Soil Application of a Formulated Biocontrol Rhizobacterium, Pseudomonas chlororaphis PCL1606, Induces Soil Suppressiveness by Impacting Specific Microbial Communities.

Biocontrol bacteria can be used for plant protection against some plant diseases. Pseudomonas chlororaphis PCL1606 (PcPCL1606) is a model bacterium isolated from the avocado rhizosphere with strong antifungal antagonism mediated by the production of 2-hexyl, 5-propil resorcinol (HPR). Additionally, PcPCL1606 has biological control against different soil-borne fungal pathogens, including the causal agent of the white root rot of many woody crops and avocado in the Mediterranean area, Rosellinia necatrix. The objective of this study was to assess whether the semicommercial application of PcPCL1606 to soil can potentially affect avocado soil and rhizosphere microbial communities and their activities in natural conditions and under R. necatrix infection. To test the putative effects of PcPCL1606 on soil eukaryotic and prokaryotic communities, a formulated PcPCL1606 was prepared and applied to the soil of avocado plants growing in mesocosm experiments, and the communities were analyzed by using 16S/ITS metagenomics. PcPCL1606 survived until the end of the experiments. The effect of PcPCL1606 application on prokaryotic communities in soil and rhizosphere samples from natural soil was not detectable, and very minor changes were observed in eukaryotic communities. In the infested soils, the presence of R. necatrix strongly impacted the soil and rhizosphere microbial communities. However, after PcPCL1606 was applied to soil infested with R. necatrix, the prokaryotic community reacted by increasing the relative abundance of few families with protective features against fungal soilborne pathogens and organic matter decomposition (Chitinophagaceae, Cytophagaceae), but no new prokaryotic families were detected. The treatment of PcPCL1606 impacted the fungal profile, which strongly reduced the presence of R. necatrix in avocado soil and rhizosphere, minimizing its effect on the rest of the microbial communities. The bacterial treatment of formulated PcPCL1606 on avocado soils infested with R. necatrix resulted in biological control of the pathogen. This suppressiveness phenotype was analyzed, and PcPCL1606 has a key role in suppressiveness induction; in addition, this phenotype was strongly dependent on the production of HPR.

Functional analysis of three putative galactofuranosyltransferases with redundant functions in galactofuranosylation in Aspergillus niger.

Galactofuranose (Galf)-containing glycostructures are important to secure the integrity of the fungal cell wall. Golgi-localized Galf-transferases (Gfs) have been identified in Aspergillus nidulans and Aspergillus fumigatus. BLASTp searches identified three putative Galf-transferases in Aspergillus niger. Phylogenetic analysis showed that they group in three distinct groups. Characterization of the three Galf-transferases in A. niger by constructing single, double, and triple mutants revealed that gfsA is most important for Galf biosynthesis. The growth phenotypes of the ΔgfsA mutant are less severe than that of the ΔgfsAC mutant, indicating that GfsA and GfsC have redundant functions. Deletion of gfsB did not result in any growth defect and combining ΔgfsB with other deletion mutants did not exacerbate the growth phenotype. RT-qPCR experiments showed that induction of the agsA gene was higher in the ΔgfsAC and ΔgfsABC compared to the single mutants, indicating a severe cell wall stress response after multiple gfs gene deletions.

Rhizosphere-Associated Pseudomonas Suppress Local Root Immune Responses by Gluconic Acid-Mediated Lowering of Environmental pH.

The root microbiome consists of commensal, pathogenic, and plant-beneficial microbes [1]. Most members of the root microbiome possess microbe-associated molecular patterns (MAMPs) similar to those of plant pathogens [2]. Their recognition can lead to the activation of host immunity and suppression of plant growth due to growth-defense tradeoffs [3, 4]. We found that 42% of the tested root microbiota, including the plant growth-promoting rhizobacteria Pseudomonas capeferrum WCS358 [5, 6] and Pseudomonas simiae WCS417 [6, 7], are able to quench local Arabidopsis thaliana root immune responses that are triggered by flg22 [8], an immunogenic epitope of the MAMP flagellin [9], suggesting that this is an important function of the root microbiome. In a screen for WCS358 mutants that lost their capacity to suppress flg22-induced CYP71A12pro:GUS MAMP-reporter gene expression, we identified the bacterial genes pqqF and cyoB in WCS358, which are required for the production of gluconic acid and its derivative 2-keto gluconic acid. Both WCS358 mutants are impaired in the production of these organic acids and consequently lowered their extracellular pH to a lesser extent than wild-type WCS358. Acidification of the plant growth medium similarly suppressed flg22-induced CYP71A12pro:GUS and MYB51pro:GUS expression, and the flg22-mediated oxidative burst, suggesting a role for rhizobacterial gluconic acid-mediated modulation of the extracellular pH in the suppression of root immunity. Rhizosphere population densities of the mutants were significantly reduced compared to wild-type. Collectively, these findings show that suppression of immune responses is an important function of the root microbiome, as it facilitates colonization by beneficial root microbiota.

