Regulation, Integrase-Dependent Excision, and Horizontal Transfer of Genomic Islands in Legionella pneumophila

Legionella pneumophila is a Gram-negative freshwater agent which multiplies in specialized nutrient-rich vacuoles of amoebae. When replicating in human alveolar macrophages, Legionella can cause Legionnaires' disease. Recently, we identified a new type of conjugation/type IVA secretion system (T4ASS) in L. pneumophila Corby (named trb-tra). Analogous versions of trb-tra are localized on the genomic islands Trb-1 and Trb-2. Both can exist as an episomal circular form, and Trb-1 can be transferred horizontally to other Legionella strains by conjugation. In our current work, we discovered the importance of a site-specific integrase (Int-1, lpc2818) for the excision and conjugation process of Trb-1. Furthermore, we identified the genes lvrRABC (lpc2813 to lpc2816) to be involved in the regulation of Trb-1 excision. In addition, we demonstrated for the first time that a Legionella genomic island (LGI) of L. pneumophila Corby (LpcGI-2) encodes a functional type IV secretion system. The island can be transferred horizontally by conjugation and is integrated site specifically into the genome of the transconjugants. LpcGI-2 generates three different episomal forms. The predominant episomal form, form A, is generated integrase dependently (Lpc1833) and transferred by conjugation in a pilT-dependent manner. Therefore, the genomic islands Trb-1 and LpcGI-2 should be classified as integrative and conjugative elements (ICEs). Coculture studies of L. pneumophila wild-type and mutant strains revealed that the int-1 and lvrRABC genes (located on Trb-1) as well as lpc1833 and pilT (located on LpcGI-2) do not influence the in vivo fitness of L. pneumophila in Acanthamoeba castellanii

The application of flow cytometry in microbiological monitoring during winemaking: two case studies

In this work, we exploit a general flow cytometry technique involved in the differentiation of live and dead yeast cells for two applications in winemaking. The discrimination of yeast populations is achieved using two fluorescent dyes that measure the metabolic activity and membrane integrity of the yeast. This analytical approach is first applied for quality control of active dry yeast. Results are discussed in comparison with the Codex Oenologique International (International Oenological Codex) of the International Organisation of Vine and Wine (OIV), demonstrating that analysis using flow cytometry is a valuable alternative, given the ease of execution and the high quality of results obtained in terms of reproducibility, repeatability, and confidence interval. In the second case, we apply flow cytometry as a technique for monitoring the production of sparkling wines using the “Champenoise” method, and describe the evolution of yeast through the production process. In this case, results are directly compared with those obtained with the two methods (plate counts and direct microscopic count) listed in the OIV standards, in order to ensure a thorough understanding of the improvements related to the use of flow cytometr

FliA expression analysis and influence of the regulatory proteins RpoN, FleQ and FliA on virulence and in vivo fitness in Legionella pneumophila

In Legionella pneumophila, the regulation of the flagellum and the expression of virulence traits are linked. FleQ, RpoN and FliA are the major regulators of the flagellar regulon. We demonstrated here that all three regulatory proteins mentioned (FleQ, RpoN and FliA) are necessary for full in vivo fitness of L. pneumophila strains Corby and Paris. In this study, we clarified the role of FleQ for fliA expression from the level of mRNA toward protein translation. FleQ enhanced fliA expression, but FleQ and RpoN were not necessary for basal expression. In addition, we identified the initiation site of fliA in L. pneumophila and found a putative σ(70) promoter element localized upstream. The initiation site was not influenced in the ΔfleQ or ΔrpoN mutant strain. We demonstrated that there is no significant difference in the regulation of fliA between strains Corby and Paris, but the FleQ-dependent induction of fliA transcription in the exponential phase is stronger in strain Paris than in strain Corby. In addition, we showed for the first time the presence of a straight hook at the pole of the non-flagellated ΔfliA and ΔfliD mutant strains by electron microscopy, indicating the presence of an intact basal body in these strains