Several bacterial proteins are non-covalently anchored to the cell surface via an S-layer homology (SLH) domain. Previous studies have suggested that this cell surface display mechanism involves a noncovalent interaction between the SLH domain and peptidoglycan-associated polymers. Here we report the characterization of a two-gene operon, csaAB, for cell surface anchoring, in Bacillus anthracis. Its distal open reading frame (csaB) is required for the retention of SLH-containing proteins on the cell wall. Biochemical analysis of cell wall components showed that CsaB was involved in the addition of a pyruvyl group to a peptidoglycan-associated polysaccharide fraction, and that this modi®cation was necessary for binding of the SLH domain. The csaAB operon is present in several bacterial species that synthesize SLHcontaining proteins. This observation and the presence of pyruvate in the cell wall of the corresponding bacteria suggest that the mechanism described in this study is widespread among bacteria.

Directional control of bacterial motility is regulated by dynamic polarity inversions driven by pole-to-pole oscillation of a Ras family small G-protein and its associated GTPase-activating protein.

The bacterium Myxococcus xanthus has two motility systems: S motility, which is powered by type IV pilus retraction, and A motility, which is powered by unknown mechanism(s). We found that A motility involved transient adhesion complexes that remained at fixed positions relative to the substratum as cells moved forward. Complexes assembled at leading cell poles and dispersed at the rear of the cells. When cells reversed direction, the A-motility clusters relocalized to the new leading poles together with S-motility proteins. The Frz chemosensory system coordinated the two motility systems. The dynamics of protein cluster localization suggest that intracellular motors and force transmission by dynamic focal adhesions can power bacterial motility.During the exhibition of gliding motility, bacteria move across solid surfaces without the use of flagella (1). Glidingmotility is important for biofilm formation and bacterial virulence. Motility in Myxococcus xanthus, a Gram-negative rod-shaped bacterium, relies on two separate but coordinated motility engines. S motility is powered by type IV pili that are assembled at the leading cell pole; movement is produced as the pili bind to surface exopolysaccharides and are retracted, thereby pulling the cell forward (2). A motility, on the other hand, is not associated with pili or other obvious structures and is not well understood.To investigate the A-motility system, we studied AglZ, a protein that is essential for A motility but dispensable for S motility (fig. S1, A and B) (3). AglZ is similar to FrzS, an Smotility protein that oscillates from one cell pole to the other when cells reverse direction (4) ( fig. S1A) S2, B and C). We followed AglZ-YFP localization using time-lapse video microscopy: In fully motile cells, AglZ-YFP was localized in ordered clusters spanning the cell length; in stalled cells, it was localized at the leading cell pole (fig . S3).To study the link between the localization of AglZ-YFP and motility, we focused our observations on AglZ-YFP in fully motile cells. These cells showed an ordered array of AglZ-YFP clusters spanning the cell body (Fig. 1A). As cells moved forward, AglZ-YFP clusters maintained fixed positions with respect to the agar substrate rather than to their relative positions in the cell (Fig. 1A). We analyzed the clusters by taking line scans of the fluorescence intensity along motility paths for successive movie frames (Fig. 1B). To identify the position of peaks in multiple frames, we calculated a thresholded line-scan average (Fig. 1B) (5). This analysis not only located the positions of clusters shown in Fig. 1A, but also had the sensitivity to find other common peaks that were difficult to identify when viewing images with the eye. In every cell that was examined (n = 30), the AglZ-YFP clusters remained fixed relative to the substratum. The only AglZ-YFP clusters that moved relative to the cell body were located at the leading pole, which suggests that new sites were assembled at that pole. The number of clusters pe...

Contractile tails are composed of an inner tube wrapped by an outer sheath assembled in an extended, metastable conformation that stores mechanical energy necessary for its contraction. Contraction is used to propel the rigid inner tube towards target cells for DNA or toxin delivery. Although recent studies have revealed the structure of the contractile sheath of the type VI secretion system, the mechanisms by which its polymerization is controlled and coordinated with the assembly of the inner tube remain unknown. Here we show that the starfish-like TssA dodecameric complex interacts with tube and sheath components. Fluorescence microscopy experiments in enteroaggregative Escherichia coli reveal that TssA binds first to the type VI secretion system membrane core complex and then initiates tail polymerization. TssA remains at the tip of the growing structure and incorporates new tube and sheath blocks. On the basis of these results, we propose that TssA primes and coordinates tail tube and sheath biogenesis.

Protein-directed intracellular transport has not been observed in bacteria despite the existence of dynamic protein localization and a complex cytoskeleton. However, protein trafficking has clear potential uses for important cellular processes such as growth, development, chromosome segregation, and motility. Conflicting models have been proposed to explain Myxococcus xanthus motility on solid surfaces, some favoring secretion engines at the rear of cells and others evoking an unknown class of molecular motors distributed along the cell body. Through a combination of fluorescence imaging, force microscopy, and genetic manipulation, we show that membrane-bound cytoplasmic complexes consisting of motor and regulatory proteins are directionally transported down the axis of a cell at constant velocity. This intracellular motion is transmitted to the exterior of the cell and converted to traction forces on the substrate. Thus, this study demonstrates the existence of a conserved class of processive intracellular motors in bacteria and shows how these motors have been adapted to produce cell motility.murein cluster B | proton motive force

Gliding motility in the bacterium Myxococcus xanthus uses two motility engines: S-motility powered by type-IV pili and A-motility powered by uncharacterized motor proteins and focal adhesion complexes. In this paper, we identified MreB, an actin-like protein, and MglA, a small GTPase of the Ras superfamily, as essential for both motility systems. A22, an inhibitor of MreB cytoskeleton assembly, reversibly inhibited S-and A-motility, causing rapid dispersal of S-and A-motility protein clusters, FrzS and AglZ. This suggests that the MreB cytoskeleton is involved in directing the positioning of these proteins. We also found that a DmglA motility mutant showed defective localization of AglZ and FrzS clusters. Interestingly, MglA-YFP localization mimicked both FrzS and AglZ patterns and was perturbed by A22 treatment, consistent with results indicating that both MglA and MreB bind to motility complexes. We propose that MglA and the MreB cytoskeleton act together in a pathway to localize motility proteins such as AglZ and FrzS to assemble the A-motility machineries. Interestingly, M. xanthus motility systems, like eukaryotic systems, use an actin-like protein and a small GTPase spatial regulator.

Summary Various rod-shaped bacteria mysteriously glide on surfaces in the absence of appendages such as flagella or pili. In the deltaproteobacterium Myxococcus xanthus, a putative gliding motility machinery (Agl–Glt) localizes to so-called Focal Adhesion sites (FA) that form stationary contact points with the underlying surface. We discovered that the Agl–Glt machinery contains an inner-membrane motor complex that moves intracellularly along a right-handed helical path, and when it becomes stationary at FA sites, it powers a left-handed rotation of the cell around its long axis. At FA sites, force transmission requires cyclic interactions between the molecular motor and adhesion proteins of the outer membrane via a periplasmic interaction platform, which presumably involves a contractile activity of motor components and possible interactions with the peptidoglycan. This work provides the first molecular model for bacterial gliding motility.

Little is known about directed motility of bacteria that move by type IV pilus-mediated (twitching) motility. Here, we found that during periodic cell reversals of Myxoccocus xanthus, type IV pili were disassembled at one pole and reassembled at the other pole. Accompanying these reversals, FrzS, a protein required for directed motility, moved in an oscillatory pattern between the cell poles. The frequency of the oscillations was controlled by the Frz chemosensory system, which is essential for directed motility. Pole-to-pole migration of FrzS appeared to involve movement along a filament running the length of the cell. FrzS dynamics may thus regulate cell polarity during directed motility.