The biosurfactant viscosin produced by Pseudomonas fluorescens

SBW25 mediates in vitro spreading motility and plant growth

promotion1

Abdullah S. Alsohim1, Tiffany B. Taylor1, Glyn A. Barrett1, Jenna Gallie2,3,4, Xue-Xian Zhang2,

Astrid E. Altamirano-Junqueira1, Louise J. Johnson1, Paul B. Rainey2,5 and Robert W.

Jackson1,*

School of Biological Sciences, University of Reading, Whiteknights, Reading, RG6 6AJ, UK

New Zealand Institute for Advanced Study, Massey University, Auckland, New Zealand

Department of Environmental Microbiology, Eawag, 8600 Dbendorf, Switzerland

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Department of Environmental Systems Science, ETH Zrich, 8092 Zrich, Switzerland

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Max Planck Institute for Evolutionary Biology, Pln, Germany

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*Corresponding author

Accepted Article

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Keywords: Plant growth-promoting rhizobacteria (PGPR); Food security; FleQ; Plant

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colonisation

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This article has been accepted for publication and undergone full peer review but has not been through the
copyediting, typesetting, pagination and proofreading process, which may lead to differences between this
version and the Version of Record. Please cite this article as doi: 10.1111/1462-2920.12469

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Accepted Article
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Abstract

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Food security depends on enhancing production and reducing loss to pests and pathogens.

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A promising alternative to agrochemicals is the use of plant growth promoting rhizobacteria

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(PGPR), which are commonly associated with many, if not all, plant species. However,

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exploiting the benefits of PGPRs requires knowledge of bacterial function and an in-depth

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understanding of plant-bacteria associations. Motility is important for colonisation efficiency

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and microbial fitness in the plant environment, but the mechanisms employed by bacteria

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on and around plants are not well understood. We describe and investigate an atypical

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mode of motility in Pseudomonas fluorescens SBW25 that was revealed only after flagellum

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production was eliminated by deletion of the master regulator fleQ. Our results suggest this

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spidery spreading is a type of surface motility. Transposon mutagenesis of SBW25fleQ

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(SBW25Q) produced mutants, defective in viscosin production, and surface spreading was

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also abolished. Genetic analysis indicated growth-dependency, production of viscosin and

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several potential regulatory and secretory systems involved in the spidery spreading

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phenotype. Moreover, viscosin both increases efficiency of surface spreading over the plant

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root and protects germinating seedlings in soil infected with the plant pathogen Pythium.

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Thus, viscosin could be a useful target for biotechnological development of plant growth

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promotion agents.

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Introduction

Accepted Article

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Many strains of Pseudomonas fluorescens are proficient colonizers of soil and the

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phytosphere (plant environment). P. fluorescens can be genetically engineered to promote

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plant growth by producing hormones or inducing systemic resistance (Glick & Bashan,

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1997), but many naturally-occurring strains also significantly improve plant growth (Cook et

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al., 1995, Schippers et al., 1987, Weller, 1988) by improving the bioavailability of nutrients

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and antagonizing pathogenic fungi and oomycetes (Artursson et al., 2006, Sarathchandra et

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al., 1993). Antagonism can be conferred by the production of siderophores and of

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surfactants, such as viscosin and viscosinamide, as well as antimicrobial compounds, such as

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hydrogen cyanide, phenazines, pyrrolnitrin or 2,4-DAP (Haas & Dfago, 2005). Many strains

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harbour a type III protein secretion system that is expressed in the plant environment

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(Rainey 1999, Preston et al., 2001), and is implicated in plant growth promotion by

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antagonism of oomycete pathogens (Rezzonico et al., 2005, Weller et al., 2002, de Souza et

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al., 2003).

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Strains of P. fluorescens are attractive as plant growth promoting agents, but a major

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challenge is to ensure reliable and stable protection. Desirable qualities include production

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of a range of antagonistic molecules to target a variety of pathogens and, particularly,

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durability in the field, e.g., long-lasting colonization of plant surfaces and persistence in soil.

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There is natural variation in the ability of P. fluorescens to colonize plants, and variation in

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growth rates under different environmental conditions (Loon, 2007; Chiarini et al., 1998);

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the basis of these differences in ecological success has been the subject of intense study,

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and has been partially elucidated by genetic screens (Rainey, 1999; Gal et al., 2003; Giddens

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et al., 2007) and genome sequencing (Silby et al., 2009). Promoter-probe studies using in

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vivo expression technology (IVET) have led to the discovery of a wide range of mechanisms

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for colonisation of plants, ranging from specific metabolism and nutrient scavenging

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systems to motility and secretion systems and the production of compounds such as

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extracellular polysaccharide (EPS; e.g. cellulose) (Rainey, 1999; Gal et al. 2003; Silby et al.,

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2009) and secreted proteins (Silby & Levy, 2004).

Accepted Article

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A large number of regulators have also been identified by IVET as being expressed in

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the plant environment, and the interplay between regulators and plant-induced genes was

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studied using a combination of suppressor mutagenesis and IVET, termed SPyVET (Giddens

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et al., 2007). This identified both positive and negative regulatory genes of plant-induced

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genes, and also provided insight to the complexity and hierarchy of gene regulation. For

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example, the wss gene locus is under the control of at least 7 regulators. One of these, FleQ,

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negatively regulates wss expression. Mutation of fleQ in P. fluorescens SBW25 led to

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activation of wss, but is also linked to loss of flagellum biosynthesis and swimming motility.

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An analysis of bacterial movement over the surface of semi-solid agar also revealed a

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change in the surface motility phenotype, with a switch from a radial spreading colony to a

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dendritic spreading colony. We aimed to investigate the mechanism of this motility

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phenotype (termed spidery swarming in Giddens et al., 2007; here we use spidery

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spreading because spreading encompasses any motility over the surface and therefore

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avoids assumptions about motility mechanism).

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FleQ (sometimes called AdnA) is an RpoN-dependent enhancer binding protein and

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the master regulator of flagella biosynthesis (Dasgupta et al., 2003). As well as activating

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motility, it also regulates genes involved in attachment to surfaces and biofilm formation

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(DeFlaun et al., 1994; Robleto et al,. 2003; Mastropaolo et al., 2012). Mastropaolo et al.

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(2012) found that FleQ (AdnA) positively regulates 92 genes in P. fluorescens Pf0-1, 48 that
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are known motility or chemotaxis genes and 44 that are predicted to have a range of other

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functions, e.g. lipoproteins. Recent evidence has shown that FleQ is also a negative

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regulator of extracellular polysaccharide biosynthesis controlling pel genes in P. aeruginosa

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(Hickman & Harwood, 2008; Baraquet et al., 2012). The activity of FleQ in P. aeruginosa is

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modulated by cyclic di-GMP, a secondary messenger molecule that is synthesized by

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diguanylate cyclases these are thought to be influenced by environmental signals

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(Hickman & Harwood, 2008; Baraquet et al., 2012). Thus, FleQ plays a dual regulatory role

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and probably acts, via environmental signalling, as a switching mechanism for inverse

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expression of the flagellum and EPS common strategies for bacteria switching between

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planktonic and biofilm lifestyles (Coggan & Wolfgang, 2012).

Accepted Article

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Bacteria have a range of mechanisms that allow efficient motility under diverse

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environmental conditions from liquid, to semi-solid and solid surfaces. Motility mechanisms

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include flagellum-dependent swimming and swarming, or flagellum-independent

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mechanisms including twitching/gliding using type IV pili, non-social gliding (a poorly

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understood type of motility; Youderian, 1998) and sliding using reduced surface tension

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(Henrichsen, 1972; Harshey, 2003). Swimming motility is dependent on flagella and is

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observed as bacteria moving through liquids or semi-solid media, such as low percentage

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agar, and requires a high liquid presence, whereas swarming is the active spread of bacteria

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over surface like semi-solid agar, and depends on flagella, pili and the presence of a water

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film or surfactant (such as LPS or rhamnolipids) to enable motility. Sliding motility is

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independent of flagella, relies on a growing culture in combination with reduced surface

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tension between the cell and the surface (Henrichsen, 1972; Kinsinger et al., 2003), and is

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usually dependent on production of a biosurfactant. Biosurfactants are a vast and diverse

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range of metabolites of great ecological importance, as well as economic value in the food,
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drug and cosmetics industries (Mandal et al., 2013). Biosurfactants are involved in bacterial

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virulence, biofilm formation and defence against predators and pathogens in addition to

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having a role in multiple motility mechanisms. It was recently discovered that P. aeruginosa

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exhibited sliding motility over swarming plates when both pili and flagella expression were

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deleted, and biosurfactant expression was suggested as a major factor regulating this sliding

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motility (Murray & Kazmierczak, 2008). Biosurfactants have also been implicated in flagella-

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mediated motility, perhaps by acting as a lubricant of the flagellar propeller (Burch et al.,

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2012). Although biosurfactants include many types of molecule, the lipopeptides, which

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include an oligopeptide and a lipid tail, are a particularly important and well-studied family

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(Raaijmakers et al., 2010). P. fluorescens strain SBW25 is known to produce the potent anti-

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microbial lipopeptide viscosin (Laycock et al., 1991).

Accepted Article

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The observation that the fleQ mutant of SBW25 retains some motility, albeit

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phenotypically different to that of the wild-type, led us to hypothesize that fleQ deletion

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exposed a FleQ-independent surface motility. To investigate the mechanism of spidery

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spreading, we used a genetic screen to identify the genes underlying it, and show that the

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phenotype requires the surfactant viscosin, consistent with its being a form of sliding

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motility. Moreover, we show that viscosin is important for plant root colonisation and plays

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a further role in protecting plant roots from an oomycete pathogen.

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Results

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FleQ is required for radial surface spreading by SBW25

Accepted Article

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Our first aim was to characterize the change in surface spreading motility observed by

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Giddens et al. (2007) after mutation of fleQ. In that study, the change in motility phenotype

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was observed with strain NR9.5, which carried an IVET construct integrated into the

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chromosome and a transposon insertion in fleQ. To eliminate any possible genetic side

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effects with the presence of these constructs, we made a deletion of the fleQ gene in

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SBW25 (SBW25Q) and determined that the phenotypic consequences of the gene deletion

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matched the phenotypes previously seen with strain NR9.5 (Giddens et al., 2007). The loss

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of FleQ activity was confirmed by the associated loss of swimming motility through semi-

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solid agar (data not shown) and production of aflagellate cells (Figure 1a). Importantly,

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SBW25Q exhibited spidery spreading (Figure 1b) identical to that seen with NR9.5 by

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Giddens et al. (2007).

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To confirm the phenotypic changes were due to loss of FleQ activity, the fleQ

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mutation was complemented by ectopic expression of fleQ from a plasmid resulting in a

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surface motility phenotype similar to the wild-type (Figure 1a and b): In addition,

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SBW25Q(pFleQ) was observed to produce flagella and regained the ability to swim through

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agar in the swimming assay. Twitching motility was not affected by the fleQ mutation (data

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not shown). Taken together, these experiments show that mutation of fleQ alters bacterial

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surface motility. To clarify this phenotype further, we also tested the wildtype and SBW25Q

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on media that have been used in other surface motility studies. These included 0.6%

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standard succinate medium (SSM) and 0.6% LB: neither strain was observed to move over

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the surface of the media after 3 days incubation. We also tested both strains on 0.25% SSM;
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in both cases the strains moved over the surface of the agar, exhibiting a similar dendritic

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spread with very white tendril, different from the spidery spreading phenotype observed

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with SBW25Q on 0.25% LB (Supplementary Figure S1). Taken together, these experiments

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show that the SBW25Q phenotype on 0.25% LB is a novel observation that is linked to agar

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content and medium composition. This suggests that spidery spreading is an alternative

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form of surface motility, independent of FleQ regulation (and flagella) that is unmasked

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upon inactivation of fleQ.

Accepted Article

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Mutagenesis of SBW25Q reveals that spidery spreading requires the surfactant viscosin

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To determine the genetic basis of spidery spreading, a transposon mutagenesis of

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SBW25Q with IS--Km/hah was performed to identify mutants that either reverted to wild-

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type surface spreading or exhibited loss or change of surface motility. To ensure surface

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motility phenotypes were reproducible and comparable to the control strains (SBW25 and

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SBW25Q) we performed motility assays on 0.25% LB agar. During the course of these

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experiments, a clear liquid film was observed to be emanating from the point of inoculation,

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eventually forming a radial film moving in front of the bacterial colony, for both SBW25 and

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SBW25Q. Quantification of the liquid film and the spreading of the colony indicated that the

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liquid film moved across the agar surface at a similar rate for SBW25 and SBW25Q (data not

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shown), whereas the SBW25Q spidery spreader colony spread much more slowly than

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SBW25 (Supplementary Figure S2).

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A total of 2000 random mutants were individually inoculated, in duplicate, by sterile

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wire into the centre of 0.25% LB agar plates and plates were incubated for 48 h. Each

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mutant was assessed every 6-12 h against control strains SBW25 and SBW25Q. Twenty-

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seven mutants (AR1-26, AR28) exhibited altered surface motility compared to the controls

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and all 27 were retested in triplicate: no mutant exhibited a reversion to the wild-type

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phenotype, four mutants (AR1, AR2, AR8 and AR11) were completely unable to move from

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the point of inoculation, and the remaining 23 mutants exhibited altered patterns of surface

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motility (Figure 2). Some of these latter mutants exhibited the same spidery spreader

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pattern, but moved over the surface faster than SBW25Q, while others showed altered

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colony patterns when spreading over the surface.

