Gerda E. M. Lamers

Simultaneous Imaging of Pseudomonas fluorescens WCS365 Populations Expressing Three Different Autofluorescent Proteins in the Rhizosphere: New Perspectives for Studying Microbial Communities

To visualize simultaneously different populations of pseudomonads in the rhizosphere at the single cell level in a noninvasive way, a set of four rhizosphere-stable plasmids was constructed expressing three different derivatives of the green fluorescent protein (GFP), namely enhanced cyan (ECFP), enhanced green (EGFP), enhanced yellow (EYFP), and the recently published red fluorescent protein (RFP; DsRed). Upon tomato seedling inoculation with Pseudomonas fluorescens WCS365 populations, each expressing a different autofluorescent protein followed by plant growth for 5 days, the rhizosphere was inspected using confocal laser scanning microscopy. We were able to visualize simultaneously and clearly distinguish from each other up to three different bacterial populations. Microcolonies consisting of mixed populations were frequently observed at the base of the root system, whereas microcolonies further toward the root tip predominantly consisted of a single population, suggesting a dynamic behavior of microcolonies over time. Since the cloning vector pME6010 has a broad host range for gram-negative bacteria, the constructed plasmids can be used for many purposes. In particular, they will be of great value for the analysis of microbial communities, for example in processes such as biocontrol, biofertilization, biostimulation, competition for niches, colonization, and biofilm formation.

Characterization of two Pseudomonas putida lipopeptide biosurfactants, putisolvin I and II, which inhibit biofilm formation and break down existing biofilms

Pseudomonas putida strain PCL1445 was isolated from roots of plants, grown on a site polluted with polycyclic aromatic hydrocarbons. PCL1445 produces biosurfactant activity at the end of the exponential growth phase. High‐performance liquid chromatography (HPLC) analysis of supernatant extracts of PCL1445 showed two peaks with surface‐tension reducing activity, tentatively assigned as biosurfactants putisolvin I and putisolvin II and was followed by structural analyses. A transposon mutant of PCL1445, strain PCL1436, which lacks the two surface‐active peaks appeared to be mutated in an open reading frame (ORF) with amino acid homology to various lipopeptide synthetases. Structural analyses of the two biosurfactants of PCL1445 revealed that both are novel cyclic lipodepsipeptides with a hexanoic lipid chain connected to the N‐terminus of a 12‐amino‐acid peptide moiety, in which the C‐terminal carboxylic acid group forms an ester with the hydroxyl side‐chain of Ser9. The difference between the two structures is located in the second amino acid from the C‐terminus, being valine for putisolvin I, and leucine/isoleucine for putisolvin II. We show that these novel compounds lower the surface tension and influence the biofilm development on polyvinyl chloride (PVC). Biofilm formation of the bio‐synthetic mutant PCL1436 was strongly increased containing more cells, which formed aggregates earlier as compared with wild‐type PCL1445 biofilms. Using purified putisolvin I and II it was shown that biofilm formation of different Pseudomonas strains was inhibited and most interestingly, that both putisolvins are also able to break down existing Pseudomonas biofilms.

Novel aspects of tomato root colonization and infection by Fusarium oxysporum f. sp. radicis-lycopersici revealed by confocal laser scanning microscopic analysis using the green fluorescent protein as a marker

The fungus Fusarium oxysporum f. sp. radicis-lycopersici is the causal agent of tomato foot and root rot disease. The green fluorescent protein (GFP) was used to mark this fungus in order to visualize and analyze the colonization and infection processes in vivo. Transformation of F oxysporum f. sp. radicis-lycopersici was very efficient and gfp expression was stable for at least nine subcultures. Microscopic analysis of the transformants revealed homogeneity of the fluorescent signal, which was clearly visible in the hyphae as well as in the chlamydospores and conidia. To our knowledge, this is the first report in which this is shown. The transformation did not affect the pathogenicity. Using confocal laser scanning microscopy, colonization, infection, and disease development on tomato roots were visualized in detail and several new aspects of these processes were observed, such as (i) the complete colonization pattern of the tomato root system; (ii) the very first steps of contact between the fungus and the host, which takes place at the root hair zone by mingling and by the attachment of hyphae to the root hairs; (iii) the preferential colonization sites on the root surface, which are the grooves along the junctions of the epidermal cells; and (iv) the absence of specific infection sites, such as sites of emergence of secondary roots, root tips, or wounded tissue, and the absence of specific infection structures, such as appressoria. The results of this work prove that the use of GFP as a marker for F. oxysporum f. sp. radicis-lycopersici is a convenient, fast, and effective approach for studying plant-fungus interactions.

