Bacterial conjugation | EPFL Graph Search

Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. This takes place through a pilus. It is a parasexual mode of reproduction in bacteria. It is a mechanism of horizontal gene transfer as are transformation and transduction although these two other mechanisms do not involve cell-to-cell contact. Classical E. coli bacterial conjugation is often regarded as the bacterial equivalent of sexual reproduction or mating since it involves the exchange of genetic material. However, it is not sexual reproduction, since no exchange of gamete occurs, and indeed no generation of a new organism: instead an existing organism is transformed. During classical E. coli conjugation the donor cell provides a conjugative or mobilizable genetic element that is most often a plasmid or transposon. Most conjugative plasmids have systems ensuring that the recipient cell does not already contain a sim

Genetic manipulation of Vibrio cholerae by combining natural transformation with FLP recombination

Melanie Blokesch, Olga de Souza Silva

Even though Vibrio cholerae is a well-known human pathogen, it is also a normal member of aquatic habitats. Within this environment it often forms biofilms on the chitin-containing exoskeleton of crustaceans and their molts. Chitin not only serves as nutrient source but also induces a developmental program called natural competence. Naturally competent bacteria take up free DNA and integrate it into their genome by homologous recombination, thereby becoming naturally transformed. In this study, we made use of the knowledge on the environmental lifestyle of V. cholerae to genetically manipulate its genome. We achieved this by combining the methods of chitin-induced natural transformation and Flp recombination. Using this approach, we disrupted several genes by insertion of FRT-site-flanked antibiotic-resistance cassettes. The cassettes were subsequently excised by induction of the Flp recombinase, which acts on the FRT sites. This method represents a simplified and faster alternative to standard gene deletion techniques, which often depend on bacterial conjugation and the availability of suicide vectors.

Elsevier2010

The double-edged toxins

Mario Gonzalo Garcia Arraez

Many insect species are associated with endosymbiotic bacteria which have the particularity of living within host tissues. Endosymbionts benefit from this stable and nutritious environment, while providing ecological advantages to their host, such as protection against parasites or thermal tolerance. Spiroplasma poulsonii is an endosymbiotic bacterium that infects natural populations of Drosophila melanogaster. Spiroplasma poulsonii lacks a cell wall, a fact that renders it invisible to the host immune system and allows it to thrive in the host hemolymph. It invades the female germline by co-opting the hostâs yolk transport and uptake machinery, which ensures its vertical transmission. Its efficient transmission is associated with a phenotype called male-killing, whereby infected male embryos die during their early development while infected females survive. The mechanisms that ensure the stability of Drosophila-Spiroplasma symbiosis are increasingly well understood, but the bacterial genes involved remain poorly known because of the intractability of Spiroplasma. This project aims at better characterizing the Drosophila-Spiroplasma interaction, with particular focus on the bacterial side. In the first part, I developed a method to cultivate Spiroplasma poulsonii in vitro by optimizing a commercially available medium. This culture method allowed comparing the transcriptome of in vitro grown versus host-grown Spiroplasma, enabling us to identify putative genes involved in the interaction with the host. Interestingly, inside its insect host, S. poulsonii up-regulates genes coding for toxins of the Ribosomal Inactivating Protein (RIP) family. RIPs were previously known for their role in host protection against macro-parasites, such as wasps and nematodes. Their up-regulation in unparasitized hosts compared to culture was thus peculiar, raising the question what effect these RIPs have on host biology. Thus, in the second part, I studied the function of S. poulsonii RIPs in the absence of parasites. I showed that two of them are constantly expressed within the host and provide evidence that these toxins shorten host life span and increase embryonic mortality. Interestingly, the expression of RIPs was more toxic to male embryos than females, suggesting that RIPs contribute to S. poulsonii-induced male-killing. Last, I studied the D. melanogaster response to RIPs, and how the host mitigates the deleterious effects of these toxins by up-regulating the cytosolic chaperone Heat-Shock-Protein 70B (HSP70B). This protein carries out essential functions in protein homeostasis under normal and stressful conditions such as folding, refolding, or increasing the half-life of proteins. Interestingly, up-regulation of Hsp70B in the presence of RIPs results in an increased lifespan and in a better tolerance to heat stress, which may be ecologically advantageous. Altogether, this work illustrates how Spiroplasma-derived RIP toxins can differentially affect Drosophila depending on the ecological context, ranging from beneficial upon parasite infection or heat shock to detrimental in the absence of such environmental pressures.

EPFL2018

Identification of genes required for host-cell adhesion and antibiotic persistence of uropathogenic Escherichia coli via high-content screening

Thomas Marie Simonet

Uropathogenic Escherichia coli (UPEC) are the most common bacterial pathogens causing urinary tract infections (UTIs). Driven by the development of antibiotic resistant UPEC strains, UTIs have become a major public health issue and generate substantial healthcare costs. Even in the absence of antibiotic resistance, UTIs are notoriously difficult to treat and have high rates of recurrence, mostly due to: 1. the ability of UPEC to adhere to and invade bladder epithelial cells, which protects them from the action of antimicrobial agents and the immune system 2. the presence in antibiotic-susceptible UPEC populations of a small fraction of so-called persister cells, which are killed at a slower rate than the bulk of the population and can therefore survive prolonged antibiotic exposureThese two features allow some bacteria to remain viable in a protected niche for the whole duration of the antibiotic treatment, potentially leading to infection relapse when the treatment is terminated.High-content screening (HCS), which uses microscopy as a screening tool, allows the visualization of bacterial phenotypes at the single-cell level and can therefore provide a powerful and unbiased tool to understand the genetic basis of these phenotypes. The present thesis describes two complementary high-content screening approaches aimed at identifying UPEC mutants with altered adhesion to bladder cells and with altered antibiotic persistence respectively.In a first chapter, we report preparatory steps that led to the construction of a transposon insertion library of UPEC mutants that is suitable for high-content imaging, using the CFT073 clinical UTI isolate as background strain.In the second chapter, we performed fluorescence microscopy-based high-content screening to identify mutants with defects in early adhesion to bladder epithelial cells. We recovered 82 mutants with decreased adhesion and 54 mutants with increased adhesion. Unexpectedly, nine low-adhesion ÂÂhitsÂÂ mapped to the two P pili operons encoded by CFT073, which are thought to mediate adhesion to kidney cells rather than bladder cells. Additionally, six high-adhesion hits mapped to the operon coding for F1C pili, and we show that this phenotype is linked to increased P pili synthesis. These results therefore reveal a critical role for P pili in UPEC adhesion to bladder epithelial cells, which may inform the development of novel anti-adhesion therapies to prevent UTI recurrence.In the third chapter, we combined microfluidics with time-lapse microscopy to identify mutants with altered persistence to fosfomycin (FOS) âÂÂ a cell-wall inhibitor used as first-line agent in the treatment of UTIs âÂÂ in synthetic human urine (SHU), which mimics the physiology of UPEC in real urine. We recovered four mutants with decreased persistence, three of which had defects in lipopolysaccharide (LPS) synthesis. These results identify the outer membrane of UPEC as a key component for survival to FOS treatment, thus opening up new avenues in the fight against antibiotic persistence.

EPFL2022