Accepted Article

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To identify the location of the transposon in the genomes of these mutants,

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Arbitrarily-Primed PCR analysis was used to pinpoint the precise sequences of nucleotides

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spanning the insertion. These were mapped to the chromosome of SBW25 using the

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available genome sequence (Table 1). A variety of different genes, including those encoding

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putative enzymes and GGDEF signalling regulators, accounted for some of the 23 mutants

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with altered surface motility. A subset of cell membrane/wall associated proteins and

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lipoproteins were among these as well as some genes involved in metabolic processes

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including production of amino acids. These may indicate that cell replication is a key

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requirement for expression of the spidery spreader phenotype. However, the transposons in

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the 4 sessile mutants (AR1, AR2, AR8 and AR11) were all found to be inserted independently

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within genes PFLU2552 (viscB) and PFLU2553 (viscC), which are non-ribosomal peptide

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synthetase (NRPS) genes involved in making the lipopeptide surfactant viscosin. Of the 27

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mutants, only these four failed to produce a liquid film on the agar surface or froth in

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shaken cultures. Notably, these viscosin mutants and SBW25C, a viscosin knockout mutant,

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were affected in their ability to spread over 0.25% SSM agar, providing further evidence for

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the novel phenotype seen on 0.25% LB. While we were able to complement the deleted fleQ

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gene, the large size of the NRPS genes precluded individual cloning of the genes for
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complementation and a genomic cosmid clone containing the genes has not been found in

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the SBW25 cosmid library. We were therefore unable to create a complemented mutant for

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viscosin production, but the four independent insertions in two genes provide compelling

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proof of their involvement.

Accepted Article

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SBW25 concurrently employs flagellum-dependent and flagellum-independent viscosin-

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dependent surface motility

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A previous study reported that an SBW25 viscosin mutant (SBW25C) was unable to

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move over 0.6% SSM agar by swarming (de Bruijn et al., 2007) suggesting that surface

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motility is dependent on both flagella and viscosin production. Since SBW25C carries a

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transposon in the viscC NRPS, the mutation is not predicted to affect the flagellum

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biosynthesis system. However, the sessile phenotype observed with SBW25C by de Bruijn et

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al. (2007) suggested that mutation of viscC does indeed impact flagellum-dependent

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motility i.e. viscosin is essential for the surface spreading phenotype. We therefore

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complemented strain AR1 (Fla-, Visc-) with plasmid pFleQ to first examine bacterial surface

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motility on LB agar (0.25% and 0.6%). The complemented strain, termed AR1(pFleQ),

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exhibited surface motility producing a radial colony spreading from the inoculation point on

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0.25% LB agar, although the spread was slower than that of the wild-type (Figure 3). No

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liquid film was observed on the plate, the complemented strain produced flagella (data not

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shown), and we observed slow surface spreading on 0.6% LB agar (data not shown). We can

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therefore conclude that AR1(pFleQ) moves over the surface of 0.25% LB agar by flagellum-

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dependent motility without the need for viscosin. We next tested SBW25C on 0.25% LB

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agar; like AR1(pFleQ), this strain also spread across the agar surface (Figure 3), but without a

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liquid film being apparent. We suggest that the liquid film observed moving in front of the

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bacterial colony is viscosin, meaning that SBW25C is Fla+ Visc-, and that SBW25C can still

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move slowly over surfaces without viscosin. Notably, there was a subtle difference in the

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structure of the colony edge of the different strains on 0.25% LB agar. The wild-type SBW25

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colony edge was erose whereas AR1(pFleQ) and SBW25C colony edges were entire (Figure

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3): SBW25Q is the only strain that produces spidery tendrils. Finally, we tested the

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phenotype of SBW25C on SSM (0.25% and 0.6%) compared to the other strains. On 0.6%

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SSM, SBW25C did not move from the point of inoculation, confirming the previous

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observation made by de Bruijn et al. (2007), but we also did not see movement of SBW25 on

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0.6% SSM agar in contrast to de Bruijn et al. (2007); this is discussed further below.

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However, SBW25C did spread over the surface of 0.25% SSM and a difference in colony

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phenotype was also seen, with SBW25C (Fla+ Visc-) exhibiting a circular colony spread

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compared to the dendritic spread of wildtype (Fla+ Visc+) and SBW25Q (Fla+, Visc-) on this

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particular medium (Supplementary Figure 1). When considered together, it is clear that agar

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concentration and medium components affect surface motility behaviour. Moreover, these

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data indicate that both flagellum- and viscosin-dependent surface spreading motility

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contribute to colony spreading phenotype, which are most notable on 0.25% LB: SBW25

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wild-type has an erose colony edge when both flagella and viscosin operate together,

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compared to the entire colony edge phenotype when the flagellum alone is operating

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(SBW25C), or the spidery spreader colony phenotype when viscosin operates alone

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(SBW25Q). Wild-type cells apparently use both motility systems concurrently; this favours

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an explanation for the observed phenotype of fleQ deletion mutants in which deletion

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exposes an alternative motility system independent of FleQ. The slower spreading of

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AR1(pFleQ) and SBW25C relative to SBW25Q(pFleQ) shows that viscosin, while not

Accepted Article

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necessary for movement, increases speed of spread over a semi-solid surface. We therefore

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investigated whether these motility differences in vitro had effects in planta.

Accepted Article

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The flagellum is essential for root surface migration while viscosin can aid surface migration

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and is a key factor for SBW25 plant growth promotion

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An important question emerging from this study relates to the biological function of

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viscosin, which has previously been reported to lyse zoospores of the oomycete pathogen

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Phytopththora infestans as well as being involved in motility (de Bruijn et al., 2007). Thus,

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we tested (i) if viscosin contributes to bacterial motility over the root surface and aimed to

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resolve its contribution relative to flagellum-dependent motility and (ii) if viscosin is

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antagonistic to the soil oomycete pathogen Pythium by assessing the effects of the different

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bacteria in protecting plants grown in the presence of the oomycete. We first tested aim (i)

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and grew sugar beet seedlings in sterile vermiculite and in vitro on water agar plates kept

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vertically. After germination and seedling development (8 days), a 2 l drop of 105 bacterial

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suspension (SBW25 (Fla+, Visc+), SBW25Q (Fla-, Visc+), AR1 (Fla-, Visc-) and SBW25C (Fla+,

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Visc-)) was applied to the seedling hypocotyl. Bacteria were recovered after 1 and 3 days,

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but all strains were found to have reached the root tip at the end of the experiment (data

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not shown). We therefore repeated the experiment, but the seedlings were laid flat to

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prevent any gravitropic or moisture-aided bacterial distribution over the root surface. We

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postulated that if motility is important for bacterial spread over the root surface, then this

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design should resolve the individual contributions of the flagellum and viscosin without

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compounding effects of gravity or moisture. The distal 1 cm of the plant root was sampled

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at time zero, and after 1 and 3 days. Wild-type SBW25 was observed to colonize the root tip

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after 3 days whereas neither SBW25Q nor AR1 were detected. SBW25C was observed to

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colonize the root tip, but was slower to reach the tip (Figure 4; General Linear Model (GLM),

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Strain as main effect: F3, 32 = 3.353, P = 0.031). Taken together, these data clearly show that

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the flagellum is essential for bacterial movement over the root surface whereas viscosin is

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dispensable; however, viscosin appears to play a role in aiding the spread of motile (Fla+)

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bacteria (GLM, Strains categorised by flagella / viscosin production as main effect: Flagella,

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F1, 32 = 9.761. P = 0.004; Viscosin, F1, 32 = 0.148, P = 0.703).

