Quorum sensing-related activities of beneficial and pathogenic bacteria have important implications for plant and human health.

Eukaryotic organisms coevolved with microbes from the environment forming holobiotic meta-genomic units. Members of host-associated microbiomes have commensalic, beneficial/symbiotic, or pathogenic phenotypes. More than 100 years ago, Lorenz Hiltner, pioneer of soil microbiology, introduced the term 'Rhizosphere' to characterize the observation that a high density of saprophytic, beneficial, and pathogenic microbes are attracted by root exudates. The balance between these types of microbes decide about the health of the host. Nowadays we know, that for the interaction of microbes with all eukaryotic hosts similar principles and processes of cooperative and competitive functions are in action. Small diffusible molecules like (phyto)hormones, volatiles and quorum sensing signals are examples for mediators of interspecies and cross-kingdom interactions. Quorum sensing of bacteria is mediated by different autoinducible metabolites in a density-dependent manner. In this perspective publication, the role of QS-related activities for the health of hosts will be discussed focussing mostly on N-acyl-homoserine lactones (AHL). It is also considered that in some cases very close phylogenetic relations exist between plant beneficial and opportunistic human pathogenic bacteria. Based on a genome and system-targeted new understanding, sociomicrobiological solutions are possible for the biocontrol of diseases and the health improvement of eukaryotic hosts.

Biological Control of Phytopathogens: Mechanisms and Applications.

According to the inherent ecological mechanisms within community structures, organismic interactions are mediated by chemical structures and signaling molecules as well as enzymatic activities targeting the vital activities of microbial competitors [...].

Importance of N-Acyl-Homoserine Lactone-Based Quorum Sensing and Quorum Quenching in Pathogen Control and Plant Growth Promotion.

The biological control of plant pathogens is linked to the composition and activity of the plant microbiome. Plant-associated microbiomes co-evolved with land plants, leading to plant holobionts with plant-beneficial microbes but also with plant pathogens. A diverse range of plant-beneficial microbes assists plants to reach their optimal development and growth under both abiotic and biotic stress conditions. Communication within the plant holobiont plays an important role, and besides plant hormonal interactions, quorum-sensing signalling of plant-associated microbes plays a central role. Quorum-sensing (QS) autoinducers, such as N-acyl-homoserine lactones (AHL) of Gram-negative bacteria, cause a pronounced interkingdom signalling effect on plants, provoking priming processes of pathogen defence and insect pest control. However, plant pathogenic bacteria also use QS signalling to optimise their virulence; these QS activities can be controlled by quorum quenching (QQ) and quorum-sensing inhibition (QSI) approaches by accompanying microbes and also by plants. Plant growth-promoting bacteria (PGPB) have also been shown to demonstrate QQ activity. In addition, some PGPB only harbour genes for AHL receptors, so-called luxR-solo genes, which can contribute to plant growth promotion and biological control. The presence of autoinducer solo receptors may reflect ongoing microevolution processes in microbe-plant interactions. Different aspects of QS systems in bacteria-plant interactions of plant-beneficial and pathogenic bacteria will be discussed, and practical applications of bacteria with AHL-producing or -quenching activity; QS signal molecules stimulating pathogen control and plant growth promotion will also be presented.

Genome-based reclassification of Azospirillum brasilense Sp245 as the type strain of Azospirillum baldaniorum sp. nov.