Interactions in the tomato rhizosphere of two Pseudomonas biocontrol strains with the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici.

The fungus Fusarium oxysporum f. sp. radicis-lycopersici causes foot and root rot of tomato plants, which can be controlled by the bacteria Pseudomonas fluorescens WCS365 and P. chlororaphis PCL1391. Induced systemic resistance is thought to be involved in biocontrol by P. fluorescens WCS365. The antifungal metabolite phenazine-1-carboxamide (PCN), as well as efficient root colonization, are essential in the mechanism of biocontrol by P. chlororaphis PCL1391. To understand the effects of bacterial strains WCS365 and PCL1391 on the fungus in the tomato rhizosphere, microscopic analyses were performed using different autofluorescent proteins as markers. Tomato seedlings were inoculated with biocontrol bacteria and planted in an F. oxysporum f. sp. radicis-lycopersici-infested gnotobiotic sand system. Confocal laser scanning microscope analyses of the interactions in the tomato rhizosphere revealed that i) the microbes effectively compete for the same niche, and presumably also for root exudate nutrients; ii) the presence of either of the two bacteria negatively affects infection of the tomato root by the fungus; iii) both biocontrol bacteria colonize the hyphae extensively, which may represent a new mechanism in biocontrol by these pseudomonads; and iv) the production of PCN by P. chlororaphis PCL1391 negatively affects hyphal growth and branching, which presumably affects the colonization and infecting ability of the fungus.

A novel polar surface polysaccharide from Rhizobium leguminosarum binds host plant lectin

Rhizobium bacteria produce different surface polysaccharides which are either secreted in the growth medium or contribute to a capsule surrounding the cell. Here, we describe isolation and partial characterization of a novel high molecular weight surface polysaccharide from a strain of Rhizobium leguminosarum that nodulates Pisum sativum (pea) and Vicia sativa (vetch) roots. Carbohydrate analysis showed that the polysaccharide consists for 95% of mannose and glucose, with minor amounts of galactose and rhamnose. Lectin precipitation analysis revealed high binding affinity of pea and vetch lectin for this polysaccharide, in contrast to the other known capsular and extracellular polysaccharides of this strain. Expression of the polysaccharide was independent of the presence of a Sym plasmid or the nod gene inducer naringenin. Incubation of R. leguminosarum with labelled pea lectin showed that this polysaccharide is exclusively localized on one of the poles of the bacterial cell. Vetch roots incubated with rhizobia and labelled pea lectin revealed that this bacterial pole is involved in attachment to the root surface. A mutant strain deficient in the production of this polysaccharide was impaired in attachment and root hair infection under slightly acidic conditions, in contrast to the situation at slightly alkaline conditions. Our data are consistent with the hypothesis that rhizobia can use (at least) two mechanisms for docking at the root surface, with use of a lectin–glycan mechanism under slightly acidic conditions.

Neutrophil-mediated experimental metastasis is enhanced by VEGFR inhibition in a zebrafish xenograft model

Inhibition of VEGF signalling effectively suppresses localized tumour growth but accelerates tumour invasiveness and micrometastasis by unknown mechanisms. To study the dynamic and reciprocal interactions between tumour cells and their microenvironment during these processes, we established a xenograft model by injecting tumour cells into the blood circulation of transparent zebrafish embryos. This reproducibly results in rapid simultaneous formation of a localized tumour and experimental micrometastasis, allowing time‐resolved imaging of both processes at single‐cell resolution within 1 week. The tumour vasculature was initiated de novo by remodelling of primitive endothelial cells into a functional network. Roles of myeloid cells in critical tumourigenesis steps such as vascularization and invasion were revealed by genetic and pharmaceutical approaches. We discovered that the physiological migration of neutrophils controlled tumour invasion by conditioning the collagen matrix and forming the metastatic niche, as detected by two‐photon confocal microscopy and second harmonic generation. Administration of VEGFR inhibitors blocked tumour vascularization and a localized tumour growth but enhanced migration of neutrophils, which in turn promoted tumour invasion and formation of micrometastasis. This demonstrates the in vivo cooperation between VEGF signalling and myeloid cells in metastasis and provides a new mechanism underlying the recent findings that VEGFR targeting can promote tumour invasiveness. Copyright

Characterization of Mucosal Biofilms on Human Adenoid Tissues

Objectives: To demonstrate the presence of mucosal biofilm in adenoid tissue using double staining for visualization of both the bacterial matrix and the bacterial cells. To identify bacterial species present on the surface of the studied adenoids.

