Annu. Rev. Microbiol. 2003. 57:24973 doi: 10.1146/annurev.micro.57.030502.091014 Copyright c 2003 by Annual Reviews.

All rights reserved

BACTERIAL MOTILITY ON A SURFACE:

Many Ways to a Common Goal
Rasika M. Harshey
Section of Molecular Genetics and Microbiology, Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, 78712; email: rasika@uts.cc.utexas.edu

Key Words swarming, gliding, twitching, sliding, biolms, agella, pili, fruiting body s Abstract When free-living bacteria colonize biotic or abiotic surfaces, the resultant changes in physiology and morphology have important consequences on their growth, development, and survival. Surface motility, biolm formation, fruiting body development, and host invasion are some of the manifestations of functional responses to surface colonization. Bacteria may sense the growth surface either directly through physical contact or indirectly by sensing the proximity of fellow bacteria. Extracellular signals that elicit new gene expression include autoinducers, amino acids, peptides, proteins, and carbohydrates. This review focuses mainly on surface motility and makes comparisons to features shared by other surface phenomenon.

CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TYPES OF SURFACE MOTILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Swimming and Swarming with Flagella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Twitching: Gliding with Pili or Social Gliding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gliding Without Pili . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sliding or Spreading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . INITIATION OF MOTILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conditions Favoring Motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signals and Signal Transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Specialized Cells/Organelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MIGRATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanism of Movement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of Motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MOTILITY IN OTHER SURFACE PHENOMENON: SOME COMMON THEMES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biolm Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fruiting Body Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pattern Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0066-4227/03/1013-0249$14.00 250 250 250 253 254 255 255 255 257 259 260 260 262 264 264 265 266

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HARSHEY Host Invasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266 CONCLUSIONS AND FUTURE PROSPECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

INTRODUCTION
Bacteria colonize surfaces in many different environments. Depending on availability of nutrients and surface conditions, bacteria can remain localized, move out to colonize larger areas, invade host tissue, or elaborate fruiting bodies to produce spores and await more favorable seasons. As a group, bacteria living in surface colonies have several advantages over single cells. They can optimize (a) growth and survival by having distinct cell types perform specialized functions, (b) access to nutrients, and (c) defense mechanisms for protection from dessication and antagonists. In the laboratory bacterial colonies are spatially organized, with distinctive morphological zones and biochemical expression patterns (7, 80). Bacteria interact dynamically with the surface to detect environmental signals as well as the presence of other bacteria (28). They secrete polysaccharides that constitute important layers called biolms, which promote bacterial adhesion, survival, and movement (69, 89). This review focuses primarily on bacterial movement over surfaces and how these studies relate to other surface phenomenon, drawing from separate reviews that appeared recently on swarming (24, 27, 59), twitching (57, 93), gliding (58), biolm formation (69, 89), pattern formation (7, 54), and fruiting body formation (83).

TYPES OF SURFACE MOTILITY

Over a quarter century ago J. Henrichsen (36) surveyed surface motility in hundreds of strains from 40 bacterial species belonging to 18 different genera. He identied six different categories: swimming, swarming, gliding, twitching, sliding, and darting. Only swimming and swarming could be correlated with the presence of agella. We have since learned a lot more about the rst four forms of motility. Swimming and swarming are indeed dependent on agella (33, 48); twitching has been shown to require type IV pili, as do some forms of gliding (57); the mechanism of some other forms of gliding still remain a mystery (58); and sliding/spreading are forms of passive translocation (49, 50). Nothing is known about darting. Table 1 summarizes the main features of the various types of surface motility discussed below.

Swimming and Swarming with Flagella

Swimming in liquid medium in Escherichia coli and Salmonella typhimurium has been studied in great detail over the past three decades (48). Typically, ve to eight peritrichous agella randomly emerging from the cell surface are each driven by a motor at their base. The motor acts as a reversible rotary device, using transmembrane proton potential (proton motive force) as the energy source. Each agellum is composed of a long helical lament, a short curved structure

BACTERIAL MOTILITY ON A SURFACE TABLE 1 Main features of various types of surface motility
Types of motility Swarming Motive organelles Flagella Cell differentiation Yes Colony expansion rates (m/s) 210 Bacterial generaa

251

Function Surface colonization

Aeromonas, Azospirillum, Bacillus, Clostridium, Escherichia, Proteus, Pseudomonas, Rhodospirillum, Salmonella, Serratia, Vibrio, Yersinia Aeromonas, Acinetobacter, Azoarcus, Bacteroides, Branhamella, Comomonas, Dichelobacter, Eikenella, Kingella, Legionella, Moraxella, Myxococcus, Neisseria, Pasteurella, Pseudomonas, Ralstonia, Shewanella, Streptococcus, Suttonella, Synechocystis, Vibrio, Wolinella Anabaena, Cytophaga, Flavobacterium, Flexibacter, Mycoplasma, Myxococcus, Phormidium, Saprospira, Stigmatella Acinetobacter, Alcaligenes, Bacillus, Escherichia, Flavobacterium, Mycobacterium, Serratia, Streptococcus, Vibrio

Twitching/ social gliding/ retractile motility

Type IV pili

No

0.060.3

Surface colonization, biolm formation, fruiting body development, b phage infection, transformation, conjugation

Gliding/ adventurous gliding

Not known

No

0.02510

Surface colonization

Sliding/ spreading

None

No

0.036

Surface colonization

Not all species in a genera display the indicated motility. See review articles cited in text. Type IV pili function, and not twitching motility per se, is required for phage infection, transformation and conjugation.

called the hook, and a basal body consisting of a central rod and several rings. The basal body is embedded in the cell surface, whereas the hook and lament are external to the cell. The agellar lament is normally a left-handed helix of variable length (typically 5 to 10 m), with a diameter of 20 nm. Motor rotation in the counterclockwise (CCW) sense causes a helical wave to travel from proximal to distal and to exert a pushing motion on the cell. Mechanical and hydrodynamic forces cause the agellar laments to coalesce into a bundle, usually around the long axis of the cell body. The concerted action of the agellar bundle results in running or smooth swimming, pushing the bacterium along relatively linear trajectories at speeds of up to 40 m/s. When motors rotate in the clockwise (CW) sense, the laments are placed under a right-handed torsional load, which initiates a polymorphic transition to a right-handed waveform. This transition results in tumbling because the agellar laments now have a poorly dened orientation, causing the cell to reorient its travel direction more or less randomly. The chemotaxis signal transduction network modulates the switch between the CCW and CW modes (88). In a constant environment a cell typically moves in a random walk of runs of approximately 1 s interspersed by tumbles of 0.1 s.