Aspergillus fumigatus establishes infection in zebrafish by germination of phagocytized conidia, while Aspergillus niger relies on extracellular germination.

Among opportunistically pathogenic filamentous fungi of the Aspergillus genus, Aspergillus fumigatus stands out as a drastically more prevalent cause of infection than others. Utilizing the zebrafish embryo model, we applied a combination of non-invasive real-time imaging and genetic approaches to compare the infectious development of A. fumigatus with that of the less pathogenic A. niger. We found that both species evoke similar immune cell migratory responses, but A. fumigatus is more efficiently phagocytized than A. niger. Though efficiently phagocytized, A. fumigatus conidia retains the ability to germinate and form hyphae from inside macrophages leading to serious infection even at relatively low infectious burdens. By contrast, A. niger appears to rely on extracellular germination, and rapid hyphal growth to establish infection. Despite these differences in the mechanism of infection between the species, galactofuranose mutant strains of both A. fumigatus and A. niger display attenuated pathogenesis. However, deficiency in this cell wall component has a stronger impact on A. niger, which is dependent on rapid extracellular hyphal growth. In conclusion, we uncover differences in the interaction of the two fungal species with innate immune cells, noticeable from very early stages of infection, which drive a divergence in their route to establishing infections.

Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus.

BACKGROUND: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus. RESULTS: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli. CONCLUSIONS: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.

Transcriptomic and molecular genetic analysis of the cell wall salvage response of Aspergillus niger to the absence of galactofuranose synthesis.

The biosynthesis of cell surface-located galactofuranose (Galf)-containing glycostructures such as galactomannan, N-glycans and O-glycans in filamentous fungi is important to secure the integrity of the cell wall. UgmA encodes an UDP-galactopyranose mutase, which is essential for the formation of Galf. Consequently, the ΔugmA mutant lacks Galf-containing molecules. Our previous work in Aspergillus niger work suggested that loss of function of ugmA results in activation of the cell wall integrity (CWI) pathway which is characterized by increased expression of the agsA gene, encoding an α-glucan synthase. In this study, the transcriptional response of the ΔugmA mutant was further linked to the CWI pathway by showing the induced and constitutive phosphorylation of the CWI-MAP kinase in the ΔugmA mutant. To identify genes involved in cell wall remodelling in response to the absence of galactofuranose biosynthesis, a genome-wide expression analysis was performed using RNAseq. Over 400 genes were higher expressed in the ΔugmA mutant compared to the wild-type. These include genes that encode enzymes involved in chitin (gfaB, gnsA, chsA) and α-glucan synthesis (agsA), and in ß-glucan remodelling (bgxA, gelF and dfgC), and also include several glycosylphosphatidylinositol (GPI)-anchored cell wall protein-encoding genes. In silico analysis of the 1-kb promoter regions of the up-regulated genes in the ΔugmA mutant indicated overrepresentation of genes with RlmA, MsnA, PacC and SteA-binding sites. The importance of these transcription factors for survival of the ΔugmA mutant was analysed by constructing the respective double mutants. The ΔugmA/ΔrlmA and ΔugmA/ΔmsnA double mutants showed strong synthetic growth defects, indicating the importance of these transcription factors to maintain cell wall integrity in the absence of Galf biosynthesis.

Identification and functional analysis of two Golgi-localized UDP-galactofuranose transporters with overlapping functions in Aspergillus niger.

