A new class of Caulobacter crescentus flagellar genes

Abstract

Eight Caulobacter crescentus flagellar genes, flmA, flmB, flmC, flmD, flmE, flmF, flmG, and flmH, have been cloned and characterized. These eight genes are clustered in pairs (flmAB, flmCD, flmEF, and flmGH) that appear to be structurally organized as operons. Homology comparisons suggest that the proteins encoded by the flm genes may be involved in posttranslational modification of flagellins or proteins that interact with flagellin monomers prior to their assembly into a flagellar filament. Expression of the flmAB, flmEF, and flmGH operons was shown to occur primarily in predivisional cells. In contrast, the flmCD operon was expressed throughout the cell cycle, with only a twofold increase in predivisional cells. The expression of the three temporally regulated operons was subject to positive regulation by the CtrA response regulator protein. Mutations in class II and III flagellar genes had no significant effect on the expression of the flm genes. Furthermore, the flm genes did not affect the expression of class II or class III flagellar genes. However, mutations in the flm genes did result in reduced synthesis of the class IV flagellin proteins. Taken together, these data indicate that the flm operons belong to a new class of flagellar genes.

FIG. 1

Analysis of the flmCD and flmEF regions. Shown is a restriction map of plasmid pSCC2, which contains a 14-kb EcoRI DNA fragment of the C. crescentus chromosome. Subclones from this region in plasmid pRK2L1 or R300B were tested for the ability to complement the motility defect of strains SC305 (flmD148), SC175 (flmE102), SC3898 (flmC148 recA526 zzz::Tn5), and SC3899 (flmE102 recA526 zzz:Tn5). +, complementation of the motility defect; −, failure to complement. Solid arrows represent predicted open reading frames and direction of transcription. Abbreviations: A, SacI; B, BamHI; E, EcoRI; N, NcoI; P, HpaI; S, SalI.

FIG. 2

(A) Analysis of the flmAB region. Genetic organization of plasmid pSCC7 harboring the 5.0-kb EcoRI fragment is represented. Solid arrows represent open reading frames and direction of transcription. The ability to complement strain SC229 (flmA104) is shown. (B) Organization of the flmGH region. Solid arrows represent open reading frames and direction of transcription. + and − denote the ability and inability, respectively to swarm in a semisolid medium. Abbreviations: B, BamHI; C, ClaI; E, EcoRI; H, HindIII; P, HpaI; S, SalI.

FIG. 3

Cell cycle expression of the flmA, flmC, flmE, and flmG operons. Synchronized populations of Caulobacter strains SC3971, SC3973, SC4016, and SC4250 were pulse-labeled with [³⁵S]methionine at 15-min intervals during the cell cycle. (A) Immunoprecipitation of labeled proteins with CAT, β-galactosidase, or flagellin antibodies. A cartoon showing progress through the cell cycle is shown at the top. The cell cycle-dependent expression of the flagellin genes is shown as a control. (B) Quantification of these data by using a Alpha Innotech photodocumentation system. Percentage of maximal expression of each sample is shown as a function of cell division units. One cell division unit is equivalent to a generation time of 180 min. Closed squares represent 25-kDa flagellin expression (recovered from SC3973 cells carrying the flmE::cat fusion); open squares represent expression of the flmA::lacZ, flmC::cat, flmE::cat, or flmG::cat fusion.