Accepted Article

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Our second hypothesis posited that viscosin plays a role in plant growth promotion

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through antagonism of oomycete root pathogens. Since SBW25 is known to protect plants

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from oospores and hyphae of the oomycete Pythium (Naseby et al., 2001) and viscosin is

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known to lyse oomycete zoospores (de Bruijn et al., 2007), we focused on testing the in vivo

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effects by assessing sugar beet seedling germination and root and shoot weight and length.

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Seeds were soaked in different bacterial suspensions or buffer alone and sown into soil with

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or without Pythium. In soil without Pythium, the presence of the different bacterial strains

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had no discernible effect on seedling germination or development compared to seedlings

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grown in the absence of bacteria (data not shown); this demonstrates that viscosin alone

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does not promote plant growth. When untreated seeds were sown into soil containing

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Pythium, there was a negative impact on root development (Figure 5a). However, seeds

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soaked in bacteria before sowing showed genotype-specific effects. Specifically, the type of

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bacterial strain inoculated significantly affected the root length (GLM: F7,78 = 4.520, P <

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0.001) but not the shoot length (GLM: F7,78 = 1.419, P = 0.210) after 14 days growth.

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Subsequent Tukey-Kramer HSD tests were conducted as multiple comparisons of means.

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The average root length in the No Bacteria treatment group was 2.07 0.79 s.d., and

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treating seeds with wild-type SBW25 increased root length more than 2-fold (SBW25 vs. No
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Bacteria, P < 0.001). The flagellum mutant (SBW25Q) was also able to improve root growth

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compared to seeds that were not treated with bacteria (SBW25Q vs. No Bacteria, P = 0.054)

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to a level that was not significantly different from SBW25 (SBW25 vs. SBW25Q, P = 0.288). In

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comparison, the SBW25C viscosin mutant did not show recovery of root growth, with root

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length similar to that of the bacteria absent treatment (SBW25C vs. No Bacteria, P = 0.686).

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Likewise, the flagellum-viscosin mutants (AR1, AR2, AR8 and AR11) exhibited a mean root

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length that was not significantly different from the treatment without bacteria (for all

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flagellum-viscosin mutants vs. No Bacteria, P > 0.05). In other words, only the viscosin

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producing strains (SBW25 and SBW25Q) significantly promoted root growth in the presence

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of Pythium, whereas the difference between viscosin knockouts and the no bacteria

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treatment was not significant (Figure 5a). This indicated viscosin production is important for

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protection of the plant in the presence of Pythium. Although there appears to be a slight

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reduction in shoot length in flagellum-viscosin mutants, this effect is not significant (Figure

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5a; GLM: Flagellum/viscosin production as main effect, F4, 81 = 74.193; P = 0.068).

Accepted Article

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Soaking the seeds in the different mutant types before planting also had an effect on

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the number of seedlings that successfully germinated in soil supplemented with Pythium

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(SBW25 vs. No Bacteria, P = 0.035; Figure 5b). When mutants were sorted based on whether

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they produced viscosin, we found those seeds that had been soaked in mutants that

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expressed viscosin (i.e. SBW25 and SBW25Q) produced on average 30% more seedlings than

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those that were unable to produce viscosin (T-test: t19 = 2.78; P = 0.012), whereas flagellum

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expression was not found to have a significant effect (P > 0.05). However, we acknowledge

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that the interaction between a PGPR and the plant host and any benefits it provides is likely

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to be more complex than one metabolite, because although the seeds treated with viscosin

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mutants led to a reduced number of germinated seedlings, seedling germination appears to

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be better than if no bacteria were applied at all.

Accepted Article

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Discussion

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Plant growth promoting rhizobacteria (PGPR) are used in agriculture and are gaining

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recognition as key future components of integrated pest management to replace or

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complement particular chemotherapeutic plant treatments (Mendes et al., 2011). However,

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there are concerns surrounding the use of some PGPR strains in terms of their durability and

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efficacy (de Weger et al., 1995). Fundamental to this issue is an understanding of the

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dynamics of bacterial function in the natural environment. The synergy of fundamental

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biology with applied science helps to unravel the different mechanisms employed by PGPR,

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their relative contributions to the agricultural application, and ultimately, their contribution

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to ecological performance. This, of course, requires extensive analysis of bacterial traits and

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empirical testing in the plant environment. In this work, we extended functional analysis of

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the PGPR P. fluorescens SBW25 emanating from a previous gene regulation study (Giddens

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et al., 2007) and found that viscosin plays important roles in motility and plant protection.

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Our results also raise important questions concerning the use of motility nomenclature and

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the conditions used for testing different motility mechanisms.

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Although the focus for this paper is on viscosin, several other genes were identified by

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the mutagenesis that affected surface spreading motility. A range of quantitatively and

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qualitatively different spreading phenotypes were seen (Figure 2). The AR6, AR12 and AR22

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transposon sites were individually situated within putative GGDEF/EAL regulators. These

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regulators are commonly found amongst sequenced bacterial genomes and are involved in

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the production and breakdown of the bacterial intracellular second messenger cyclic-di15
This article is protected by copyright. All rights reserved.

GMP (Kulesekara et al., 2006; Paul et al., 2004; Simm et al., 2004). They are involved in

339

several cellular mechanisms including the biosynthesis of exopolysaccharides, the formation

340

of biofilms and surface motility (Ryjenkov et al., 2005). Phenotypic shifts from sessile to

341

motile strains in Salmonella enterica serovar Typhimurium, E. coli and P. aeruginosa have

342

been attributed to the activity of GGDEF and EAL proteins (Simm et al., 2004).

Accepted Article

338

343

Whilst AR13 and AR18 both contained independent mutations in the same gene,

344

PFLU0735, the mutants were phenotypically distinct, with AR13 displaying increased

345

motility. This may be due to the exact location of transposon insertion, but could be the

346

result of a secondary mutation in the chromosome. PFLU0735 is of unknown function, but is

347

closely linked to PFLU0734, which encodes a putative lipoprotein. In Gram-negative

348

bacteria, lipoproteins that are most often located at the bacterial outer membrane are

349

frequently associated with many aspects of pathogenicity including motility and invasion

350

(Kovacs-Simon et al., 2011; Zhang et al., 1998). For example, mutation of Flavobacterium

351

johnsoniae lipoprotein GldJ severely impaired gliding motility over agar and glass (Braun &

352

McBride, 2005). Interestingly, the transposon in strain AR9, which moves faster than the

353

SBW25Q progenitor, sits intergenically between a putative LysR regulator and PFLU0129,

354

which is part of a putative lipoprotein transporter system. While this may affect an

355

intergenic transcript, the orientation of the transposon is such that its outward facing

356

terminal promoters could be activating PFLU0129. Thus, AR9 may be moving faster because

357

it is secreting more surfactant. This could be a good target cluster for engineering secretion

358

of surfactant to maximize biocontrol activity.

359

The biosurfactant viscosin has been characterized by several other research groups

360

and has been implicated as important for motility (de Bruijn et al., 2007), as a biocontrol

361

agent that lyses oomycete zoospores (de Bruijn et al., 2007), for protozoan grazing defence
16
This article is protected by copyright. All rights reserved.