BACKGROUND: Galactofuranose (Galf)-containing glycoconjugates are present in numerous microbes, including filamentous fungi where they are important for morphology, virulence and maintaining cell wall integrity. The incorporation of Galf-residues into galactomannan, galactomannoproteins and glycolipids is carried out by Golgi-localized Galf transferases. The nucleotide sugar donor used by these transferases (UDP-Galf) is produced in the cytoplasm and has to be transported to the lumen of the Golgi by a dedicated nucleotide sugar transporter. METHODS: Based on homology with recently identified UDP-Galf-transporters in A. fumigatus and A. nidulans, two putative UDP-Galf-transporters in A. niger were found. Their function and localization was determined by gene deletions and GFP-tagging studies, respectively. RESULTS: The two putative UDP-Galf-transporters in A. niger are homologous to each other and are predicted to contain eleven transmembrane domains (UgtA) or ten transmembrane domains (UgtB) due to a reduced length of the C-terminal part of the UgtB protein. The presence of two putative UDP-Galf-transporters in the genome was not unique for A. niger. From the twenty Aspergillus species analysed, nine species contained two additional putative UDP-Galf-transporters. Three of the nine species were outside the Aspergillus section nigri, indication an early duplication of UDP-Galf-transporters and subsequent loss of the UgtB copy in several aspergilli. Deletion analysis of the single and double mutants in A. niger indicated that the two putative UDP-Galf-transporters (named UgtA and UgtB) have a redundant function in UDP-Galf-transport as only the double mutant displayed a Galf-negative phenotype. The Galf-negative phenotype of the double mutant could be complemented by expressing either CFP-UgtA or CFP-UgtB fusion proteins from their endogenous promoters, indicating that both CFP-tagged proteins are functional. Both Ugt proteins co-localize with each other as well as with the GDP-mannose nucleotide transporter, as was demonstrated by fluorescence microscopy, thereby confirming their predicted localization in the Golgi. CONCLUSION: A. niger contains two genes encoding UDP-Galf-transporters. Deletion and localization studies indicate that UgtA and UgtB have redundant functions in the biosynthesis of Galf-containing glycoconjugates.

A new vector for efficient gene targeting to the pyrG locus in Aspergillus niger.

BACKGROUND: The possibility for efficient gene targeting for the controlled integration of DNA constructs is an important tool in fungal genetics. FINDINGS: In this study, we report a new targeting vector based on the pyrG marker in Aspergillus niger. The DNA sequence to be targeted is surrounded by two fragments of the pyrG gene to allow homologous recombination of the recombinant DNA at the pyrG locus. The 5' end of the targeting cassette contains a non-functional truncated pyrG open reading frame (first 112 bases deleted) and the 3' untranslated region (3' UTR). At the 3' end, the targeting cassette consists of the 3' flanking region of the pyrG gene. A unique NotI site between the flanks allows the insertion of a gene of interest. The linearized targeting cassette is transformed to the A. niger pyrG mutant strain AB4.1 or a derivative thereof. By using a constitutively expressed luciferase reporter gene (mluc) as an example, it is shown that the targeting system is efficient as 4 out of 6 (67%) AB4.1 transformants and 51 out of 66 (77%) MA169.4 (ku70- ) transformants contained the reporter gene at the pyrG locus. A luciferase (lux) activity assay, performed with independently obtained transformants in which the mluc reporter was integrated at the pyrG locus, showed comparable and reproducible lux activities. CONCLUSION: The new pyrG targeting vector is an important improvement to the existing method for gene targeting in A. niger. Although the vector is specific for A. niger, the presented design and approach is easily applicable for constructing integration vectors for other fungi.

Induction of A. fumigatus-specific CD4-positive T cells in patients recovering from invasive aspergillosis.

After allogeneic stem cell transplantation patients are at risk of invasive aspergillosis, especially during the period of neutropenia. Recent data suggest that impaired T-cell immune reconstitution after transplantation plays an important role in this increased risk. In this study we investigated whether Aspergillus-specific T cells are involved in the recovery from invasive aspergillosis by analyzing the Aspergillus-specific T-cell response in patients with invasive aspergillosis. In nine patients whose Aspergillus infection improved, we identified Crf1- or Catalase1-specific T cells on the basis of CD154 expression and interferon-γ production following stimulation with overlapping peptides of the A. fumigatus proteins Crf1 and Catalase1. These Aspergillus-specific T cells were induced at the moment of regression of the aspergillus lesions. Crf1- and Catalase1-specific T cells, sorted on the basis of CD154 expression at the peak of the immune response, had a T helper-1 phenotype and recognized a variety of T-cell epitopes. In contrast, in two patients with progressive invasive aspergillosis, no Crf1- or Catalase1-specific T cells were identified. These data indicate that the presence of Aspergillus-specific T cells with a T helper-1 phenotype correlates with the clearance of aspergillus infection.

