Bacillus mycoides: novel tools for studying the mechanisms of its interaction with plants

Plantengroei bevorderende rhizobacteriën (PBR) is een groep bacteriën die de wortels van de plant koloniseren en de plantengroei kunnen bevorderen. Inzicht in de moleculaire mechanismen van hoe planten en PBR naast elkaar kunnen bestaan ​​en van elkaar kunnen profiteren, kan nieuwe strategieën bieden om de gezondheid en productiviteit van planten te verbeteren. Dit kan leiden to duurzame alternatieven voor klassieke bestrijdingsmiddelen. Bacillus mycoides is een bacterie die veel aanwezig is in gezonde aardappelwortels en een sterk groei bevorderend potentieel heeft voor planten. In dit onderzoek hebben we het moleculaire mechanisme van de B. mycoides-plant interacties onderzocht. Hiertoe werd een multidisciplinaire benadering toegepast en werden nieuwe moleculair genetische hulpmiddelen ontwikkeld. De vergelijkende genoom analyses onthulden dat er een vermeende endofytische clade is binnen de B. mycoides-soort. Fluorescente eiwitten werden gebruikt om een endofytische en een bodemstam te labelen. Hun wortelkolonisatiepatronen werden bestudeerd met behulp van een confocale laserscanningmicroscoop, waaruit bleek dat de endofytische stam een ​​beter vermogen tot wortelkolonisatie heeft dan de bodemstam. Uit de transcriptoomanalyse bleek dat de endofytische stam een ​​breder scala aan substraten in de rhizosfeer-nis kan gebruiken dan de bodemstam. Er werd ontdekt dat sideroforen geproduceerd door B. mycoides de groei van planten met een ijzertekort bevorderen en een belangrijke rol spelen bij de interactie tussen deze bacteriën en planten

Characterization of plant growth-promoting rhizobacteria from perennial ryegrass and genome mining of novel antimicrobial gene clusters

Background Plant growth-promoting rhizobacteria (PGPR) are good alternatives for chemical fertilizers and pesticides, which cause severe environmental problems worldwide. Even though many studies focus on PGPR, most of them are limited in plant-microbe interaction studies and neglect the pathogens affecting ruminants that consume plants. In this study, we expand the view to the food chain of grass-ruminant-human. We aimed to find biocontrol strains that can antagonize grass pathogens and mammalian pathogens originated from grass, thus protecting this food chain. Furthermore, we deeply mined into bacterial genomes for novel biosynthetic gene clusters (BGCs) that can contribute to biocontrol. Results We screened 90 bacterial strains from the rhizosphere of healthy Dutch perennial ryegrass and characterized seven strains (B. subtilis subsp. subtilis MG27, B. velezensis MG33 and MG43, B. pumilus MG52 and MG84, B. altitudinis MG75, and B. laterosporus MG64) that showed a stimulatory effect on grass growth and pathogen antagonism on both phytopathogens and mammalian pathogens. Genome-mining of the seven strains discovered abundant BGCs, with some known, but also several potential novel ones. Further analysis revealed potential intact and novel BGCs, including two NRPSs, four NRPS-PKS hybrids, and five bacteriocins. Conclusion Abundant potential novel BGCs were discovered in functional protective isolates, especially in B. pumilus, B. altitudinis and Brevibacillus strains, indicating their great potential for the production of novel secondary metabolites. Our report serves as a basis to further identify and characterize these compounds and study their antagonistic effects against plant and mammalian pathogens

Development of an efficient electroporation method for rhizobacterial Bacillus mycoides strains

In order to develop a method for electroporation of environmental Bacillus mycoides strains, we optimized several conditions that affect the electroporation efficiency of this bacterium. By combining the optimized conditions, the electroporation efficiency of strain EC18 was improved to (1.3 ± 0.6) ×10(5) cfu/μg DNA, which is about 10(3)-fold increase in comparison with a previously reported value. The method was further validated on various B. mycoides strains, yielding reasonable transformation efficiencies. Furthermore, we confirmed that restriction/modification is the main barrier for electroporation of this bacterium. To the best of our knowledge, this is the first systematic investigation of various parameters of electroporation of B. mycoides. The electroporation method reported will allow for efficient genetic manipulation of this bacterium

Plant-Microbe Interaction:Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant growth-promoting rhizobacteria (PGPR) as biofertilizers or biocontrol agents has become an effective alternative to the use of conventional fertilizers and can increase crop productivity at low cost. Plant-microbe interactions depend upon host plant-secreted signals and a reaction hereon by their associated bacteria. However, the molecular mechanisms of how beneficial bacteria respond to their associated plant-derived signals are not fully understood. Assessing the transcriptomic response of bacteria to root exudates is a powerful approach to determine the bacterial gene expression and regulation under rhizospheric conditions. Such knowledge is necessary to understand the underlying mechanisms involved in plant-microbe interactions. This paper describes a detailed protocol to study the transcriptomic response of B. mycoides EC18, a strain isolated from the potato endosphere, to potato root exudates. With the help of recent high-throughput sequencing technology, this protocol can be performed in several weeks and produce massive datasets. First, we collect the root exudates under sterile conditions, after which they are added to B. mycoides cultures. The RNA from these cultures is isolated using a phenol/chloroform method combined with a commercial kit and subjected to quality control by an automated electrophoresis instrument. After sequencing, data analysis is performed with the web-based T-REx pipeline and a group of differentially expressed genes is identified. This method is a useful tool to facilitate new discoveries on the bacterial genes involved in plant-microbe interactions

