Abstract
Identification and characterization of bacterial genes that are induced during the disease process are important in understanding the molecular mechanism of disease and can be useful in designing antimicrobial drugs to control the disease. The identification of in vivo induced (ivi) genes of an Escherichia coli septicemia strain by using antibiotic-based in vivo expression technology is described. Bacterial clones resistant to chloramphenicol in vivo were recovered from the livers of infected mice. Most of the ivi clones were sensitive to chloramphenicol when grown in vitro. Using reverse transcription-PCR, it was demonstrated that selected ivi clones expressed cat in the livers of infected mice but not during in vitro growth. A total of 750 colonies were recovered after three successive rounds of in vivo selection, and 168 isolated ivi clones were sequenced. The sequence analysis revealed that 37 clones encoded hypothetical proteins found in E. coli K-12, whereas 10 clones contained genes that had no significant homology to DNA sequences in GenBank. Two clones were found to contain transposon-related functions. Other clones contained genes required for amino acid metabolism, anaerobic respiration, DNA repair, the heat shock response, and the cellular repressor of the SOS response. In addition, one clone contained the aerobactin biosynthesis gene iucA. Mutations were introduced in to seven of the identified ivi genes. An in vivo mouse challenge-competition assay was used to determine if the mutants were attenuated. The results suggested that these ivi genes were important for survival in vivo, and three of the seven mutant ivi clones were required for successful infection of mice.