Functional genome analysis of the plant-growth promoting bacterium Bacillus amyloliquefaciens strain FZB42

Bacillus amyloliquefaciens FZB42 ist ein im Boden weit verbreitetes Bakterium. Es kolonisiert Pflanzenwurzeln und werde als Biodünger verwendet, da sie in der Lage sind, Pflanzenwachstum zu fördern. Der domestizierte Stamm B. subtilis 168 ist eng verwand mit B. amyloliquefaciens FZB42, fördert jedoch kein Pflanzenwachstum. Als ein erster Ansatz zur Ermittlung von Gendifferenzen zwischen FZB42 und B. subtilis 168 - wobei zum damaligen Zeitpunkt nur die Genomsequenz letzteren Organismus bekannt war - wurde die Supression Subtractive Hybridisation (SSH) angewandt. Hierdurch wurden mehrere einzigartige Gene in B. amyloliquefaziens identifiziert. Unterdessen beteiligte sich unser Labor in Kollaboration mit dem GenoMik Network in Göttingen an einem Projekt, dessen Ziel die komplette Sequenzierung des Genoms von B. amyloliquefaciens war. Der Hauptanteil der Arbeit, sowie die Koordination des gesamten Projekts wurden von Xiao-Hua Chen und mir selbst durchgeführt. B. amyloliquefaciens FZB42 besitzt die srf, fen, pks1 (bae), bac und dhb Operons, welche für die Synthese von Surfactin, Fengycin, Bacillaene, Bacilysin und Bacillibactin verantwortlich sind und die ebenfalls im Genom von B. subtilis 168 enthalten sind. Das Genom von B. amyloliquefaciens FZB42, beinhaltet die bmy Gencluster, die die Synthese von Bacillomycin D kontrolliert. Ein weiteres in dieser Arbeit verfolgtes Ziel war die Identifizierung der regulatorischen Wege, die die Expression von Bacillomycin D steuern. Es wurde gezeigt, dass globale Regulatoren, wie beispielsweise DegU, DegQ und ComA, die alternativen Sigmafaktoren sigB und sigH und ein neuartiges Rap-Protein die transkriptionale Aktivität des in dieser Arbeit identifizierten Hauptpromotors des bmy-Operons beeinflussen. Es wurde gezeigt, dass DegU seine Effekte nach direkter Bindung an zwei unterschiedliche Regionen im bmy-Promotor ausübt. Es wurde außerdem gezeigt, dass DegU abgesehen von der Aktivierung des Hauptpromoters des bmy-Operons eine zusätzliche, vermutlich eine post-transkriptionale Rolle spielt. Auf ähnliche Weise erwies sich YczE, ein Membranprotein unbekannter Funktion, das neben sfp kodiert liegt, als essentiell für die Bacillomycin D-Produktion. Der Effekt wurde auf einem post-transkriptionalen Level ausgeübt und war unabhängig von DegU.Bacillus amyloliquefaciens FZB42 is widely distributed in the soil. It colonizes the plant roots and is used as bio-fertilizer, since they can promote plant growth.The domesticated strain of B. subtilis 168 is closely related to B. amyloliquefaciens FZB42, but does not promote plant growth. As a first approach to detect gene differentiation between FZB42 and B. subtilis 168, and since only the genome sequence of the latter was known at that point, Suppression Subtractive Hybridization (SSH) was employed. Thereby, several unique genes of B. amyloliquefaciens FZB42 could be identified. Meanwhile, our laboratory became engaged in a project aiming to define the complete genome sequence of B. amyloliquefaciens FZB42, in collaboration with the GenoMik Network in Göttingen. The major part of the work and the co-ordination of the whole process were performed by Xiao-Hua Chen and myself. B. amyloliquefaciens FZB42 possesses the srf, fen, pks1 (bae), bac and dhb operons, which are also shared by B. subtilis 168. In addition, the genome of B. amyloliquefaciens FZB42 contains the bmy gene clusters, which controls the synthesis of bacillomycin D. A further issue pursued in this work was to identify the regulatory pathways that govern the expression of bacillomycin D. Global regulators, such as DegU, DegQ and ComA, the alternative sigma factors, sigB and sigH, and a novel Rap protein were found to affect the transcriptional activation of the main promoter of the bmy operon identified in this work. In particular, DegU was shown to mediate its effects, after binding directly to two sites at the bmy promoter region. DegU was shown to play an additional role on bacillomycin D production, presumably a post-transcriptional one. Similarly, YczE, a membrane protein of unknown function, encoded adjacently to sfp proved to be essential for bacillomycin D production, but dispensable for the production of the rest peptide antibiotics produced by B. amyloliquefaciens FZB42. The effect was mediated at a post-transcriptional level and was independent of DegU

