bioRxiv preprint doi: https://doi.org/10.1101/335265; this version posted May 31, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Multiple Flagellin Proteins Have Distinct and Synergistic Roles 2 in Agrobacterium tumefaciens Motility 3 4 Bitan Moharia, Melene A. Thompsona, Jonathan C Trinidadb and Clay Fuquaa* 5 6 aDepartment of Biology 7 Indiana University 8 Bloomington, Indiana, USA 9 10 bDepartment of Chemistry 11 Indiana University 12 Bloomington, Indiana, USA 13 14 Running Head: Multiple flagellins of A. tumefaciens 15 16 Keywords: Flagella, motility, flagellin, Agrobacterium tumefaciens 17 18 19 *Address for correspondence: Clay Fuqua, Dept. Biol., 1001 E. 3rd St., Jordan Hall 142, 20 Indiana Univ., Bloomington, IN 47405-1847. Tel: 812-856-6005, FAX: 812-855-6705, E- 21 mail: [email protected] 22 23 Document Word count: 8,583 (primary text not including abstract, acknowledgements, 24 references, legends): 25 26 Figures: 10 27 28 Tables: 2 29 30 1 bioRxiv preprint doi: https://doi.org/10.1101/335265; this version posted May 31, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 31 Abstract 32 Rotary flagella propel bacteria through liquid and across semi-solid environments. 33 Flagella are composed of the basal body that constitutes the motor for rotation, the 34 curved hook that connects to the basal body, and the flagellar filament that propels the 35 cell. Flagellar filaments can be comprised of a single flagellin protein such as in 36 Escherichia coli or with multiple flagellins such is in Agrobacterium tumefaciens. The 37 four distinct flagellins FlaA, FlaB, FlaC and FlaD produced by wild type A. tumefaciens, 38 are not redundant in function, but have specific properties. FlaA and FlaB are much 39 more abundant than FlaC and FlaD and are readily observable in mature flagellar 40 filaments, when either FlaA or FlaB is fluorescently labeled. Cells having FlaA with any 41 one of the other three flagellins can generate functional filaments and thus are motile, 42 but FlaA alone cannot constitute a functional filament. In flaA mutants that manifest 43 swimming deficiencies, there are multiple ways by which these mutations can be 44 phenotypically suppressed. These suppressor mutations primarily occur within or 45 upstream of the flaB flagellin gene or in the transcriptional factor sciP regulating flagellar 46 expression. The helical conformation of the flagellar filament appears to require a key 47 asparagine residue present in FlaA and absent in other flagellins. However, FlaB can be 48 spontaneously mutated to render helical flagella in absence of FlaA, reflecting their 49 overall similarity and perhaps the subtle differences in the specific functions they have 50 evolved to fulfill. 2 bioRxiv preprint doi: https://doi.org/10.1101/335265; this version posted May 31, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 51 Importance 52 Flagellins are abundant bacterial proteins comprising the flagellar filaments that propel 53 bacterial movement. Several members of the Alphaproteobacterial group express 54 multiple flagellins, in contrast to model systems such as Escherichia coli that has only 55 one flagellin protein. The plant pathogen Agrobacterium tumefaciens has four flagellins, 56 the abundant and readily detected FlaA and FlaB, and lower levels of FlaC and FlaD. 57 Mutational analysis reveals that FlaA requires at least one of the other flagellins to 58 function - flaA mutants produce non-helical flagella and cannot swim efficiently. 59 Suppressor mutations can rescue this swimming defect through mutations in the 60 remaining flagellins, including structural changes imparting flagellar helical shape, and 61 putative regulators. Our findings shed light on how multiple flagellins contribute to 62 motility. 63 3 bioRxiv preprint doi: https://doi.org/10.1101/335265; this version posted May 31, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 64 Introduction 65 Flagella typically function as a means of bacterial locomotion in liquid environments, by 66 a discontinuous pattern of swimming comprised of straight runs and directional changes 67 known as tumbles. Other forms of motility include swarming, which also requires 68 flagella, and gliding, twitching, and sliding which do not. Flagella are extracellular helical 69 filaments with a curved hook serving as a universal joint between the filament and the 70 rotary basal body embedded in the cellular membrane (1). The basal body generates 