Genetic and physical analyses of Caulobacter crescentus trp genes

Abstract

Caulobacter crescentus trp mutants were identified from a collection of auxotrophs. Precursor feeding experiments, accumulation studies, and complementation experiments resulted in the identification of six genes corresponding to trpA, trpB, trpC, trpD, trpE, and trpF. Genetic mapping experiments demonstrated that the trp genes were in two clusters, trpCDE and trpFBA, and a 5.4-kilobase restriction fragment from the C. crescentus chromosome was isolated that contained the trpFBA gene cluster. Complementation experiments with clones containing the 5.4-kilobase fragment indicated that trpF was expressed in Escherichia coli and that all three genes were expressed in Pseudomonas putida. This expression was lost in both organisms when the pBR322 tet gene promoter was inactivated, indicating that all three genes were transcribed in the same orientation from the tet promoter. Thus, the C. crescentus promoters do not seem to be expressed in E. coli or P. putida. Complementation of the C. crescentus trp mutants indicated that the tet promoter was not necessary for expression in C. crescentus and suggested that at least two native promoters were present for expression of the trpF, trpB, and trpA genes. Taken together, these results indicate that C. crescentus promoters may have structures that are significantly different from the promoters of other gram-negative species.* Corresponding author. 279that the trp genes are located in two widely separated regions of the C. crescentus chromosome. In addition, we have isolated a 5.4-kilobase (kb) fragment of C. crescentus DNA, and we demonstrate that it contains the trpFBA gene cluster.

The Caulobacter crescentus flaFG region regulates synthesis and assembly of flagellin proteins encoded by two genetically unlinked gene clusters

“…Analyses of spontaneous flagellar mutations at other locations on the chromosome have failed to identify deletions among the mutations at any of these loci (12,17,18,24). Furthermore, analyses of spontaneous ilv and op mutations resulted in the identification of only a single deletion that occurred in the trpFBA region (32,35). Therefore, spontaneously occurring deletions do not occur at a high frequency in C. crescentus.…”

7 The Molecular Biology of Tryptophan Synthase: A Model for Protein–Protein Interaction

“…The trp mutant hosts are mostly E. coli derivatives, as used in the cloning of Bacillus stearothermophilus trpBA (Ishiwata et al, 1989), Brevibacterium lactofermentum trpE (Matsui, Miwa and Sano, 1987), Caulobacter crescentus trpF (Winkler et al, 1984), Clostridium thermocellum trpEG (Sato et al, 1989), Lactobacillus casei trpDCFBA (Natori, Kano and Imamoto, 1990), Methanococcus voltae trpFBA (Sibold and Henriquet, 1988), Streptomyces griseus trpCBA (Rivero-Lezcano et al, 1990) and Thermus thermophilus trpEG (Sato et al, 1988). In cases where problems with an E. coli host were anticipated or encountered, other hosts were used.…”