MATERIALS AND METHODS

1. 1 2 3 Comparative gene analysis of stress susceptible and tolerant Campylobacter jejuni Takamiya, M.1, Rasmussen, M2, Ozen, A.3, Nielsen, S1, Ussery, D.3 and S. Knøchel1 1 Department of Food Sciences, Faculty of Life Sciences (KU-LIFE), University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark. 2 Department of Biology, Faculty of Science (KU-NAT), University of Copenhagen, Ole Maaløes Vej 5, K-2200 Copenhagen N, Denmark. 3 Center for Biological Sequence Analysis (CBS), Department of Systems Biology, Technical University of Denmark (DTU), Building 208, DK-2800 Lyngby, Denmark. BACKGROUND AND AIM MATERIALS AND METHODS • Campylobacter jejuni, a Gram-negative, spiral shaped bacterium is one of the leading bacterial causes of food-borne human gastroenteritis often linked to the improper handling and consumption of poultry, contaminated milk and untreated water. The organism is regarded as fastidious and sensitive but nevertheless it may survive food-processing related stresses like cold, salt, reactive oxygen species (ROS) etc. both in the external environment and the gastrointestinal tract. We have previously observed variations in the sensitivity of strain to acids (Birk, 2009) so we were interested in identifying robust and sensitive strains and elucidate the genomic differences between these. • The aim of this study was to: • Investigate the stress (such as oxidation, NaCl, chilling (4°C), and drying) tolerance behaviour of selcted C.jejuni strains. • Obtain the full sequence of selected strains • Perform a comparative gene analysis on robust and sensitive C.jejuni strains Strains: C. jejuni strains 305, 327, DFVF1099 and NCTC11168 were selected based on previous data. Survival in BHI broth at 0.1, 1, and 10 mM H2O2: late exponential phase, 37°C. Survival during chilling (4ºC) in Bolton broth at 0.5, 1.5, and 2.5% NaCl inoculation level at T = 0 was 6.95 ± 0.51 (SD) log10 CFU/ml. Sampling at 2, 3, and 4 weeks at 4ºC. Genome sequencing and analysis:three C.jejuni genomes were pyrosequenced using the Genome Sequencer FLX System (454 Life Sciences). Draft assemblies ensured a coverage >10. De novo contig assembly was done using CLC Genomics Workbench software (CLC Bio, Aarhus, Denmark). The assembly resulted in a total of 333 contigs (1,81 Mb) for 305, 71 contigs (1,73 Mb) for DFVF1099, and 48 contigs (1,62 Mb) for 327, respectively. Genome annotation was done using PFunc (v. 0.1.1.), and manual curation for GenBank submission is currently in progress using Sequin (v.10). CONCLUSION • A stress susceptible (327, poultry) and two robust strains (305 and DFVF1099, poultry) relative to the NCTC11168 strain were identified • Comparative proteomic analysis with publicly available strains revealed the presence of major gaps in our sensitive strain 327 among others. Genes absent in 327 using NCTC11168 as reference revealed the absence of genes encoding for e.g. capsule locus (lipo-oligosaccharide synthesis, LOS) and marA (multiple antibiotic resistanceDNA-binding transcriptional activator also involved in global stress response). RESULTS Survival to elevated conc. of H2O2 at 37°C Survival of C.jejuni to food-related environments A.during elevated NaCl and chilling (4°C) B. during chilling (4°C) Figure 2.Survival of C. jejuni during A) elevated NaCl conc. at 4°C storage for 2 weeks in Bolton broth, and B) during chilling (4°C). Results are shown as log reduction and the error bars show minimum and maximum values. Limited strain differences were seen during drying (data not shown). Figure 1. Survival of C.jejuni strains 305, 327, DFVF1099, and NCTC11168 during 0, 1, and 10 mM of H202 in Brain Heart Infusion (BHI) broth at 37°C. BLAST Atlas comparing 305, 327 and DFVF1099 with publicly available C.jejuni genomes (13) Initial analysis has shown that the sensitive strain 327 resembles a previously sequenced strain while the robust strains 305 and DFVF1099 are closer to NCTC11168. Major gaps shown on BlastAtlas are indicated by numbers (1 to 3) and some of the genes missing on 327 are indicated below: Gap1: Transcriptional regulator mar A (multiple antibiotic resistance), which mediates global stress response in E.coli and affects the expression of iron-sulfur cluster proteins involved in sensing O2 and iron. Gap2: genes cj1415c to cj1441c (as part of the C. jejuni capsule locus (Cj1413c - Cj1448c) Gap3: genes involved in lipooligosaccharide (LOS) synthesis (Cj1131c -Cj1152c):wlaN (beta-1,3 galactosyltransferase) cstIII (alpha-2,3 sialyltransferase ), Cj1136-Cj1138 (putative glycosyltransferases). References Birk et al., J.Food Prot. (2010). 73:258-265. Barbosa et al., J.Bacteriol (2000). 182: 3467-3474. Thybert et al.,BMC Genomics (2008) 9:637-649. Hallin et al., Bioinformatics (2004) 20: 3682-3686. Figure 3. Genome Blast Atlas of C.jejuni NCTC11168 as the reference strain compared to a set of 13 sequenced C.jejuni genomes. The legend indicates which genome is represented in the lanes. The lanes inside the red BLAST lanes represent the Genome Atlas of C.jejuni strains DFVF1099, 305, 327 and NCTC11158. The Genome Blast Atlas was developed by Hallin et al., 2004.