Minimum requirements of flagellation and motility for the infection of 3 Agrobacterium sp . H 13 - 3 by the flagellotropic bacteriophage 7 - 7 - 1

31 The flagellotropic phage 7-7-1 specifically adsorbs to Agrobacterium sp. H13-3 (formerly 32 Rhizobium lupini H13-3) flagella for efficient host infection. The Agrobacterium sp. H13-3 33 flagellum is complex and consists of three flagellin proteins, the primary flagellin FlaA which is 34 essential for motility and the secondary flagellins FlaB and FlaD which have minor functions in 35 motility. Using quantitative infectivity assays, we show that absence of FlaD had no effect on 36 phage infection, while absence of FlaB resulted in a 2.5-fold increase in infectivity. A flaA 37 deletion strain which produces straight and severely truncated flagella experienced a 38 significantly reduced infectivity, similar to a flaBflaD strain which produces a low number of 39 straight flagella. A strain lacking all three flagellin genes is phage resistant. In addition to 40 flagellation, flagellar rotation is required for infection. A strain that is non-motile due to an in41 frame deletion in the gene encoding the motor component MotA is resistant to phage infection. 42 We also generated two strains with point mutations in the motA gene resulting in replacement of 43 the conserved charged residue Glu98, which is important for modulation of rotary speed. A 44 substitution to the neutral Gln caused the flagellar motor to rotate at a constant fast speed 45 allowing a 2.2 fold enhanced infectivity. An exchange to the positively charged Lys caused a 46 jiggly motility phenotype with very slow flagellar rotation, which significantly reduced the 47 efficiency of infection. In conclusion, flagellar number and length, as well as speed of flagellar 48 rotation are important determinants for infection by phage 7-7-1. 49 50 51 on S etem er 2, 2017 by gest ht://aem .sm .rg/ D ow nladed fom INTRODUCTION 52 Agrobacterium sp. H13-3, formerly known as Rhizobium lupini H13-3, was isolated from the 53 rhizosphere of Lupinus luteus (11). Its complete genome has been recently sequenced, and 54 subsequent genome structure and phylogenetic analyses identified the strain as non-pathogenic 55 Agrobacterium (44). Agrobacterium sp. H13-3 is motile by means of peritrichously inserted 56 flagella. Based on the microscopic appearance, there are two types of flagellar filaments in 57 bacteria, plain and complex. The plain filaments of Escherichia coli have a smooth surface 58 structure and are capable of switching from left-handed to right-handed helicity (22). The 59 complex filaments of Agrobacterium sp. H13-3 and the related soil bacterium Sinorhizobium 60 meliloti exhibit a dominant pattern of alternating ridges and grooves, and do not switch 61 handedness (18; 42; 43). The E. coli filament consists of a single type of flagellin, while the 62 Agrobacterium sp. H13-3 filament consists of three related flagellin subunits, FlaA, FlaB, and 63 FlaD that are assembled as functional heterodimers (35). The Cand N-terminal regions of 64 flagellins are required for assembly of the flagellum and are highly conserved among the three 65 subunits. The central domains, defining the diverse surface of the filament, exhibit more 66 sequence variability. FlaA is the primary flagellin and a flaA deletion mutant produces severely 67 truncated, straight flagella that appear unusually fragile. Since flaB and flaD deletion mutants 68 exhibit a minor reduction in swimming speed and have normal flagellar appearance, they are 69 considered secondary flagellins. However, a strain carrying deletions in both secondary flagellin 70 genes is non-motile and produces a very low number of straight flagella. In conclusion, FlaA and 71 at least one secondary Fla protein are required for the assembly of a flagellar filament that can 72 promote motility (35). 73 Bacterial flagella are driven by a rotary motor at the flagellar base which is energized by a proton 74 gradient across the cytoplasmic membrane (23). The flagellar motors of E. coli switch their sense 75 on S etem er 2, 2017 by gest ht://aem .sm .rg/ D ow nladed fom of rotation, which enables them to change direction (21). The complex filaments are more rigid, 76 because interflagellin bonds lock the filament in a right-handed helical conformation (6). The 77 rigidity of these filaments allows propulsion with a greater force in viscous media (12). 78 Concomitantly, Agrobacterium sp. H13-3 and S. meliloti flagellar motors rotate exclusively 79 clockwise, and direct the swimming path by modulating their rotary speed (1; 34). This 80 swimming behavior requires the activity of two novel motility proteins, MotC and MotE, which 81 are present in addition to the ubiquitous proton channel forming MotA and MotB. MotC binds to 82 the periplasmic portion of MotB, whereas MotE serves as its periplasmic chaperone (8). 83 Flagellotropic bacteriophages specifically target bacterial flagella and require motility of its host 84 for a productive infection. They adsorb to the host’s flagella and then proceed to the cell 85 membrane where the phage DNA is injected. This phage family is represented by χ of E. coli 86 and Serratia marcescens, ФCB13 and ФCbK of Caulobacter crescentus, PBS1 and SP3 of 87 Bacillus subtilis, ФAT1 of Erwinia carotovora (10), Ф OT8 of Serratia sp. ATCC 39006 and 88 Pantoea agglomerans, and 7-7-1 of Agrobacterium sp. H13-3 (9; 13; 15; 16; 19; 28; 30-32). 89 Since not the mere presence of flagella, but their rotation is essential for infection, a “nut and 90 bolt” model for the translocation of phage along the filament was suggested. The tail fiber of the 91 E. coli χ phage fits the right-handed helical grooves on the flagellar surface, and counter92 clockwise rotation would force the phage to follow the grooves to the base of the flagellar 93 filament (3; 31). Bacteriophage 7-7-1 was isolated from composed soil in Germany in the 1970s 94 (20). It is a member of the Myoviridae family consisting of a hexagonal head and a tail separated 95 by a neck (5). The diameter of the head is 68 nm and the phage tail has a length of 135 nm and a 96 diameter of 20 nm. The tail is contractile and ends in bushy fibers (19; 20). 7-7-1 is a lytic phage 97 which specifically infects Agrobacterium sp. H13-3, but not the closely related S. meliloti that 98 on S etem er 2, 2017 by gest ht://aem .sm .rg/ D ow nladed fom also possesses complex flagella (19). The eclipse period lasts approximately 60 minutes and the 99 complete cycle of phage propagation takes 80 minutes with a burst size of 120 particles per 100 bacterial cell (19; 41). Phage resistant Agrobacterium sp. H13-3 mutants were selected after 101 chemical mutagenesis and most of these were non-flagellated. In addition, a small percentage of 102 mutants were found to be non-motile (19). Therefore, flagella and motility seem to be essential 103 for the infection of Agrobacterium sp. H13-3 by phage 7-7-1. However, it is yet unknown how 104 the phage attaches to the flagellum and reaches the bacterial cell surface. 105 In this study, we determined flagellation and flagellar rotation as a requirement for infection of 106 Agrobacterium sp. H13-3 by bacteriophage 7-7-1. Defined deletion and amino acid exchange 107 mutants of Agrobacterium sp. H13-3 were constructed by allelic exchange and infectivity of 108 bacteriophage 7-7-1 for the resulting flagellar and motility mutants was quantified. Requirements 109 for the infection of Agrobacterium sp. H13-3 are discussed. 110 111 on S etem er 2, 2017 by gest ht://aem .sm .rg/ D ow nladed fom MATERIALS AND METHODS 112 Bacterial strains and plasmids 113 Derivatives of E. coli K-12 and Agrobacterium sp. H13-3 (17), Agrobacterium and Rhizobium 114 strains, and the plasmids used are listed in Table 1. 115