Azospirillum sp. strain Sp245T, originally identified as belonging to Azospirillum brasilense, is recognized as a plant-growth-promoting rhizobacterium due to its ability to fix atmospheric nitrogen and to produce plant-beneficial compounds. Azospirillum sp. Sp245T and other related strains were isolated from the root surfaces of different plants in Brazil. Cells are Gram-negative, curved or slightly curved rods, and motile with polar and lateral flagella. Their growth temperature varies between 20 to 38 °C and their carbon source utilization is similar to other Azospirillum species. A preliminary 16S rRNA sequence analysis showed that the new species is closely related to A. brasilense Sp7T and A. formosense CC-Nfb-7T. Housekeeping genes revealed that Azospirillum sp. Sp245T, BR 12001 and Vi22 form a separate cluster from strain A. formosense CC-Nfb-7T, and a group of strains closely related to A. brasilense Sp7T. Overall genome relatedness index (OGRI) analyses estimated based on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between Azospirillum sp. Sp245T and its close relatives to other Azospirillum species type strains, such as A. brasilense Sp7T and A. formosense CC-Nfb-7T , revealed values lower than the limit of species circumscription. Moreover, core-proteome phylogeny including 1079 common shared proteins showed the independent clusterization of A. brasilense Sp7T, A. formosense CC-Nfb-7T and Azospirillum sp. Sp245T, a finding that was corroborated by the genome clustering of OGRI values and housekeeping phylogenies. The DNA G+C content of the cluster of Sp245T was 68.4-68.6 %. Based on the phylogenetic, genomic, phenotypical and physiological analysis, we propose that strain Sp245T together with the strains Vi22 and BR12001 represent a novel species of the genus Azospirillum, for which the name Azospirillum baldaniorum sp. nov. is proposed. The type strain is Sp245T (=BR 11005T=IBPPM 219T) (GCF_007827915.1, GCF_000237365.1, and GCF_003119195.2).

Assessment of the structural and functional diversities of plant microbiota: Achievements and challenges - A review.

Analyses of the spatial localization and the functions of bacteria in host plant habitats through in situ identification by immunological and molecular genetic techniques combined with high resolving microscopic tools and 3D-image analysis contributed substantially to a better understanding of the functional interplay of the microbiota in plants. Among the molecular genetic methods, 16S-rRNA genes were of central importance to reconstruct the phylogeny of newly isolated bacteria and to localize them in situ. However, they usually do not allow resolution for phylogenetic affiliations below genus level. Especially, the separation of opportunistic human pathogens from plant beneficial strains, currently allocated to the same species, needs genome-based resolving techniques. Whole bacterial genome sequences allow to discriminate phylogenetically closely related strains. In addition, complete genome sequences enable strain-specific monitoring for biotechnologically relevant strains. In this mini-review we present high resolving approaches for analysis of the composition and key functions of plant microbiota, focusing on interactions of diazotrophic plant growth promoting bacteria, like Azospirillum brasilense, with non-legume host plants. Combining high resolving microscopic analyses with specific immunological detection methods and molecular genetic tools, including especially transcriptome analyses of both the bacterial and plant partners, enables new insights into key traits of beneficial bacteria-plant interactions in holobiontic systems.

Detection of the Bacterial Quorum-Sensing Signaling Molecules N-Acyl-Homoserine Lactones (HSL) and N-Acyl-Homoserine (HS) with an Enzyme-Linked Immunosorbent Assay (ELISA) and via Ultrahigh-Performance Liquid Chromatography Coupled to Mass Spectrometry (UHPLC-MS).

Quick and reliable quantitative methods requiring low amounts of sample volume are needed for the detection of N-acyl-homoserine lactones (HSL) and their degradation products N-acyl-homoserines (HS) in order to elucidate the occurrence and dynamics of these prevalent quorum-sensing molecules of Gram-negative bacteria in natural samples and laboratory model experiments. A combination of ELISA and UHPLC-MS is presented here which has proven to meet these requirements. Both methods can not only precisely detect and quantify HSLs but also their degradation products HS and thereby enable studying signaling dynamics in quorum sensing, which have been identified to play an essential role in bacterial communication.

Sulfonolipids as novel metabolite markers of Alistipes and Odoribacter affected by high-fat diets.

The gut microbiota generates a huge pool of unknown metabolites, and their identification and characterization is a key challenge in metabolomics. However, there are still gaps on the studies of gut microbiota and their chemical structures. In this investigation, an unusual class of bacterial sulfonolipids (SLs) is detected in mouse cecum, which was originally found in environmental microbes. We have performed a detailed molecular level characterization of this class of lipids by combining high-resolution mass spectrometry and liquid chromatography analysis. Eighteen SLs that differ in their capnoid and fatty acid chain compositions were identified. The SL called "sulfobacin B" was isolated, characterized, and was significantly increased in mice fed with high-fat diets. To reveal bacterial producers of SLs, metagenome analysis was acquired and only two bacterial genera, i.e., Alistipes and Odoribacter, were revealed to be responsible for their production. This knowledge enables explaining a part of the molecular complexity introduced by microbes to the mammalian gastrointestinal tract and can be used as chemotaxonomic evidence in gut microbiota.