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When a cell nds itself swimming toward increasing attractant or decreasing repellent concentrations, it tumbles less frequently, thus biasing its random walk in the preferred direction. Whereas swimming is an individual endeavor, swarming is the movement of a group of bacteria. Hauser rst described swarming in Proteus species in 1885 [see (97) for a review of earlier literature]. It has since been shown to be widespread among agellated bacteria, suggesting that this mode of surface translocation must play an important role in the colonization of natural environments by microorganisms (33) (Table 1). In the laboratory the concentration of the agar used to solidify the medium can be critical for swarming (0.5%2%), likely owing to a certain level of wetness required for movement. More agella are needed for swarming on a surface than for swimming in liquid media, perhaps because of surface friction and/or higher viscosity of the slime encasing a swarmer colony. While many bacteria have only one kind of agella for both swimming and swarming, some species have separate and distinct agella for the two modes of motility (59). Growth of a bacterial colony on swarm medium leads to differentiation of vegetative cells into swarmer cells, which are hyperagellated and generally longer than their vegetative counterparts. Continuous outward movement provides a constant supply of fresh nutrients until the surface is fully colonized. Exceptions are Proteus species, which swarm in temporal cycles (4). Isolated swarmer cells generally do not move unless the agar concentration is low or the agar is supplemented with surfactant. Association of cells in a group likely facilitates movement by increasing uid retention (54). If a drop of uid is added to the surface of a swarming colony, the bacteria will disperse readily and individual bacteria can be seen swimming efciently, usually in the smooth swimming mode, i.e., without tumbling. Swarming may be viewed as a specialized case of swimming on a surface. Being in a at two-dimensional environment, swarmer cells can move only forward or backward. They are generally seen moving only in one direction and reverse only on impact with another group of cells or barriers such as the outer edge of the colony. Nothing is known about the mechanics of motor rotation on the surface, although several agellar bundles have been observed along the length of elongated swarmer cells of Proteus sp. (67). The rate of surface colonization via swarming equals or often exceeds the rate at which swimmer cells of the same species colonize swim agar (0.2%0.35%), where individual bacteria swim in the submerged water-lled spaces of the agar. The most active swarming occurs near the periphery of the colony, where the longest and most agellated cells are found. Cells in the interior of the colony are less motile and appear to dedifferentiate to a vegetative morphology. Bacteria that can swarm on higher agar concentration (1%2%) show a more striking swarmer cell morphology, with longer cells and an increased number of agella. Individual cells in the colony move at speeds ranging from 2 to 10 m/sec, and the swarm front advances at similar rates, making this the fastest mode of surface translocation. Often, multiple spearheaded rafts emerge at various points along the advancing front (Figure 1a). The spearheads can meander and merge with other

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Figure 1 Advancing edges of colonies exhibiting (a) swarming, (b) twitching, (c) gliding, and (d ) sliding. The organisms shown are P. mirabilis, P. aeruginosa, Cytophaga hutch, and S. marcescens, respectively. Cell lengths range from 2 m (in panel a) to 10 m (in panel c). Mark McBride (University of Wisconsin at Milwaukee) kindly provided photo of C. hutch.

groups, leading to the formation of a network of trails within which cells move in both directions, sometimes pausing and reversing direction but always following the long axis of the cell. Growth eventually produces a conuent lawn of cells. Transfer of swarmer cells from the surface to the liquid medium results in a rapid return to vegetative morphology. Swarming has been used loosely in the literature to describe outward migration of either swimming bacteria in a chemotaxis gradient or gliding bacteria as they move out in a group. In order to avoid confusion, I suggest this terminology be restricted to describe agella-dependent surface motility.

Twitching: Gliding with Pili or Social Gliding

Twitching was originally dened as an intermittent and jerky movement predominantly displayed by single cells (36). This form of motility occurs in a wide variety of bacteria (36, 57, 93) (Table 1), requires a moist surface, and enables cells to move both forward and backward at speeds of a few tenths of a micrometer per second. Henrichsen & Blom (37) suggested the possible involvement of polar pili in twitching. On the basis of observations regarding phage infection of Pseudomonas aeruginosa, Bradley (14) proposed that retraction of polar pili was the driving force of twitching motility. It is now clear that active extension and

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retraction of type IV pili is involved in twitching motility (63, 84, 90). These pili are about 6 nm in diameter and up to 4 m in length; they are typically found at one or both cell poles (12, 95). Radial expansion rates of colonies via twitching motility can approach 0.3 m/s (57). This form of movement underlies the phenomenon of social gliding and formation of fruiting bodies (81) and is also involved in biolm formation (70). Functional type IV pili are required for bacteriophage infection (13, 42), transformation (99), conjugation (102), and host cell responses (62). Given that the historical distinction between social gliding motility and twitching motility is now a matter of semantics, Mattick (57) has suggested the use of a more neutral and descriptively accurate new term such as retractile motility to describe motility mediated by type IV pilus extension and retraction. Like swarmer cells, rafts or spearhead-like clusters of aggregated cells in P. aeruginosa (Figure 1b) and in Myxococcus xanthus have been observed during twitching motility and social gliding motility, respectively. Within the rafts, cells are highly aligned in close cell-cell contact. The rafts move radially outward, following the long axis of the cells. Cells from one group join into another and form a lattice, much like swarming bacteria. Such cells at rst move end forward toward the other cells until they touch with their poles and then rapidly snap into an aligned position, which accounts for the characteristic jerky twitching motion observed with this form of motility (57). Similar trails and cell reversals are also observed in social gliding motility in M. xanthus (81). Although twitching motility is primarily a social activity, individual cells can show limited movement when in contact with inert substrates or on agar at low concentrations (57). Twitching motility can both promote outward movement of colonies under nutrient-rich conditions, as well as bring cells together to form fruiting bodies under conditions of nutrient depletion.

