A DNase encoded by integrated element CJIE1 inhibits natural transformation of Campylobacter jejuni

Abstract

The species Campylobacter jejuni is considered naturally competent for DNA uptake and displays strong genetic diversity. Nevertheless, nonnaturally transformable strains and several relatively stable clonal lineages exist. In the present study, the molecular mechanism responsible for the nonnatural transformability of a subset of C. jejuni strains was investigated. Comparative genome hybridization indicated that C. jejuni Mu-like prophage integrated element 1 (CJIE1) was more abundant in nonnaturally transformable C. jejuni strains than in naturally transformable strains. Analysis of CJIE1 indicated the presence of dns (CJE0256), which is annotated as a gene encoding an extracellular DNase. DNase assays using a defined dns mutant and a dns-negative strain expressing Dns from a plasmid indicated that Dns is an endogenous DNase. The DNA-hydrolyzing activity directly correlated with the natural transformability of the knockout mutant and the dns-negative strain expressing Dns from a plasmid. Analysis of a broader set of strains indicated that the majority of nonnaturally transformable strains expressed DNase activity, while all naturally competent strains lacked this activity. The inhibition of natural transformation in C. jejuni via endogenous DNase activity may contribute to the formation of stable lineages in the C. jejuni population.

FIG. 1.

Comparison of C. jejuni strains by cluster analysis of comparative genomic indexing results. The genes are presented in the order of their positions on the genome of C. jejuni NCTC 11168 and are followed by the genes of C. jejuni RM1221 that are absent from C. jejuni NCTC 11168. For frames of reference, the lipooligosaccharide biosynthesis locus (Cj1136-Cj1144c) and the capsular biosynthesis locus (Cj1415c-Cj1442c) from C. jejuni NCTC 11168 are indicated. The Mu-like prophage insertion element (CJIE1) from C. jejuni RM1221 is also indicated. The gene status is color coded as follows: blue, present; yellow, variable or unknown; red, absent; gray, no data. For cutoffs for absence and presence predictions, see Materials and Methods. An average linkage hierarchical clustering of the C. jejuni strains was compiled in GeneSpring, version 7.3, from the comparative genome hybridization data for each element with the standard correlation and bootstrapping. A scale for distance scores is on the right.

FIG. 2.

Determination of the DNase activity of a C. jejuni dns knockout mutant (C356dns::cat) and a Dns expression construct [CNET002(pWM1007Pr1492dns)]. Phage lambda DNA (500 ng) was incubated (1 h, 37°C) with fractions isolated from C. jejuni and analyzed by agarose gel electrophoresis. Lane 1, nonnaturally transformable dns-positive strain C356; lane 2, dns knockout strain C356dns::cat; lane 3, naturally transformable strain CNET002; lane 4, CNET002 with Dns expression construct [CNET002(pWM1007Pr1492dns)]; lane 5, CNET002 containing the empty expression plasmid [CNET002(pWM1007Pr1492)]; lane +, positive control (DNA treated with 1 μg DNase I); lane −, negative control (mock-treated DNA).

FIG. 3.

Determination of the DNase activity of naturally transformable C. jejuni strains and dns-positive nonnaturally transformable strains. DNase activity was assayed as described in the legend to Fig. 2. Fractions were isolated from naturally transformable C. jejuni strains C013199, C011338, C019168, C013500, and GB18 (lanes 1 to 5, respectively) and from dns-positive nonnaturally transformable C. jejuni strains C011300, C019165, C012446, C012599, CCUG10950, 21.97, 233.95, 260.94, 308.95, and 386.95 (lanes 6 to 15, respectively). Lane +, positive control (DNA treated with 1 μg DNase I); lane −, negative control (mock-treated DNA).

FIG. 4.

Determination of the DNase activity of nonnaturally transformable dns-negative C. jejuni strains. DNase activity was assayed as described in the legend to Fig. 2. The nonnaturally transformable dns-negative strains tested were C017289, C011672, D3141, D3226, D3468, CNET007, 07479, 12795850312, and CNET005 (lanes 1 to 9, respectively). Lane +, positive control (DNA treated with 1 μg DNase I); lane −, negative control (mock-treated DNA).