The Rhizobacterium Bacillus amyloliquefaciens subsp. plantarum NAU-B3 Contains a Large Inversion within the Central Portion of the Genome

Wu H, Qiao J, Blom J, et al. The Rhizobacterium Bacillus amyloliquefaciens subsp. plantarum NAU-B3 Contains a Large Inversion within the Central Portion of the Genome. Genome announcements. 2013;1(6):e00941-13.The genome of rhizobacterium Bacillus amyloliquefaciens subsp. plantarum strain NAU-B3 is 4,196,170 bp in size and harbors 4,001 genes. Nine giant gene clusters are dedicated to the nonribosomal synthesis of antimicrobial lipopeptides and polyketides. Remarkably, NAU_B3 contains a large inversion within the central portion of the genome

Randomly barcoded transposon mutant libraries for gut commensals II: Applying libraries for functional genetics.

The critical role of the intestinal microbiota in human health and disease is well recognized. Nevertheless, there are still large gaps in our understanding of the functions and mechanisms encoded in the genomes of most members of the gut microbiota. Genome-scale libraries of transposon mutants are a powerful tool to help us address this gap. Recent advances in barcoded transposon mutagenesis have dramatically lowered the cost of mutant fitness determination in hundreds of in vitro and in vivo experimental conditions. In an accompanying review, we discuss recent advances and caveats for the construction of pooled and arrayed barcoded transposon mutant libraries in human gut commensals. In this review, we discuss how these libraries can be used across a wide range of applications, the technical aspects involved, and expectations for such screens

Randomly barcoded transposon mutant libraries for gut commensals I: Strategies for efficient library construction.

Randomly barcoded transposon mutant libraries are powerful tools for studying gene function and organization, assessing gene essentiality and pathways, discovering potential therapeutic targets, and understanding the physiology of gut bacteria and their interactions with the host. However, construction of high-quality libraries with uniform representation can be challenging. In this review, we survey various strategies for barcoded library construction, including transposition systems, methods of transposon delivery, optimal library size, and transconjugant selection schemes. We discuss the advantages and limitations of each approach, as well as factors to consider when selecting a strategy. In addition, we highlight experimental and computational advances in arraying condensed libraries from mutant pools. We focus on examples of successful library construction in gut bacteria and their application to gene function studies and drug discovery. Given the need for understanding gene function and organization in gut bacteria, we provide a comprehensive guide for researchers to construct randomly barcoded transposon mutant libraries

Коло Марусі Чурай

In this article Marusya Churay*s (a character famous in story and song) life history is researched. On the basis of real events and historical facts the author tells about people who were related to the life of this personality

Induction of natural competence in Bacillus cereus ATCC14579

Natural competence is the ability of certain microbes to take up exogenous DNA from the environment and integrate it in their genome. Competence development has been described for a variety of bacteria, but has so far not been shown to occur in Bacillus cereus. However, orthologues of most proteins involved in natural DNA uptake in Bacillus subtilis could be identified in B. cereus. Here, we report that B. cereus ATCC14579 can become naturally competent. When expressing the B. subtilis ComK protein using an IPTG-inducible system in B. cereus ATCC14579, cells grown in minimal medium displayed natural competence, as either genomic DNA or plasmid DNA was shown to be taken up by the cells and integrated into the genome or stably maintained respectively. This work proves that a sufficient structural system for DNA uptake exists in B. cereus. Bacillus cereus can be employed as a model system to investigate the mechanism of DNA uptake in related bacteria such as Bacillus anthracis and Bacillus thuringiensis. Moreover, natural competence provides an important tool for biotechnology, as it will allow more efficient transformation of B. cereus and related organisms, e. g. to knockout genes in a high-throughput way.</p

