Expanding the Helicobacter pylori genetic toolbox: modification of an endogenous plasmid for use as a transcriptional reporter and complementation vector.
Helicobacter pylori is an important human pathogen. However, the study of this organism is often limited by a relative shortage of genetic tools. In an effort to expand the methods available for genetic study, an endogenous H. pylori plasmid was modified for use as a transcriptional reporter and as a complementation vector. This was accomplished by addition of an Escherichia coli origin of replication, a kanamycin resistance cassette, a promoterless gfpmut3 gene, and a functional multiple cloning site to form pTM117. The promoters of amiE and pfr, two well-characterized Fur-regulated promoters, were fused to the promoterless gfpmut3, and green fluorescent protein (GFP) expression of the fusions in wild-type and delta fur strains was analyzed by flow cytometry under iron-replete and iron-depleted conditions. GFP expression was altered as expected based on current knowledge of Fur regulation of these promoters. RNase protection assays were used to determine the ability of this plasmid to serve as a complementation vector by analyzing amiE, pfr, and fur expression in wild-type and delta fur strains carrying a wild-type copy of fur on the plasmid. Proper regulation of these genes was restored in the delta fur background under high- and low-iron conditions, signifying complementation of both iron-bound and apo Fur regulation. These studies show the potential of pTM117 as a molecular tool for genetic analysis of H. pylori.
Carpenter
Beth M
BM
Department of Microbiology and Immunology, Uniformed Services University of the Heath Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814, USA.
McDaniel
Timothy K
TK
Whitmire
Jeannette M
JM
Gancz
Hanan
H
Guidotti
Silvia
S
Censini
Stefano
S
Merrell
D Scott
DS
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016480:Helicobacter pylori / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*
D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins