Mutational Analysis of the Rhizobium lupini H13-3 and Sinorhizobium meliloti Flagellin Genes: Importance of Flagellin A for Flagellar Filament Structure and Transcriptional Regulation

Abstract

Motile bacteria are propelled by helical flagellar filaments connected by a proximal hook to the basal body holding the flagellar rotary motor (for reviews, see references 1, 20, and 41). Traditionally, flagellar filaments are classified by their electron microscopical appearance into two types, named plain and complex (22,33). The plain filaments of Escherichia coli and Salmonella spp. have a smooth surface structure with faint striations, whereas the complex filaments of soil bacteria, like Rhizobium lupini H13-3 and Sinorhizobium meliloti, exhibit a prominent helical pattern of alternating ridges and grooves (16,32,38). Unlike the flexible plain filaments, which are capable of switching from left-handed to right-handed helicity (21), the complex filaments are more rigid and do not switch handedness. Pairwise helical perturbations result in a subunit composed of a dimer of flagellin (39). Interflagellin bonds are believed to lock the complex filament in a rigid, right-handed helical conformation suitable for propulsion in viscous media (4, 8). Concomitantly, the flagellar motor of Rhizobium rotates entirely clockwise and does not reverse its sense of rotation (9). It has been shown that swimming S. meliloti cells respond to tactic stimuli by modulating their flagellar rotary speed (37) and that two novel motor proteins may be essential players in speed control (27). Hence, directional changes in the tracks of swimming S. meliloti cells-imperative for any chemotactic response-are a consequence of individual flagella rotating at different speeds (31). It thus appears that complex flagellar filaments and the new mode of directional control of swimming cells have evolved in response to the specific condition of swimming in viscous fluids prevailing in the soil biotope.The flagellar filament consists of an assembly of about 20,000 flagellin subunits, whose molecular mass typically ranges from 40 to 60 kDa (20). Flagellins are three-domain proteins, with the N-and C-terminal domains being responsible for the quarternary interactions between subunits and the central, surfaceexposed domain performing no obvious structural role but containing all of the potent antigenic epitopes. We have previously shown that the S. meliloti genome contains four genes, flaA, flaB, flaC, and flaD, encoding four related flagellin subunits (28, 36), and we report here three flagellin genes, flaA, flaB, and flaD, as constituents of the R. lupini H13-3 flagellar regulon. The latter strain was chosen because the first 13-Å-resolution, three-dimensional density map has been generated from its complex filament using low-dose electron micrographs of negatively stained specimens (4). This constellation may provide specific handles for future sequence-structure analysis.In an effort to understand the process of assembling the complex filaments of the related soil bacteria R. lupini H13-3 and S. meliloti and to elucidate the contribution of single subunits to the filament structure, we have taken a genetic approach. Mutational analyses revealed th...

Regulation of type 1 fimbriae synthesis and biofilm formation by the transcriptional regulator LrhA of Escherichia coli

“…The cells were allowed to sediment to carbon-coated grids for 1-2 min. After negative staining with 1 % uranyl acetate, the grids were examined with a Philips CM12 transmission electron microscope (Scharf et al, 2001).…”

Sinorhizobial chemotaxis: a departure from the enterobacterial paradigm a aTo Professor Wolfram Heumann on his 87th birthday.

“…Unlike E. coli, the soil bacterium S. meliloti is driven by rather rigid, righthanded helical filaments, which facilitate efficient swimming in a viscous environment (Go$ tz et al, 1982). Whereas the ' plain ' enterobacterial flagellar filaments are assembled from monomeric flagellin subunits, the ' complex ' rhizobial filaments consist of four related flagellin subunits that are assembled as functional heterodimers (Scharf et al, 2001). The complex flagellar fine structure is dominated by three helical ribbons that lock the filament in right-handedness, thus blocking a switch of hand (Cohen-Krausz & Trachtenberg, 1998).…”

“…In S. meliloti and the related Rhizobium lupini H13-3 (Scharf et al, 2001), rotary speed variation of the flagellar motor has a molecular corollary in two new motility proteins, MotC and MotD (Platzer et al, 1997), present in addition to the ubiquitous MotA\MotB transmembrane proteins that form the energizing proton channels (Fig. 1).…”

Regulation of flagellar, motility and chemotaxis genes in Rhizobium leguminosarum by the VisN/R-Rem cascade

“…It has been suggested that the direction of movement is reversed by changes in flagellar rotational speed (Sourjik & Schmitt, 1996;Miller et al, 2007). This type of flagellar movement is thought to be an adaptation of the rhizobia to the specific condition of swimming in viscous fluids that is prevalent in the soil (Scharf et al, 2001). …”