1887

Abstract

serovar 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 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 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 , and five SPI-2 genes as well as seven non-polar deletions validated the screen. No invasion deficiencies of the mutants were observed. The -- cluster containing the genes for cobalamin synthesis and 1,2-propanediol degradation was shown to be required for 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 -- 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 fitness .

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2007-04-01
2020-01-24
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