Gliding Without Pili

Gliding is dened as a smooth movement of cells, generally along the long axis of the cell (36), and is particularly seen in three large bacterial groups, the myxobacteria (motility rates range from 0.025 to 0.1 m/s), the cyanobacteria (speeds approaching 10 m/s), and the Cytophaga-Flavobacterium group (24 m/s) (58) (Table 1). In M. xanthus this form of motility is also known as adventurous motility. The advancing edges of these gliding bacteria look similar to those of swarming and twitching bacteria, and movement is associated with reversals (Figure 1c). Many different models have been proposed to explain gliding motility, including propulsion by transport of macromolecules such as polysaccharides, movement of outer membrane components by protein complexes in the cytoplasmic membrane, as well as contraction of brils in cell walls. However, there is no clear evidence supporting any specic model, and it is possible that several different mechanisms are involved. Mycoplasmas are parasitic bacteria that lack the peptidoglycan layer (77a). Many of these have a distinct cell polarity and glide on glass and other solid surfaces. Attachment organelles that protrude from the membrane around the necks of these bacteria (81a) are proposed to play a role in gliding motility [see (33a)].

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Sliding or Spreading
Sliding is produced by the expansive forces of a growing colony in combination with reduced surface tension and has been observed in many bacteria (36) (Table 1). Sheets of cells packed in different directions can spread outward as a unit, suggesting that this is not an active form of movement (Figure 1d ). Spreading of the colony front can range from relatively slow (0.03 m/s in Mycobacterium smegmatis; 49) to moderately fast (26 m/s in Serratia marcescens; R.M. Harshey, unpublished data). There is a strong correlation between the production of surfactants such as lipopeptides, lipopolysaccharides (LPS), and glycolipids and the sliding/spreading phenomenon (16, 50, 79). Although passive, this mode of translocation likely plays a signicant role in bacterial surface colonization (78).

INITIATION OF MOTILITY Conditions Favoring Motility

MOISTURE Surface motility is generally dependent on moist conditions. This is particularly evident for swarming in species of Serratia (1, 23), Escherichia (34), Salmonella (34), Aeromonas (33), Bacillus (33, 79a), Yersinia (33, 103), and Pseudomonas (45a, 76). These bacteria will swarm optimally on 0.5%0.7% agar and generally not swarm at agar concentrations above 1%. The peculiar behavior of E. coli K-12 strains in requiring a specic commercial source (Eiken) of agar for swarming (34) is likely due to superior wettability of this agar (91). This was deduced from the behavior of swarming mutants of S. marcescens defective in production of the surfactant serrawettin, as well as a large number of swarming mutants of S. typhimurium defective in LPS biosynthesis, which did not swarm on normal agar but could be rescued for swarming on Eiken agar (50, 91). The latter observation is in keeping with the absence of the LPS O-antigen layer in K-12 strains, as well as the ability of E. coli with an intact O-antigen to swarm on agar other than Eiken (34). Similar observations have been made with Bacillus subtilis mutants defective in surfactin production (65). External addition of surfactin can rescue the swarming defects of not only these B. subtilis mutants, but also LPS mutants of S. typhimurium, suggesting that LPS normally acts as a surfactant (91). Surfactants such as serrawettin and surfactin are exolipids known to lower surface tension and improve surface wettability, allowing liquids to spread on hydrophobic surfaces (52, 55). These and other exolipids not only promote swarming motility (24, 45a, 65) but also allow cells to spread even in the absence of active motility (50, 52) and are likely to play an important role in microbial colonization of hydrophobic surfaces. Some species of swarming bacteria such as those of Proteus (36, 77), Vibrio (59), Rhodospirillum (74), and Azospirillum (3, 36) can swarm on higher agar concentrations (1.5%2%). These bacteria likely excrete special forms of surfactants/ polysaccharides to trap sufcient moisture for swarming on these relatively less moist surfaces. Indeed, Proteus mirabilis excretes an acidic polysaccharide

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Figure 2 Slime trail in the wake of a spearhead of Bacillus sp. swarming on agar. Scenes were recorded at 1-minute intervals, left to right. Reprinted with permission from Reference 51.

proposed to create an appropriate uid microenvironment for swarming (27, 77). Polysaccharides and surfactants are components of what is generally referred to as slime surrounding motile surface colonies (Figure 2). Twitching is usually also dependent on the availability of moisture (36). It is often studied by stabbing a culture through the agar to the bottom of the plate so cells can move in the lm of water between the agar and the petri dish (57). Twitching motility takes place on both organic and inorganic surfaces, including agar gels, epithelial cells, plastics, glass, and metals. Social, or S, motility in myxobacteria can work under drier conditions such as 1.5% agar. Surface-active lubricating compounds are also part of the extracellular slime released by these bacteria (82). Gliding without pili is likely to encompass several different phenomena, so it is hard to make generalizations. Most gliders (myxobacteria, Cytophaga, lamentous cyanobacteria, lysobacters) move well on media solidied with 1.5% agar. However, strain variation is extensive, with some strains showing motility on media with less agar (1%) and some others on media with more agar (2%). Gliding bacteria also excrete surface-active molecules required for movement and have unusual sulfonolipids in their outer membranes that promote gliding (31, 32). Like swarming and twitching, sliding is also dependent on agar concentration, is optimally observed between 0.3%0.7% agar, and is critically dependent on surface-active compounds such as peptidolipids, glycolipids, and LPS (16, 49, 50).

