Use of digitized video microscopy with a fluorogenic enzyme substrate to demonstrate cell- and compartment-specific gene expression in Salmonella enteritidis and Bacillus subtilis.
A rapid and sensitive method for detection of cell- and compartment-specific gene expression in individual cells of both Gram-negative and Gram-positive microorganisms is described. The method combines the use of gene fusions to lacZ, and a fluorogenic beta-galactosidase substrate, fluorescein-di-(beta-D-galactopyranoside), with digitized video microscopy. All of the reporter constructs tested were successfully detected. Secondary staining of the cells with a nucleic acid-specific dye, propidium iodide, allowed cells devoid of nucleic acid to be identified, while cell nucleoid shape and the morphological stage of development could be correlated with the location of beta-galactosidase activity. The double-staining procedure was used to show that gene expression can be induced in non-culturable cells of Salmonella enteritidis produced by carbon/nitrogen starvation. The resolution was sufficient to distinguish between cells at different morphological stages of sporulation in Bacillus subtilis. This highly sensitive and rapid method may have many other applications in basic and applied microbiology.
Lewis
P J
PJ
Sir William Dunn School of Pathology, University of Oxford, UK.
Nwoguh
C E
CE
Barer
M R
MR
Harwood
C R
CR
Errington
J
J
D001412:Bacillus subtilis / Q000166:cytology / Q000502:physiology*; D002451:Cell Compartmentation; D004790:Enzyme Induction / Q000187:drug effects; D005452:Fluoresceins / Q000378:metabolism*; D005697:Galactosides / Q000378:metabolism*; D015870:Gene Expression; D017930:Genes, Reporter; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008853:Microscopy / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D018715:Microscopy, Video / Q000379:methods; D009024:Morphogenesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D012477:Salmonella enteritidis / Q000502:physiology*; D013171:Spores, Bacterial / Q000166:cytology / Q000502:physiology; D001616:beta-Galactosidase / Q000032:analysis
D005452:Fluoresceins; D005697:Galactosides; D011994:Recombinant Proteins; C038848:fluorescein-digalactoside; D007544:Isopropyl Thiogalactoside; D001616:beta-Galactosidase