Identification of the UDP-glucose-4-epimerase required for galactofuranose biosynthesis and galactose metabolism in A. niger.

BACKGROUND: Galactofuranose (Galf)-containing glycoconjugates are important to secure the integrity of the cell wall of filamentous fungi. Mutations that prevent the biosynthesis of Galf-containing molecules compromise cell wall integrity. In response to cell wall weakening, the cell wall integrity (CWI)-pathway is activated to reinforce the strength of the cell wall. Activation of CWI-pathway in Aspergillus niger is characterized by the specific induction of the agsA gene, which encodes a cell wall α-glucan synthase. RESULTS: In this study, we screened a collection of cell wall mutants with an induced expression of agsA for defects in Galf biosynthesis using a with anti-Galf antibody (L10). From this collection of mutants, we previously identified mutants in the UDP-galactopyranose mutase encoding gene (ugmA). Here, we have identified six additional UDP-galactopyranose mutase (ugmA) mutants and one mutant (named mutant #41) in an additional complementation group that displayed strongly reduced Galf-levels in the cell wall. By using a whole genome sequencing approach, 21 SNPs in coding regions were identified between mutant #41 and its parental strain which changed the amino acid sequence of the encoded proteins. One of these mutations was in gene An14g03820, which codes for a putative UDP-glucose-4-epimerase (UgeA). The A to G mutation in this gene causes an amino acid change of Asn to Asp at position 191 in the UgeA protein. Targeted deletion of ugeA resulted in an even more severe reduction of Galf in N-linked glucans, indicating that the UgeA protein in mutant #41 is partially active. The ugeA gene is also required for growth on galactose despite the presence of two UgeA homologs in the A. niger genome. CONCLUSION: By using a classical mutant screen and whole genome sequencing of a new Galf-deficient mutant, the UDP-glucose-4-epimerase gene (ugeA) has been identified. UgeA is required for the biosynthesis of Galf as well as for galactose metabolism in Aspergillus niger.

Fungal α-arabinofuranosidases of glycosyl hydrolase families 51 and 54 show a dual arabinofuranosyl- and galactofuranosyl-hydrolyzing activity.

Aspergillus niger possesses a galactofuranosidase activity, however, the corresponding enzyme or gene encoding this enzyme has never been identified. As evidence is mounting that enzymes exist with affinity for both arabinofuranose and galactofuranose, we investigated the possibility that α-L-arabinofuranosidases, encoded by the abfA and abfB genes, are responsible for the galactofuranosidase activity of A. niger. Characterization of the recombinant AbfA and AbfB proteins revealed that both enzymes do not only hydrolyze p-nitrophenyl-α-L-arabinofuranoside (pNp-α-Araf) but are also capable of hydrolyzing p-nitrophenyl-ß-D-galactofuranoside (pNp-ß-Galf). Molecular modeling of the AbfB protein with pNp-ß-Galf confirmed the possibility for AbfB to interact with this substrate, similarly as with pNp-α-Araf. We also show that galactomannan, a cell wall compound of A. niger, containing ß-linked terminal and internal galactofuranosyl moieties, can be degraded by an enzyme activity that is present in the supernatant of inulin-grown A. niger. Interestingly, purified AbfA and AbfB did not show this hydrolyzing activity toward A. nigergalactomannan. In summary, our studies demonstrate that AbfA and AbfB, α-L-arabinofuranosidases from different families, both contain a galactofuranose (Galf)-hydrolyzing activity. In addition, our data support the presence of a Galf-hydrolase activity expressed by A. niger that is capable of degrading fungal galactomannan.

Vacuolar H(+)-ATPase plays a key role in cell wall biosynthesis of Aspergillus niger.