Exploring plant-microbe interactions of the rhizobacteria Bacillus subtilis and Bacillus mycoides by use of the CRISPR-Cas9 system

Bacillus subtilis HS3 and Bacillus mycoides EC18 are two rhizosphere-associated bacteria with plant growth-promoting activity. The CRISPR-Cas9 system was implemented to study various aspects of plant-microbe interaction mechanisms of these two environmental isolates. The results show that fengycin and surfactin are involved in the antifungal activity of B. subtilis HS3. Moreover, this strain emits several other volatile organic compounds than 2,3-butanediol, contributing to plant growth promotion. Confocal laser scanning microscopy observations of the GFP-labelled strain showed that HS3 selectively colonizes root hairs of grass (Lolium perenne) in a hydroponic system. For B. mycoides EC18, we found that the wild-type EC18 strain and a Delta asbA (petropectin-deficient) mutant, but not the Delta dhbB (bacillibactin-deficient) and ADKO (asbA and dhbB double knockout) mutants, can increase the plant biomass and total chlorophyll. All the mutant strains have a reduced colonization capability on Chinese cabbage (Brassica rapa) roots, at the root tip and root hair region compared with the wild-type strain. These results indicate that the siderophore, bacillibactin, is involved in the plant growth promoting activity and could affect the root colonization of B. mycoides. Collectively, the CRISPR-Cas9 system we developed for environmental isolates is broadly applicable and will facilitate deciphering the mechanisms of Bacillus-plant interactions. (c) 2018 The Authors

Comparative Transcriptomics of Bacillus mycoides Strains in Response to Potato-Root Exudates Reveals Different Genetic Adaptation of Endophytic and Soil Isolates

Plant root secreted compounds alter the gene expression of associated microorganisms by acting as signal molecules that either stimulate or repel the interaction with beneficial or harmful species, respectively. However, it is still unclear whether two distinct groups of beneficial bacteria, non-plant-associated (soil) strains and plant-associated (endophytic) strains, respond uniformly or variably to the exposure with root exudates. Therefore, Bacillus mycoides, a potential biocontrol agent and plant growth-promoting bacterium, was isolated from the endosphere of potatoes and from soil of the same geographical region. Confocal fluorescence microscopy of plants inoculated with GFP-tagged B. mycoides strains showed that the endosphere isolate EC18 had a stronger plant colonization ability and competed more successfully for the colonization sites than the soil isolate SB8. To dissect these phenotypic differences, the genomes of the two strains were sequenced and the transcriptome response to potato root exudates was compared. The global transcriptome profiles evidenced that the endophytic isolate responded more pronounced than the soil-derived isolate and a higher number of significant differentially expressed genes were detected. Both isolates responded with the alteration of expression of an overlapping set of genes, which had previously been reported to be involved in plant–microbe interactions; including organic substance metabolism, oxidative reduction, and transmembrane transport. Notably, several genes were specifically upregulated in the endosphere isolate EC18, while being oppositely downregulated in the soil isolate SB8. These genes mainly encoded membrane proteins, transcriptional regulators or were involved in amino acid metabolism and biosynthesis. By contrast, several genes upregulated in the soil isolate SB8 and downregulated in the endosphere isolate EC18 were related to sugar transport, which might coincide with the different nutrient availability in the two environments. Altogether, the presented transcriptome profiles provide highly improved insights into the life strategies of plant-associated endophytes and soil isolates of B. mycoides

Optimized fluorescent proteins for the rhizosphere-associated bacterium <i>Bacillus mycoides</i> with endophytic and biocontrol agent potential

Tracking of fluorescent protein (FP)-labelled rhizobacteria is a key prerequisite to gain insights into plant-bacteria interaction mechanisms. However, the performance of FPs mostly has to be optimized for the bacterial host and for the environment of intended application. We report on the construction of mutational libraries of the superfolder green fluorescent protein sfGFP and the red fluorescent protein mKate2 in the bacterium B. mycoides, which next to its potential as plant-biocontrol agent occasionally enters an endophytic lifestyle. By fluorescence-activated cell sorting and comparison of signal intensities at the colony and single-cell level, the variants sfGFP(SPS6) and mKate (KPS12) with significantly increased brightness were isolated. Their high applicability for plant-bacteria interaction studies was shown by confocal laser scanning microscopy tracking of FP-tagged B. mycoides strains after inoculation to Chinese cabbage plants in a hydroponic system. During the process of colonization, strain EC18 rapidly attached to plant roots and formed a multicellular matrix, especially at the branching regions of the root hair, which probably constitute entrance sites to establish an endophytic lifestyle. The universal applicability of the novels FPs was proven by expression from a weak promoter, dual-labelling of B. mycoides, and by excellent expression and detectability in additional soil- and rhizosphere-associated Bacillus species