title: Identification of novel genes in genomic islands that contribute to Salmonella typhimurium replication in macrophages.
abstract: Salmonella enterica serovar Typhimurium (S. typhimurium) survives and proliferates within macrophage cells. A mutant library of strain ATCC 14028 based on gene disruption by homologous recombination was screened in order to identify genes that are required for wild-type-like intracellular replication. Randomly generated chromosomal fragments from the genome of S. typhimurium were cloned into a temperature-sensitive vector, and approximately 8000 individual mutant clones were obtained by insertional-duplication mutagenesis (IDM) upon selection at non-permissive temperature. Large-scale screening for replication defects in mouse macrophages, but not during growth in rich or minimal medium, revealed a set of attenuated mutants that were further characterized by PCR amplification and sequencing of the mutagenic fragments. Following analysis of a Salmonella genome map with the annotated positions of vector insertions, an accumulation of 33 attenuating insertions within genes of ten non-collinear regions was found. Insertions in virK, gipA and five SPI-2 genes as well as seven non-polar deletions validated the screen. No invasion deficiencies of the mutants were observed. The cob-cbi-pdu cluster containing the genes for cobalamin synthesis and 1,2-propanediol degradation was shown to be required for Salmonella replication within macrophages. These data gave rise to a model of eukaryotic glycoconjugates and phospholipids as alternative carbon, nitrogen and energy sources for intracellularly replicating bacteria. The contribution of as yet unknown components of SPI-6 and the Gifsy-1 and Gifsy-2 prophage islands to intracellular replication is reported, as well as the fivefold reduced intracellular growth rate of a mutant with a deletion of STM1677, which probably encodes a LysR-like transcriptional regulator. The intracellular replication rate of three double mutants, each lacking two gene products of the cob-cbi-pdu cluster or the Gifsy-1 prophage, was shown to be lower than that of the respective single mutants, suggesting that additive effects of subtle intracellular advantages contribute to Salmonella fitness in vivo.
authors: Institute of Food Science and Nutrition, ETH Zürich, Schmelzbergstr. 7, 8092 Zürich, Switzerland.|Zentralinstitut für Ernährungs- und Lebensmittelforschung (ZIEL), Abteilung Mikrobiologie, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany.
authors: Jochen
authors: J
authors: Klumpp
authors: Zentralinstitut für Ernährungs- und Lebensmittelforschung (ZIEL), Abteilung Mikrobiologie, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany.
authors: Thilo M
authors: TM
authors: Fuchs
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D004261:DNA Replication; D004268:DNA-Binding Proteins; D017353:Gene Deletion; D015723:Gene Library; D005798:Genes, Bacterial; D044404:Genomic Islands; D008264:Macrophages / Q000382:microbiology*; D051379:Mice; D016254:Mutagenesis, Insertional; D012486:Salmonella typhimurium / Q000235:genetics* / Q000254:growth & development*; D015534:Trans-Activators
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C084166:PocR protein, Salmonella; D015534:Trans-Activators