The identification of suitable targets is crucial for the discovery and development of new antifungals. Since the fungal cell wall is an essential organelle, the identification of genes involved in cell wall biosynthesis is expected to help discover new antifungal targets. From our previously obtained collection of cell wall mutants with a constitutively active cell wall stress response pathway, we selected a thermosensitive, osmotic-remediable mutant with decreased resistance to SDS for complementation analysis. The phenotypes of this mutant were complemented by a gene encoding a protein with high sequence similarity to subunit d of the eukaryotic Vacuolar-H(+)-ATPase (VmaD). Genetic analysis of this thermosensitive mutant revealed that the conditional mutant allele encodes a protein that lacks 12 amino acids at the C-terminus due to a point mutation that introduces a stop codon. Deletion of the entire gene resulted in very poor growth. The conditional mutant displayed several phenotypes that are typical for V-ATPase mutants, including increased sensitivity to zinc ions and reduced acidification of the vacuole as observed by quinacrine staining. Treatment of Aspergillus niger with the V-ATPase inhibitor bafilomycinB(1) induced the expression of agsA and other cell wall related genes. Furthermore genes involved in cell wall reassembly like fksA, agsA and phiA were clearly up-regulated in the conditional mutant. Our results indicate that the ATP-driven transport of protons and acidification of the vacuole is crucial for the strength of the fungal cell wall and that reduced activity of the V-ATPase induces the cell wall stress response pathway.

Biofilms on tracheoesophageal voice prostheses: a confocal laser scanning microscopy demonstration of mixed bacterial and yeast biofilms.

The aim of this study was to demonstrate the presence of yeast and bacterial biofilms on the surface of tracheoesophageal voice prostheses (TVPs) by a double-staining technique with confocal laser scanning microscopy (CLSM). Biofilms of 12 removed TVPs were visualized by scanning electron microscopy, then stained with ConA-FITC and propidium iodide for CLSM. Microbial identification was by partial 16S rRNA gene analysis and ITS-2 sequence analysis. Microbial biofilms on the TVPs consisted of bacteria and filamentous cells. Bacterial cells were attached to the filamentous and unicellular yeast cells, thus forming a network. Sequence analyses of six voice prostheses identified the presence of a variety of bacterial and yeast species. In vivo studies showed that Klebsiella oxytoca and Micrococcus luteus efficiently attached to Candida albicans. CLSM with double fluorescence staining can be used to demonstrate biofilm formations composed of a mixture of yeast and bacterial cells on the surface of TVPs.

Do biofilm formation and interactions with human cells explain the clinical success of Acinetobacter baumannii?

BACKGROUND: The dramatic increase in antibiotic resistance and the recent manifestation in war trauma patients underscore the threat of Acinetobacter baumannii as a nosocomial pathogen. Despite numerous reports documenting its epidemicity, little is known about the pathogenicity of A. baumannii. The aim of this study was to obtain insight into the factors that might explain the clinical success of A. baumannii. METHODOLOGY/PRINCIPAL FINDINGS: We compared biofilm formation, adherence to and inflammatory cytokine induction by human cells for a large panel of well-described strains of A. baumannii and compared these features to that of other, clinically less relevant Acinetobacter species. Results revealed that biofilm formation and adherence to airway epithelial cells varied widely within the various species, but did not differ among the species. However, airway epithelial cells and cultured human macrophages produced significantly less inflammatory cytokines upon exposure to A. baumannii strains than to strains of A. junii, a species infrequently causing infection. CONCLUSION/SIGNIFICANCE: The induction of a weak inflammatory response may provide a clue to the persistence of A. baumannii in patients.

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.

Maggot excretions/secretions are differentially effective against biofilms of Staphylococcus aureus and Pseudomonas aeruginosa.

OBJECTIVES: Lucilia sericata maggots are successfully used for treating chronic wounds. As the healing process in these wounds is complicated by bacteria, particularly when residing in biofilms that protect them from antibiotics and the immune system, we assessed the effects of maggot excretions/secretions (ES) on Staphylococcus aureus and Pseudomonas aeruginosa biofilms, the clinically most relevant species. METHODS: We assessed the effects of ES on biofilms using microtitre plate assays, on bacterial viability using in vitro killing and radial diffusion assays, and on quorum sensing systems using specific reporter bacteria. RESULTS: As little as 0.2 microg of ES prevented S. aureus biofilm formation and 2 microg of ES rapidly degraded biofilms. In contrast, ES initially promoted P. aeruginosa biofilm formation, but after 10 h the biofilms collapsed. Degradation of P. aeruginosa biofilms started after 10 h and required 10-fold more ES than S. aureus biofilms. Boiling of ES abrogated their effects on S. aureus, but not on P. aeruginosa, biofilms, indicating that different molecules within ES are responsible for the observed effects. Modulation of biofilms by ES did not involve bacterial killing or effects on quorum sensing systems. CONCLUSIONS: Maggot ES are differentially effective against biofilms of S. aureus and P. aeruginosa.

