of the system. The deletion was created using the two-step allelic exchange strategy described by Zhang and Rainey (2007) with modifications to the selection procedure to make it suitable for BBc6R8 (details available on request); the strains, constructs and primers used are given in Table S1. Mutants were verified by antibiotic resistance profile and sequencing of the deleted region. Two confirmed mutants, designated BBc6R8DrscRST-(D) and BBc6R8DrscRST-(J) were chosen for use in subsequent assays to control for possible non-specific changes (such as point mutations elsewhere in the genome) which could potentially have been introduced during the mutagenesis procedure and affected bacterial fitness. We have previously observed differences in the in vitro growth of T3SS mutants of other Pseudomonas strains (Jackson et al., 2005; G. Preston, unpubl. obs.) suggesting that a thorough characterization of T3SS mutant growth phenotypes under different growth conditions is prudent. Accordingly, we performed a detailed comparison of the in vitro growth of the two T3SS mutants to the parental strain in eight laboratory media, representing a range of nutrient conditions (Fig. S1). Neither mutant
exhibited a substantial difference in growth compared with the parental strain and while some minor differences were observed in some of the growth media tested, there was no consistent pattern to these differences between the three bacterial strains. In motility assays to assess the swimming ability of BBc6R8 and the T3SS mutants (performed according to Jones et al., 2007) no significant difference was detected between the three strains (Fig. S2). Taken together, the growth and motility data point to the absence of pleiotropic effects associated with rscRST deletion in strain BBc6R8. BBc6R8 T3SS mutants retain the ability to promote fungal growth in vitro The effect of the two T3SS mutants, BBc6R8DrscRST-(D) and BBc6R8DrscRST-(J), on the growth of L. bicolor S238N was monitored following the method developed by Deveau and colleagues (2007) in which bacteria and fungus are co-cultivated in vitro and the influence of the presence of the bacteria on rate of hyphal growth is measured periodically. The parental strain BBc6R8 and both the T3SS mutants similarly induced the in vitro growth of L. bicolor mycelium after 40 days of incubation (Fig. 3A). Microscopic observation of the growing mycelium indicated that mutation of the BBc6R8 T3SS did not alter the morphology of the fungus when compared with the parental strain. Moreover, we observed that both T3SS mutants retain the ability of the parental strain to colonize the fungal hyphae once the cell–cell contact occurs (data not shown). These results demonstrate that the BBc6R8 T3SS gene cluster is not required for the in vitro fungal growth-promoting effect of BBc6R8 in our bioassay, which is in accordance with recent results from Deveau and colleagues (2010) that indicate a metabolic effect, such as bacterial thiamine production, may be responsible. The mycorrhizal helper effect is lost in T3SS mutants To test the impact of T3SS mutation on the mycorrhiza helper effect we examined the ability of the two T3SS mutants, BBc6R8DrscRST-(D) and BBc6R8DrscRST-(J), to promote the ectomycorrhizal Douglas–L. bicolor symbiosis in a glasshouse microcosm previously described by Frey-Klett and colleagues (1997). Briefly, pre-treated Douglas fir seeds were sown in 4-cm-diameter containers filled with an autoclaved peat–vermiculite mix, inoculated with 2.5% (v/v) L. bicolor S238N. Five millilitres of an aqueous suspension of BBc6R8, BBc6R8DrscRST-(D) or BBc6R8DrscRST-(J) (108 cfu ml-1) was individually inoculated onto 40 microcosms, each with one Douglas fir seedling; sterile water was used in control treatments. Twenty seedlings per treatment were randomly sampled 12 weeks after inoculation and blinded to remove
