RESUMO
To enable specific and tightly controlled gene expression both in vitro and during the intracellular lifecycle of the pathogen Listeria monocytogenes, a TetR-dependent genetic induction system was developed. Highest concentration of cytoplasmic TetR and best repression of tetO-controlled genes was obtained by tetR expression from the synthetic promoter Pt17 . Anhydrotetracycline (ATc) as inducer permitted concentration-dependent, fine-tuned expression of genes under control of the tetO operator and a suitable promoter. The actin-polymerizing ActA protein represents a major virulence factor of L. monocytogenes, required for actin-based motility and cell-to-cell spread in infected host cells. To be able to observe its spatial and temporal distribution on intracellular L. monocytogenes cells, conditional mutants featuring actA placed under TetR control were used to infect PtK2 epithelial cells. Following induction at different time intervals, the subsequent recruitment of actin by L. monocytogenes could be monitored. We found that cells displayed functional ActA after approximately 15 min, while formation of polarized actin tail was complete after 90-120 min. At this point, intracellular motility of the induced mutants was indistinguishable from wild-type bacteria. Interestingly, de novo ActA synthesis in intracellular Listeria also demonstrated the temporal, asymmetric redistribution of the membrane-anchored proteins from the lateral walls toward the cell poles.
Assuntos
Proteínas de Bactérias/metabolismo , Listeria monocytogenes/genética , Proteínas de Membrana/metabolismo , Actinas/metabolismo , Animais , Proteínas de Bactérias/genética , Técnicas de Cultura de Células , Movimento Celular , Citoplasma/metabolismo , Dipodomys , Regulação Bacteriana da Expressão Gênica/genética , Listeria monocytogenes/metabolismo , Proteínas de Membrana/genética , Ratos , Análise Espaço-Temporal , Resistência a Tetraciclina/genética , Fatores de Virulência/metabolismoRESUMO
We present the complete de novo assembled genome sequences of Listeria monocytogenes strains WSLC 1001 (ATCC 19112) and WSLC 1042 (ATCC 23074) and Listeria ivanovii WSLC 3009, three strains frequently used for the propagation and study of bacteriophages because they are presumed to be free of inducible prophages.
RESUMO
Acid production from rhamnose is a characteristic phenotype of Listeria monocytogenes. We report the identification of the rhamnose transport and utilization operon located at lmo2846 to lmo2851, including the rhamnose-dependent promoter P(rha). Expression of reporter genes under control of P(rha) on a single copy integration vector demonstrated its suitability for inducible gene expression in L. monocytogenes. Transcription initiation from P(rha) is dose dependent, and a concentration as low as 100 µM rhamnose was found sufficient for induction. Moreover, P(rha) is subject to glucose catabolite repression, which provides additional options for strict control of expression. Infection of human THP1 macrophages revealed that P(rha) is repressed in intracellular L. monocytogenes, which is explained by the absence of rhamnose in the cytosol and possible interference by catabolite repression. The P(rha) promoter provides a novel and useful tool for triggering gene expression in extracellular L. monocytogenes, whereas intracellular conditions prevent transcription from this promoter.
