Motility defects in Campylobacter jejuni defined gene deletion mutants caused by second-site mutations

Abstract

Genetic variation due to mutation and phase variation has a considerable impact on the commensal and pathogenic behaviours of Campylobacter jejuni. In this study, we provide an example of how second-site mutations can interfere with gene function analysis in C. jejuni. Deletion of the flagellin B gene (flaB) in C. jejuni M1 resulted in mutant clones with inconsistent motility phenotypes. From the flaB mutant clones picked for further analysis, two were motile, one showed intermediate motility and two displayed severely attenuated motility. To determine the molecular basis of this differential motility, a genome resequencing approach was used. Second-site mutations were identified in the severely attenuated and intermediate motility flaB mutant clones: a TA-dinucleotide deletion in fliW and an A deletion in flgD, respectively. Restoration of WT fliW, using a newly developed genetic complementation system, confirmed that the second-site fliW mutation caused the motility defect as opposed to the primary deletion of flaB. This study highlights the importance of (i) screening multiple defined gene deletion mutant clones, (ii) genetic complementation of the gene deletion and ideally (iii) screening for second-site mutations that might interfere with the pathways/mechanisms under study.

The Minor Flagellin of Campylobacter jejuni (FlaB) Confers Defensive Properties against Bacteriophage Infection

“…We also detected no mutations in the flaA gene, as further supported by findings from motility tests. Analysis of the motility of the flaB mutant strains revealed no major reduction in swarming motility (Figure 2), which is in accordance with previous observations (Wassenaar et al, 1991; de Vries et al, 2015). …”

“…Further, nucleotide deletions in fliW and flgD have also been found in connection with motility loss. This phenomenon was observed upon analysis of second-site mutations in a flaB knock-out mutant strain (de Vries et al, 2015). To exclude that secondary mutation in connection with flaB disruption were responsible for the observed increase in bacteriophage sensitivity; we tested five independent clones, which all carried the selective marker in the desired position.…”

“…However, propagation on a flaB mutant of PT14 produced clear lysis zones that persisted even after prolonged incubation for 24 h (Figure 1). Five individual clones, which all carried the desired disruption of flaB were tested in spot test assays to exclude effects of secondary mutations (de Vries et al, 2015). All showed identical phenotypes toward phage-induced lysis on soft agar lawns and motility (Supplementary Figure S2).…”

“…Information regarding the function of the minor flagellin is sparse, although several studies have characterized flaB as not essential for motility (Guerry et al, 1991; Wassenaar et al, 1993; de Vries et al, 2015). These observations lead to the question as to why a paralogous structural gene has emerged and has been preserved throughout all known Campylobacter species.…”

Genomic variations leading to alterations in cell morphology of Campylobacter spp

“…For example, differences within the STM library could be derived from differences in growth or transformation micro-environments while the STM library was being made. Collectively, the presence of these mutations warns against assumptions that isolates within any C. jejuni population are genetically identical (also addressed in refs 46, 47, 48, 49) or that any site-directed genotypic changes to C. jejuni are responsible for observed phenotypic changes. To overcome this uncertainty during phenotypic characterisation, the precedent should be maintained that C. jejuni isolates should be assessed by genome sequencing, by appropriate complementation analyses of mutants, and/or by performing laboratory manipulations and physiological assays on a panel of isolates.…”