Gene cassettes unique to epidemic-associated lineages of Listeria monocytogenes serotype 4b

by 1977- Yue, Lili

YUE, LILI. Gene cassettes unique to epidemic-associated lineages of Listeria monocytogenes serotype 4b. (Under the direction of Dr. Sophia Kathariou) Listeria monocytogenes is responsible for severe foodborne infections, with high mortality and morbidity. A clonal group of L. monocytogenes strains of serotype 4b designated Epidemic Clone I (ECI) has been responsible for numerous outbreaks of foodborne illness in the United States and elsewhere. A number of genes and gene cassettes unique to ECI strains have been identified through molecular approaches, and through the recently completed genome sequencing on ECI strain. However, the functions of most of these ECI specific sequences remain unclear. The purpose of this research has been to perform a comparative genomic analysis of the regions harboring four ECI specific gene cassettes, designated region 144, 133, 17B and 85, among five different Listeria strains for which genome sequencing projects have been undertaken. The strains included ECI strain F2365 (serotype 4b), ECII strain H7858 (serotype 4b), serotype 1/2a strains EGD-e and F6854, and Listeria innocua CLIP 11262. The genomic organization of the region harboring these cassettes in ECI was investigated in the different genomes, and transcriptional analysis by reverse-transcriptase polymerase chain reaction was pursued with three regions, 144, 133 and 17B. The comparative genomic organization data revealed that all four ECI specific regions have features typical of genomic islands (GEIs), being present in the genome of ECI strains but absent from the genome of other serotype 4b strains, suggesting horizontal insertion / deletion events and possible roles in pathogenicity and metabolism of the organism. Transcriptional data suggested that the six ECI specific open reading frames in region 133 were co-transcribed as a unit separate from adjacent genes which were highly conserved among different strains. To obtain information on the possible functions of the ECI specific genes and gene cassettes, a mutational approach was pursued. A deletion mutant of the ECI specific sequence in region 133 was constructed in two different ECI strains, and the mutants were characterized bacteriologically and in terms of phenotypic microarrays. The deletion mutant F2365 ?133 was characterized in terms of basic bacteriological features including hemolytic activity, phage susceptibility, motility, cell morphology, growth at 37ºC, 25ºC and 4ºC, bacitracin resistance, surface antigen detection, and with a panel of phenotypic microarrays. The parental strain F2365 was observed to grow better than the deletion mutant F2365 ?133 at 4ºC, and the Phenotypic Microarrays identified certain differences in substrate utilization between the mutant and the wild type parental strain. The findings suggest that the ECI specific cassette in region 133 may contribute to bacterial growth at cold temperature and to the metabolism of certain carbon and nitrogen sources. Future studies employing animal and cell culture models will be needed to evaluate the possible impact of the ECI specific cassette in region 133 in virulence and pathogenesis of the bacteria. Gene cassettes unique to epidemic-associated lineages of Listeria monocytogenes serotype 4b By Lili Yue A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Master of Science