Complete Genome Sequence of Industrial Biocontrol Strain Paenibacillus polymyxa HY96-2 and Further Analysis of Its Biocontrol Mechanism

Paenibacillus polymyxa (formerly known as Bacillus polymyxa) has been extensively studied for agricultural applications as a plant-growth-promoting rhizobacterium and is also an important biocontrol agent. Our team has developed the P. polymyxa strain HY96-2 from the tomato rhizosphere as the first microbial biopesticide based on P. polymyxa for controlling plant diseases around the world, leading to the commercialization of this microbial biopesticide in China. However, further research is essential for understanding its precise biocontrol mechanisms. In this paper, we report the complete genome sequence of HY96-2 and the results of a comparative genomic analysis between different P. polymyxa strains. The complete genome size of HY96-2 was found to be 5.75 Mb and 5207 coding sequences were predicted. HY96-2 was compared with seven other P. polymyxa strains for which complete genome sequences have been published, using phylogenetic tree, pan-genome, and nucleic acid co-linearity analysis. In addition, the genes and gene clusters involved in biofilm formation, antibiotic synthesis, and systemic resistance inducer production were compared between strain HY96-2 and two other strains, namely, SC2 and E681. The results revealed that all three of the P. polymyxa strains have the ability to control plant diseases via the mechanisms of colonization (biofilm formation), antagonism (antibiotic production), and induced resistance (systemic resistance inducer production). However, the variation of the corresponding genes or gene clusters between the three strains may lead to different antimicrobial spectra and biocontrol efficacies. Two possible pathways of biofilm formation in P. polymyxa were reported for the first time after searching the KEGG database. This study provides a scientific basis for the further optimization of the field applications and quality standards of industrial microbial biopesticides based on HY96-2. It may also serve as a reference for studying the differences in antimicrobial spectra and biocontrol capability between different biocontrol agents

Identification and classification of known and putative antimicrobial compounds produced by a wide variety of Bacillales species

BACKGROUND: Gram-positive bacteria of the Bacillales are important producers of antimicrobial compounds that might be utilized for medical, food or agricultural applications. Thanks to the wide availability of whole genome sequence data and the development of specific genome mining tools, novel antimicrobial compounds, either ribosomally- or non-ribosomally produced, of various Bacillales species can be predicted and classified. Here, we provide a classification scheme of known and putative antimicrobial compounds in the specific context of Bacillales species. RESULTS: We identify and describe known and putative bacteriocins, non-ribosomally synthesized peptides (NRPs), polyketides (PKs) and other antimicrobials from 328 whole-genome sequenced strains of 57 species of Bacillales by using web based genome-mining prediction tools. We provide a classification scheme for these bacteriocins, update the findings of NRPs and PKs and investigate their characteristics and suitability for biocontrol by describing per class their genetic organization and structure. Moreover, we highlight the potential of several known and novel antimicrobials from various species of Bacillales. CONCLUSIONS: Our extended classification of antimicrobial compounds demonstrates that Bacillales provide a rich source of novel antimicrobials that can now readily be tapped experimentally, since many new gene clusters are identified