(Mazzola et al., 2009) and in altering soil water characteristics (Fechtner et al., 2011). In this

363

study, we have found that viscosin production is important in spidery spreading, which

364

operates independently of the flagellum; we conclude spidery spreading is a type of sliding

365

motility (Henrichsen, 1972). We cannot rule out the possibility that pili are also employed in

366

the sliding phenotype, as they have been implicated in swarming of P. aeruginosa (Kohler et

367

al., 2000). However, the evidence suggests pili are not involved in spidery spreading, as no

368

pil mutants were found in the genetic screen, and we also observed the normal twitching

369

motility phenotype for SBW25Q.

Accepted Article

362

370

Biosurfactants have been implicated in surface spreading in other bacteria as well as

371

SBW25 (Burch et al., 2012; Raaijmakers et al., 2010; Tremblay et al., 2007). In effect, the

372

biosurfactant reduces the surface tension of the agar, and, as the bacteria divide, they slide

373

over the biosurfactant moving outward from the point of inoculation. Sliding motility is

374

slower than swarming motility, which we suggest accounts for the relatively slow spread of

375

SBW25Q compared to the wild-type (Supplementary Figure S2), i.e. we suggest that its

376

motility relies on cell division rather than active propulsion by the flagellum. This correlates

377

with the observation that some of the SBW25Q transposon mutants (e.g. in parA, hisF,

378

ampD, xerD) exhibited slower surface spreading, which may be due to their function in cell

379

division and metabolism.

380

Since we observed that SBW25 produced viscosin and SBW25C (Fla+, Visc-) was still

381

able to move over a surface, we can conclude that both flagellum-dependent motility and

382

viscosin-dependent motility are in operation on the agar plates. This was also reflected in

383

the subtle differences in the colony edges of the different strains. The microbial mechanics

384

behind swarming and sliding motility have been a point of contention for many years, and

385

surfactants have been implicated in both (Murray & Kazmierczak, 2008). Here, we provide
17
This article is protected by copyright. All rights reserved.

direct evidence that biosurfactant viscosin production is crucial in the expression of the

387

sliding motility phenotype seen when flagella function is lost. An interesting observation

388

that arises from our work is that viscosin spreads in a radial pattern for both SBW25 and

389

SBW25Q. This raises the question of why SBW25Q spreads dendritically. Tremblay et al.

390

(2007) proposed that this effect was mediated by self-produced di-rhamnolipid attractants

391

and 3-(3-hydroxyalkanoyloxy) alkanoic acid repellents. Whether a similar mechanism

392

operates in P. fluorescens in the absence of fleQ-dependent swarming motility remains to be

393

seen.

Accepted Article

386

394

One of the unexpected outcomes of our experiments has been the observation of

395

different motility phenotypes when using the same medium for surface motility

396

experiments as used in other experiments in the literature. In our experiments we have

397

observed SBW25 movement through 0.25% LB agar (which we interpret as swimming) and

398

over the surface of 0.25% LB agar, but not over the surface of 0.6% LB or SSM agar. The

399

literature would suggest that lack of movement over 0.6% agar should result in the

400

conclusion that the bacterium cannot swarm. However, we know from testing AR1(pFleQ)

401

and SBW25C, which do not produce viscosin and do produce flagella, that these strains can

402

move over the surface of 0.25% agar. We suggest this is likely to be swarming motility rather

403

than swimming motility and thus the strains are actually able to move by swarming, but

404

clearly this needs further analysis. Evidently, the conditions used for carrying out motility

405

experiments must be very carefully controlled to avoid misinterpretation. For example, the

406

apparent disparity between our experimental observations and those of de Bruijn et al.

407

(2007) suggesting a viscosin mutants cannot spread over a surface can be explained simply

408

as a difference in methodology: to investigate the spidery spreading phenotype, we used LB

409

0.25% soft-agar plates rather than 0.6% standard succinate medium which is not conducive
18
This article is protected by copyright. All rights reserved.

to either spidery spreading or swimming. However, for comparison, we performed motility

411

assays on 0.25% SSM, 0.6% SSM and 0.6% LB media (Supplementary Figure S1); these assays

412

confirmed that although SBW25C does have functioning flagella, it cannot move over the

413

surface of higher agar concentration. In doing this, wildtype SBW25 (our control) swarming

414

motility was not observed at agar concentrations of 0.6% in our experiment, but was

415

observed on 0.6% SSM agar by de Bruijn et al. (2007). The most likely explanation will be

416

due to subtle differences in the culture medium used in each study. In our initial

417

experiments at the start of our study, we found that subtle alterations in medium

418

preparation, such as using different agars that have different water retention properties,

419

was sufficient to change the SBW25Q phenotype. We also observed that surface motility on

420

LB was reduced and eventually abolished as we increased the agar concentration (data not

421

shown, full media details given in Supplementary Table S1). The high sensitivity of motility

422

phenotypes to culture medium and growth conditions could explain the disparity in

423

observations. It may be that differences in preparation of SSM medium, including source of

424

agar and volume of medium in plates, is enough to distinguish the phenotypes in the two

425

studies. Importantly, the observations from the two studies suggest that flagellum-based

426

swarming is insufficient for surface movement over drier surfaces, and so the role of

427

viscosin in promoting surface spreading in drier conditions is a good target for future

428

research.

Accepted Article

410

429

Our results also suggest an ecological role for viscosin-mediated motility. Mutation

430

of fleQ completely abolished the ability of SBW25 to reach the root tip, confirming a result

431

previously observed in competition experiments with P. fluorescens F113 (Capdevila et al.,

432

2004). This suggests fleQ-flagellum-dependent swarming motility plays a key role in motility

433

over a horizontal root surface; however, as FleQ plays a regulatory role, it is possible that
19
This article is protected by copyright. All rights reserved.

differences could be due to phenotypes other than motility that manifest in the mutant. The

435

mutation of viscosin did not prevent SBW25 from reaching the root tip, but the viscosin-

436

mutant was slower to reach the root tip. This suggests viscosin enhances bacterial surface

437

spreading when employing flagella, but is not essential. These experiments indicate that

438

expression of the flagellum and viscosin may aid horizontal surface spread, and may

439

therefore be important for spreading over lateral roots. Indeed, sliding motility linked to

440

bacterial cell division has been proposed as the method by which Rhizobia move within

441

legume root infection threads (Gage & Margolin, 2000; Fournier et al., 2008). However, it

442

appears they are less important for spreading in a vertical root system. This difference may

443

go some way toward explaining conflicting results of other studies on the importance of

444

flagella to P. fluorescens root colonisation; for example, while de Weger et al. (1987) found

445

flagella were essential, and Turnbull et al. (2001) found an advantage to motility in root

446

attachment, the results of Bowers and Parke (1993) suggested passive movement with

447

water flow played a more important role than motility.

Accepted Article

434

448

Previous studies have observed that viscosin exhibits anti-fungal and anti-oomycete

449

activity by inducing encystment of Pythium zoospores and adversely affecting mycelia of

450

Rhizoctonia solani and Pythium ultimum, both in vitro and in planta (Nielsen et al., 1999; de

451

Souza et al., 2003). SBW25 is also known to protect plants from the root pathogen P.

452

ultimum, although the underlying basis for this was not determined (Naseby et al., 2001);

453

our results suggest that viscosin production contributes to plant protection in SBW25, but is

454

not the only factor involved.

455

Our results paint a complex picture in which a single substance, viscosin, fulfils

456

multiple functions, including motility and plant protection; correspondingly, each of these

457

functions is the result of multiple, possibly partially redundant, genetic pathways. We would
20
This article is protected by copyright. All rights reserved.

suggest that motility is the primary function of viscosin, as swift colonisation of growing

459

roots provides an obvious selective advantage to the bacterium, and that the ability to lyse

460

oomycete zoospores is an incidental by-product that happens to benefit plants. However,

461

there may not be a clear-cut distinction between the primary and secondary functions of a

462

gene: selection on each role is likely to vary depending on the environmental and social

463

conditions experienced by a bacterial population. Rhizosphere communities are capable of

464

rapid evolutionary change (Kiers & Denison, 2008), so we must understand these selective

465

processes if we wish to optimize the agricultural benefits of PGPRs and improve the stability

466

of agriculturally beneficial traits.

Accepted Article

458

467

In summary, we found that viscosin plays a role in P. fluorescens motility, both in

468

vitro and in planta, that is additional to the flagellum-based behaviour. This showcases the

469

flexibility of motility systems used by P. fluorescens and contributes to our understanding of

470

the many motility variants we observe in natural populations of soil microbes (Achouak et

471

al., 2004). We have also observed the beneficial impact of viscosin on plant health by

472

suppressing the detrimental effects of a root pathogen. The dual function of viscosin

473

production in motility and anti-fungal/oomycete action identifies it as a critical trait in terms

474

of P. fluorescens function as a biocontrol agent. Biosurfactants may be useful targets for

475

formulation as a seed or plant treatment compound, or the viscosin genes for a synthetic

476

biology approach to creating optimal PGPR.

477
478

Experimental procedures

479
480

Strains, plasmids and growth conditions

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This article is protected by copyright. All rights reserved.

The principal strains and plasmids used are shown in Table 2. Pseudomonas

Accepted Article

481
482

fluorescens strains were routinely grown in lysogeny broth (LB) or agar (1.5%) at 27 oC for 16

483

h or 48 h, respectively. Escherichia coli was grown in LB broth or agar at 37oC for 16 h or 24

484

h, respectively. Antibiotics and supplements were included at the following final

485

concentrationsg ml-1): kanamycin, 50; X-gal (5-bromo-4-chloro-3-indolyl--D-

486

galactopyranoside), 40; nitrofurantoin, 100; tetracycline, 15. CFC (Oxoid) was used

487

according to manufacturers instructions. The comparison strains used in this study are:

488

wild-type P. fluorescens SBW25, and its derived fleQ deletion mutant (fleQ), hereafter

489

SBW25Q (details of constructions given below; the phenotype of this strain is flagellum

490

negative (Fla-) but viscosin positive (Visc+)) - these were used as control strains. In addition

491

we used the mutant SBW25viscC::TnMod-OKm (hereafter SBW25C, gratefully obtained from

492

Raaijmakers and de Bruijn; Fla+, Visc-), a functional viscosin knock out, for comparison

493

against viscosin mutants isolated from the transposon library. Biparental matings were done

494

by mixing 750 l S17-1pir carrying either plasmid pSCR001 or pFleQ and 750 l P.

495

fluorescens strain, spinning cells at 13000 rpm to obtain a pellet, which was transferred to

496

an LB plate. After 16 h incubation at 30oC, the pellet was streaked onto the relevant

497

selective medium.

498
499

500

Molecular analyses
When designing deletion of fleQ, we were mindful of our lack of knowledge of

501

potential cis-encoding regulatory sites located outside of fleQ, which might be important for

502

flanking genes (particularly the regulators fleS and fleR); therefore, partial deletion of the

503

fleQ gene PFLU4443 was done. To do this we removed the ATPase and most of the receiver

22
This article is protected by copyright. All rights reserved.

domain by stitching together flanking regions of fleQ by SOE-PCR (splicing by overlapping

505

extension using the polymerase chain reaction). The resulting 1.64 kb PCR product was first

506

cloned into pCR8/GW/TOPO using the TA cloning kit from Invitrogen. After its DNA

507

sequence was verified, the DNA fragment was cloned into the BglII site of pUIC3 (ref. Rainey

508

1999). The resultant construct pUIC3-140 was then conjugated into SBW25 with the help of

509

pRK2013, and transconjugants were selected on LB supplemented with tetracycline and X-

510

Gal. The double crossover mutant was selected using the previously described method of

511

cycloserine enrichment (Zhang and Rainey, 2007). White colonies were picked and assessed

512

by PCR to confirm double-crossover deletion of fleQ. This confirmed that the entire 54

513

interaction domain and most of the receiver domain were deleted in the strain, which we

514

named SBW25Q (Table 2). Transposon mutagenesis of SBW25Q with IS--Km/hah

515

transposon was carried out by conjugation of S17-7pir (pSCR001) with SBW25Q;

516

subsequent Arbitrarily-Primed PCR and genome analysis was done to determine the location

517

of transposon insertions (Giddens et al., 2007).

Accepted Article

504

518
519

520

Motility assays
Motility assays for the observation of swarming and sliding motility used full strength

521

LB with 0.25% agar. Agar plates were always carefully made by pipetting 30 ml of molten

522

agar into 88 mm Petri dishes and the plates allowed to set at room temperature for 4 h.

523

Plates were then placed in a laminar flow hood and lids were then removed for 30 min.

524

Assays were conducted by dipping a sterile wire into a single colony and stabbing the wire

525

into the centre of an agar plate so that the wire touched the bottom of the plate. The agar

526

plates were incubated on a bench without stacking in a walk-in incubator maintained at

23
This article is protected by copyright. All rights reserved.

27oC and without light. Motile bacteria were observed as large colonies that moved across

528

the top of the agar. Swimming was assessed by monitoring bacterial movement through

529

one-tenth strength LB 0.25 % agar; bacterial movement was observed as a halo effect with

530

no bacterial movement over the surface. Twitching motility was assessed by stab

531

inoculation of a single colony to the bottom of a petri dish through a 1% full strength LB agar

532

layer, and subsequently visualising bacterial movement across the Petri dish at the dish-agar

533

interface.

Accepted Article

527

534

For the root migration experiments, initial experiments were carried out by growing

535

sugar beet seedlings in sterile vermiculite microcosms after Rainey (1999) and on 1.5%

536

water agar plates with the plates kept vertical to encourage normal root growth on the agar.

537

A 2 l drop of 105 bacterial suspension was applied directly to the hypocotyl region on the

538

agar plates and to the vermiculite-root interface in the microcosms. The latter method was

539

adapted so that the plates and therefore the seedlings were laid flat before bacterial

540

application to the hypocotyl. In all experiments, bacterial locomotion was assessed by

541

dissecting the 1cm of the root from the tip (a distance of about 5 cm from the hypocotyl) to

542

enumerate the bacteria. The root tissue was crushed in 500 l phosphate buffered saline

543

with an Eppendorf pestle, made up to 1ml, serially diluted and plated onto the relevant

544

medium.

