Bacteria Use Type IV Pili to Walk
Upright and Detach from Surfaces
Maxsim L. Gibiansky,1* Jacinta C. Conrad,2* Fan Jin,1Vernita D. Gordon,1
Dominick A. Motto,4Margie A. Mathewson,3Wiktor G. Stopka,3Daria C. Zelasko,3
Joshua D. Shrout,4Gerard C. L. Wong1,3†
aeruginosa, a biofilm-forming pathogen respon-
sible for lethal infections in cystic fibrosis (2), the
motility appendages type IV pili (TFP) mediate
the “twitching” motility mode observed in bio-
films (3). Comparatively little is known about the
transition from planktonic to surface-associated
states that initiates biofilm formation. Here, we
converted microscopy movies into searchable
databases of bacterial behavior by using particle-
time scales of all visible bacterial trajectories (4).
Shortly after attachment, before microcolony
formation, we observed two TFP-driven surface
tants, whose movement is strictly TFP-dependent,
“crawled” along their body axis with high direc-
tional persistence when oriented horizontally,
parallel to the surface, and “walked” omnidirec-
tionally with low directional persistence when
attached vertically by one end (Fig. 1, A and B).
Bacteria reversibly transitioned between these
mechanisms. By contrast, Myxococcus xanthus
acterial biofilms are multicellular surface-
bound communities with important hu-
slowly jiggles vertically before transitioning to a
horizontal orientation for lateral crawling (5).
Each mechanism confers advantages for sur-
horizontal bacteria could be divided into crawling
superdiffusive and surface-anchored subdiffusive
subpopulations (fig. S1). The average persistence
length, Lp, over which trajectories appear straight
was shorter for walking bacteria (2 mm) than for
crawling bacteria (6 mm); the former was similar
walking was caused by pulls of splayed TFP.
Walking bacteria exhibited a higher instantaneous
velocity [mean 71 T 2 nm/s (SEM) versus 41 T 2
nm/s], but crawling bacteria moved further on long
time scales because of the Lp. Crawling enabled
directional motion; walking enabled rapid local
exploration. These trends were preserved in wild-
type (WT) bacteria.
Bacterial orientation played a key role in life
cycle events. In 99% of 214 WT division events,
the majority (67%) of the other daughters left the
division site by detaching, walking, or crawling.
TFP governed this motility; TFP-deficient bacte-
search engine to locate all detachment events, we
observed that detaching bacteria were over-
whelmingly oriented out of plane (Fig. 1C). We
found that TFP facilitated detachment by tilting
from horizontal to vertical orientations; the influ-
ence of prevailing conditions was weak. TFP-
deficientDpilAbacteria were defective inmaking
this transition. This suggests a physical onset of
reversible polar attachment to irreversible longi-
tudinal attachment (8).
Bacteria lacking TFP neither crawled nor
achieved vertical orientations for walking and
detachment. DpilA biofilms contained heteroge-
neous bacterial clusters (6) whose positions were
determined by initial attachment sites (Fig. 1D).
Divisions (Nd= 79) outnumbered attachments
(Na= 18) during 1 hour of cluster formation,
TFP-competent WT could actively walk, crawl,
redistribute, and detach; despite a similar number
of divisions (Nd= 95), the WT biofilm did not
contain clusters, indicating the DpilA biofilm
morphology is caused by motility defects rather
than adhesion defects.
References and Notes
1. J. W. Costerton, P. S. Stewart, E. P. Greenberg,
Science 284, 1318 (1999).
2. T. Tolker-Nielsen et al., J. Bacteriol. 182, 6482
3. J. S. Mattick, Annu. Rev. Microbiol. 56, 289 (2002).
4. Materials and methods are available as supporting
material on Science Online.
5. H. Sun, D. R. Zusman, W. Shi, Curr. Biol. 10, 1143
6. M. Klausen et al., Mol. Microbiol. 48, 1511 (2003).
7. J. M. Skerker, H. C. Berg, Proc. Natl. Acad. Sci. U.S.A. 98,
8. G. A. O’Toole, H. B. Kaplan, R. Kolter, Annu. Rev.
Microbiol. 54, 49 (2000).
9. We thank S. Lee, G. O’Toole, T. Yahr, and M. Parsek
for critically reading the manuscript. G.C.L.W.
and co-workers are funded by the NIH, the NSF,
and the Cystic Fibrosis Foundation.
Supporting Online Material
Materials and Methods
25 June 2010; accepted 2 August 2010
1Department of Bioengineering, California Nano Systems In-
stitute, University of California, Los Angeles, CA 90024, USA.
2Department of Chemical and Biomolecular Engineering, Uni-
versity of Houston, Houston, TX 77204, USA.3Department of
Materials Science and Engineering, University of Illinois, Urbana-
Champaign, IL 61801, USA.4Department of Civil Engineering
and Geological Sciences, University of Notre Dame, Notre
Dame, IN 46556, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail:
% of detaching bacteria
Number of bacteria
Fig. 1. TFP-mediated (A) vertical walking (top) and horizontal crawling (bottom) trajectories in
projected length (L) of N = 70,073 bacterial images showing populations of vertical (red) and horizontal
(blue) bacteria. (C) Out-of-plane (vertical or tilted) orientations facilitate detachment. (D) DpilA biofilms
had clusters (top); WT biofilms were uniform (bottom).
VOL 3308 OCTOBER 2010
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Bacterial biofilms are structured multicellular communities involved in a broad range of infections. Knowing
how free-swimming bacteria adapt their motility mechanisms near surfaces is crucial for understanding the
transition between planktonic and biofilm phenotypes. By translating microscopy movies into searchable
databases of bacterial behavior, we identified fundamental type IV pili–driven mechanisms for Pseudomonas
aeruginosa surface motility involved in distinct foraging strategies. Bacteria stood upright and “walked” with
trajectories optimized for two-dimensional surface exploration. Vertical orientation facilitated surface
detachment and could influence biofilm morphology.
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on November 13, 2010