Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: February 1, 2003
Publication Date: May 18, 2003
Citation: Wonderling, L.D., Uhlich, G.A., Cooke, P.H., Luchansky, J.B. 2003. Identification of two putative regulatory genes required for motility of listeria monocytogenes strain f2365. American Society of Microbiology.Abstract # K126, p. 381. Technical Abstract: Based on whole-genome comparative analysis of L. monocytogenes strains EGD (serotype 1/2a) and F2365 (serotype 4b), 15 ORFS were predicted to encode proteins of the CRP/FNR family of regulatory proteins. This suggested an over-representation of this protein family in L. monocytogenes, since B. subtilis and E. coli possess only 1 and 2 CRP/FNR-type proteins, respectively. To determine if these putative regulatory proteins have any functional role in L. monocytogenes strain F2365 (food isolate, 1985 Mexican-style cheese outbreak), 13 of the 15 genes were mutated on the chromosome of strain F2365 by transpositional insertion. Subsequent phenotypic screening of the 13 mutant strains included a "swarming" assay, where the wild type and mutant cultures were grown on soft agar plates to investigate motility. When cultured at 37ºC, the wild type and all 13 mutant strains displayed non-swarming phenotypes. However, when cultured at 25ºC, two of the mutant strains, referred to as strains MFS1366 and MFS1372, displayed a non-swarming phenotype, in contrast to the other 11 mutant and wild type strains which displayed zones of swarming. To further investigate the non-swarming phenotype, the wild type and strains MFS1366 and MFS1372 were examined for flagella formation by transmission electron microscopy. When cells were cultured at 25ºC, 88% (23 of 26) of the wild type cells were flagellated compared to 15% (4 of 27) and 29% (8 of 28) of the cells for strains MFS1366 and MFS1372, respectively. In addition, wild type cells possessed an average of 2.19 flagella per cell compared to 0.22 (MFS1366) and 0.29 (MFS1372) flagella per cell for the two mutant strains. These results indicate that at least two genes predicted to be proteins of the CRP/FNR family in L. monocytogenes strain F2365 are required for flagella formation at 25ºC. Further studies will investigate if these two regulatory proteins have any role in L. monocytogenes survival in food, where flagella may allow surface attachment and/or biofilm formation.