Design and construction of a Haemophilus influenzae conjugal expression system.
Haemophilus influenzae is a fastidious Gram-negative coccobacilli that is a common commensal in the human upper respiratory tract. However, certain strains of this bacterium, including those considered to be nontypeable (NTHi), can cause human diseases ranging from otitis media to meningitis. Although naturally competent, NTHi take up plasmids by transformation very inefficiently, if at all. Many clinical isolates have also proven refractory to the introduction of currently available shuttle vectors via electroporation. Further, it has been difficult to determine protein expression from these vectors, unless specific antisera has been raised or a phenotype conferred. To address these problems, we have designed and constructed a set of broad host range vectors that are transferable via intergeneric conjugation with an Escherichia coli strain carrying chromosomally-encoded transfer functions. These vectors provide a site for cloning promoter::MCS regions and carry genes encoding resistance to one of two different antibiotics. This conjugal system allows the expression of marker genes in NTHi strains, enabling researchers to track the microbe's progress either in vivo using the infant rat model of infection, or in vitro through invasions of human tissue culture cell lines.
Daines
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Department of Molecular Microbiology and Immunology. University of Missouri - Columbia, M616 Medical Science Building, DCO44.00, Columbia, MO 65212, USA. dainesd@health.missouri.edu
Smith
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D015153:Blotting, Western; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D006193:Haemophilus influenzae / Q000235:genetics*; D006639:Histidine / Q000235:genetics; D007763:Lac Operon; D009876:Operon; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000096:biosynthesis
D011993:Recombinant Fusion Proteins; D006639:Histidine; D014364:Tryptophan; D015500:Chloramphenicol O-Acetyltransferase