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MHB effect lost in T3SS mutants 5 Fig. 3. A. In vitro fungal growth promotion assay. BBc6R8 and the BBc6R8DrscRST deletion mutants (D) and (J) promote the in vitro radial growth of Laccaria bicolor S238N to the same extent compared with the control (no MHB, water inoculum). The diameter of the fungal colony was measured in two perpendicular directions regularly; data for day 16 (white bars) and 40 (grey bars) are shown for comparison. Letters represent a significant difference according to a one-factor (bacterial treatment) ANOVA (F = 64.4, P < 0.001, n = 6) and the Bonferroni–Dunn test; error bars indicate the standard deviation. The experiment was performed twice with similar results. B. Biomass of plants from the glasshouse assay 12 weeks after bacterial inoculation, determined after separating aerial and root compartments (which were washed free of soil) and drying at 60°C until a stable dry mass was obtained. Bars represent the average dry mass (n = 20) of root (grey) and aerial (white) portions of the plants. Error bars indicate the standard deviation of the total mean (root + aerial mass). As indicated by the letters, no statistically significant difference between treatments was found for any of the mass parameters according to one-factor (bacterial treatment) ANOVA (root F = 0.99, P = 0.403; aerial F = 1.76, P = 0.163; total F = 1.58, P = 0.202). C. Effect of P. fluorescens BBc6R8 and BBc6R8DrscRST deletion mutants (D) and (J) on mycorrhiza formation in the glasshouse experiment. The sampled roots were dried at 60°C and the mycorrhizal index for each treatment calculated (number of mycorrhizal roots per gram of root dry mass). The mycorrhizal index values are shown for the control treatment (no MHB, water inoculum) and the treatments inoculated with either BBc6R8 or the two T3SS mutants. The mycorrhizal helper effect exhibited by BBc6R8 is absent in both T3SS mutant treatments. Letters indicate a significant difference according to one-factor (bacterial treatment) ANOVA (F = 4.59, P = 0.0052; n = 20) and Bonferroni–Dunn test; error bars indicate standard deviation. Statistical analyses were performed using SuperANOVA 1.11.
potential subjective bias in assessment of levels of mycorrhization. Measurements of plant dry mass (Fig. 3B) and plant height (data not shown) found no significant difference between the averages for the treatments at this time point. Prior to dry mass determination the root system was cut into 1-cm-long pieces and a random sample of 100 sections for each root system was taken, for which the percentage of short roots forming mycorrhizas was determined stereomicroscopically. As expected, P. fluorescens BBc6R8 significantly increased the mycorrhizal index compared with the treatment inoculated with L. bicolor alone (Fig. 3C), confirming the MHB effect of this bacterial strain. In contrast, both T3SS mutants were unable to promote the Douglas fir– L. bicolor symbiosis. The fact that both mutants were no longer able to promote mycorrhiza formation emphasizes the consistency of the mutant phenotype we observed.
Based on our results, the BBc6R8 T3SS genes can be considered the first mycorrhiza helper effect-related genes identified through mutagenesis. However, the mechanism by which the T3SS is able to modulate the ectomycorrhizal symbiosis remains to be elucidated. Type III secretion plays an important role in host–pathogen specificity in other systems and it is possible that the T3SS may be involved in the fungus specificity demonstrated by some MHBs, in which ectomycorrhiza establishment by some fungi but not others is promoted (Garbaye and Duponnois, 1992). This effect could be mediated through its action on either the fungal or plant partner in the symbiosis. Several mechanisms have been postulated for the mycorrhiza helper effect demonstrated by a variety of bacteria in different mycorrhizal symbioses (Frey-Klett et al., 2007). A plausible hypothesis is that the BBc6R8 T3SS may be involved in suppressing root innate immune responses, which has been suggested to be involved in a Streptomyces MHB (Lehr et al., 2007). The T3SS of phytopathogenic bacteria such as P. syringae suppress plant defence responses to the pathogen due to recognition of pathogen-derived molecules (Jones and Dangl, 2006). A similar suppression in Douglas fir roots in the presence of BBc6R8 could facilitate formation of the ectomycorrhizal complex, which results from an intimate association between plant cells and fungal hyphae. A role
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A. M. Cusano et al.