Folic Acid-Modified Mesoporous Silica Nanoparticles for Cellular and Nuclear Targeted Drug Delivery

Site-specific stimuli responsive nanomaterials are an important breakthrough for the improvement of modern therapies in nanomedicine. Mesoporous silica nanoparticles are good candidate for the development of targeted delivery system as their surface can be easily modified with functional groups in order to achieve controlled and specific release. We designed a drug delivery system based on mesoporous silica nanoparticles modified with folic acid as a specific targeting moiety. The functionalization forms a nanovalve system in which the surface is modified with an aliphatic chain. This stalk tethers a cyclodextrin with the specific role to prevent undesired release of the cargo. To avoid any movement of the cyclodextrin the folic acid is placed at the end of the chain. The release kinetics were investigated with UV/VIS spectroscopy and cellular uptake was extensively studied using flow cytometry. Through this study we demonstrated the biocompatibility of folic acid modified MSNs and the effective release of an encapsulated anticancer drug using TUNEL and Western Blot assays. Chapter 3: Folic Acid Modified Mesoporous Silica Nanoparticles for Cellular and Nuclear Targeted Drug Delivery

Genetic tools for tagging Gram-negative bacteria with mCherry for visualization in vitro and in natural habitats, biofilm and pathogenicity studies.

Live-cell imaging techniques are essential to gain a better understanding of microbial functioning in natural systems, for example in biofilms. Autofluorescent proteins, such as the green fluorescent protein (GFP) and the red fluorescent protein (DsRed), are valuable tools for studying microbial communities in their natural environment. Because of the functional limitations of DsRed such as slow maturation and low photostability, new and improved variants were created such as mCherry. In this study, we developed genetic tools for labeling Gram-negative bacteria in order to visualize them in vitro and in their natural environment without the necessity of antibiotic pressure for maintenance. mcherry was cloned into two broad host-range cloning vectors and a pBK-miniTn7 transposon under the constitutive expression of the tac promoter. The applicability of the different constructs was shown in Escherichia coli, various Pseudomonas spp. and Edwardsiella tarda. The expression of mcherry was qualitatively analyzed by fluorescence microscopy and quantified by fluorometry. The suitability of the constructs for visualizing microbial communities was shown for biofilms formed on glass and tomato roots. In addition, it is shown that mCherry in combination with GFP is a suitable marker for studying mixed microbial communities.

NODZ OF BRADYRHIZOBIUM EXTENDS THE NODULATION HOST RANGE OF RHIZOBIUM BY ADDING A FUCOSYL RESIDUE TO NODULATION SIGNALS

The nodulation genes of rhizobia are involved in the production of the lipo‐chitin oligosaccharides (LCO), which are signal molecules required for nodule formation. A mutation in nodZ of Bradyrhizobium japonicum results in the synthesis of nodulation signals lacking the wild‐type 2‐O‐methylfucose residue at the reducing‐terminal N‐acetylglucosamine. This phenotype is correlated with a defective nodulation of siratro (Macroptilium atropurpureum). Here we show that transfer of nodZ to Rhizobium leguminosarum biovar (bv) viciae, which produces LCOs that are not modified at the reducing‐terminal N‐acetylglucosamine, results in production of LCOs with a fucosyl residue on C‐6 of the reducing‐terminal N‐acetylglucosamine. This finding, together with in vitro enzymatic assays, indicates that the product of nodZ functions as a fucosyltransferase. The transconjugant R. leguminosarum strain producing fucosylated LCOs acquires the capacity to nodulate M. atropurpureumGlycine sojaVigna unguiculata and Leucaena leucocephala. Therefore, nodZ extends the narrow host range of R. leguminosarum bv. viciae to include various tropical legumes. However, microscopic analysis of nodules induced on siratro shows that these nodules do not contain bacteroids, showing that transfer of nodZ does not allow R. leguminosarum to engage in a nitrogen‐fixing symbiosis with this plant.