Gene expression regulation in the plant growth promoting Bacillus atrophaeus UCMB-5137 stimulated by maize root exudates

Liberata Mwita, Wai Yin Chan, Theresa Pretorius, Sylvester L. Lyantagaye, Svitlana V. Lapa, Lilia V. Avdeeva, Oleg N. Reva
2016 Gene  
Despite successful use of Plant Growth Promoting Rhizobacteria (PGPR) in agriculture, little is known about specific mechanisms of gene regulation facilitating the effective communication between bacteria and plants during plant colonization. Active PGPR strain B. atrophaeus UCMB-5137 was studied in this research. RNA sequencing profiles were generated in experiments where root exudate stimulations were used to mimic interactions between bacteria and plants. It was found that the gene
more » ... in B. atrophaeus UCMB-5137 in response to the root exudate stimuli differed from the reported gene regulation at similar conditions in B. amyloliquefaciens FZB42, which was considered as a paradigm PGPR. This difference was explained by hypersensitivity of UCMB-5137 to the root exudate stimuli impelling it to a sessile root colonization behavior through the CcpA-CodY-AbrB regulation. It was found that the transcriptional factor DegU also could play an important role in gene regulations during plant colonization. A significant stress caused by the root exudates on in vitro cultivated B. atrophaeus UCMB-5137 was noticed and discussed. Multiple cases of conflicted gene regulations showed scantiness of our knowledge on the regulatory network in Bacillus. Some of these conflicted regulations could be explained by interference of non-coding RNA (ncRNA). Search through differential expressed intergenic regions revealed 49 putative loci of ncRNA regulated by the root exudate stimuli. Possible target mRNA were predicted and a general regulatory network of B. atrophaeus UCMB-5137 genome was designed. Highlights  Plant colonizing PGPR Bacillus responded differently to the root exudate stimuli;  In UCMB-5137 the CcpA-CodY-AbrB regulation caused fast cell immobilization;  DegU regulon is important for plant colonization behavior in PGPR Bacillus;  ncRNA involved in regulation of plant colonization were identified;  A comprehensive model of gene regulation in UCMB-5137 was developed; Promoting Rhizobacteria; (p)ppGpp, guanosine pentaphosphate; rRNA, ribosomal RNA. in Ukraine in 1989 and at first, based solely on the phenotype; it was identified as B. subtilis. In an array of bioassays, B. atrophaeus UCMB-5137 showed ability to protect plants and crops from bacterial and fungal phytopathogens, and to promote the plant growth (Lapa & Reva, 2005). Complete genome sequence of B. atrophaeus UCMB-5137 has been achieved recently (Chan et al., 2013). B. atrophaeus is a common soil inhabitant. Spores of B. atrophaeus have been used in biotechnology to control sterilization processes because of the resistance of the spores to extreme temperatures and chemical detergents (Pinzón-Arango et al., 2009) . Except for UCMB-5137, the strains of B. atrophaeus were not reported in the literature as active plant associated growth promoters or protectors, in contrast to the strains of the closely related species B. amyloliquefaciens and B. subtilis. This brought our interest to study this bioactive strain UCMB-5137 to contribute to the knowledge on the gene regulation in PGPR Bacillus. Current knowledge on this issue is biased towards the paradigm examples of B. amyloliquefaciens ssp. plantarum (Reva et al., 2004; Chen et al., 2007; Fan et al., 2011; 2013;. In various studies it was demonstrated that the maize root exudates were useful to mimic in vitro interactions between different plants and PGPR by initiating a range of biological responses within the bacteria (Mark et al., 2005; Broeckling et al., 2008; Fan et al., 2012; Kierul et al., 2015). It was therefore interesting to study further the gene expression regulation in B. atrophaeus UCMB-5137 under the standard laboratory conditions by simulation with the root exudates, to compare the gene expression profile with the published results for other PGPR Bacillus. Differential transcription regulation stimulated by the root exudates in B. atrophaeus UCMB-5137 and B. amyloliquefaciens FZB42 revealed substantial differences between these bacteria. To identify other transcription factors and genes specifically involved in root colonization by B. atrophaeus UCMB-5137, the obtained gene regulation profiles were superimposed over known regulatory networks in B. subtilis (Kohlstedt et al., 2014; Michna