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NUTRIENTS The abundance of nutrients is generally also an important factor in motility. Swarming is not observed in many organisms on minimal media unless casamino acids are included (23, 34, 77, 91). This likely reects the higher metabolic cost of increased agellar synthesis. Some bacteria such as Vibrio parahaemolyticus, however, can swarm on minimal media (59). Biomass production appears to be maximized during swarming (77), which also occurs under anaerobic conditions in E. coli and S. typhimurium (91). Twitching motility is also dependent on high nutrients (57), although fruiting body formation, which requires social motility, is a starvation response (83). For bacteria that glide without pili, as a general rule, nutrient-poor conditions favor motility and colony spreading, although there is a lot of strain variation. Cytophaga spp., avobacteria, and relatives glide well on agar with no added nutrients or in water or buffer on a glass slide. Sliding has been reported to occur on both rich and minimal media (49, 50). SLIME Polysaccharides and surface-active compounds are major components of slime. In addition, autoinducers such as homoserine lactones (HSLs), amino acids, and peptides (28, 64), and proteins such as proteases, lipases, and virulence determinants (23, 27, 48b, 79a, 82, 83), are included in the slime in various bacteria, where they serve multifarious roles. Polysaccharides and surfactants not only offer protection from dessication by water retention, but also promote motility by providing a hydrated milieu within which agella and pili function (Figure 2). They allow the cells to spread in the absence of motility and have been implicated in signaling as well (91). Amino acids, peptides, and HSLs serve as cell density signals that regulate expression of many genes including those involved in production of surfactants and virulence determinants (24, 27, 28, 45a). Some proteins also serve as signals, such as the C-signal required for fruiting body formation in M. xanthus (83). TEMPERATURE In S. marcescens, swimming and swarming motility as well as sliding are inhibited at temperatures above 32 C (1, 50, 56). Inhibition of sliding is likely due to the absence of serrawettin synthesis at the higher temperatures (55). Although absence of surfactant synthesis would be expected to affect swarming, even swimming motility is inhibited at 37 C (1). Interestingly, pigment production in this bacterium is also temperature sensitive (98). Surface translocation in most other bacteria occurs in the normal temperature ranges.

Signals and Signal Transduction

Both physical and chemical signals have been implicated in initiating the development of swarmer cells, which have distinct cell morphology (longer and more agellated cells) compared with their broth-grown counterparts. In V. parahaemolyticus all conditions that slow down polar agellar rotation lead to swarmer cell induction (59). These include increasing viscosity, using antibodies to inhibit agellar function, and decreasing motor speed by controlled use of a sodium

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channelblocking drug (43). Thus, the polar agellum appears to act as a mechanosensor, although nothing is known about how such a signal is transduced to program gene expression. Iron starvation is also reported to be a signal for swarmer cell differentiation in this organism. Amassing a critical cell density is closely linked to initiation of swarming in most organisms. In Serratia liquefaciens two quorumsensing regulators, N-acyl HSLs, control swarming (24). They do so by binding to a transcription activator that regulates the production of the surfactant serrawettin required for colony expansion; they do not control swarmer cell differentiation per se. Quorum sensing systems responsible for production of rhamnolipid appear to facilitate swarming in P. aeruginosa (45a). Peptides and amino acids in the external slime have been implicated as signals in Proteus sp. (27), while polysaccharides have been proposed to perform this role in bacteria such as S. typhimurium and E. coli (91). How any of these putative signals are transduced to program swarmer cell development is not known. In E. coli and S. typhimurium mutations in the chemotaxis signaling pathway inhibit swarmer cell development, although chemotaxis is not required for movement (18, 34). In P. mirabilis, two-component regulators such as RcsC-RcsB have been implicated in signal transduction (9). In P. mirabilis, which undergoes multiple cycles of swarmer cell migration, it has been suggested that both initiation and cessation of migration (consolidation) are a function of swarmer cell age. This model predicts that both events are governed by population dynamics rather than by responses to nutrient depletion or accumulation of chemotactic repellents (77). On the basis of properties of an LPS mutant that excretes copious amounts of slime and swarms at lower cell densities, it has been proposed that the cell density requirement may be related to slime accumulation (91). In this model slime buildup is initially constitutive. Multiple sources of polysaccharides (O-antigen sloughing, capsular polysaccharides, glycolipid secretion) contribute to the initial slime, whose accumulation is dependent on cell metabolism and growth. The concentration of the polysaccharide signal in the slime is thus directly related to growth. Unknown pathways detect the signal to elicit swarmer cell differentiation and secretion of more slime. This model is proposed to account for the periodic swarmer cell migration in P. mirabilis (91). As swarmer cells move out they carry the slime with them, thus depleting the signal from the center and diluting it from the edge of the colony. The consolidation phase and the subsequent waves of swarming can be explained by reaccumulation of slime and signal in a time-dependent (and hence age-dependent) manner. Unlike swarming cells, twitching and gliding bacteria do not undergo surfaceinduced morphological changes. Twitching motility is inuenced by cell density, cell contact-dependent intercellular signals, as well as nutritional status (86, 93, 94, 101). The environmental signals that control twitching motility are not well understood (57). Phosphatidylethanolamine has been implicated as a signal in P. aeruginosa as well as in M. xanthus (44). In Synechocystis sp. PCC6803, light controls motility and production of extracellular type IV pili via a phototactic gene cluster where signaling is linked to chromophore-binding photoreceptor domains (10). Chemosensory systems or gene clusters showing homologies to these systems

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in M. xanthus and P. aeruginosa are involved in controlling reversal frequencies as well as in bril and type IV pili biogenesis, but sensory input and output details are still lacking (57).

