Irene de Bruijn

Deciphering the rhizosphere microbiome for disease-suppressive bacteria.

A common plant pathogen induces the growth of disease-suppressive microbes in local soil communities. Disease-suppressive soils are exceptional ecosystems in which crop plants suffer less from specific soil-borne pathogens than expected owing to the activities of other soil microorganisms. For most disease-suppressive soils, the microbes and mechanisms involved in pathogen control are unknown. By coupling PhyloChip-based metagenomics of the rhizosphere microbiome with culture-dependent functional analyses, we identified key bacterial taxa and genes involved in suppression of a fungal root pathogen. More than 33,000 bacterial and archaeal species were detected, with Proteobacteria, Firmicutes, and Actinobacteria consistently associated with disease suppression. Members of the γ-Proteobacteria were shown to have disease-suppressive activity governed by nonribosomal peptide synthetases. Our data indicate that upon attack by a fungal root pathogen, plants can exploit microbial consortia from soil for protection against infections.

Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics

Lipopeptides constitute a structurally diverse group of metabolites produced by various bacterial and fungal genera. In the past decades, research on lipopeptides has been fueled by their antimicrobial, antitumour, immunosuppressant and surfactant activities. However, the natural functions of lipopeptides in the lifestyles of the producing microorganisms have received considerably less attention. The substantial structural diversity of lipopeptides suggests that these metabolites have different natural roles, some of which may be unique to the biology of the producing organism. This review gives a detailed overview of the versatile functions of lipopeptides in the biology of Pseudomonas and Bacillus species, and highlights their role in competitive interactions with coexisting organisms, including bacteria, fungi, oomycetes, protozoa, nematodes and plants. Their functions in cell motility, leading to colonization of novel habitats, and in the formation and development of highly structured biofilms are discussed in detail. Finally, this review provides an update on lipopeptide detection and discovery as well as on novel regulatory mechanisms and genes involved in lipopeptide biosynthesis in these two bacterial genera.

Stimulation of chitin synthesis rescues Candida albicans from echinocandins

Echinocandins are a new generation of novel antifungal agent that inhibit cell wall β(1,3)-glucan synthesis and are normally cidal for the human pathogen Candida albicans. Treatment of C. albicans with low levels of echinocandins stimulated chitin synthase (CHS) gene expression, increased Chs activity, elevated chitin content and reduced efficacy of these drugs. Elevation of chitin synthesis was mediated via the PKC, HOG, and Ca2+-calcineurin signalling pathways. Stimulation of Chs2p and Chs8p by activators of these pathways enabled cells to survive otherwise lethal concentrations of echinocandins, even in the absence of Chs3p and the normally essential Chs1p, which synthesize the chitinous septal ring and primary septum of the fungus. Under such conditions, a novel proximally offset septum was synthesized that restored the capacity for cell division, sustained the viability of the cell, and abrogated morphological and growth defects associated with echinocandin treatment and the chs mutations. These findings anticipate potential resistance mechanisms to echinocandins. However, echinocandins and chitin synthase inhibitors synergized strongly, highlighting the potential for combination therapies with greatly enhanced cidal activity.

The PKC, HOG and Ca2+ signalling pathways co-ordinately regulate chitin synthesis in Candida albicans

Chitin is an essential component of the fungal cell wall and its synthesis is under tight spatial and temporal regulation. The fungal human pathogen Candida albicans has a four member chitin synthase gene family comprising of CHS1 (class II), CHS2 (class I), CHS3 (class IV) and CHS8 (class I). LacZ reporters were fused to each CHS promoter to examine the transcriptional regulation of chitin synthesis. Each CHS promoter had a unique regulatory profile and responded to the addition of cell wall damaging agents, to mutations in specific CHS genes and exogenous Ca2+. The regulation of both CHS gene expression and chitin synthesis was co‐ordinated by the PKC, HOG MAP kinase and Ca2+/calcineurin signalling pathways. Activation of these pathways also resulted in increased chitin synthase activity in vitro and elevated cell wall chitin content. Combinations of treatments that activated multiple pathways resulted in synergistic increases in CHS expression and in cell wall chitin content. Therefore, at least three pathways co‐ordinately regulate chitin synthesis and activation of chitin synthesis operates at both transcriptional and post‐transcriptional levels.

Genome-based discovery, structure prediction and functional analysis of cyclic lipopeptide antibiotics in Pseudomonas species

Analysis of microbial genome sequences have revealed numerous genes involved in antibiotic biosynthesis. In Pseudomonads, several gene clusters encoding non‐ribosomal peptide synthetases (NRPSs) were predicted to be involved in the synthesis of cyclic lipopeptide (CLP) antibiotics. Most of these predictions, however, are untested and the association between genome sequence and biological function of the predicted metabolite is lacking. Here we report the genome‐based identification of previously unknown CLP gene clusters in plant pathogenic Pseudomonas syringae strains B728a and DC3000 and in plant beneficial Pseudomonas fluorescens Pf0‐1 and SBW25. For P. fluorescens SBW25, a model strain in studying bacterial evolution and adaptation, the structure of the CLP with a predicted 9‐amino acid peptide moiety was confirmed by chemical analyses. Mutagenesis confirmed that the three identified NRPS genes are essential for CLP synthesis in strain SBW25. CLP production was shown to play a key role in motility, biofilm formation and in activity of SBW25 against zoospores of Phytophthora infestans. This is the first time that an antimicrobial metabolite is identified from strain SBW25. The results indicate that genome mining may enable the discovery of unknown gene clusters and traits that are highly relevant in the lifestyle of plant beneficial and plant pathogenic bacteria.