71 power and rotates the flagellum and the hook is important for rendering the optimum 72 angle for propulsion. Flagellar filaments are composed of up to 30,000 subunits of the 73 protein flagellin (2). For bacteria with multiple flagella, these form a bundle to propel 74 forward movement and unbundle to promote cellular re-orientation (3). Flagellar rotation 75 in enteric bacteria such as Escherichia coli and Salmonella typhimurium is bidirectional; 76 (counterclockwise promoting bundling and straight swimming, and reversals to 77 clockwise rotation disrupting the flagellar bundle, and causing tumbles). However, in 78 members of the family Rhizobiaceae, including Sinorhizobium meliloti and 79 Agrobacterium tumefaciens, the rotation is unidirectional and always clockwise, with 80 disruption of bundles occurring due to discordance of flagellar rotation rates (4). The 81 helical shape of the flagellar filament is of utmost importance for these dynamic aspects 82 of propulsion (5). 83 Bacterial filaments can be categorized as plain or complex depending on whether 84 they are made of one or multiple kinds of flagellin protein (6). In well studied systems, 85 such as peritrichously flagellated E. coli, there are multiple filaments comprised of the 86 FliC flagellin protein (7, 8). Similarly the alphaproteobacterium Rhodobacter 4 bioRxiv preprint doi: https://doi.org/10.1101/335265; this version posted May 31, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 87 sphaeroides has one flagellum (laterally positioned on the cell) that is made up of a 88 single flagellin protein (9, 10). On the other hand, examples of bacteria with filaments 89 made of multiple flagellins include Caulobacter crescentus, which has a single polar 90 flagellum, but remarkably six flagellin proteins (11). Within the Rhizobiaceae family 91 Sinorhizobium meliloti, Rhizobium leguminosarum, Agrobacterium sp. H13-3, and 92 Agrobacterium tumefaciens all encode multiple flagellins. A. tumefaciens mutated in 93 three of its four flagellin genes is reduced in virulence by about 38% and Agrobacterium 94 sp. H13-3 lacking all of its three flagellin genes is resistant to flagella-specific phage 95 infection (12-16). Flagella with simple, single flagellin filaments exhibit structural 96 polymorphisms during the course of normal flagellar propulsion and rotational switching, 97 whereas those with complex filaments do not exhibit these polymorphisms unless they 98 are exposed to extreme conditions of pH and ionic strengths (13, 17). Studies in C. 99 crescentus with six different flagellin genes suggest that multiple flagellins can have a 100 certain level of redundancy (11). In some cases there can be a single predominant 101 flagellin required for flagellar function, such as those for S. meliloti, Agrobacterium sp. 102 H13-3 and Rhizobium leguminosarum (12, 14). Multiple flagellins can also be 103 differentially regulated as in spirochetes, Vibrio cholerae, and C. crescentus (11, 18, 104 19). 105 A. tumefaciens has a lophotrichous arrangement of 5-6 flagellar filaments 106 hypothesized to be comprised of four flagellins. The flagellins all share significant 107 sequence similarity (Table 1; Fig. S1). The functions and coordination of the flagellins 108 are however, not well studied. FlaA, FlaB and FlaC are encoded in a single gene cluster 109 and are of similar molecular mass: (FlaA, 306 aa; FlaB, 320 aa; FlaC, 313 aa). In 5 bioRxiv preprint doi: https://doi.org/10.1101/335265; this version posted May 31, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 110 contrast, FlaD is the most dissimilar, located roughly 15 kb distal to the flaABC cluster 111 and is oriented in the opposite direction, relative to the other flagellins (Fig. S2). FlaD is 112 430 aa, with a large internal segment that is not shared with the other flagellins. FlaA is 113 reported to be the major flagellin, as disruption of the gene severely compromised 114 motility, reportedly resulting in vestigial stubs instead of flagellar filaments (16, 20). 115 Other single flagellin mutants were shown to be attenuated in motility that produced 116 filaments with some structural abnormalities. Mutants were generated either via 117 transposon or antibiotic cassette insertions, and thus are prone to polar effects on 118 downstream genes and partial gene copies, confounding the results reported for these 119 flagellins and making it difficult to evaluate individual gene function (16, 20).