Specialized Cells/Organelles
Only swarming bacteria are known to change morphology upon propagation on a surface. Some bacteria have distinct polar and peritrichous agella for swimming and swarming motility, respectively. Examples of these include V. parahaemolyticus, V. alginolyticus, Rhodospirillum centenum, and Azospirillum brasilense (59). Others possess a single peritrichous system for both forms of motility. Examples include Proteus, Serratia, Salmonella, Yersinia, and Escherichia species (33). P. aeruginosa has been reported to swarm using multiple polar agella (45a, 76). The structure of the polar agellum has been studied in some detail in V. parahaemolyticus (59). In this marine organism sodium motive force is used to power the polar agellar motor, which achieves swimming speeds up to 60 m/s. The agellum is composed of subunits encoded by multiple genes and is sheathed by an apparent extension of the outer cell membrane. The mechanism that controls agellum rotation within the sheath is not understood. The lateral agella used for swarming are not sheathed, and the lament is polymerized from a single agellin subunit. Lateral agellar motors are powered by proton motive force, and although the dual agellar systems possess no shared structural components, movement is coordinated by shared chemotaxis machinery (60). The best-studied peritrichous agellar system of a swarming bacterium is that of S. typhimurium (48). Although a high-resolution crystal structure is not available, much has been learned from three-dimensional reconstructions from electron micrographs. A single subunit called agellin is arranged in a helical conformation with 11 subunits per turn in the outer lament. The lament is attached to the hook-basal-body complex that spans the bacterial membrane. Assembly of the structure is a sequential process that begins with insertion of the MS ring into the inner membrane. The individual extracytoplasmic agellar subunits are secreted through the MS ring after assembly of an associated type III secretion system. Rotation of the hook-basal-body structure is achieved via the motor force generators which act as stators against the C ring, a ring composed of three structural proteins associated with the cytoplasmic face of the MS ring (48a). One of these structural proteins, FliM, interacts with the phosphorylated form of the chemotaxis response regulator CheY to control the direction of switching. Thus, the direction of movement is regulated by the chemotactic output. Regulation of agellar assembly involves a combination of transcriptional, translational, and posttranslational regulatory mechanisms (2). Type IV pili are composed primarily of a single small protein subunit termed pilin, which is arranged in helical conformation with ve subunits per turn and which may be glycosylated and/or phosphorylated in different species (57). The three-dimensional crystal structure of Neisseria gonorrhoeae MS11 pilin has been

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solved (71). A large number of proteins showing similarities to those in the type II secretion pathway are involved in assembly of the pilus (57). Different pili may have different binding specicities for different types of cells mediated both by structural features of the pilin itself and/or by the presence of tip-exposed binding sites or tip-associated adhesins, which may be quite specic to the species and its ecology (57). In bacteria that glide without pili, a specic motor has yet to be identied. In some lamentous cyanobacteria junction pores with a diameter of 14 to 16 nm have been found near septa that separate cells of a lament (58). It has been proposed that polysaccharide extrusion may provide the propulsive force for gliding in these bacteria. The mechanisms responsible for myxobacterial adventurous gliding or for Cytophaga-Flavobacterium gliding are still a matter of speculation. Freeze-fracture electron microscopy has identied several 50-nm spikes used as attachment organelles in many gliding mycoplasmas (81a). In Mycoplasma mobile, two large proteins with distinct functions in gliding have been localized to these organelles, which are proposed to comprise the motor [see (33a)].

MIGRATION Mechanism of Movement

Swarming motility involves movement of cells in groups aligned along their long axis in multicellular rafts (Figure 1a). The observation that isolated cells rarely move could be related to the amount of slime encasing a group of cells versus individual cells because wetting agents in the slime provide a hydrated environment for agellar function (77, 91) (Figure 2). This notion is consistent with inactivity of cells at the edge of the advancing swarm front, while vigorous motility is evident just behind the front (6). The mechanism of agellar rotation on a surface is almost entirely unknown, although darkeld images showing multiple agellar bundles have been reported on swarmer cells of Proteus sp. (67). Swarmer cells of S. marcescens, S. typhimurium, and E. coli show prolonged smooth swimming when suspended in a drop of liquid, suggesting that their motors may only turn CCW on the surface (R.M. Harshey, unpublished data). It is not known if CW rotation causes cells to pause, because they cannot tumble on a at surface. It is possible that CCW-rotating agella, because of their exible hooks, form bundles that can push the cell in either direction. Components of the chemotaxis system are critical for swarming migration in V. parahaemolyticus and R. centenum but not for swarmer cell development (59). In S. marcescens, S. typhimurium, and E. coli, however, they are apparently required for development (34). In the latter two organisms chemotaxis per se is not required for swarming (18). It is not clear whether the chemotaxis system controls swarmer cell development directly or whether it does so indirectly by controlling movement.
SWARMING MOTILITY TWITCHING MOTILITY

Twitching motility, like swarming, is also colonial in nature and involves cell-cell contact (Figure 1b). Isolated cells rarely move, except