D-tryptophan from probiotic bacteria influences the gut microbiome and allergic airway disease.

BACKGROUND: Chronic immune diseases, such as asthma, are highly prevalent. Currently available pharmaceuticals improve symptoms but cannot cure the disease. This prompted demands for alternatives to pharmaceuticals, such as probiotics, for the prevention of allergic disease. However, clinical trials have produced inconsistent results. This is at least partly explained by the highly complex crosstalk among probiotic bacteria, the host's microbiota, and immune cells. The identification of a bioactive substance from probiotic bacteria could circumvent this difficulty. OBJECTIVE: We sought to identify and characterize a bioactive probiotic metabolite for potential prevention of allergic airway disease. METHODS: Probiotic supernatants were screened for their ability to concordantly decrease the constitutive CCL17 secretion of a human Hodgkin lymphoma cell line and prevent upregulation of costimulatory molecules of LPS-stimulated human dendritic cells. RESULTS: Supernatants from 13 of 37 tested probiotic strains showed immunoactivity. Bioassay-guided chromatographic fractionation of 2 supernatants according to polarity, followed by total ion chromatography and mass spectrometry, yielded C11H12N2O2 as the molecular formula of a bioactive substance. Proton nuclear magnetic resonance and enantiomeric separation identified D-tryptophan. In contrast, L-tryptophan and 11 other D-amino acids were inactive. Feeding D-tryptophan to mice before experimental asthma induction increased numbers of lung and gut regulatory T cells, decreased lung TH2 responses, and ameliorated allergic airway inflammation and hyperresponsiveness. Allergic airway inflammation reduced gut microbial diversity, which was increased by D-tryptophan. CONCLUSIONS: D-tryptophan is a newly identified product from probiotic bacteria. Our findings support the concept that defined bacterial products can be exploited in novel preventative strategies for chronic immune diseases.

Systemic Responses of Barley to the 3-hydroxy-decanoyl-homoserine Lactone Producing Plant Beneficial Endophyte Acidovorax radicis N35.

Quorum sensing auto-inducers of the N-acyl homoserine lactone (AHL) type produced by Gram-negative bacteria have different effects on plants including stimulation on root growth and/or priming or acquirement of systemic resistance in plants. In this communication the influence of AHL production of the plant growth promoting endophytic rhizosphere bacterium Acidovorax radicis N35 on barley seedlings was investigated. A. radicis N35 produces 3-hydroxy-C10-homoserine lactone (3-OH-C10-HSL) as the major AHL compound. To study the influence of this QS autoinducer on the interaction with barley, the araI-biosynthesis gene was deleted. The comparison of inoculation effects of the A. radicis N35 wild type and the araI mutant resulted in remarkable differences. While the N35 wild type colonized plant roots effectively in microcolonies, the araI mutant occurred at the root surface as single cells. Furthermore, in a mixed inoculum the wild type was much more prevalent in colonization than the araI mutant documenting that the araI mutation affected root colonization. Nevertheless, a significant plant growth promoting effect could be shown after inoculation of barley with the wild type and the araI mutant in soil after 2 months cultivation. While A. radicis N35 wild type showed only a very weak induction of early defense responses in plant RNA expression analysis, the araI mutant caused increased expression of flavonoid biosynthesis genes. This was corroborated by the accumulation of several flavonoid compounds such as saponarin and lutonarin in leaves of root inoculated barley seedlings. Thus, although the exact role of the flavonoids in this plant response is not clear yet, it can be concluded, that the synthesis of AHLs by A. radicis has implications on the perception by the host plant barley and thereby contributes to the establishment and function of the bacteria-plant interaction.

A distinct microbiota composition is associated with protection from food allergy in an oral mouse immunization model.