for type III secretion in the interaction of a phytopathogen with a plant root system has not been demonstrated, although phytopathogenic P. syringae apparently possess other mechanisms to suppress innate immune responses triggered by microbe associated molecular patterns (Millet et al., 2010). Utilization of the T3SS in the delivery of nodulation proteins into root cells has, however, been described in Rhizobium (Kambara et al., 2009) which inhabits a niche more similar to that of BBc6R8 and other P. fluorescens. The potential impact of type III secretion on plant root exudates or hormone production, in a manner akin to changes occurring in plant leaves due to T3SE activity, may also be responsible for the promotion of ectomycorrhiza formation (Torto-Alalibo et al., 2009). On the fungal side of the symbiosis, there is currently no direct evidence for T3SE delivery into fungal cells, although the presence of T3SS genes in bacteria associated with fungi has been reported and a role for type III secretion in bacterial– fungal interactions speculated (Mazurier et al., 2004; Leveau and Preston, 2007; Warmink and van Elsas, 2008; Bonfante and Anca, 2009). Thus further studies, including a thorough assessment of spacial and temporal T3SS expression throughout the mycorrhization process, are required to determine whether the BBc6R8 T3SS targets the plant or fungal partner in the symbiosis. Examination of the effect of BBc6R8 T3SS mutants on Douglas fir seedlings in the absence of the fungal partner may also provide a route to revealing the mechanisms of the T3SS within this system. Despite this, our findings represent an important advance towards understanding of the mechanisms by which mycorrhiza helper bacteria can exert their beneficial effect. Furthermore, our novel results extend the concept of type III secretion beyond its typically ascribed roles in pathogenic or symbiotic interactions, since in this context it facilitates a mutually beneficial interaction between two different eukaryotic systems. Acknowledgements We thank F. Martin for helpful suggestions and support in genome sequencing and analysis, A. Sarniguet for useful discussions and B. Palin for assistance. This study was supported by INRA, IFR110, The Lorraine Region, The French Ministry of Research, The French Ministry of Foreign Affairs, The Royal Society and The British Council.
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Supporting information Additional Supporting Information may be found in the online version of this article: Fig. S1. In vitro growth comparison of BBc6R8 (solid line) and mutants BBc6R8DrscRST-(D) (dashed line) and -(J) (dotted line). Each strain was resuspended in sterile dH2O from a 24 h overnight culture grown on King’s B agar and the OD600 adjusted to 0.1 using a spectrophotometer. Each strain was then inoculated (75 ml) into wells of a 96-well plate containing an equal volume of growth media at 2¥ the working concentration. Plates were incubated at 28°C (shaking every 15 min for 10 s) in a Tecan Infinite M200 96-well plate reader and optical density in each well monitored. Each graph represents a different nutrient regime as indicated: King’s B (King et al., 1954), Luria Bertani (Sambrook and Russell, 2001), Potato dextrose broth (Sigma), SM (Sussmann, 1966), M9-glucose (Sambrook and Russell, 2001), P20 pH 5.5 and pH 7.0 (thiamine omitted, Pachlewski and Pachlewska, 1974) and nutrient broth (Downes and Ito, 2001). Error bars represent the standard deviation of the mean; n = 8. Arrows indicate representative time points at which a significant difference (P < 0.05) between the growth
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of the strains was detected. The experiment was performed twice with similar results. Fig. S2. Comparison of BBc6R8 and T3SS mutant motility. Bacteria from an overnight culture grown on King’s B agar were stabbed into the centre of 9 cm Petri dishes containing motility agar (0.3% agar, 10% LB plates) and incubated at room temperature (~23°C). The diameter of the bacterial swarm was measured through the inoculation point 36 h after inoculation. No significant difference was found between the different treatments by one-way ANOVA (F = 1.99, P = 0.158,
n = 9). The experiment was performed twice with similar results. Table S1. Strains, plasmids and oligonucleotides used in this study. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports