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when within a certain distance of others, which corresponds to the length of type IV pili (57). The motive force for twitching motility is pili retraction. This was rst deduced by Bradley (14) and recently conrmed by studies in N. gonorrhoeae using optical tweezers (63), in M. xanthus using a tethering assay (90), and in P. aeruginosa using uorescently labeled pili (84). In N. gonorrhoeae cells were pulled at speeds of around 1 m/s with forces ranging up to 90 pN. Retraction events were sporadic, separated by 120 s. P. aeruginosa pili were shown to extend and retract at approximately 0.5 m/s with a force of approximately 10 pN. Individual pili on the same cell appeared to extend and retract independently. Cell movement was not associated with extension per se, but rather by retraction of pili after they had attached to the substratum at their distal tip. In M. xanthus, rates of twitching motility were around 0.4 m/s. Tethered wild-type cells appeared to be retracted toward the surface via one pole and be pulled away from the initial attachment site by extrusion of pili from the other pole by a similar tether-and-retract step. These studies indicated that reversals of movement involve alternating the activity of pili from one cell pole to the other, the frequency of which was correlated with attachment time and is controlled by the frz chemosensory system. Retraction of pili to bring cells into close alignment may involve specic recognition and possibly sensing by the pili of receptors present on the surface of neighboring cells. In M. xanthus, the extracellular polysaccharide brils serve as receptors [see (33a, 101)]. Sensing tensional or torsional stress on the lament after attachment could trigger retraction, although retraction of individual pili is reported to occur independently regardless of whether pili are attached in P. aeruginosa (84). The mechanism of pilus retraction is thought to be lament disassembly mediated by PilT (57), a process that has been estimated to occur at around 1000 pilin subunits per sec (63). PilT is a nucleotide-binding protein and a member of the AAA family of motor proteins. PilT is suggested to form a hexameric ATPase that surrounds the base of the pilus, providing an axial rotary power stroke analogous to that provided by F1 ATPase (40).
GLIDING WITHOUT PILI Although no clear mechanism is at hand, mutants that are impaired in gliding motility provide food for thought (58). For example, adventurous motility in M. xanthus is affected by mutations in outer membrane lipoproteins. Similarities of these proteins to those that use proton motive force to transport molecules across the outer membrane suggest that they may effect motility by propulsion of macromolecules such as polysaccharides. Although unusual chain-like strands that wrap helically around the cell have been observed by transmission electron microscopy in cell walls of some myxobacteria and gliding bacteria of the Cytophaga-Flavobacterium group, their role in motility is not known. Some cyanobacteria rotate as they glide. An extracellular protein is thought to form helical bers on the cell surface that may serve as a passive screw thread. Some lamentous cyanobacteria are proposed to glide by contraction of brils present in their cell walls (58). Members of the Cytophaga-Flavobacterium group glide

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over glass surfaces at rates of approximately 2 to 4 m/s and occasionally reverse (Figure 1c). They also frequently attach to a glass slide by one pole and rotate. Even when suspended in liquid, cells can attach to added latex spheres, which are seen to propel along the cell surface. A sphere may travel the length of a cell, around the pole, and down the opposite side. Two spheres may be propelled in different directions and may pass one another while going in opposite directions. A sphere may reverse direction, cross the width of the cell, and even follow a helical path. Some spheres become temporarily trapped near the cell pole and rotate in place. Sphere movement appears to be mediated by the same machinery that causes cell movement, as judged by the effect of mutations. The energy for movement is likely proton motive force. Polysaccharide extrusion is unlikely to account for this form of motility. A large number of nonmotile mutants of Flavobacterium johnsoniae are decient in sulfonolipid synthesis, whose role is not understood. Genes with sequence similarities to ABC transporters are also required for motility in this organism. Whether these transport polysaccharide slime or proteins that make up the motility machinery is unclear. An unusual gliding mechanism has recently come to light in M. mobile [see (33a)]. Mycoplasmas do not have agella, pili, or a chemosensory system (77a). M. mobile glides continuously on glass, exerting a force up to 27 pN. Two large proteins with distinct functions have been localized to the attachment organelles that protrude from the neck of this organism and grab the glass surface (33a). A mechanical cycle of gliding is proposed, where one of these proteins is involved in attachment while the other is responsible for the stroke or force generation that achieves gliding speeds of 24.5 m/s.

Regulation of Motility
SWARMING

Swarmer cells need to upregulate the number of agella in order to move. In E. coli and S. typhimurium the agellar genes are expressed in a hierarchical manner (48). The master hDC operon encodes transcription factors that control a second level of genes that specify the hook-basal-body complex as well as the transcription factor FliA, which in turn regulates late genes encoding the agellar lament, the motor proteins, and the chemotaxis components. Expression of the hDC operon is inuenced by environmental signals such as glucose availability, osmolarity, heat shock, acetyl phosphate, as well as by cell cycle regulation. While the details might differ, it is likely that other swarming bacteria fundamentally share this hierarchy of agellar gene expression as well (24, 27, 59). In P. mirabilis the swarmer cells express 30-fold more hDC mRNA than the vegetative cells do, and swarmer cell differentiation appears to be primarily controlled at this level (27). Because FlhDC proteins also appear to repress cell division in various organisms (23, 29, 73), it is reasonable to think that hDC is the primary site for swarm signal action. In this organism the leucine-responsive regulatory protein Lrp positively regulates hDC expression (27). Several additional regulatory proteins have also been identied, some of which apparently network with the

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hDC operon to coordinately control the expression of virulence genes along with swarmer cell differentiation (26). FlhD and FlhC proteins are also rapidly proteolysed following differentiation (20). In V. parahaemolyticus mutation of the LonS protease results in a constitutive swarmer cell phenotype (87). In S. liquefaciens the hDC operon controls the expression of at least 62 proteins (30). In addition, the swrI gene controls the synthesis of the HSLs, which regulate 28 other genes. The FlhDC and SwrI regulons are independent and control different aspects of swarming, swarmer cell differentiation and migration, respectively. Even though overexpression of the hDC operon can produce cells that resemble swarmer cells in both Serratia liquefaciens and Salmonella typhimurium, no signicant increase in the transcription of this operon could be observed in cells swarming on the surface in either organism [(92); Q. Wang & R.M. Harshey, unpublished results for S. typhimurium]. The chemotaxis system is important for regulating swarming movement in all organisms. In E. coli and S. typhimurium this system operates via methyl-accepting chemotaxis proteins (MCPs), which induce autophosphorylation of a central histidine kinase. The kinase phosphorylates a response regulator (CheY) that controls motor reversals by binding to a switch protein complex at the base of the motor (88). Other chemotaxis proteins in the pathway act as adaptors between the methylaccepting chemotaxis proteins and the kinase, or they modulate the methylation state of the methyl-accepting chemotaxis proteins in order to reset the system to its prestimulus state. In the swimming mode the steady-state level of phosphoCheY controls the steady-state pattern of runs and tumbles. In S. marcescens, S. typhimurium, and E. coli chemotaxis components are apparently required for development, although in the latter two organisms, chemotaxis per se is not required for swarming (34). It is not clear whether the chemotaxis signaling system controls swarmer cell development by controlling gene expression in these organisms; such a function has not yet been directly ascribed to the response regulator CheY. An alternative possibility is that the chemotaxis system controls movement on the surface, and that movement somehow indirectly controls development.
TWITCHING Biogenesis of type IV pili in both P. aeruginosa and M. xanthus is inuenced by typical and atypical two-component signaling pairs of regulatory proteins (57). The signals to which these proteins respond are not known, but they could include nutritional cues such as amino acids as well as cell density. The frz and chp chemosensory systems control twitching motility in M. xanthus and P. aeruginosa, respectively. These systems appear to be more complex than those of E. coli and S. typhimurium. For example, P. aeruginosa has four complete chemosensory systems encoded in its genome (22), one each for swimming and twitching motility and two of unknown function [see also (33a)]. In M. xanthus, the frizzy or frz system controls twitching, and a second dif chemosensory system controls the expression of extracellular brils that are required for social motility (86, 93, 101). The frz system controls the frequency of cell reversal and therefore the direction and pattern of twitching motility both in normal vegetative growth and