The heat shock genes dnaK, dnaJ, and grpE are involved in regulation of putisolvin biosynthesis in Pseudomonas putida PCL1445.

Pseudomonas putida PCL1445 produces two cyclic lipopeptides, putisolvins I and II, which possess surfactant activity and play an important role in biofilm formation and degradation. In order to identify genes and traits that are involved in the regulation of putisolvin production of PCL1445, a Tn5luxAB library was generated and mutants were selected for the lack of biosurfactant production using a drop-collapsing assay. Sequence analysis of the Tn5luxAB flanking region of one biosurfactant mutant, strain PCL1627, showed that the transposon had inserted in a dnaK homologue which is located downstream of grpE and upstream of dnaJ. Analysis of putisolvin production and expression studies indicate that dnaK, together with the dnaJ and grpE heat shock genes, takes part in the positive regulation (directly or indirectly) of putisolvin biosynthesis at the transcriptional level. Growth of PCL1445 at low temperature resulted in an increased level of putisolvins, and mutant analyses showed that this requires dnaK and dnaJ but not grpE. In addition, putisolvin biosynthesis of PCL1445 was found to be dependent on the GacA/GacS two-component signaling system. Expression analysis indicated that dnaK is positively regulated by GacA/GacS.

Role of chemotaxis toward fusaric acid in colonization of hyphae of Fusarium oxysporum f. sp. radicis-lycopersici by Pseudomonas fluorescens WCS365.

Pseudomonas fluorescens WCS365 is an excellent competitive colonizer of tomato root tips after bacterization of seed or seedlings. The strain controls tomato foot and root rot (TFRR) caused by the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici. Under biocontrol conditions, fungal hyphae were shown to be colonized by WCS365 bacteria. Because chemotaxis is required for root colonization by WCS365 cells, we studied whether chemotaxis also is required for hyphae colonization. To that end, an in vitro assay was developed to study hyphae colonization by bacteria. The results indicated that cells of the cheA mutant FAJ2060 colonize hyphae less efficiently than cells of wild-type strain WCS365, when single strains were analyzed as well as when both strains were applied together. Cells of WCS365 show a chemotactic response toward the spent growth medium of F. oxysporum f. sp. radicis-lycopersici, but those of its cheA mutant, FAJ2060, did not. Fusaric acid, a secondary metabolite secreted by Fusarium strains, appeared to be an excellent chemo-attractant. Supernatant fluids of a number of Fusarium strains secreting different levels of fusaric acid were tested as chemo-attractants. A positive correlation was found between chemo-attractant activity and fusaric acid level. No chemotactic response was observed toward the low fusaric acid-producer FO242. Nevertheless, the hyphae of FO242 still were colonized by WCS365, suggesting that other metabolites also play a role in this process. The possible function of hyphae colonization for the bacterium is discussed.

Rhizoremediation: a beneficial plant-microbe interaction.

Worldwide, contamination of soil and ground water is a severe problem. The negative effects of pollutants on the environment and on human health are diverse and depend on the nature of the pollution. The search for alternative methods for excavation and incineration to clean polluted sites resulted in the application of bioremediation techniques. In this review, we describe some generally accepted bioremediation tools and subsequently focus on the combination of two approaches, phytoremediation and bioaugmentation, resulting in rhizoremediation. During rhizoremediation, exudates derived from the plant can help to stimulate the survival and action of bacteria, which subsequently results in a more efficient degradation of pollutants. The root system of plants can help to spread bacteria through soil and help to penetrate otherwise impermeable soil layers. The inoculation of pollutant-degrading bacteria on plant seed can be an important additive to improve the efficiency of phytoremediation or bioaugmentation.

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.