Conditional surface structure mutants of Caulobacter crescentus temperature-sensitive flagella formation due to an altered flagellin monomer

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Two classes of temperature-sensitive surface structure mutants of Caulobacter crescentus have been isolated. Mutants in one class were unable to form flagella at the restrictive temperature (37°C) and mutants in the second class were unable to form a stalk structure at 37°C. All of the mutants appeared normal at the permissive temperature (23°C). Analysis of the conditional flagella mutant revealed that the temperature-sensitive lesion resided in the structural flagellin monomer, since (1) extracts of mutant cells grown at the restrictive temperature contained material cross-reacting with anti-flagellin antibody although flagella were not assembled. (2) Mutant flagellin prepared from cells grown at the permissive temperature was heat-denatured to a different conformation than wild-type flagellin, as demonstrated by lateral shifts in microcomplement fixation assays. (3) Amino acid analysis showed the presence of tyrosine in the mutant flagellin, whereas none was detected in the wild-type control. (4) Chymotryptic peptide maps of mutant and wild-type flagellin showed the appearance of two new peptides in the former case. (5) A spontaneous revertant regained both motility and wild-type flagellin amino acid composition and chymotryptic peptide maps. An alteration in the primary structure of the mutant flagellin therefore results in a temperature-dependent alteration in flagellin conformation, perhaps accounting for the inability to assemble a flagellar structure at the cell surface. Comparison of immunoprecipitates of intracellular and extracellular mutant flagellin, as well as flagellin concentrations, further showed that the inability to assemble a flagellar structure interfered with the regulatory processes controlling both the amount and structural integrity of the intracellular flagellin at the restrictive temperature.