Cultures of wild-type Caulobacter crescentus and strains with fla mutations representing 24 genes were pulse-labeled with 14C-amino acids and analyzed by immunoprecipitation to study the synthesis of flagellar components. Most fla mutants synthesize flagellin proteins at a reduced rate, suggesting the existence of some mechanism to prevent the accumulation of unpolymerized flagellin subunits. Two strains contain deletions that appear to remove a region necessary for this regulation. The hook protein does not seem to be subject to this type of regulation and, in addition, appears to be synthesized as a faster-sedimenting precursor. Mutations in a number of genes result in the appearance of degradation products of either the flagellin or the hook proteins. Mutations in flaA, -X, -Y, or -Z result in the production of filaments (stubs) that contain altered ratios of the flagellin proteins. In some flaA mutants, other flagellin-related proteins were assembled into the stub structures in addition to the flagellins normally present. Taken together, these analyses have begun to provide insight into the roles of individual fla genes in flagellum biogenesis in C. crescentus.

Intact bacterial flagella possessing a membrane-free hook and basal complex were purified from Caulobacter crescentus CB15, as well as from mutants which synthesize incomplete flagella. The basal body consisted of five rings mounted on a rod. Two rings were in the hook-proximal upper set, and three rings (two narrow and one wide) were in the lower set. The diameters of the two upper rings differed, being 32 and 21 nm, respectively. The lower rings were all approximately 21 nm in diameter, although they varied significantly in width. During the normal course of the C. crescentus cell cycle, the polar flagellum with hook and rod was shed into the culture medium without the basal rings. Similarly, hooks with attached rods were shed from nonflagellate mutants, and these structures also lacked the basal rings. The hook structure was purified from nonflagellated mutants and found to be composed of a 70,000-molecular-weight protein component.

A total of 69 spontaneous nonmotile mutants were isolated from the dimorphic bacterium Caulobacter crescentus. The majority of the mutants were unable to assemble a flagellar filament (Fla-), although eight were able to synthesize a short stub of a flagellum. A third mutant class assembled flagella of normal morphology but were nonmotile (Mot-). Genetic analysis by phiCr30-mediated transduction revealed 27 linkage groups for the fla and stub-forming mutations, and three linkage groups for the mot mutations. Intracellular flagellin detected by immunodiffusion was at the limit of detectability in most of the Fla- and stub-forming mutants but normal in the Mot- mutants. The Fla- and stub-forming mutants also showed decreased sensitivity to the swarmer-specific phages phiCbK and phiCb5 and phiCr40. One additional strain was totally resistant to phiCbK, and the mutation in this strain has been designated pleA. Each of the mutants containing mot mutations showed wild-type sensitivity to all of the phages tested.

Caulobacter crescentus has a penicillinase which precludes the use of penicillin for mutant enrichment. However, two other antibiotics, fosfomycin and D-cycloserine, can be enrich for C. crescentus mutants. In enrichment procedures for C. crescentus auxotrophs, spontaneously derived mutants occur at a frequency of 5-10% among the survivors of an enrichment procedure. Consequently, large numbers of mutants are readily obtained without any need for mutagenesis. These mutants are heterogeneous both with regard to the type of mutation and to the nutritional requirement. A similar procedure has been used to isolate temperature-sensitive mutants.

Summary We have isolated 38 viruses capable of infecting Caulobacter crescentus. Twenty-eight of these isolates were DNA phages with a prolate cylindrical head and a long flexible tail. Each of them infected only one of the Caulobacter cell types, the swarmer cell, and many proved to be capable of establishing a lysogenic relationship with their host. Despite structural similarity and host cell specificity, the majority of these DNA phages appeared to be genetically distinct as shown by EcoR1 restriction patterns of their DNA and by host range on phage resistant and lysogenic strains. Two isolates were small RNA phages, structurally similar to the Escherichia coli RNA phages, which specifically adsorbed to pili on the Caulobacter swarmer cell. The remaining eight isolates were DNA phages with polyhedral heads ranging from 60 to 160 nm in diameter. Six had contractile tails ranging from 50 to 160 nm in length, while two had long flexible non-contractile tails. Each of these isolates was capable of attaching to all Caulobacter cell types and two were capable of mediating generalized transduction between strains of Caulobacter (Ely & Johnson, 1977).

Two closely related bacteriophage, varphiCr30 and varphiCr35, are the first bacteriophage shown to mediate generalized transduction in Caulobacter crescentus. Unlike most other transducing phage, they are virulent and do not form any sort of lysogenic relationship with their host. However, they are rather inefficient at adsorption, so that transductants have a good chance of survival. The phage particles have a head 80 nm in diameter and a contractile tail 140 nm in length. Procedures for growth and transduction with varphiCr30 are relatively simple; thus, it will be of great value for the genetic analysis of C. crescentus.