Analysis of the complete genome sequence of Brevibacterium frigoritolerans ZB201705 isolated from drought- and salt-stressed rhizosphere soil of maize
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To analyze the complete genome sequence of the Brevibacterium frigoritolerans ZB201705, a Brevibacterium strain was isolated from the maize rhizosphere in drought- and salt-stressed soil, and the activity of the strain under simulated drought and high salt conditions was assessed.
We used a combination of the PacBio RS and Illumina sequencing platforms to obtain the complete genome sequence of B. frigoritolerans ZB201705.
The genome consists of 5,475,560 bp in a linear chromosome with no gaps, 4,391 protein-coding sequences, 39 ribosomal RNAs, and 81 transfer RNAs. The genome analysis revealed many putative gene clusters involved in defense mechanisms. In addition, an activity analysis of the strain under high-salt and simulated drought conditions helped clarify its potential tolerance to these abiotic stresses.
Our data revealed the complete genome sequence of the new isolated strain, and showed that it produces many proteins involved in drought and salt stress responses, suggesting that B. frigoritolerans ZB201705 may be a potential factor to increase crop yield under abiotic stresses. The information provided here on the genome of B. frigoritolerans ZB201705 provides valuable insight into rhizobacteria-mediated plant salt and drought tolerance and rhizobacteria-based solutions for agriculture under abiotic stress.
KeywordsB. frigoritolerans Comparison Classification Abiotic stress Survival rates
The genus Brevibacterium was suggested by Breed (1953) based on Brevibacterium linens as the type species. Thereafter, many species of Brevibacterium were isolated and studied. The genus includes at least 49 recognized species according to (www.bacterio.net/brevibacterium.html) (Choi et al. 2018). Brevibacterium has been isolated from various environments such as food (e.g., fermented seafood [Choi et al. 2013] and cheese [Leclercq-Perlat et al. 2000]), humans (e.g., clinical specimens [Wauters et al. 2001] and the human body [Wauters et al. 2003]), and saline environments (e.g., saline soil [Tang et al. 2008], beach sediment [Lee 2006], marine environments [Lee 2008], salt-lake sediment [Guan et al. 2010]), and other environment such as soil of ginseng field (Jung et al. 2018).
Some Brevibacterium, as plant growth-promoting bacteria (PGPB), can improve plant growth under different environmental conditions. B. frigoritolerans strain Imbl 2.1 has potential for bioremediation of phorate both in liquid cultures and agricultural soils (Jariyal et al. 2015). Recent study indicated that Brevibacterium linens RS16 can confer salt tolerance to Oryza sativa (Chatterjee et al. 2018).
Maize is one of the most important foods, feed, and industrial crops worldwide, and is highly sensitive to drought and salt (Morari et al. 2015). To identify beneficial rhizobacteria that promote maize growth, we isolated a Brevibacterium strain, B. frigoritolerans ZB201705, from the maize rhizosphere in drought- and salt-stressed soil and analyzed its complete genome sequence. In addition, we assessed the activity of the strain under high-salt and simulated drought conditions. The information provided here will provide valuable insight into PGPB for agriculture under abiotic stress.
Materials and methods
Sample collection and bacteria isolation
Brevibacterium frigoritolerans ZB201705 was isolated from drought-stressed saline rhizosphere soil of maize in Bayan Nur, Inner Mongolia Autonomous Region, China (40° 13′–42° 28′, E 105° 12′–109° 53′). Soil samples were collected at a depth of 5–10 cm in the maize rhizosphere. Five points were chosen according to the “S” form five-spot sampling method (Lu et al. 2013). Then, 0.1 kg of soil was collected at each point and pooled into one composite soil sample.
To isolate bacteria, soil samples were placed in paper bags and stored at 4 °C. Next, 100 mL of sterile water and 0.01 kg of soil were transferred to a Waring blender. The sample was homogenized for 1 min and the supernatant was collected. The supernatant was centrifuged for 10 min at 5000×g and re-dissolved in 0.5 mL of sterile water. Samples diluted to 10−3, 10−4, and 10−5 were streaked onto soil extract agar plates containing 5 g/L of beef extract, 10 g/L of peptone, 5 g/L of NaCl, 100 g/L of mannitol, 15 g/L of agar, and 1 L of distilled water (pH 7.2). After 2 days of incubation at 30 °C, colonies of different morphologies and sizes were frozen in 15% glycerol and stored at −80 °C.
Identification of sequence similarity
To identify the colony strains, the bacteria were first identified by Gram staining. Then, a similarity search with the 16S rRNA gene nucleotide sequence (accession number MH490935) was performed using the online program EzBioCloud (Yoon et al. 2017) and two housekeeping genes (recA and atpD) were used to identify the strains. Finally, the average nucleotide identity (ANI) was calculated using JSpeciesWS (Richter et al. 2016) to identify the interspecific relationships among strains.