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during fruiting body formation (94). FrzCD undergoes methylation and demethylation during social motility and fruiting body formation, but unlike well-known members of the methylated chemotaxis protein family, it is a soluble cytoplasmic protein rather than a membrane protein (83). Slime from developing cells was shown to cause methylation of FrzCD, suggesting that an extracellular, quorumdependent signal could be involved (81). Like frz, the chp system in P. aeruginosa also appears to control the speed and frequency of cell reversal in twitching motility. Some Chp mutants show defects in the expression of many virulence factors, suggesting that this system is involved in the regulation and integration of a range of cellular responses, including mbriae and alginate production during surface and host colonization (57). In contrast, N. gonorrhoeae and N. meningitidis do not have any chemosensory systems encoded in their genomes. Not much is known about regulation of twitching motility in these obligate pathogens other than that pilin expression is inuenced by two genes that appear to encode an unusual twocomponent regulatory system responsive to GTP.

MOTILITY IN OTHER SURFACE PHENOMENON: SOME COMMON THEMES Biolm Formation

Biolms are communities of microorganisms attached to a surface (89). They can comprise a single microbial species or multiple species and can form on a range of biotic and abiotic surfaces. They are responsible for a variety of human infections and are more resistant to antibiotics and host immune responses (21). Rapid progress is being made in understanding their formation owing to the development of simple screens for isolation of biolm-defective mutants (69). P. aeruginosa biolms have been studied extensively, although P. uorescens, E. coli, and V. cholerae have also been studied. Among gram-positive bacteria, Staphylococcus epidermidis, S. aureus, and the enterococci have been studied. Bacteria seem to initiate biolm development in response to environmental cues such as nutrient availability. They continue to develop as long as fresh nutrients are provided, but they detach from the surface and return to a planktonic mode of growth when deprived of nutrients. Once attached, a common feature is the expression of large quantities of exopolysaccharides (EPS), which not only promote adherence but also protect the bacteria from dessication and, because of their anionic nature, help hold minerals and nutrients near the cell (68). Confocal scanning laser microscopy has allowed visualization of fully hydrated biolms. The structure of a mature P. aeruginosa biolm comprises mushroom-shaped microcolonies of bacteria that are surrounded by an extracellular polysaccharide matrix and separated by uid-lled channels, reminiscent of fruiting bodies in M. xanthus. In P. aeruginosa and V. cholerae, agella and type IV pili were shown to play an important role in events leading to attachment (69). LPS is important depending on the hydrophobicity of the surface. Mutants with various LPS defects show

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differential attachment to hydrophilic versus hydrophobic surfaces, which could either be due to exposure of lipid moieties or proteins on the cell surface. Proteases have also been shown to have a role in development, although their mechanism is not known. Flagella are important in swimming along the surface until an appropriate location for initial contact is found. After attachment, bacteria move by type IV pilimediated motility, which appears to be dependent on contact with other cells. Type IV pili mutants fail to form the characteristic mushroom-like mounds, much as these mutants fail to form fruiting bodies in M. xanthus (83). Surface contact appears to induce synthesis of the EPS and to downregulate agellar biosynthesis. Like the type IV pili mutants, those that interfere with production of one of the two HSLs made by P. aeruginosa also fail to develop the mushroom structures. Interestingly, while the wild-type strain is resistant to biocides, a strain unable to produce acyl-HSL was reported to become extremely sensitive to the biocide sodium dodecyl sulfate despite wild-type levels of EPS in the colony. HSLs in P. cepacia have been recently implicated in late stages of biolm development (38). Microarray analysis showed that gene expression in biolm cells of P. aeruginosa is similar to that in free-living cells, but there are a small number of signicant differences, which include expression of genes for some types of antibiotic resistance (96). In E. coli, motility but not chemotaxis was seen to be important for initial attachment (69). Type I pili were also important. In S. typhimurium an inverse relationship was observed between swarming and biolm formation (65). Thus, surfactants that promote swarming motility were found to inhibit biolm formation and vice versa. In summary, biolm formation shares several features with bacterial surface translocation. These include motility mediated by agella and type IV pili, cell density-mediated events such as quorum sensing, secretion of extracellular polysaccharides, and formation of structures that resemble fruiting bodies.

Fruiting Body Formation

A comparison between fruiting body formation and biolm formation has recently been made (69). When deprived of nutrients on an appropriate surface, M. xanthus cells use social gliding motility, or S motility, to aggregate into multicellular structures called fruiting bodies within which cells sporulate. Biolm formation also occurs in response to nutritional signals and on a surface. Three cell surface structurestype IV pili, extracellular matrix brils, and LPS O-antigenare all critical for S motility. Similarly, type IV pili and EPS production are important for biolm formation. Both phenomena require cell-cell contact. Like biolms of P. aeruginosa that require acyl-HSLs, the formation of fruiting bodies also requires extracellular signaling molecules known as A-signal (a mixture of amino acids) and C-signal (a protein) (83). S motility in M. xanthus is controlled by the frz and dif chemosensory systems (90, 100). Whereas the dif system is essential for S motility, the frz system is not. The dif system has been implicated in the biogenesis of the bril material (101). Chemotaxis is apparently not required for biolm formation (72), although it is required for the extent of migration in swarming and

