An asterisk above the bars indicate statistically significant differences in mRNA levels between the C. sakazakii ES5 wt and mutant (P < 0.05). Conclusions By using a transposon knock out approach we were able to identify structural and regulatory genes in Cronobacter sakazakii ES5, deletion of which resulted in a dramatically

reduced capability to survive in serum. Additionally, several mutants were found displaying an enhanced survival Rabusertib mouse in serum as compared to the wild type. Analysis of the genetic elements possibly responsible for this phenotype revealed genes coding for chaperone-like proteins, regulatory (repressor) elements as well as genes for structures or components representing immunogenic targets. The deletion of the ybaJ element which is part of the antitoxin-toxin pair YbaJ-Hha resulted

in an abolished expression of a key element of the type selleck inhibitor 1 fimbriae. The absence of the latter most likely accounted for the enhanced survival of this mutant in human serum. Methods Bacterial strains and culture conditions Cronobacter sakazakii strain E5, a clinical strain was used in this study. Wild Ceramide glucosyltransferase type and mutant strains, E. coli DH5 alpha

as well as plasmids and primers that were included and constructed during the transposon library screening, the mutant complementation (BF4) and the expression (21_G1) experiments are summarized in Table 2. All strains were incubated at 37°C in Luria–Bertani (LB) broth, over night with gentle shaking. When appropriate, antibiotics were used at the following concentrations: kanamycin at 50 μg ml-1 and tetracyclin at 50 μg ml-1. Table 2 Material used in this study Strains/plasmids/primers Genotype/characteristic(s)/sequences Source or reference Strains     Cronobacter sakazakii       ES5 (wild type) Human isolate Hartmann et al., 2010, Johler et al., 2010 [11, 13]   BF4 (mutant) ΔESA_04103, KanR Hartmann et al., 2010 [13]   BF4_pCCR9 BF4 harboring pCCR9, KanR, TetR This study   BF4_pCCR9::ESA_04103 BF4 harboring pCCR9:: ESA_04103, KanR, TetR This study   21_G1 (mutant) ΔybaJ, KanR This study Escherichia coli DH5 alpha F– Φ 80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK–, mK+) phoA supE44 λ– thi-1 gyrA96 relA1 Epicentre Plasmids       pUC19 High copy cloning/expression vector AmpR Epicentre   pCCR9 Low copy cloning/expression vector, TetR ATM/ATR phosphorylation Randegger et al.