Dataset Information

Xenocin export by the flagellar type III pathway in Xenorhabdus nematophila.

ABSTRACT: The xenocin operon of Xenorhabdus nematophila consists of xciA and ximB genes encoding a 64-kDa xenocin and 42-kDa immunity protein to kill competing microbes in the insect larva. The catalytic domain of xenocin has RNase activity and is responsible for its cytotoxicity. Under SOS conditions, xenocin is produced with immunity protein as a complex. Here, we show that xenocin and immunity protein complex are exported through the flagellar type III system of X. nematophila. Secretion of xenocin complex was abolished in an flhA strain but not in an fliC strain. The xenocin operon is not linked to the flagellar operon transcriptionally. The immunity protein is produced alone from a second, constitutive promoter and is targeted to the periplasm in a flagellum-independent manner. For stable expression of xenocin, coexpression of immunity protein was necessary. To examine the role of immunity protein in xenocin export, an enzymatically inactive protein was produced by site-directed mutagenesis in the active site of the catalytic domain. Toxicity was abolished in D535A and H538A variants of xenocin, which were expressed alone without an immunity domain and secreted in the culture supernatant through flagellar export. Secretion of xenocin through the flagellar pathway has important implications in the evolutionary success of X. nematophila.

SUBMITTER: Singh P

PROVIDER: S-EPMC3624530 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Xenocin export by the flagellar type III pathway in Xenorhabdus nematophila.

Singh Preeti P Park Dongjin D Forst Steven S Banerjee Nirupama N

Journal of bacteriology 20130118 7

The xenocin operon of Xenorhabdus nematophila consists of xciA and ximB genes encoding a 64-kDa xenocin and 42-kDa immunity protein to kill competing microbes in the insect larva. The catalytic domain of xenocin has RNase activity and is responsible for its cytotoxicity. Under SOS conditions, xenocin is produced with immunity protein as a complex. Here, we show that xenocin and immunity protein complex are exported through the flagellar type III system of X. nematophila. Secretion of xenocin com ...[more]

PMID: 23335409

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Project description:The bacterial type III export apparatus is found in the flagellum and in the needle complex of some pathogenic Gram-negative bacteria. In the needle complex its function is to secrete effector proteins for infection into Eukaryotic cells. In the bacterial flagellum it exports specific proteins for the building of the flagellum during its assembly. The export apparatus is composed of about five membrane proteins and three soluble proteins. The mechanism of the export apparatus is not fully understood. The five membrane proteins are well conserved and essential. Here a cross-complementation assay was performed: substituting in the flagellar system of Salmonella one of these membrane proteins, FlhB, by the FlhB ortholog from Aquifex aeolicus (an evolutionary distant hyperthermophilic bacteria) or a chimeric protein (AquSalFlhB) made by the combination of the trans-membrane domain of A. aeolicus FlhB with the cytoplasmic domain of Salmonella FlhB dramatically reduced numbers of flagella and motility. From cells expressing the chimeric AquSalFlhB protein, suppressor mutants with enhanced motility were isolated and the mutations were identified using whole genome sequencing. Gain-of-function mutations were found in the gene encoding FlhA, another membrane protein of the type III export apparatus. Also, mutations were identified in genes encoding 4-hydroxybenzoate octaprenyltransferase, ubiquinone/menaquinone biosynthesis methyltransferase, and 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, which are required for ubiquinone biosynthesis. The mutations were shown by reversed-phase high performance liquid chromatography to reduce the quinone pool of the cytoplasmic membrane. Ubiquinone biosynthesis could be restored for the strain bearing a mutated gene for 4-hydroxybenzoate octaprenyltransferase by the addition of excess exogenous 4-hydroxybenzoate. Restoring the level of ubiquinone reduced flagella biogenesis with the AquSalFlhB chimera demonstrating that the respiratory chain quinone pool is responsible for this phenomenon.

Dataset Information

Xenocin export by the flagellar type III pathway in Xenorhabdus nematophila.

Publications

Xenocin export by the flagellar type III pathway in Xenorhabdus nematophila.

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