Genomic analysis of Paenibacillus larvae and its relation to virulence

Paenibacillus larvae is a Gram-positive sporulating bacterium that causes American foulbrood (AFB). It is one of the most dangerous bacterial pathogens of the honeybee (Apis mellifera). P. larvae spores are highly infectious to bee larvae and resist physicochemical influences. P. larvae is subtyped using repPCR with ERIC primers (Enterobacterial Repetitive Integrance Consensus) into five genotypes (ERIC I-V), which possess different colony morphology, metabolism and especially virulence. There is a significant genetic variability among isolates of P. larvae, which may contribute to differences in virulence. P. larvae isolates used in this work were obtained from clinical cases of American foulbrood as well as from a debris collected from bee hives with no American foulbrood symptoms from all over the Czech Republic in cooperation with the Beekeeping Research Institute, s.r.o., Dol. The isolates were obtained from larvae and hive debris. Both virulet and avirulet strains were sequenced using the SMRT (single molecule real time) method on the Sequel platform (PacBio). This method is suitable for Whole Genome Sequencing (WGS), because it allows sequencing of long reads with high accuracy, eliminating the effect of a large number of repetitive sequences during the genome assembly. Furthermore,...Paenibacillus larvae je grampozitivní sporulující bakterie, která je původcem moru včelího plodu. Jedná se o jednoho z nejvýznamnějších bakteriálních patogenů včely medonosné (Apis mellifera). Spory P. larvae jsou vysoce infekční pro včelí larvu a odolávají fyzikálně-chemickým vlivům. P. larvae se subtypizuje za využití repPCR s ERIC primery (Enterobacterial Repetitive Integrance Consensus). Dosud bylo popsáno 5 genotypů ERIC I-V, které se liší morfologií kolonií, metabolismem a především virulencí. Rovněž je mezi jednotlivými izoláty P. larvae významná genetická variabilita, která se může podílet na rozdílech ve virulenci. Izoláty P. larvae použité v této práci byly získány z klinických případů moru včelího plodu z celé České republiky ve spolupráci s Výzkumným ústavem včelařským, s.r.o., Dol. Jednalo se o bakteriální kultury získané kultivací infikovaných larev a měli. Také byly použity izoláty z měli získané ze včelstev bez klinických příznaků moru včelího plodu. Metodou SMRT (Single Molecule Real Time) na platformě Sequel (PacBio) byly sekvenovány virulentní i pravděpodobně avirulentní izoláty. Použitá metoda je vhodná pro celogenomové sekvenování bakteriálních genomů, protože umožňuje sekvenování dlouhých úseků DNA s vysokou přesností. Tím je eliminován efekt velkého množství repetitivních...Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologiePřírodovědecká fakultaFaculty of Scienc

Antimicrobials of Bacillus species: mining and engineering

Bacillus sp. have been successfully used to suppress various bacterial and fungal pathogens. Due to the wide availability of whole genome sequence data and the development of genome mining tools, novel antimicrobials are being discovered and updated,;not only bacteriocins, but also NRPs and PKs. A new classification system of known and putative antimicrobial compounds of Bacillus by genome mining is presented in Chapter 2. Importantly, predicting, isolating and screening of Bacillus strains with antimicrobial activity from natural sources keeps showing surprises and underlines the wealth of antimicrobial power these organisms have. Chapter 3 describes the bacterium Bacillus sp. BH072, isolated from a honey sample, with antifungal activities against molds. In order to explain the antifungal activity, the peptides of the fermentation broth were isolated and identified by a combination of several purification methods, and their genes were identified and analyzed. Subsequently in Chapter 4, the genome of Bacillus sp. BH072 was completely sequenced and it was identified to be Bacillus amyloliquefaciens. BAGEL3 mining results showed that the whole mersacidin operon is present in the genome of BH072, while no mersacidin product was obtained by direct production. Considering that the nisin synthetic machinery with additional modification enzymes brings a new opportunity to produce a broader range of lanthipeptides with various modifications, Chapter 5 describes production of class I and II hybrid lantibiotics (nisin and mersacidin) using the nisin leader sequence and NisBC together with GdmD in Lactococcus lactis

Analysis of paenilarvin as putative virulence factor during pathogenesis.

<p>(A) At the age of 12 hours after egg hatching, honey bee larvae were infected with <i>P</i>. <i>larvae</i> DSM25430 wt and DSM25430 Δ<i>itu</i>. Daily mortality due to <i>P</i>. <i>larvae</i> infection was recorded and total mortality after 15 days was determined. Bars represent the percentage of exposed larvae that died from American Foulbrood after 15 days. Bars represent mean values ± SD of three independent exposure bioassays for each group and 30 larvae per group. No significant difference in total mortality (Student´s t-test; p-value = 0.89) was observed. (B) Daily mortality due to <i>P</i>. <i>larvae</i> infection was recorded and cumulative mortality per day was calculated. Curves represent mean cumulative mortality ± SD for each day of 3 replicates with 30 larvae each. Analysis of the data by two-way ANOVA did not reveal any significant difference between the two curves (p-value = 0.15).</p