et al., 2015). Possible involvement of non-coding RNA (ncRNA) in gene regulations during plant colonization was studied. Materials and methods Root exudate preparation Root exudates were extracted from maize roots as it was described by Fan et al. (2012). Seeds of the maize breed 5Q-751BR were surface sterilized by treating the seeds with 70% ethanol for 3 min and then with 5% (v/v) sodium hypochlorite for 3 min followed by rinsing 5 times with sterile distilled water. The seeds were germinated at 28°C until the main root was at least 2 cm long before transferring into test tubes with sterilized water in a way that only the roots were submerged into water. The test tubes were kept in plant growth chamber (16-h light/8-h dark) at 24°C for 8 days. Water aliquots were collected daily from the third to eighth day and the tubes were refilled with the same amount of fresh sterilized water. Each collection was kept separate, and 100 µL from each sample were spread on the nutrient agar to check for contamination. Contaminated root exudate samples were discarded and the clean samples were pooled, freeze dried and stored at minus 20°C. The lyophilized exudates were weighted, dissolved in 100 µL of water and centrifuged. The supernatant was filter sterilized. Concentrations of the root exudates were adjusted to 0.25 g/L and the solutions were kept as the stock at minus 80°C. 3 Bacterial growth conditions and RNA preparation Bacillus atrophaeus UCMB-5137 was obtained from the Ukrainian collection of microorganisms at Danylo Bacterial cultures were inoculated from a frozen stock culture and incubated at 37°C overnight on solid Luria agar medium (MERCK). Colonies from the overnight culture were inoculated into 1C medium and cultivated at 24°C for 14 hours with shaking at 180 rpm. Composition of the 1C medium was the same as suggested by Fan et al. (2012): 0.7% w/v pancreatic digest of casein, 0.3% w/v papain digest of soya flour, 0.5% w/v NaCl and 0.1% glucose (all the mentioned reagents were purchased from MERCK). 1 ml aliquots of the overnight cultures were inoculated into conical flasks with 20 ml of fresh 1C medium in control, and supplemented with 0.25 mg/ml of the maize root exudates in treatment. Control and treatment cultures were grown at 24°C for 14 hours 30 min with shaking at 180 rpm to achieve the transition to stationary growth phase, which was controlled by the medium opacity of OD 600 ≈ 1.0 units. Growth curve analysis showed that the bacterial cultures reached the transition to stationary phase in average after 14 hr 30 min of cultivation at 24°C with shaking (OD 600 ≈ 1.0 units) at both treatment and control conditions. Bacterial cells were harvested for the total RNA extraction by mixing with 2 volumes of the ice cold killing buffer (20 mM Tris-HCl from BDH Laboratory, 5 mM MgCl 2 from MERCK and 20 mM NaN 3 from SIGMA; pH was adjusted to 7.5) (Völker et al., 1994). The mixture was centrifuged at 5,000 rpm for 3 minutes at room temperature. The final pellet was washed with 1 ml of the killing buffer and immediately frozen at minus 80°C until the RNA extraction. Six RNA samples (3 controls and 3 treatments) were obtained from three independent experiments. However, one sample of the treated bacteria did not pass the RNA quality control and was discarded. Total RNA extraction and sequencing Total RNA were isolated using ZR Fungal/Bacteria RNA Mini Prep kit from Zymo research Corp. according to the manufacturer's instruction. Concentration and quality of RNA samples were checked by NanoDrop. Ribolock Ribonuclease inhibitor (Thermo Scientific) was added to prevent RNA degradation. Paired-end RNA sequencing was performed on the MiSeq Illumina platform in Inqaba Biotech (Pretoria, South Africa, RNA-Seq datasets and the results of the statistical analysis by CLC Genomics Workbench 7 were deposited in NCBI GEO database under the accession number GSE68543. The RNA-Seq reads were trimmed from the adapter sequences and mapped against the predicted coding and noncoding loci of the reference genome sequence of B. atrophaeus UCMB-5137 (CP011802) using CLC Genomics Workbench 7.0.3 (currently this program is distributed by QIAGEN Estimated Degree of Gene Expression statistics approach (Magoc et al., 2013) was used to identify up-and down-regulated genes. Different cutoff values of the fold change and p-values were applied as explained below. Complete genome sequence of B. atrophaeus UCMB-5137
doi:10.1016/j.gene.2016.05.045 pmid:27259668 fatcat:hwivczylzfcgjom3ydqycjqtke