Agrobacterium tumefaciens possesses a fourth flagellin gene located in a large gene cluster concerned with flagellar structure, assembly and motility

Deakin, William J.; Parker, Victoria; Wright, Emma L.; Ashcroft, Kevin J.; Loake, Gary J.; Shaw, Charles R.

doi:10.1099/13500872-145-6-1397

Cited by 45 publications

(47 citation statements)

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“…Five of the selected clones (SR3c6, SR3c11, SR3c14, SR3c17, and SR3cb11) encode peptides with significant similarity to parts of the N termini of A. tumefaciens flagellins FlaA, FlaB, and FlaC (data not shown). With molecular masses that range from 31.7 kDa for FlaA to 33 kDa for FlaB of A. tumefaciens (9), it is likely that flagellins of USDA257 were copurified with NopP during the initial protein extraction from SDS-PAGE gels (21). The finding that USDA257 is motile on YEM or RMS swarm plates supports this contention (W. J. Deakin, unpublished data).…”

Section: Discussionsupporting

confidence: 75%

Characterization of NopP, a Type III Secreted Effector of Rhizobium sp. Strain NGR234

et al. 2004

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The type three secretion system (TTSS) encoded by pNGR234a, the symbiotic plasmid of Rhizobium sp. strain NGR234, is responsible for the flavonoid-and NodD1-dependent secretion of nodulation outer proteins (Nops). Abolition of secretion of all or specific Nops significantly alters the nodulation ability of NGR234 on many of its hosts. In the closely related strain Rhizobium fredii USDA257, inactivation of the TTSS modifies the host range of the mutant so that it includes the improved Glycine max variety McCall. To assess the impact of individual TTSS-secreted proteins on symbioses with legumes, various attempts were made to identify nop genes. Amino-terminal sequencing of peptides purified from gels was used to characterize NopA, NopL, and NopX, but it failed to identify SR3, a TTSS-dependent product of USDA257. By using phage display and antibodies that recognize SR3, the corresponding protein of NGR234 was identified as NopP. NopP, like NopL, is an effector secreted by the TTSS of NGR234, and depending on the legume host, it may have a deleterious or beneficial effect on nodulation or it may have little effect.A variety of prokaryotic organisms are capable of enzymatically reducing atmospheric nitrogen to ammonia. This process, known as biological nitrogen fixation, can be performed either by free-living bacteria (e.g., Klebsiella pneumoniae) or by symbiotic bacteria. Nitrogen-fixing symbioses between plants belonging to the family Leguminosae and soil bacteria collectively called rhizobia contribute substantially to plant productivity. Ultimately, these associations lead to the formation of specialized structures called nodules on the stems or roots of host plants, where infecting rhizobia differentiate into bacteroids that reduce atmospheric nitrogen to ammonia. The ammonia is incorporated into amino acids that are supplied to the host, which return the favor by supplying the microsymbionts with photosynthates. Nodulation, the process that leads to bacterial colonization of root or stem nodules, is highly selective; a continuous exchange of molecular signals between the two symbionts enables the host to distinguish compatible rhizobia from potential pathogens. Initially, lipochitooligosaccharidic nodulation factors (Nod factors) that are secreted by rhizobia in response to plant flavonoids are essential for infection. Later, additional bacterial signals, such as surface polysaccharides or secreted proteins, are also required for efficient nodulation of specific hosts (7,35).Among the known microsymbionts, Rhizobium sp. strain NGR234 has the rare ability to nodulate more than 112 genera of legumes (37). The closely related strain Rhizobium fredii USDA257 forms nodules on a smaller subset of plants (Ͼ79 genera), but it fixes nitrogen with Glycine max and Glycine soja, two hosts that fail to establish effective symbioses with NGR234. Early work showed that mutations in the cultivar specificity locus nolXWBTUV modified the host range of USDA257 so that it included the improved soybean variety McCall (14,28). Fla...