Distinctive expansion of potential virulence genes in the genome of the oomycete fish pathogen Saprolegnia parasitica.

Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinklers, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.

Protozoan-Induced Regulation of Cyclic Lipopeptide Biosynthesis Is an Effective Predation Defense Mechanism for Pseudomonas fluorescens

ABSTRACT Environmental bacteria are exposed to a myriad of biotic interactions that influence their function and survival. The grazing activity of protozoan predators significantly impacts the dynamics, diversification, and evolution of bacterial communities in soil ecosystems. To evade protozoan predation, bacteria employ various defense strategies. Soil-dwelling Pseudomonas fluorescens strains SS101 and SBW25 produce the cyclic lipopeptide surfactants (CLPs) massetolide and viscosin, respectively, in a quorum-sensing-independent manner. In this study, CLP production was shown to protect these bacteria from protozoan predation as, compared to CLP-deficient mutants, strains SS101 and SBW25 exhibited resistance to grazing by Naegleria americana in vitro and superior persistence in soil in the presence of this bacterial predator. In the wheat rhizosphere, CLP-producing strains had a direct deleterious impact on the survival of N. americana. In vitro assays further showed that N. americana was three times more sensitive to viscosin than to massetolide and that exposure of strain SS101 or SBW25 to this protozoan resulted in upregulation of CLP biosynthesis genes. Enhanced expression of the massABC and viscABC genes did not require physical contact between the two organisms as gene expression levels were up to threefold higher in bacterial cells harvested 1 cm from feeding protozoans than in cells collected 4 cm from feeding protozoans. These findings document a new natural function of CLPs and highlight that bacterium-protozoan interactions can result in activation of an antipredator response in prey populations.

Host-targeting protein 1 (SpHtp1) from the oomycete Saprolegnia parasitica translocates specifically into fish cells in a tyrosine-O-sulphate–dependent manner

The eukaryotic oomycetes, or water molds, contain several species that are devastating pathogens of plants and animals. During infection, oomycetes translocate effector proteins into host cells, where they interfere with host-defense responses. For several oomycete effectors (i.e., the RxLR-effectors) it has been shown that their N-terminal polypeptides are important for the delivery into the host. Here we demonstrate that the putative RxLR-like effector, host-targeting protein 1 (SpHtp1), from the fish pathogen Saprolegnia parasitica translocates specifically inside host cells. We further demonstrate that cell-surface binding and uptake of this effector protein is mediated by an interaction with tyrosine-O-sulfate–modified cell-surface molecules and not via phospholipids, as has been reported for RxLR-effectors from plant pathogenic oomycetes. These results reveal an effector translocation route based on tyrosine-O-sulfate binding, which could be highly relevant for a wide range of host–microbe interactions.

The putative RxLR effector protein SpHtp1 from the fish pathogenic oomycete Saprolegnia parasitica is translocated into fish cells

The fish pathogenic oomycete Saprolegnia parasitica causes the disease Saprolegniosis in salmonids and other freshwater fish, resulting in considerable economic losses in aquaculture. Very little is known about the molecular and cellular mechanisms underlying the infection process of fish pathogenic oomycetes. In order to investigate the interaction in detail, an in vitro infection assay using an Oncorhynchus mykiss (rainbow trout) cell line (RTG-2) was developed. In a zoospore/cyst cDNA library, we identified the ORF SpHtp1, which encodes a secreted protein containing an RxLR motif. Detailed expression analysis indicated that SpHtp1 is highly expressed in zoospores/cysts from S. parasitica and in the very early stages of infection on RTG-2 cells, when compared with in vitro-grown mycelium. Moreover, the protein, SpHtp1, was found to translocate into the RTG-2 trout cells, during the interaction with S. parasitica, and also when the RTG-2 cells were treated with recombinant SpHtp1 fused to a C-terminal His-tag. These findings suggest that protein translocation could play an important role in Saprolegniosis.

Comparative genomics and metabolic profiling of the genus Lysobacter.

BackgroundLysobacter species are Gram-negative bacteria widely distributed in soil, plant and freshwater habitats. Lysobacter owes its name to the lytic effects on other microorganisms. To better understand their ecology and interactions with other (micro)organisms, five Lysobacter strains representing the four species L. enzymogenes, L. capsici, L. gummosus and L. antibioticus were subjected to genomics and metabolomics analyses.ResultsComparative genomics revealed a diverse genome content among the Lysobacter species with a core genome of 2,891 and a pangenome of 10,028 coding sequences. Genes encoding type I, II, III, IV, V secretion systems and type IV pili were highly conserved in all five genomes, whereas type VI secretion systems were only found in L. enzymogenes and L. gummosus. Genes encoding components of the flagellar apparatus were absent in the two sequenced L. antibioticus strains. The genomes contained a large number of genes encoding extracellular enzymes including chitinases, glucanases and peptidases. Various nonribosomal peptide synthase (NRPS) and polyketide synthase (PKS) gene clusters encoding putative bioactive metabolites were identified but only few of these clusters were shared between the different species. Metabolic profiling by imaging mass spectrometry complemented, in part, the in silico genome analyses and allowed visualisation of the spatial distribution patterns of several secondary metabolites produced by or induced in Lysobacter species during interactions with the soil-borne fungus Rhizoctonia solani.ConclusionsOur work shows that mining the genomes of Lysobacter species in combination with metabolic profiling provides novel insights into the genomic and metabolic potential of this widely distributed but understudied and versatile bacterial genus.