Natural transformation of Campylobacter jejuni requires components of a type II secretion system

Abstract

The human pathogen Campylobacter jejuni is one of more than 40 naturally competent bacterial species able to import macromolecular DNA from the environment and incorporate it into their genomes. However, in C. jejuni little is known about the genes involved in this process. We used random transposon mutagenesis to identify genes that are required for the transformation of this organism. We isolated mutants with insertions in 11 different genes; most of the mutants are affected in the DNA uptake stage of transformation, whereas two mutants are affected in steps subsequent to DNA uptake, such as recombination into the chromosome or in DNA transport across the inner membrane. Several of these genes encode proteins homologous to those involved in type II secretion systems, biogenesis of type IV pili, and competence for natural transformation in gram-positive and gram-negative species. Other genes identified in our screen encode proteins unique to C. jejuni or are homologous to proteins that have not been shown to play a role in the transformation in other bacteria.

FIG. 1.

Regions encompassing genes identified as playing a role in transformation and location of solo in these genes. Triangles represent solo insertion sites. Black arrows indicate the direction of transcription.

FIG. 2.

Alignment of N-terminal regions of CtsG, CtsT, and Cj1078 to pre-pilins and pre-pilin-like proteins from S. pneumoniae (45), N. gonorrhoeae (65), K. pneumoniae (47), P. aeruginosa (41, 44), and B. subtilis (1, 6). The processing site is indicated by an arrow. The conserved G, F, and E residues are boxed, and residues conserved among a number of the prepilins are indicated in boldface.

FIG. 3.

Transformation efficiency of in-frame deletion mutants and complemented constructs. The data represent the average of three samples per strain from one experiment. Experiments were repeated at least three times. (A) Transformation efficiency of C. jejuni solo mutants complemented with pECO102 or derivative containing the coding sequence for the appropriate gene; (B) transformation efficiency of C. jejuni deletion mutants; (C) transformation efficiency of C. jejuni deletion mutants complemented with pECO102 or derivative containing the coding sequence from deleted gene.

FIG. 4.

DNA uptake assays. The data are the average of three samples from one experiment, which was repeated at least three times. Values are normalized to the wild-type levels. Wild-type 81-176 samples with no added radiolabeled DNA serve as a control. Since C. jejuni does not take up E. coli DNA (62), we performed DNA uptake experiments with radiolabeled DH5α DNA which served as a negative control. (A) Uptake of radiolabeled 81-176 chromosomal DNA by C. jejuni 81-176 and mutants isolated in the operon spanning from ctsR to ctsF; (B) uptake of radiolabeled 81-176 chromosomal DNA by 81-176 and solo mutants from other locations in the genome.

FIG. 5.

Transformation efficiency of type IV secretion system mutants of pVir. The results are from one experiment conducted in triplicate, and the experiment was repeated at least three times.