The GTPase FtsZ forms the cell division scaffold in bacteria, which mediates the recruitment of the other components of the divisome. Streptomycetes undergo two different forms of cell division.... Show moreThe GTPase FtsZ forms the cell division scaffold in bacteria, which mediates the recruitment of the other components of the divisome. Streptomycetes undergo two different forms of cell division. Septa without detectable peptidoglycan divide the highly compartmentalised young hyphae during early vegetative growth, and cross-walls are formed that dissect the hyphae into long multinucleoid compartments in the substrate mycelium, while ladders of septa are formed in the aerial hyphae that lead to chains of uninucleoid spores. In a previous study, we analysed the phosphoproteome of Streptomyces coelicolor and showed that FtsZ is phosphorylated at Ser 317 and Ser389. Substituting Ser-Ser for either Glu-Glu (mimicking phosphorylation) or Ala-Ala (mimicking non-phosphorylation) hinted at changes in antibiotic production. Here we analyse development, colony morphology, spore resistance, and antibiotic production in FtsZ knockout mutants expressing FtsZ alleles mimicking Ser319 and Ser387 phosphorylation and non-phosphorylation: AA (no phosphorylation), AE, EA (mixed), and EE (double phosphorylation). The FtsZ-eGFP AE, EA and EE alleles were not able to form observable FtsZ-eGFP ladders when they were expressed in the S. coelicolor wild-type strain, whereas the AA allele could form apparently normal eGFP Z-ladders. The FtsZ mutant expressing the FtsZ EE or EA or AE alleles is able to sporulate indicating that the mutant alleles are able to form functional Z-rings leading to sporulation when the wild-type FtsZ gene is absent. The four mutants were pleiotropically affected in colony morphogenesis, antibiotic production, substrate mycelium differentiation and sporulation (sporulation timing and spore resistance) which may be an indirect result of the effect in sporulation Z-ladder formation. Each mutant showed a distinctive phenotype in antibiotic production, single colony morphology, and sporulation (sporulation timing and spore resistance) indicating that the different FtsZ phosphomimetic alleles led to different phenotypes. Taken together, our data provide evidence for a pleiotropic effect of FtsZ phosphorylation in colony morphology, antibiotic production, and sporulation. Show less
Fluorescence microscopy is a valuable tool to study a broad variety of bacterial cell components and dynamics thereof. For Clostridioides difficile, the fluorescent proteins CFPopt, mCherry(Opt)... Show moreFluorescence microscopy is a valuable tool to study a broad variety of bacterial cell components and dynamics thereof. For Clostridioides difficile, the fluorescent proteins CFPopt, mCherry(Opt) and phiLOV2.1, and the self-labelling tags SNAP(Cd) and HaloTag, hereafter collectively referred as fluorescent systems, have been described to explore different cellular pathways. In this study, we sought to characterize previously used fluorescent systems in C. difficile cells. We performed single cell analyses using fluorescence microscopy of exponentially growing C. difficile cells harbouring different fluorescent systems, either expressing these separately in the cytosol or fused to the C-terminus of HupA, under defined conditions. We show that the intrinsic fluorescence of C. difficile cells increases during growth, independent of sigB or spo0A. However, when C. difficile cells are exposed to environmental oxygen autofluorescence is enhanced. Cytosolic overexpression of the different fluorescent systems alone, using the same expression signals, showed heterogeneous expression of the fluorescent systems. High levels of mCherry(Opt) were toxic for C. difficile cells limiting the applicability of this fluorophore as a transcriptional reporter. When fused to HupA, a C. difficile histone-like protein, the fluorescent systems behaved similarly and did not affect the HupA overproduction phenotype. The present study compares several commonly used fluorescent systems for application as transcriptional or translational reporters in microscopy and summarizes the limitations and key challenges for live-cell imaging of C. difficile. Due to independence of molecular oxygen and fluorescent signal, SNAP(Cd) appears the most suitable candidate for live-cell imaging in C. difficile to date. Show less
The interaction between soil bacteria belonging to the genera Rhizobium, Bradyrhizobium and Azorhizobium and leguminous plants results in the induction of a new plant organ, the root nodule. After... Show moreThe interaction between soil bacteria belonging to the genera Rhizobium, Bradyrhizobium and Azorhizobium and leguminous plants results in the induction of a new plant organ, the root nodule. After invading these root nodules via infection threads the bacteria start to fix atmospheric nitrogen into ammonia which is beneficial for the host plant. This symbiotic interaction is highly host-specific in that each rhizobial strain is able to associate with only a limited number of host plant species. The subject of this presentation is the molecular mechanism by which the bacterium determines its host-specific characteristics. This mechanism appears to be based on at least two stages of molecular signaling between the bacterium and the plant host. In the first stage, flavonoids secreted by the plant root induce, in a host specific way, the transcription of bacterial genes which are involved in nodulation, the so-called nod genes. This leads to the second step of the signaling system: the production and secretion of lipo-oligosaccharide molecules by the Rhizobium bacteria. These signal molecules, which are acylated forms of small fragments of chitin, have various discernable effects on the roots of the host plants. One of these effects is the dedifferentiation of groups of cells located in the cortex which leads to the formation of nodule meristems. In their mitogenic activity the bacterial signals resemble several well-known plant hormones like auxins and cytokinins. However, there are two major differences: (i) the bacterial signals lead to the induction of a specific organ and (ii) they are host-specific in that only the signals produced by compatible bacteria are able to induce meristems. The nod genes determine this stage of host specificity by their essential role in the biosynthesis of the signal molecules. They appear to encode enzymes which are involved in the processes of fatty acid biosynthesis, fatty acid transfer, chitin synthesis and chitin modification. I will illustrate the statement that the nod gene products are ideal model enzymes for the study of these important processes because they are not needed in the free-living state of the bacteria. Show less
