Regulation of flagellar motility during biofilm formation

Abstract

Many bacteria swim in liquid or swarm over solid surfaces by synthesizing rotary flagella. The same bacteria that are motile also commonly form nonmotile multicellular aggregates called biofilms. Biofilms are an important part of the lifestyle of pathogenic bacteria, and it is assumed that there is a motility-to-biofilm transition wherein the inhibition of motility promotes biofilm formation. The transition is largely inferred from regulatory mutants that reveal the opposite regulation of the two phenotypes. Here, we review the regulation of motility during biofilm formation in Bacillus, Pseudomonas, Vibrio, and Escherichia, and we conclude that the motility-to-biofilm transition, if necessary, likely involves two steps. In the short term, flagella are functionally regulated to either inhibit rotation or modulate the basal flagellar reversal frequency. Over the long term, flagellar gene transcription is inhibited and in the absence of de novo synthesis, flagella are diluted to extinction through growth. Both short-term and long-term motility inhibition is likely important to stabilize cell aggregates and optimize resource investment. We emphasize the newly discovered flagellar functional regulators and speculate that others await discovery in the context of biofilm formation.

Keywords: EpsE; YcgR; brake; c-di-GMP; clutch; motor.

Figure 1. Functional regulation of the flagellum

Schematic of the Gram-positive and Gram-negative flagellar structures with the filament (green), hook (light blue), basal body (pink) and motor (tan) indicated. The site of clutch or brake motility inhibition is indicated in red. Micrographs of EpsE-GFP and YcgR-GFP localizing as puncta at the flagellum are reprinted from Blair et al., 2008 and Boehm et al., 2010 respectively. Scale bars are 2 μm.

Figure 2. General steps of the motility to biofilm transition

We propose a model for biofilm formation that incorporates two cell types: those that are non-motile and those that are initially motile. Flagella that are rotating are labeled in green, functional inhibition of the flagellum is labeled in red, decreased transcription of flagella genes is denoted by labeling the flagellum blue, and the extracellular matrix is shown in purple. Non-motile cells increase transcription of and synthesize the extracellular matrix components (purple), while motile cells first inhibit motility functionally at the flagella basal body (red), then decrease transcription of flagella genes (blue) while increasing expression of the extracellular matrix components (purple). Finally, both types of cells form a biofilm together.

Figure 3. Network of B. subtilis biofilm-related motility regulation

Transcriptional regulation is labeled in blue, functional regulation labeled in red, and the biofilm matrix products in purple. Green circle highlights the flagellum. Solid lines indicate a confirmed direct interaction while dashed lines indicate that the interaction is poorly understood and could be indirect or direct.

Figure 4. Network of P. aeruginosa biofilm-related motility regulation

Transcriptional regulation is labeled in blue, functional regulation labeled in red, and the biofilm matrix products in purple. Green circle highlights the flagellum. C-di-GMP in superscript indicates that the protein needs to be bound to c-di-GMP to carry out the indicated activity. Solid lines indicate a confirmed direct interaction while dashed lines indicate that the interaction is poorly understood and could be indirect or direct.

Figure 5. Network of V. cholerae and V. parahaemolyticus biofilm-related motility regulation

Transcriptional regulation is labeled in blue, functional regulation labeled in red, and the biofilm matrix products in purple. Green circle highlights the flagellum. C-di-GMP in superscript indicates that the protein needs to be bound to c-di-GMP to carry out the indicated activity. Solid lines indicate a confirmed direct interaction while dashed lines indicate that the interaction is poorly understood and could be indirect or direct.

Figure 6. Network of E. coli biofilm-related motility regulation

Transcriptional regulation is labeled in blue, functional regulation labeled in red, and the biofilm matrix products in purple. Green circle highlights the flagellum. C-di-GMP in superscript indicates that the protein needs to be bound to c-di-GMP to carry out the indicated activity. Solid lines indicate a confirmed direct interaction while dashed lines indicate that the interaction is poorly understood and could be indirect or direct.