Chromosomal integration vectors allowing flexible expression of foreign genes in Campylobacter jejuni.
BACKGROUND
Campylobacter jejuni is a major cause of human gastroenteritis yet there is limited knowledge of how disease is caused. Molecular genetic approaches are vital for research into the virulence mechanisms of this important pathogen. Vectors that allow expression of genes in C. jejuni via recombination onto the chromosome are particularly useful for genetic complementation of insertional knockout mutants and more generally for expression of genes in particular C. jejuni host backgrounds.


METHODS
A series of three vectors that allow integration of genes onto the C. jejuni chromosome were constructed by standard cloning techniques with expression driven from three different strong promoters. Following integration onto the C. jejuni chromosome expression levels were quantified by fluorescence measurements and cells visualized by fluorescence microscopy.


RESULTS
We have created plasmid, pCJC1, designed for recombination-mediated delivery of genes onto the C. jejuni chromosome. This plasmid contains a chloramphenicol resistance cassette (cat) with upstream and downstream restriction sites, flanked by regions of the C. jejuni pseudogene Cj0223. Cloning of genes immediately upstream or downstream of the cat gene allows their subsequent introduction onto the C. jejuni chromosome within the pseudogene. Gene expression can be driven from the native gene promoter if included, or alternatively from the cat promoter if the gene is cloned downstream of, and in the same transcriptional orientation as cat. To provide increased and variable expression of genes from the C. jejuni chromosome we modified pCJC1 through incorporation of three relatively strong promoters from the porA, ureI and flaA genes of C. jejuni, Helicobacter pylori and Helicobacter pullorum respectively. These promoters along with their associated ribosome binding sites were cloned upstream of the cat gene on pCJC1 to create plasmids pCJC2, pCJC3 and pCJC4. To test their effectiveness, a green fluorescent protein (gfp) reporter gene was inserted downstream of each of the three promoters and following integration of promoter-gene fusions onto the C. jejuni host chromosome, expression levels were quantified. Expression from the porA promoter produced the highest fluorescence, from flaA intermediate levels and from ureI the lowest. Expression of gfp from the porA promoter enabled visualization by fluorescent microscopy of intracellular C. jejuni cells following invasion of HeLa cells.


CONCLUSIONS
The plasmids constructed allow stable chromosomal expression of genes in C. jejuni and, depending on the promoter used, different expression levels were obtained making these plasmids useful tools for genetic complementation and high level expression.
Faculty of Life Sciences, University of Manchester, Michael Smith Building, Manchester, M13 9PT, UK. Adrian.Jervis@manchester.ac.uk.|Pathogen Molecular Biology Unit, London School of Hygiene and Tropical Medicine, London, WC1E 7HT, UK. Adrian.Jervis@manchester.ac.uk.
Adrian J
AJ
Jervis
Faculty of Life Sciences, University of Manchester, Michael Smith Building, Manchester, M13 9PT, UK. j.a.butler@sheffield.ac.uk.
Jonathan A
JA
Butler
Pathogen Molecular Biology Unit, London School of Hygiene and Tropical Medicine, London, WC1E 7HT, UK. brendan.wren@lshtm.ac.uk.
Brendan W
BW
Wren
Faculty of Life Sciences, University of Manchester, Michael Smith Building, Manchester, M13 9PT, UK. james.d.linton@manchester.ac.uk.
Dennis
D
Linton
D016123:Campylobacter jejuni / Q000235:genetics*; D005470:Fluorometry; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D042822:Genomic Instability; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA
D011994:Recombinant Proteins