In our mouse model, gastric acid-suppression is associated with antigen-specific IgE and anaphylaxis development. We repeatedly observed non-responder animals protected from food allergy. Here, we aimed to analyse reasons for this protection. Ten out of 64 mice, subjected to oral ovalbumin (OVA) immunizations under gastric acid-suppression, were non-responders without OVA-specific IgE or IgG1 elevation, indicating protection from allergy. In these non-responders, allergen challenges confirmed reduced antigen uptake and lack of anaphylactic symptoms, while in allergic mice high levels of mouse mast-cell protease-1 and a body temperature reduction, indicative for anaphylaxis, were determined. Upon OVA stimulation, significantly lower IL-4, IL-5, IL-10 and IL-13 levels were detected in non-responders, while IL-22 was significantly higher. Comparison of fecal microbiota revealed differences of bacterial communities on single bacterial Operational-Taxonomic-Unit level between the groups, indicating protection from food allergy being associated with a distinct microbiota composition in a non-responding phenotype in this mouse model.

Beneficial effects of bacteria-plant communication based on quorum sensing molecules of the N-acyl homoserine lactone group.

Bacterial quorum sensing (QS) mechanisms play a crucial role in the proper performance and ecological fitness of bacterial populations. Many key physiological processes are regulated in a QS-dependent manner by auto-inducers, like the N-acyl homoserine lactones (AHLs) in numerous Gram-negative bacteria. In addition, also the interaction between bacteria and eukaryotic hosts can be regulated by AHLs. Those mechanisms gained much attention, because of the positive effects of different AHL molecules on plants. This positive impact ranges from growth promotion to induced resistance and is quite contrasting to the rather negative effects observed in the interactions between bacterial AHL molecules and animals. Only very recently, we began to understand the molecular mechanisms underpinning plant responses to AHL molecules. In this review, we gathered the latest information in this research field. The first part gives an overview of the bacterial aspects of quorum sensing. Later we focus on the impact of AHLs on plant growth and AHL-priming, as one of the most understood phenomena in respect to the inter-kingdom interactions based on AHL-quorum sensing molecules. Finally, we discuss the potential benefits of the understanding of bacteria-plant interaction for the future agricultural applications.

Pollen-Associated Microbiome Correlates with Pollution Parameters and the Allergenicity of Pollen.

Pollen allergies have been rapidly increasing over the last decades. Many allergenic proteins and non-allergenic adjuvant compounds of pollen are involved in the plant defense against environmental or microbial stress. The first aim of this study was to analyze and compare the colonizing microbes on allergenic pollen. The second aim was to investigate detectable correlations between pollen microbiota and parameters of air pollution or pollen allergenicity. To reach these aims, bacterial and fungal DNA was isolated from pollen samples of timothy grass (Phleum pratense, n = 20) and birch trees (Betula pendula, n = 55). With this isolated DNA, a terminal restriction fragment length polymorphism analysis was performed. One result was that the microbial diversity on birch tree and timothy grass pollen samples (Shannon/Simpson diversity indices) was partly significantly correlated to allergenicity parameters (Bet v 1/Phl p 5, pollen-associated lipid mediators). Furthermore, the microbial diversity on birch pollen samples was correlated to on-site air pollution (nitrogen dioxide (NO2), ammonia (NH3), and ozone (O3)). What is more, a significant negative correlation was observed between the microbial diversity on birch pollen and the measured NO2 concentrations on the corresponding trees. Our results showed that the microbial composition of pollen was correlated to environmental exposure parameters alongside with a differential expression of allergen and pollen-associated lipid mediators. This might translate into altered allergenicity of pollen due to environmental and microbial stress.

Non-pathogenic Rhizobium radiobacter F4 deploys plant beneficial activity independent of its host Piriformospora indica.