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Section: Discussionsupporting

confidence: 75%

Characterization of NopP, a Type III Secreted Effector of Rhizobium sp. Strain NGR234

et al. 2004

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“…These were used as probes for plasmid rescue of two genomic, 10.7-and 8.7-kb regions containing flaA-flaB (pRU2351) and flaD (pRU2352), respectively. The R. lupini fla genes were completely sequenced and identified by the similarity of the encoded proteins to other flagellins, in particular those of S. meliloti (28,36) and Agrobacterium tumefaciens (3,5). Adjacent portions of the two clones were sequenced only to the extent where flagellar and motility genes could be identified and arranged on the map by similarity to their S. meliloti paralogues (36).…”

Section: Resultsmentioning

confidence: 99%

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

Scharf

Schuster-Wolff-Bühring

Rachel

et al. 2001

J Bacteriol

103

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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...

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“…Next, we examined the two subpopulations of tatC mutants for production of flagella. A. tumefaciens carries four flagellin genes in its genome, two of which produce abundant levels of flagellin proteins, migrating at ϳ31 and 33 kDa, that are easily visualized by silver staining of extracellular material (19). The 33-kDa FlaA protein is essential for motility and production of wild-type flagella (19).…”

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confidence: 99%

Agrobacterium tumefaciens Twin-Arginine-Dependent Translocation Is Important for Virulence, Flagellation, and Chemotaxis but Not Type IV Secretion

Ding

Christie

2003

J Bacteriol

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This study characterized the contribution of the twin-arginine translocation (TAT) pathway to growth, motility, and virulence of the phytopathogen Agrobacterium tumefaciens. In contrast to wild-type strain A348, a tatC null mutant failed to export the green fluorescent protein fused to the trimethylamine N-oxide reductase (TorA) signal sequence or to grow on nitrate as a sole electron acceptor during anaerobic growth. The tatC mutant displayed defects in growth rate and cell division but not in cell viability, and it also released abundant levels of several proteins into the culture supernatant when grown in rich medium or in vir induction minimal medium. Nearly all A348 cells were highly motile in both rich and minimal media. By contrast, approximately 0.1% of the tatC mutant cells were motile in rich medium, and <0.01% were motile in vir induction medium. Nonmotile tatC mutant cells lacked detectable flagella, whereas motile tatC mutant cells collected from the edge of a motility halo possessed flagella but not because of reversion to a functional TAT system. Motile tatC cells failed to exhibit chemotaxis toward sugars under aerobic conditions or towards nitrate under anaerobic conditions. The tatC mutant was highly attenuated for virulence, only occasionally (ϳ15% of inoculations) inciting formation of small tumors on plants after a prolonged incubation period of 6 to 8 weeks. However, an enriched subpopulation of motile tatC mutants exhibited enhanced virulence compared to the nonmotile variants. Finally, the tatC mutant transferred T-DNA and protein effectors to plant cells and a mobilizable IncQ plasmid to agrobacterial recipients at wild-type levels. Together, our findings establish that, in addition to its role in secretion of folded cofactor-bound enzymes functioning in alternative respiration, the TAT system of A. tumefaciens is an important virulence determinant. Furthermore, this secretion pathway contributes to flagellar biogenesis and chemotactic responses but not to sensory perception of plant signals or the assembly of a type IV secretion system.Gram-negative bacteria secrete proteins across the inner membrane in an unfolded conformation by the general secretory pathway (GSP) or in a folded conformation by the secindependent twin-arginine translocation (TAT) pathway (7,49). This latter system was only recently discovered in bacteria and is structurally and functionally related to the ⌬pH-dependent protein import pathway of the plant chloroplast thyalkoid membrane (6). In bacteria, the TAT system is used predominantly for secretion of cofactor-bound enzymes to the periplasm for function in various respiratory and photosynthetic electron transport pathways (7). However, the TAT system also has been shown to direct the secretion of (i) multisubunit enzyme complexes involved in respiration (37, 58); (ii) monomeric, cofactorless proteins whose functions are unrelated to energy metabolism (2, 50, 55); and (iii) integral inner membrane proteins (42). Of particular interest is that the TAT system (40, 50...

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Agrobacterium tumefaciens possesses a fourth flagellin gene located in a large gene cluster concerned with flagellar structure, assembly and motility

Cited by 45 publications

References 53 publications

Characterization of NopP, a Type III Secreted Effector of Rhizobium sp. Strain NGR234

Characterization of NopP, a Type III Secreted Effector of Rhizobium sp. Strain NGR234

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

Agrobacterium tumefaciens Twin-Arginine-Dependent Translocation Is Important for Virulence, Flagellation, and Chemotaxis but Not Type IV Secretion

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