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twitching, and for swarmer cell development in some organisms (33). The LPS O-antigen is necessary both for swarming (91) and for normal development and social motility in M. xanthus (11). The O-antigen has been postulated to serve a surfactant function in swarming (65, 91). Surfactant synthesis is regulated by quorum sensing HSLs in S. liquefaciens and in P. aeruginosa (24, 45a). Recently, fruiting body formation in B. subtilis was found to resemble biolm formation (15) in that motile cells differentiated into aligned chains of attached cells that eventually produced fruiting bodies. Fruiting body formation depended on regulatory genes required early in sporulation and on genes evidently needed for EPS and surfactin production. Like serrawettin and rhamnolipid production in S. liquefaciens and P. aeruginosa, surfactin production in B. subtilis is regulated by a quorum sensing system where the autoinducer is a peptide (64, 66). In summary, motility appendages, EPS, surfactants, LPS O-antigen, and quorum sensing signals are common features of surface translocation, biolm formation, and fruiting body formation.

Pattern Formation
Motile microbial colonies frequently develop complex patterns (7, 54). The most striking of these are the periodic concentric rings generated by swarming movement of P. mirabilis (4, 54). These rings are the result of repeated cycles of swarming followed by consolidation, where swarmer cells revert to a vegetative form. Little is known about the mechanisms which cause the migrating swarmer cell populations to revert, although several models have been proposed (6, 77, 91). Other swarming bacteria such as Clostridium sporogenes and V. parahaemolyticus generate dendritic fractal patterns (59). Nonchemotactic E. coli expressing either the serine chemoreceptor Tsr or the aspartate chemoreceptor Tar carrying point mutations that abolish binding to their respective ligands also produce unusual swarm patterns (18). It is likely that CCW/CW switching patterns of the agella motors inuence the direction of cell movement and hence the patterns. Che mutants of S. marcescens show altered patterns, which are also inuenced by the surfactant produced by this organism (51, 53, 54, 56). These patterns are therefore different from those formed by bacteria in semisolid media that are dependent on chemotaxis (17). A variety of patterns can be induced in Bacillus and Paenibacillus strains in response to nutrient conditions, wetness of the agar, and surfactant production (7, 61). Microbial patterns have been modeled after concepts borrowed from studies of patterning from nonliving systems to suggest that cell-cell signaling, motility, and chemotaxis interact in complex ways under given nutrient and wetness conditions to generate these patterns.

Host Invasion
Host cells present microbes with surfaces for attachment and subsequent invasion (25, 41, 46). Development of convenient in vitro assays is helping us understand the initial stages of colonization. Adhesion by type I pili is a common mode of

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attachment not discussed in this review (39). Bacterial cell surface proteins and components of the extracellular slime also contribute to adherence. Motility often helps bacteria get to the surface (35). The role of type IV pili and agella in biolm formation by pathogenic bacteria was discussed above (21, 69). Motility regulons often also control the expression of virulence determinants. For example, the chemosensory gene cluster in P. aeruginosa controls the expression of virulence factors, and pilin mutants of P. aeruginosa and N. gonorrhoeae have reduced virulence (57). For the uro-pathogen Proteus, swarming behavior is closely associated with modulation of virulence characteristics and the ability to invade human uroepithelial cells (26, 27). In S. liquifaciens, Clostridium septicum, and Bacillus cereus, expression of virulence proteins was seen to be associated with the swarmer cell state (23, 48b, 79a). For V. parahaemolyticus, differentiation into swarmer cells plays an important role in adsorption and colonization of chitinaceous shells of crustaceans (8). The synthesis of virulence proteins is also inuenced by localized growth on a surface, which leads to a buildup of cell density. Quorum sensing mechanisms, wherein chemical signal molecules are detected to regulate a variety of responses, also induce the synthesis of virulence factors (5). In addition, the type III protein secretion complex, which is evolutionarily related to agella and found exclusively in gram-negative bacteria (45, 48a), provides a mechanism for delivery of bacterial effector proteins across both bacterial membranes as well as the eukaryotic plasma membrane into the host cell cytosol (19, 47, 75). Although we understand many aspects of bacterial colonization of surfaces, much remains to be learned.

CONCLUSIONS AND FUTURE PROSPECTS

In the past decade, examination of different modes of surface colonization by bacteria has led to a new appreciation and understanding of microbial physiology on the surface. A common theme to emerge from these studies is that surface appendages and secreted polysaccharides play central roles in surface colonization. Both have been proposed to signal bacteria to turn on new gene expression, although the signaling mechanisms are not yet at hand. Polysaccharides also play essential roles in bacterial colonization of plant surfaces, a topic not discussed in this review (85). More needs to be learned about the roles specic polysaccharides play in surface responses. High cell densities are more readily achieved during localized growth on a surface and lead to accumulation of autoinducers and peptides that signal new gene expression. Among these, secretion of surfactants and polysaccharides aids in both active and passive motility, and secretion of virulence determinants aids in host infection. The chemotaxis machinery controls the direction of movement and the extent of colonization. Cell-cell signaling, motility, and chemotaxis interact in complex ways under given nutrient and wetness conditions to generate interesting patterns of growth. Our knowledge of how agellar bundles propel swarmer cells on a surface is lagging behind that of how pili function in twitching motility. In the era of genomics new signaling components readily come to light, yet we are

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still in the dark about what the signals are and how they are transduced to elicit functional responses. Detailed knowledge of all these pathways should provide a greater understanding of bacterial colonization of surfaces and multiple points of intervention in bacterial infection. ACKNOWLEDGMENTS I thank Mark McBride and Tohey Matsuyama for contributing photographs, and Jim Walker and members of my laboratory for helpful comments on the manuscript. Research in my laboratory is supported by grants from the NIH (GM 57400 and GM 33247) and the Welch Foundation (F1351).
The Annual Review of Microbiology is online at http://micro.annualreviews.org

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