Clustering of Bacillus strains with potential plant growth promoting activity by searching specific genetic determinants

TORRES MANNO, M.; DAURELIO, L.D.; MENDEZ, M.B.; CHACÓN, G.; ORELLANO, E.G.; MAGNI, C.; ESPARIZ, M.

Bahía Blanca, Buenos Aires, Argentina

Congreso; VI Argentinian Conference on Bioinformatics and Computational Biology (CAB2C); 2015

Asociación Argentina de Bioinformática y Biología Computacional

Background Plant growth promoting rhizobacteria (PGPR) are benecial bacteria which have the ability to colonize the roots and either promote plant growth through direct stimulation or by biological control of plant diseases [1]. The PGPR of Bacillus spp are preferred because their long-term viability facilitates the development of commercial products [2]. At genomic level, several genes or pathways has been reported as involved in PGPR activities in Bacillus species [2, 3]. Recently, in our laboratory, a Bacillus strain of pumilus group (called Bacillus S9) has been sequenced and its capacity as PGPR observed by in planta assay. The aim of this study is to group the Bacillus S9 and other sequenced strains of pumilus group to find possible association to PGPR reported activity strains using the genomic information.Materials and method The Data Base of genes or pathways PGPR related was generated using Bacillus amyloliquefaciens FZB42 and Bacillus subtilis 168 who are model strains that contains most of PGPR pathways [2,3,4]. Genes and genome sequences were download from NCBI data base (http://www.ncbi.nlm.nih.gov). BLAST+ command line application [5] was used to align with tblastn application option. R custom developed scripts (The R Project for Statistical Computing [http://www.R-project.org]) were used to construct the list of hypothetical orthologues and to detect pathways and R´s pvclust package [6] to clustering the strains (distance manhattan, method average, bootstrap 10000).Result A total of 115 reference genes belonging to 19 pathways of B. amyloliquefaciens and B. subtilis were listed. The genomes of Bacillus S9 and 23 B. pumilus strains (INR7 and WP8 with PGPR activities) were obtained. The alignment (115 genes against 26 genomes) allow to detect 41 putative orthologues in at least one of the B. pumilus group. Such genes permit stipulate the presence or absence of 19 pathways. Considering this matrix of absence/presence of pathways the 24 genomes of B. pumilus were classified in 6 clusters, whereas strains of B. subtilis 168 and B. amyloliquefaciens FZB42 do not group generating a rooted tree (Figure 1). The strain S9 group with PGPR WP8 strain (Figure 1).Figure 1: Clustering of bacillus strains. Red numbers are the Approximately Unbiased (AU) p-value, green numbers are the Bootstrap Probability (BP) value and the grey number are the group number. Green arrows indicate the PGPR strains, yellow arrow indicate Baclillus S9. Conclusions It was possible to create a system for classifying strains of bacillus in possible PGPR. The systemcreated grouped Bacillus S9 with PGPR strains. The method will be enhanced by adding more genes in thedata base and more PGPR strains.References: 1. A. Kurmar, A. Prakash, B. N. Johri: Bacillus as PGPR in Crop Ecosystem. Bact Agrobiol Crop Ecosyst 2011:61{96.2. Chen XH, Koumoutsi A, Scholz R, Eisenreich A, Schneider K, Heinemeyer I, Morgenstern B, Voss B, Hess WR, Reva O, et al: Comparative analysis of the complete genome sequence of the plant growth - promoting bacterium Bacillus amyloliquefaciens FZB42. Nat Biotechnol 2007, 25:1007{1014.3. Gupta A, Gopal M, Thomas G V., Manikandan V, Gajewski J, Thomas G, Seshagiri S, Schuster SC, Rajesh P, Gupta R: Whole Genome Sequencing and Analysis of Plant Growth Promoting Bacteria Isolated from the Rhizosphere of Plantation Crops Coconut, Cocoa and Arecanut. PLoS One 2014, 9:e104259.4. Kloepper JW, Ryu C-M, Zhang S: Induced Systemic Resistance and Promotion of Plant Growth by Bacillus spp. Phytopathology 2004, 94:1259-1266.5. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol 1990, 215:403-410.6. Suzuki R, Shimodaira H: Pvclust: An R package for assessing the uncertainty in hierarchical clustering. Bioinformatics 2006, 22:1540-1542.