Quorum Sensing Controls FlagellarMorphogenesis in Burkholderia

glumaeBY: MOON SUN JANG, EUNHYE GOO, JAE HYUNG AN, J INWOO KIM, INGYU

HWANG

PRESENTED BY: ERIC MATHEWS AND DES ESPANTA

Rice panicle blight caused by B. glumae

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Burkholderia glumae• Burkholderia glumae is the

causative agent of rice panicle blight• Has multiple polar flagella• Has TofR/TofI QS system• Produces the phytotoxin toxoflavin

The purpose of the study is to understand the effects of temperature andquorum sensing on flagellar biogenesis and morphogenesis in B. glumae.

What is the goal?

Figure 9 from “Regulation of polar flagellum genes is mediated by quorum sensing and FlhDC in Burkholderia

glumae”

STRAINS

From Table 1 of “Regulation of polar flagellum genes is mediated by quorum sensing and FlhDC in Burkholderia glumae”

Does temperature affect flagellar numberin wild-type B. glumae?

• Cells were dipped into swim assay plates (of LB and 0.3% agar) and incubated for 20 hours at 28oC and 37oC.

• Cells were collected from the I, M and O regions and observed by Transmission Electron Microscopy.

Wild-type B. glumae

37o

C28o

C

Figure 1A

At 37oC: >77% had one or two polar flagella in all of the regions.

At 28oC,>76% had two to four polar flagella in all of the regions.

∴ Temperature does affect the number of flagella formed by wild-type B. glumae cells. As temperature gets closer to the optimal temperature for B. glumae growth (<30oC), flagellar number increases.

*n=100

Figure 1B

Does expression of flagellar biosynthesisgenes differ between 28oC and 37oC?

• By qRT-PCR, expression of flhC gene was measured and found to be at statistically equivalent levels for 28oC.

• Other flagellar genes, such as fliC and flgK, were also measured (by use of β-glucuronidase assay) at 28oC and 37oC. Results indicate higher expression levels for both genes at 28oC.

• In contrast, fliC and flgK expression levels were down for the QS mutants at 37oC, indicating that expression of these genes are dependent on QS (at 37oC).

Fig. 2

Fig. 2

Does QS and temperature influenceflagellar biogenesis and morphogenesis?

• Swim assays of BGR1, BGS2 and BGS9 were performed at 28oC and 37oC.

• At 37oC, QS mutants were largely aflagellate, indicating that QS plays a large role in flagellar biogenesis at this temperature.

• At 28oC, QS mutants were predominantly flagellated, with approximately half of these exhibiting non-polar flagella.

Figure 3

QS and flagellar morphology

• Previous studies indicate that QS affects flagellar biogenesis.

• Whether QS is directly or indirectly involved in flagellar morphology is still unclear.

• QS both positively and negatively regulates flagellar biogenesis in other organisms.

• In B. glumae, although flagellar biogenesis is independent of QS at 28oC, QS is still required for normal flagellar morphology at this temperature, showing that:

QS and temperature work together to modulate biogenesis and morphology of flagella in B. glumae.

Flagellar morphology observed under TEM

Figure 4

Fig. 6

Non-polar flagella (Peritrichous and Lateral)

BGS2 BGS9

- Sites of flagellar formation

How does lack of QS affect directionalswimming movement?

• Cell movement was recorded under a phase-contrast microscope for 30 seconds.

• As expected, at 37oC, BGR1 exhibited normal swimming movement. BGS2 and BGS9 were

largely aflagellate and were consequently non-motile.

- Addition of C8-HSL to BGS2 recovered normal swimming movement.

• At 28oC, BGR1 showed normal motility at the I region. BGS2 and BGS9 showed tumbling and

spinning motility.- Addition of C8-HSL to BGS2 recovered normal swimming movement.

• At 28oC, all strains showed normal motility in the O region. Figure 5

What is FlhF and what is its role in B. glumae?• FlhF is an SRP (Signal Recognition Particle) type GTPase that is

essential in flagellar formation.

Figure 6

Using swim assays, swimming motility of the BGR1 mutant fliA::Tn3-gusA45 was

examined at 28oC and 37oC.

plasmid carrying the fliA gene

wt phenotype recovered

At 28oC, expression of flhF gene was analyzed by qrt-PCR. BGF41 (flhC

mutant) made little to no flhF.

Fig. 6 Fig. 6

What is the relationship between QS and FlhF?

Hypothesis: QS deficiency causes mislocalization of proteins involved in flagellar formation, like FlhF in B. glumae.

• Hypothesis was supported by the mislocalization of FlhF in QS mutants.• Whether the dysfunctional FlhF directly causes abnormal flagellar formation or

mislocalizes other flagellar body proteins (thereby indirectly affecting flagellar morphology) is yet to be determined.

FlhF in other organismsThe flhF deletion mutant:• is aflagellate in V. cholerae and C. jejuni.• has randomly arranged flagella and is non-motile in P. aeruginosa.Conversely, • Overexpression of FlhF causes hyperflagellation in P. putida.

∴ FlhF expression levels and localization are important for normal flagellar formation (in polarly flagellated bacteria).

Conclusion• As established in the 2007 paper, normal polar flagellar

formation is essential for the pathogenicity of B. glumae.

• Abnormal flagellar morphology may impair swimming movement of B. glumae and hinder invasion success in rice flowers.

Therefore, this characteristic may be used to develop methods for reducing the spread of rice panicle blight and increase produce.