Abstract
Campylobacter jejuni genetic manipulation is restricted by the limited number of antibiotic resistance cassettes available for use in this diarrheal pathogen. In this study, two antibiotic resistance cassettes were developed, encoding for hygromycin B and apramycin resistance, for use in mutagenesis or for selection of gene expression and complementation constructs in C. jejuni. First, the marker genes were successfully modified to allow for insertional mutagenesis or deletion of a gene-of-interest, and were bracketed with restriction sites for the facilitation of site-specific cloning. These hygromycin B and apramycin markers are encoded by plasmids pAC1H and pAC1A, respectively. We also modified an insertional gene-delivery vector to create pRRH and pRRA, containing the hygromycin B and apramycin resistance genes, and 3 unique restriction sites for the directional introduction of genes into the conserved multi-copy rRNA gene clusters of the C. jejuni chromosome. We determined the effective antibiotic concentrations required for selection, and established that no harmful effects or fitness costs were associated with carrying hygromycin B or apramycin resistance under standard C. jejuni laboratory conditions. Using these markers, the arylsulfatase reporter gene astA was deleted, and the ability to genetically complement the astA deletion using pRRH and pRRA for astA gene insertion was demonstrated. Furthermore, the relative levels of expression from the endogenous astA promoter were compared to that of polycistronic mRNA expression from the constitutive promoter upstream of the resistance gene. The development of additional antibiotic resistance cassettes for use in Campylobacter will enable multiple gene deletion and expression combinations as well as more in-depth study of multi-gene systems important for the survival and pathogenesis of this important bacterium.
Highlights
The relative paucity of genetic techniques available for the manipulation of Campylobacter jejuni has historically been a limiting factor in the study and molecular biology of the leading cause of bacterial gastroenteritis in the developed world [1]
To construct hygromycin B (HygR) and AprR cassettes that could be used for mutagenesis, we synthesized polymerase chain reaction (PCR) ultramers to aph(70) or aac(3)IV, which included the restriction enzyme cut sites and features depicted in Fig. 1A based on the non-polar KanR cassette described by Menard, Sansonetti and Parsot [17]
When harbored by E. coli, expression of the resistance markers is driven by lac or ara inducible promoters in pGEM and pBAD respectively; induction was not required for E. coli growth in the presence of the corresponding antibiotic
Summary
The relative paucity of genetic techniques available for the manipulation of Campylobacter jejuni has historically been a limiting factor in the study and molecular biology of the leading cause of bacterial gastroenteritis in the developed world [1]. The development of the first genetic tools for C. jejuni was precipitated after the demonstration of gene transfer from Escherichia coli to C. jejuni via plasmids carrying kanamycin resistance in 1987 [2]. As our understanding of C. jejuni grows, so does the need for new markers to rapidly delete and restore complex multigene systems, and/or to simultaneously express a reporter such as green fluorescent protein (GFP), arylsulfatase, or luciferase in mutant and/or complemented strains. To address this need, we adapted current C. jejuni genetic technologies to harbor resistance genes against the antibiotics hygromycin B and apramycin
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