Neutrophil microbicidal activity: screening bacterial mutants for survival after phagocytosis using quantitative PCR.
When a constant gene replacement sequence is introduced into bacteria to produce mutants and the flanking chromosomal sequences are known, it is possible to use a quantitative polymerase chain reaction method (QPCR) to compare the concurrent survival of the different bacterial mutants under identical conditions. We describe Escherichia coli survival following neutrophil phagocytosis among three mutants deleted respectively for araB, dps or oxyR. Comparisons were made both by traditional and QPCR methods with similar results and indicate that the survival defect of an oxyR and oxyS mutant described previously can be attributed to the loss of oxyR alone. Deletion of dps, a prominent member of the regulon controlled by the oxyR gene product does not engender a survival defect. We suggest that QPCR analysis can readily compare the relative survival of 10 or more mutants concurrently. QPCR analysis would seem to be especially valuable when experimental conditions are subject to a high degree of sample to sample variability or when the stress producing system involves use of expensive or scarce resources like rare patient cells, cells from children, or the use of genetically modified animal hosts.
Rosen
Henry
H
Department of Medicine, University of Washington, Seattle, Washington, USA. hqr@u.washington.edu
Lewis
Patrick J
PJ
Nitzel
Cory M L
CM
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001770:Blood Bactericidal Activity; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics; D006801:Humans; D009154:Mutation; D009504:Neutrophils / Q000502:physiology*; D010587:Phagocytosis / Q000502:physiology*; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000379:methods; D012097:Repressor Proteins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C088010:dps protein, E coli; C499299:oxyR protein, E coli