545
546

Plant growth promotion assays

547

Five 1 cm plugs of Pythium FFP1 (a kind gift from Francesco Favaron) taken from a 5-

548

day old culture on potato dextrose agar was added to sterile sand (95 g), oats (5 g) and 20

549

ml 1/10-strength Czapek Dox, mixed and incubated at 27oC for 7 days. The Pythium mat

24
This article is protected by copyright. All rights reserved.

(oospores and hyphae) was aseptically removed from the flask, shaken to discard loose sand

551

and then mixed with SHL potting compost (Lincoln, UK) to 3% (w/w) achieving circa 104 cfu

552

ml-1 Pythium. A 12-pot tray module was filled with soil with or without Pythium. Sugar beet

553

seeds (14 seeds in each treatment group) were washed to discard the protective storage

554

coat and then soaked in 20 ml of bacterial suspension (109 cfu ml-1) for 5 min before

555

planting 1-cm deep into the compost mix. The design of the seed treatment planting with

556

different bacterial genotypes was randomized and 5 seeds were planted in each pot. In

557

addition there were 3 biological replicates in each treatment group. The plants were

558

maintained at 20oC for 14 days and the seed germination recorded daily. After 14 days the

559

plants were destructively harvested and the roots and shoots separated and assessed for

560

length and weight.

Accepted Article

550

561
562

563

Photography and Electron microscopy

P. fluorescens cells were negatively stained on copper grids with 2% uranyl acetate for

564

10 min before being soaked in water for 10 min. Cells were imaged using a Philips CM20

565

analytical transmission electron microscope. Motility plates were imaged using a Nikon

566

digital camera. Images were altered for contrast and sharpness using Adobe Photoshop CS6

567

software.

568
569

Acknowledgments

570

Qassim University provided funding to do this work; TBT is funded by a Leverhulme Trust

571

grant to LJJ and RWJ. We thank Jos Raaijmakers and Irene de Bruijn for the kind gift of

572

SBW25C and Francesco Favaron for the kind gift of Pythium FFP1.

25
This article is protected by copyright. All rights reserved.

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Accepted Article

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Accepted Article

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Accepted Article

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29
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Accepted Article

719
720
721

30
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Accepted Article

756
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Zhang, X.-X. and Rainey, P. B. (2008) Regulation of copper homeostasis in Pseudomonas

fluorescens SBW25. Environmental Microbiology, 10, 3284-3294.

Accepted Article

792
793
794

32
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Accepted Article
795

Table 1. Summary of surface motility mutants of SBW25Q.

Mutant Transposon Phenotype*
Gene
name coordinates
code
AR1
2813319Sessile
PFLU2553
2813320
AR2
2798991Sessile
PFLU2552
2798992
AR3
3280199Faster
PFLU3012
3280200

Gene
name
viscC
viscB

AR4

42903554290356

Slower

PFLU3889

AR5

51177785117779
30430003043001
67106366710637
28058022805803
139969139970

Slower

PFLU4639

Faster

PFLU2753

Altered

PFLU6127

parA

Sessile

PFLU2552

viscB

Faster

Intergenic

Faster

PFLU2912

AR6
AR7
AR8
AR9

AR10

31749083174909

Predicted gene product and

function
Non-ribosomal peptide synthase;
surfactant synthesis
Non-ribosomal peptide synthase;
surfactant synthesis; linked to viscC
Putative Cytochrome C oxidase
subunit; linked to 3011

Acyl-CoA dehydrogenase unknown

function; linked to 3890-2 (accA),
lipid biosynthesis
Hypothetical protein - Unknown
function; linked to 4638
Putative GGDEF/EAL regulator;
turnover of cyclic-di-GMP
ATPase involved in chromosome
partitioning
Same as AR2 (independent
mutation)
Between PFLU0128 and 0129;
upstream 0129, lipoprotein
transporter of unknown function
Putative membrane protein; linked
to PFLU2911, ATP-dependent DNA
ligase
33

This article is protected by copyright. All rights reserved.

Relevant
references
(De Bruijn et
al., 2007)
(De Bruijn et
al., 2007)
(Southey-Pillig
et al., 2005)
(Pitcher &
Watmough,
2004)
This study

This study
(Kulesekara et
al., 2006)
(Lasocki et al.,
2007)
(De Bruijn et
al., 2007)
(Zhang et al.,
1998)
This study

Accepted Article

AR11

28146862814687
58552995855300
836620836621

Sessile

PFLU2553

Faster

PFLU5329

Faster

PFLU0735

AR14

898973898974

Faster

PFLU0798 ampD

AR15

41632134163214
363595363596
898973898975

Faster

PFLU3766

Slower

PFLU0331

Slower

PFLU0798 ampD

836593836594
47918254791826
55037395503740

Altered

PFLU0735

Faster

PFLU4345

Slower

PFLU5008

xerD

57511695751170

Altered

PFLU5242

panC

AR12
AR13

AR16
AR17

AR18
AR19
AR20

AR21

viscC

hisF

Same as AR1 (independent

mutation)
Putative GGDEF/EAL regulator;
turnover of cyclic-di-GMP
Hypothetical protein - Unknown
function; linked to 0734
(lipoprotein)
N-acetylmuramyl-L-alanine amidase
responsible for breakdown of
MurNAc-tri-, tetra-, and
pentapeptides to release the
peptides for recycling; linked to
0799 (membrane prot)
Hypothetical protein of unknown
function
Histidine metabolism; linked to
0327-0330
Same as AR14 gene function
(identical mutation)
Same as AR13 gene function
(independent mutation)
Putative GTP cyclohydolase I
Site-specific recombinase for DNA
replication, recombination and
repair
Panthothenate synthetase for
Coenzyme metabolism; linked to
5241 (panB)
34

This article is protected by copyright. All rights reserved.

(De Bruijn et
al., 2007)
(Kulesekara et
al., 2006)
This study

(Langaee et al.,
2000, Jacobs et
al., 1995)

This study
This study
(Langaee et al.,
2000, Jacobs et
al., 1995)
This study
This study
(MartinezGranero et al.,
2005)
This study

Accepted Article

AR22
AR23
AR24

AR25
AR26
AR28

58553275855328
55304225530423
14797121479713

Slower

PFLU5329

Same as AR12 (independent

mutation)
Putative transglycosylase

Slower

PFLU5035

Faster

PFLU1343

livG

Amino acid transport system; linked

to 1342-5

3883838839
18700651870066
8968898968890

Faster

PFLU0036

trpB

Faster

PFLU1699

Altered

PFLU0797

Tryptophan synthesis; linked to

0035 (trpA)
Putative acyl-CoA thioesterase;
close to 1700
Hypothetical protein of unknown
function; close to 0798 (AR14 and
AR17)

(Kulesekara et
al., 2006)
(Suvorov et al.,
2008)
(Nazos et al.,
1985, Adams
et al., 1990)
This study
This study
This study

796
797

* Motility phenotypes are categorised here relative to their progenitor strain SBW25Q; note that all mutants shown here are therefore on

798

fleQ background. See Figure 2 for photographs.