Bioformulation of microbial biocontrol agents for a sustainable agriculture

The application of microbial based biopesticides has become a sustainable alternative to the use of chemicals to prevent yield losses due to plant pathogens. However, microbial based biopesticides are often unsuccessfully formulated and do not meet the demanding regulatory standards required by the agencies, which hinders their commercialization. Hence, an outline on the approaches to attain more effective formulations might be useful for the development of future more effective products. With this aim, this chapter reports the current state of biocontrol strategies and describes the principles of microbial biocontrol formulations. Emphasis is placed on techniques and tools available for the development and characterisation of microbial products. To provide glimpses on the possible formulations, the different existing additives, carriers, inoculation techniques and formulation types are exhaustively reviewed. Finally, requirements and principles for efficacy evaluation of plant protection products in the European Union are include

Diversity of Nonribosomal Peptide Synthetases Involved in the Biosynthesis of Lipopeptide Biosurfactants

Lipopeptide biosurfactants (LPBSs) consist of a hydrophobic fatty acid portion linked to a hydrophilic peptide chain in the molecule. With their complex and diverse structures, LPBSs exhibit various biological activities including surface activity as well as anti-cellular and anti-enzymatic activities. LPBSs are also involved in multi-cellular behaviors such as swarming motility and biofilm formation. Among the bacterial genera, Bacillus (Gram-positive) and Pseudomonas (Gram-negative) have received the most attention because they produce a wide range of effective LPBSs that are potentially useful for agricultural, chemical, food, and pharmaceutical industries. The biosynthetic mechanisms and gene regulation systems of LPBSs have been extensively analyzed over the last decade. LPBSs are generally synthesized in a ribosome-independent manner with megaenzymes called nonribosomal peptide synthetases (NRPSs). Production of active-form NRPSs requires not only transcriptional induction and translation but also post-translational modification and assemblage. The accumulated knowledge reveals the versatility and evolutionary lineage of the NRPSs system. This review provides an overview of the structural and functional diversity of LPBSs and their different biosynthetic mechanisms in Bacillus and Pseudomonas, including both typical and unique systems. Finally, successful genetic engineering of NRPSs for creating novel lipopeptides is also discussed

Expression of the Lantibiotic Mersacidin in Bacillus amyloliquefaciens FZB42

Lantibiotics are small peptide antibiotics that contain the characteristic thioether amino acids lanthionine and methyllanthionine. As ribosomally synthesized peptides, lantibiotics possess biosynthetic gene clusters which contain the structural gene (lanA) as well as the other genes which are involved in lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG). The lantibiotic mersacidin is produced by Bacillus sp. HIL Y-85,54728, which is not naturally competent

Differential Control of Yersinia pestis Biofilm Formation In Vitro and in the Flea Vector by Two c-di-GMP Diguanylate Cyclases

Yersinia pestis forms a biofilm in the foregut of its flea vector that promotes transmission by flea bite. As in many bacteria, biofilm formation in Y. pestis is controlled by intracellular levels of the bacterial second messenger c-di-GMP. Two Y. pestis diguanylate cyclase (DGC) enzymes, encoded by hmsT and y3730, and one phosphodiesterase (PDE), encoded by hmsP, have been shown to control biofilm production in vitro via their opposing c-di-GMP synthesis and degradation activities, respectively. In this study, we provide further evidence that hmsT, hmsP, and y3730 are the only three genes involved in c-di-GMP metabolism in Y. pestis and evaluated the two DGCs for their comparative roles in biofilm formation in vitro and in the flea vector. As with HmsT, the DGC activity of Y3730 depended on a catalytic GGDEF domain, but the relative contribution of the two enzymes to the biofilm phenotype was influenced strongly by the environmental niche. Deletion of y3730 had a very minor effect on in vitro biofilm formation, but resulted in greatly reduced biofilm formation in the flea. In contrast, the predominant effect of hmsT was on in vitro biofilm formation. DGC activity was also required for the Hms-independent autoaggregation phenotype of Y. pestis, but was not required for virulence in a mouse model of bubonic plague. Our results confirm that only one PDE (HmsP) and two DGCs (HmsT and Y3730) control c-di-GMP levels in Y. pestis, indicate that hmsT and y3730 are regulated post-transcriptionally to differentially control biofilm formation in vitro and in the flea vector, and identify a second c-di-GMP-regulated phenotype in Y. pestis