FleQ Coordinates Flagellum-Dependent and -Independent Motilities in Pseudomonas syringae pv. tomato DC3000

Abstract

bMotility plays an essential role in bacterial fitness and colonization in the plant environment, since it favors nutrient acquisition and avoidance of toxic substances, successful competition with other microorganisms, the ability to locate the preferred hosts, access to optimal sites within them, and dispersal in the environment during the course of transmission. In this work, we have observed that the mutation of the flagellar master regulatory gene, fleQ, alters bacterial surface motility and biosurfactant production, uncovering a new type of motility for Pseudomonas syringae pv. tomato DC3000 on semisolid surfaces. We present evidence that P. syringae pv. tomato DC3000 moves over semisolid surfaces by using at least two different types of motility, namely, swarming, which depends on the presence of flagella and syringafactin, a biosurfactant produced by this strain, and a flagellumindependent surface spreading or sliding, which also requires syringafactin. We also show that FleQ activates flagellum synthesis and negatively regulates syringafactin production in P. syringae pv. tomato DC3000. Finally, it was surprising to observe that mutants lacking flagella or syringafactin were as virulent as the wild type, and only the simultaneous loss of both flagella and syringafactin impairs the ability of P. syringae pv. tomato DC3000 to colonize tomato host plants and cause disease. Motility plays a pivotal role in the spreading of bacteria across surfaces and colonization, contributing to the formation of structured communities called biofilms (1). Efficient bacterial motility under diverse environmental conditions, from liquid to semisolid and solid surfaces, is achieved by flagellum-dependent swimming and swarming or flagellum-independent twitching, gliding, nonsocial gliding, and sliding (2, 3, 4). Swimming is a flagellum-driven motility observed in bacteria moving through liquids or semisolid media, such as low-percentage agar (0.2% to 0.4%). Twitching is a slow cell movement on surfaces mediated by the extension and retraction of type IV pili (5). Gliding, a surface movement extensively studied in myxobacteria, does not require flagella or pili but involves focal adhesion complexes, cell surfaceassociated complexes that anchor the bacterium to a substrate and might act as a motor (6). Sliding is a passive form of surface spreading by expansion that is powered by the outward pressure of bacterial growth and facilitated by compounds that reduce friction between cells and surfaces (3). Sliding or spreading by expansion has been observed in a diverse group of bacteria, such as mycobacteria, Bacillus subtilis, Vibrio cholerae, Serratia marcescens, Pseudomonas aeruginosa, or Legionella pneumophila (7-12), in which a strong correlation between sliding and production of surfactants has been established. Furthermore, sliding is easily mistaken for swarming motility and can occur when flagella are disrupted in bacteria that normally would swarm (7,8,13,14). Swarming is a rapid and coordinated movement of bacterial popul...

Comparative genomics ofPseudomonas syringaepathovartomatoreveals novel chemotaxis pathways associated with motility and plant pathogenicity

“…However, putative swarming motility was likely downregulated at 28 °C but was compensated for by twitching motility in Pto. Specifically, we found that Pto has an additional fliC- and cheA2 -dependent surface motility mechanism as previously shown (Nogales et al, 2015), which is active only at higher temperatures. pilA was essential for surface motility at 28 °C and fliC and cheA2 were essential for surface motility at 21 °C (Fig.…”

Sliding on the surface: bacterial spreading without an active motor

“…These results suggest a negative regulation of syfR and therefore also of the syf operon by FleQ. Additionally, in plant experiments the fleQ syfA double mutant showed diminished disease symptoms suggesting that sliding contributes to P. syringae colonization of the leaf surface, a habitat where flagella‐dependent movement might not be optimal (Nogales et al ., ).…”

“…P. syringae uses another lipopeptide, syringafactin to reduce the surface tension and thereby facilitate passive movement. While investigating the regulation of motility in P. syringae, Nogales et al discovered that a fleQ mutant lacking the proposed master regulator of motility can spread over semi-solid agar in a distinct pattern despite lacking flagella (Nogales et al, 2015). This spreading was proposed to be sliding motility based on its flagellum-independency.…”

“…Further, the essentiality of syringafactin was demonstrated using a double mutant (lacking the first gene of the operon encoding enzymes for syringafactin production, syfA next to fleQ) that was unable to spread. Interestingly, the fliC mutant lacking only flagellin was unable to spread in comparison to the fleQ mutant which was explained by the difference in syringafactin production level: the authors discovered that the amount of syringafactin is 40% higher in the fleQ mutant compared with fliC mutant and wild-type (Nogales et al, 2015). RNA-seq experiments revealed that the expression of the syf operon and of syfR, the transcriptional regulator presumably activating the syf operon, is upregulated in the fleQ mutant.…”

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“…syringae (Hockett et al 401 2013), surface motility of wild type Pto was not markedly affected at 28 o C compared to 21 o C. 402 However, putative swarming motility was likely downregulated at 28 o C but was compensated for 403 by twitching motility in Pto. Specifically, we found that Pto has an additional fliC-and cheA2-404 dependent surface motility mechanism as previously shown (Nogales et al 2015), which is 405 active only at higher temperatures. pilA was essential for surface motility at 28 o C and fliC and 406 cheA2 were essential for surface motility at 21 o C (Figure 5) revealing that Pto has at least two 407 genetically distinct mechanisms for surface motility, both of which are cheA1-independent.…”