Diagram of the C. crescentus flagellum. The flagellar structure is adapted from references 12 and 22. Flagellar components encoded by class II genes are light gray, components encoded by class III genes are dark gray, and the filament (class IV) is black. Class I is assigned to genes that are required for the activation of class II genes. The flagellar substructures are indicated on the right. Approximately 26 FliF protein subunits form the MS ring structure in the inner membrane (light gray with hatching). Rotation is conferred by proton flow through stator protein MotA of the force-generating unit in interaction with the rotor C ring protein FliG (22). Abbreviations: IM, inner membrane; CW, cell wall (peptidoglycan layer); OM, outer membrane.

Positions of deletions in the FliF C terminus and resulting motility phenotypes. (A) Positions of in-frame deletions Δ1, Δ15, Δ16, Δ17, Δ5, ΔI, ΔII, ΔIII, ΔIV, ΔV, and ΔVI in the cytoplasmic C terminus of FliF are marked with lines below the amino acid sequence. Deletions Δ1 and Δ5 have been described before (12). The second transmembrane domain (TM2), as proposed in reference 12, is indicated. The shaded sequences are regions that were proposed to form α-helices by the secondary-structure prediction program PDHsec. The two α-helices at the immediate C terminus are referred to in the text as helices 1 and 2, and the area between them is referred to as the loop region. The numbers beside the amino acid sequence indicate positions in the FliF protein. It has been taken into account that the experimentally proven start codon of FliF is 18 codons upstream of the one predicted in the GenBank database entry (12). (B) Semisolid agar plate assay of the motility of fliF null mutant strain LS1218 (ΔfliF) complemented with a wild-type copy of fliF (wt) or with the fliF deletion allele Δ15, Δ16, or Δ17. The fliF copy used for complementation was integrated into the chromosome at the fliF locus. The swarming capacity of strain LS1218 complemented with fliF Δ1 and Δ5 has been shown in reference 12.

Summary of all mutations in the cytoplasmic C terminus of FliF and of the corresponding phenotypic analysis described in this study. (A) A schematic diagram of the last 30 amino acids of the C terminus is shown on the left. The shaded areas are the two regions that were proposed to form α-helices by the secondary-structure prediction program PDHsec. The area between the two predicted α-helices is referred to as the loop region in the text. The corresponding mutant names are on the left. Flagellar assembly: flagella detected by electron microscopy (see Fig. 6). A plus indicates that most of the swarmer cells were flagellated, a minus indicates that no swarmer cells were flagellated, and a minus in parentheses indicates that very few swarmer cells were flagellated. Motility: cell motility determined by light microscopy. A plus indicates that swimming cells were observed, and a minus indicates that no swimming cells were present. Swarmer colony formation: motility assayed on semisolid agar plates. A plus indicates formation of a large swarmer colony of wild-type size, a minus indicates a small, compact colony, and a plus in parentheses indicates a swarmer colony of intermediate size (see Fig. 2 and 4). Filamentous morphology: cell morphology determined by light microscopy (see Fig. 5). A plus indicates that the cells were filamentous, and a minus indicates that the cells had wild-type morphology. Class III expression: activity of the flgF flagellar class III promoter (see Fig. 7). A plus indicates wild-type promoter activity, a minus indicates background promoter activity, and a plus-and-minus sign indicates intermediate promoter activity. Motile suppressors: isolation of motile suppressors from nonmotile mutants. A plus indicates that motile suppressors could be isolated, and a minus indicates that no motile suppressors were found. The numbers in parentheses are the numbers of independent suppressor strains isolated. Abbreviations: ND, not determined; NA, not applicable. (B) Immunoblot analysis of strains expressing FliF mutant forms using an anti-FliF antibody. Extracts (normalized for cell numbers) of the following strains were used: lane 1, LS1218 (ΔfliF); lane 2, NA1000 (wild type); lane 3, LS1528 (wild-type fliF); lane 4, UJ556 (fliF Δ9); lane 5, UJ857 (fliF Δ10); lane 6, UJ970 (fliF Δ13); lane 7, UJ971 (fliF Δ14); lane 8, UJ974 (fliF S3); lane 9, LS1218 (ΔfliF); lane 10, NA1000 (wild type); lane 11, UJ358 (fliF ΔIV); lane 12, UJ359 (fliF ΔV); lane 13, UJ553 (fliF Δ6); lane 14, UJ434 (fliF Δ5); lane 15, UJ554 (fliF Δ7); lane 16, UJ555 (fliF Δ8).