The Alphaproteobacterium Rhizobium radiobacter F4 (RrF4) was originally characterized as an endofungal bacterium in the beneficial endophytic Sebacinalean fungus Piriformospora indica. Although attempts to cure P. indica from RrF4 repeatedly failed, the bacterium can easily be grown in pure culture. Here, we report on RrF4's genome and the beneficial impact the free-living bacterium has on plants. In contrast to other endofungal bacteria, the genome size of RrF4 is not reduced. Instead, it shows a high degree of similarity to the plant pathogenic R. radiobacter (formerly: Agrobacterium tumefaciens) C58, except vibrant differences in both the tumor-inducing (pTi) and the accessor (pAt) plasmids, which can explain the loss of RrF4's pathogenicity. Similar to its fungal host, RrF4 colonizes plant roots without host preference and forms aggregates of attached cells and dense biofilms at the root surface of maturation zones. RrF4-colonized plants show increased biomass and enhanced resistance against bacterial leaf pathogens. Mutational analysis showed that, similar to P. indica, resistance mediated by RrF4 was dependent on the plant's jasmonate-based induced systemic resistance (ISR) pathway. Consistent with this, RrF4- and P. indica-induced pattern of defense gene expression were similar. In clear contrast to P. indica, but similar to plant growth-promoting rhizobacteria, RrF4 colonized not only the root outer cortex but also spread beyond the endodermis into the stele. On the basis of our findings, RrF4 is an efficient plant growth-promoting bacterium.

Systemic colonization of clover (Trifolium repens) by Clostridium botulinum strain 2301.

In recent years, cases of botulism in cattle and other farm animals and also in farmers increased dramatically. It was proposed, that these cases could be affiliated with the spreading of compost or other organic manures contaminated with Clostridium botulinum spores on farm land. Thus, soils and fodder plants and finally farm animals could be contaminated. Therefore, the colonization behavior and interaction of the botulinum neurotoxin (BoNT D) producing C. botulinum strain 2301 and the non-toxin producing Clostridium sporogenes strain 1739 were investigated on clover (Trifolium repens) in a field experiment as well as in phytochamber experiments applying axenic and additionally soil based systems under controlled conditions. Plants were harvested and divided into root and shoot parts for further DNA isolation and polymerase chain reaction (PCR) assays; subsamples were fixed for fluorescence in situ hybridization analysis in combination with confocal laser scanning microscopy. In addition, we observed significant differences in the growth behavior of clover plants when inoculated with clostridial spores, indicating a plant growth promoting effect. Inoculated plants showed an increased growth index (shoot size, wet and dry weight) and an enlarged root system induced by the systemic colonization of clover by C. botulinum strain 2301. To target C. botulinum and C. sporogenes, 16S rDNA directed primers were used and to specifically detect C. botulinum, BoNT D toxin genes targeted primers, using a multiplex PCR approach, were applied. Our results demonstrate an effective colonization of roots and shoots of clover by C. botulinum strain 2301 and C. sporogenes strain 1739. Detailed analysis of colonization behavior showed that C. botulinum can occur as individual cells, in cell clusters and in microcolonies within the rhizosphere, lateral roots and within the roots tissue of clover.

Zhihengliuella somnathii sp. nov., a halotolerant actinobacterium from the rhizosphere of a halophyte Salicornia brachiata.

Two novel, Gram-stain-positive, rod-shaped, halotolerent bacteria, strains JG 03(T) and JG 05 were isolated from the rhizosphere of Salicornia brachiata, an extreme halophyte. Comparative analyses of 16S rRNA gene sequences showed that they were closely related to members of the genus Zhihengliuella, with sequence similarities of 96.9-99.1%. The sequence similarity of strains JG 03(T )and JG 05 with each other was 99.4%. DNA-DNA hybridization of JG 03(T) and JG 05 with other species of the genus Zhihengliuella with validly published names showed reassociation values of 19.8%-53.4% and a value of 91.4% between each other. The peptidoglycan type of both strains was A4α and MK-9 and MK-10 were the predominant menaquinones. The predominant fatty acid in JG 03(T) was anteiso-C15 : 0 and anteiso-C17 : 0. However, iso-C15 : 0, anteiso-C15 : 0 and anteiso-C17 : 0 were the major fatty acids in strain JG 05. The DNA G+C content of strains JG 03(T) and JG 05 was 70.0 and 70.1 mol%, respectively. In nutrient broth medium both strains grew at NaCl concentrations of up to 15% (w/v). On the basis of chemotaxonomic characteristics and phylogenetic analyses, strains JG 03(T) and JG 05 should be affiliated to the genus Zhihengliuella. Strains JG 03(T) and JG 05 represent a novel species of the genus Zhihengliuella for which the name Zhihengliuella somnathii sp. nov. is proposed. The type strain is JG 03(T) ( = DSM 23187(T) = IMCC 253(T)).