799

35
This article is protected by copyright. All rights reserved.

Accepted Article
800
801

Table 2 Principal bacterial strains and plasmids

Strain
Pseudomonas fluorescens
SBW25

Relevant characteristics

Source/reference

Wild-type strain isolated from phyllosphere of sugar beet plant.

SBW25Q
SBW25C
AR1
E. coli
DH5
S17-1pir
Plasmids
pUIC3
pRK2013
pUIC3-140
pSCR001
pBBR1MCS-5
pFleQ

Unmarked deletion of fleQ; Fla , Visc

+
SBW25viscC::TnMod-OKm; Fla , Visc
+
fleQ viscC::IS--Km/hah; Fla , Visc

Rainey & Bailey,

(1996)
This work
de Bruijn et al. (2007)
This work, Table 1

F , recA, lacU169(80 lacZM15), endA, hsdR, gyrA.

r
r
- +
Tp , Sm , recA, thi, hsdR M , RP4::2-Tc::Mu::Km::Tn7, pir lysogen

Gibco-BRL
Simon et al. (1983)

Tc , Tra , Mob , R6K replicon

+
r
Helper plasmid, Tra , Km
r
pUIC3 containing 1.64 kb DNA fragment for fleQ deletion, Tc
+
r
IS--Km/hah plasmid, Mob , Km
+
r
Broad host range cloning vector, Mob , Gm
r
Full length SBW25 fleQ gene in pBBR1MCS-5, Gm

802

36
This article is protected by copyright. All rights reserved.

Rainey (1999)
Ditta et al (1980)
This work
Giddens et al. (2007)
Kovach et al. (1995)
Giddens et al. (2007)

803
804
805

Figure 1. FleQ controls production of flagella and swarming motility, but is not necessary

806

for surface motility. (a) Electron microscopy shows SBW25 produces a flagellum whereas

807

SBW25Q does not; ectopic expression of FleQ in SBW25Q rescues the mutation. Arrows

808

show the presence of flagella. (b) Spreading motility phenotypes of the wild-type (SBW25),

809

the aflagellate mutant (SBW25Q), and the complemented mutant (SBW25Q(pFleQ)).

Accepted Article

Figure Legends

810
811

Figure 2. Surface motility phenotypes of SBW25Q and 27 transposon mutants after 30

812

hours. Note mutants are derived from SBW25Q and are therefore on a fleQ background.

813

AR16, AR17 and AR23 all showed slow spreading compared to AR1, AR2, AR8 and AR11,

814

which never moved even after 48 h.

815
816

Figure 3. FleQ and viscosin can both promote surface spreading motility. Surface motility

817

phenotype of motility mutants on LB agar plates (0.25%) after 12 hours.

818
819

Figure 4. Viscosin aids bacterial spread over plant roots. Number of colony forming units

820

(CFUs/ml) across the different mutant types, shown on a log scale: wild-type (SBW25),

821

flagella knock-out (SBW25Q), viscosin knock-out (SBW25C) and flagella-viscosin knock-out

822

(AR1), detected at the root tip 3 days after inoculation at the hypocotyl. Each data point

823

represents the mean of three biological replicates and error bars are the standard error of

824

the means. Note that the minimum detection limit was 100 cfu.

825
826

Figure 5. Viscosin aids seedling germination and plant growth promotion. Bar shading

827

represents flagella/viscosin expression profile: white bars = no bacteria; grey striped bars =
37
This article is protected by copyright. All rights reserved.

flagella and viscosin production; light grey bars = viscosin expression but no flagella

829

expression; dark grey bars = flagella expression but no viscosin expression. (a) Root and

830

shoot length of sugar beet seedlings were measured in seeds that had been grown in the

831

presence of Pythium, and soaked in either the wild-type (SBW25) or one of the mutants

832

(SBW25Q, SBW25C and AR1, 11, 2 and 8) or no bacteria, prior to sowing; (b) Seedling

833

germination yield of sugar beet seeds soaked in the bacteria described in (a). Plants grown

834

in soil without added Pythium did not show significant differences in root or shoot

835

characteristics or germination yield (P>0.05). Values are the means of 3 biological replicates,

836

each containing 5 seeds. Error bars are the standard error of the means.

Accepted Article

828

837
838

Supplementary Figure S1. Motility phenotypes on different nutrients and agar

839

composition. Motility assays were conducted on 0.25% standard succinate medium (SSM)

840

to assess the surface motility phenotype on agar used by other studies. In addition, motility

841

assays were performed at 0.6% agar concentrations on SSM and LB to determine whether

842

spreading motility was still observable under higher agar concentrations. All strains shown

843

after 48 hours.

844
845

Supplementary Figure S2. Surface spreading reliant on viscosin alone is slower than

846

spreading when the flagellum is employed.

847

The area of colony spreading and growth on an LB agar plate (0.25%), for SBW25 and

848

SBW25Q was measured every 2 hours over a 26 hour period. Each data point is an individual

849

replicate (with 10 replicates for SBW25, and 9 replicates for SBW25Q), closed circles

850

represent SBW25 and open circles SBW25Q. Interpolation lines show the mean spreading

851

across the dataset at each time-point, SBW25 is represented by a solid line and SBW25Q by
38
This article is protected by copyright. All rights reserved.

a dotted line. The data shows that the rate of spreading is faster, and the time taken to start

853

rapidly spreading is quicker in SBW25 compared to SBW25Q. Therefore, this suggests that

854

surface motility that is solely dependent on viscosin has a greater dependency on cell

855

replication (i.e. cells have to grow to move over the surface of the surfactant).

Accepted Article

852

856

39
This article is protected by copyright. All rights reserved.

Accepted Article

857
858
859
860
861

Supplementary Table S1:

Microbiological media used for motility assays
Medium
Luria-Bertani (LB) broth

Standard succinate medium (SSM)

Recipe and Manufacturers details

10g/l tryptone (BD, Oxford, UK)
5g/l NaCl (Oxoid Ltd., Basingstoke, UK)
For agar plates, use Difco Agar (BD, Oxford,
UK) at 10 g/l (twitching), 6 g/l or 2.5 g/l
(spreading/swarming) depending on motility
type being tested. 1/10 strength LB with 2.5
g/l agar were used for swimming plates.
6 g/l KH2PO4 (Sigma Aldrich, Dorset, UK)
3 g/l K2HPO4 (Fisher Scientific,
Loughborough, UK)
1 g/l (NH4)2SO4 (Fisher Scientific,
Loughborough, UK)
0.2 g/l MgSO4.7H2O (Fisher Scientific,
Loughborough, UK )
4 g/l Succinic acid (Sigma Aldrich, Dorset,
UK)
Correct pH to 7.0 using KOH
For agar plates add 6 g/l or 2.5 g/l Difco
Agar (BD, Oxford, UK) depending on
motility type being tested.

862
863

40
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Accepted Article

864

865
866
867

emi_12469_f1a

41
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Accepted Article

868

869
870
871

emi_12469_f1b

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Accepted Article
872
873
874

emi_12469_f2

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Accepted Article
875
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877

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Accepted Article
878
879
880

emi_12469_f4

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Accepted Article
881
882
883

emi_12469_f5a

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Accepted Article
884
885
886

emi_12469_f5b

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