Semisolid agar plate assay used to determine the swimming capacity of fliF null mutant strain LS1218 (ΔfliF) complemented with either a wild-type copy of fliF (wt) or a copy of fliF deletion allele ΔI, ΔII, ΔIII, ΔIV, ΔV, or ΔVI. Motile strains form a large swarmer colony, and nonmotile strains form a small, compact colony.

Light microscopy analysis of fliF null mutant strain LS1218 (ΔfliF) complemented with either a wild-type copy of fliF (wt) or a copy of fliF deletion allele ΔI, ΔII, ΔIII, ΔIV, ΔV, or ΔVI. Bars, 10 μm.

Electron microscopy analysis of fliF null mutant strain LS1218 (ΔfliF) complemented with either a wild-type copy of fliF (wt) or a copy of fliF deletion allele ΔI, ΔII, ΔIII, ΔIV, ΔV, or ΔVI. Arrows indicate flagellar structures. The contrast of the flagella was manually enhanced. Bars, 1 μm.

Relative activity of the flagellar class III flgF promoter in C. crescentus fliF null mutant strain LS1218 (ΔfliF) complemented with various fliF mutant alleles. Plasmid pCM4, which contains a transcriptional fusion of the flgF promoter with the reporter gene lacZ was introduced into all mutant strains. Promoter activity was determined by measuring β-galactosidase activity. The fliF alleles are indicated below each bar. Background β-galactosidase activity was determined by using the vector control plac290 in strain NA1000. The height of each bar represents the relative promoter activity of the corresponding mutant strain compared to the activity of the C. crescentus NA1000 wild-type (wt) strain. The error bars represent the standard errors.

(A) Alignment of the N-terminal FliG sequences from C. crescentus (CC) and S. enterica serovar Typhimurium (ST) by using BLAST. The numbers on the sides are amino acid positions according to the sequences in the GenBank database. The solid line below the sequence indicates the amino acids essential for FliF binding in S. enterica serovar Typhimurium, and the broken line indicates amino acids that are required for proper motility (deletion mutants have reduced motility) (15). Arrows point to the amino acids that were altered in the motile suppressor strains of C. crescentus, and the letters above indicate the substituted amino acids. The shaded sequences are regions that were proposed to form α-helices by the secondary-structure prediction program PDHsec. (B) Swarming on semisolid agar of the motile suppressors with mutations in fliF or fliG, the original fliF mutants, and the Δ(fliF-fliG) tester strain complemented with the fliF-fliG loci of the suppressor strains. The strains shown are LS1528 (wild type [wt]), UJ1236 (ΔfliF-fliG), UJ1522 (UJ1236 with the fliF-fliG locus from wild-type strain LS1528), UJ355 (ΔI), UJ881 (motile suppressor of UJ355), UJ1512 (UJ1236 with the fliF-fliG locus from UJ881), UJ882 (motile suppressor of UJ355), UJ1513 (UJ1236 with the fliF-fliG locus from UJ882), UJ356 (ΔII), UJ887 (motile suppressor of UJ356), UJ1518 (UJ1236 with the fliF-fliG locus from UJ887), UJ858 (Δ11), UJ1471 (motile suppressor of UJ858), UJ1523 (UJ1236 with the fliF-fliG locus from UJ1471), UJ975 (S4), UJ1479 (motile suppressor of UJ975), UJ1531 (UJ1236 with the fliF-fliG locus from UJ1479), UJ1482 (motile suppressor of UJ975), and UJ1534 (UJ1236 with the fliF-fliG locus from UJ1482). Strains UJ883 and UJ1480 are not shown here because they contain the same mutations as strains UJ882 and UJ1482, respectively.