Genomic DNA preparation, genome sequencing, and assembly
For genomic DNA isolation, strains were inoculated into 50 mL of liquid medium and cultivated overnight at 30 °C in a shaker at 150 rpm. Genomic DNA was then extracted using a Rapid Bacterial Genomic DNA Isolation Kit (Sangon Biotech Shanghai Co., Ltd., Shanghai, China).
Whole-genome sequencing was performed using a combination of the PacBio RS and Illumina sequencing platforms. The Illumina PE library and PacBio Library (8–10 kb) were each constructed. First, 52 Mb of continuous long read (CLR) PacBio sequences were sequenced on the PacBio RS platform using an SMRT cell with a sequence N50 length of 12,035 bp, of which 47.2 Mb of sequences had lengths ≥ 5 kb. Then, a 400 bp Illumina sequencing library was constructed, and 2.9 Gb of paired-end sequences were sequenced on the Illumina HiSeq 2000 platform. The Illumina data were used to evaluate the complexity of the genome and were assembled using Velvet ver. 1.2.10 genome assembler with a k-mer length of 99 (Zerbino and Birney 2008).
The complete genome sequence was assembled using both the PacBio and Illumina reads. The assembly was initially produced using an in-house assembly solution, in which a de Bruijn-based assembly algorithm and CLR correction algorithm were integrated. The final circularization step was completed manually using Circos ver. 0.64 software (http://circos.ca/) (Krzywinski et al. 2009).
Prodigal software (Hyatt et al. 2010) was used to predict bacterial genes. Then, the protein sequences of the predicted genes were compared with the Clusters of Orthologous Groups (COG) protein database (http://www.ncbi.nlm.nih.gov/COG/) (Tatusov et al. 2003), and the corresponding COG annotation results were obtained. BLAST (blastx/blastp 2.2.24+) was used to compare the predicted genes with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/genes.html). Specific pathways involving the corresponding genes were obtained according to the KEGG Orthology number in the comparison results. Finally, the BLAST results were analyzed using Gene Ontology annotations (http://www.geneontology.org/) with Blast2go.
Identification of B. frigoritolerans ZB201705 abiotic stress responses
The obtained data were subjected to an analysis of variance using the general linear model software Agres and Agdata, and means were compared using the least significant difference test at a probability level ≤ 0.05.
Organism information and classification
Sequence similarity (%) with Brevibacterium frigoritolerans ZB201705
B. frigoritolerans FJAT-2396
B. psychrosaccharolyticus ATCC 23296
B. manliponensis JCM 15802
B. kochii strain BDGP4
B. novalis NBRC 102450
B. bataviensis LMG 21833
B. soli NBRC 102451
B. aquimaris TF-12
B. flexus KLBMP 4941
B. xiamenensis VV3
B. halotolerans ATCC 25096
B. subtilis subsp. inaquosorumDE111
Genome sequencing results
Genome features of Brevibacterium frigoritolerans ZB201705
% of total
Genome size (bp)
DNA coding (bp)
DNA G + C (bp)
Protein coding genes
Genes with function prediction
Activity of the strain under high salt and simulated drought conditions
Nucleotide sequence accession numbers
The complete genomic sequence and 16S rRNA gene sequence of B. frigoritolerans ZB201705 have been deposited in the GenBank database under Accessions CP030063 and MH490935.
Brevibacterium has been isolated from numerous environments, including saline environments such as saline soil, beach sediment, marine environments, and salt-lake sediment (Lee 2006; Lee 2008; Tang et al. 2008; Guan et al. 2010). However, few studies have referred specifically to B. frigoritolerans. One strain of B. frigoritolerans, Imbl 2.1, has shown potential for the bioremediation of phorate (Jariyal et al. 2015). Another strain, SMA23, may improve growth and nutrient uptake in wheat (Meena and Saharan 2017). As for plant growth-promoting bacteria (PGPB), free-living plant growth-promoting bacteria form symbiotic relationships with specific plants or bacterial endophytes that colonize some or part of the interior tissues of plants (Basu et al. 2017). Therefore, PGPB are generally used as inoculants for the biostimulation, biocontrol, and biofertilization of plants, and may improve plant growth under various environmental stress conditions (Numan et al. 2018).
The present study shows that B. frigoritolerans ZB201705 produces a large number of proteins involved in salt and drought stress responses, including DegS, DegU, CspB, and SOD. Anti-abiotic stress susceptibility tests revealed that this strain can tolerate up to 3 M d-sorbitol (~ 55%, w/v) and 5 M NaCl (~ 29%, w/v). These results suggest that B. frigoritolerans ZB201705 is a vital bacterium with potential environmental abiotic stress resistance applications, including the promotion of salt and drought tolerance in maize. The information provided here will provide valuable insight into PGPB for agriculture under abiotic stress.
This work was supported by the Beijing Academy of Agriculture and Forestry Sciences (Grant number KJCX20170404, KJCX20170203 and QNJJ201724) and the Beijing Association for Science and Technology.
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have no conflict of interest.
Research involving human participants and/or animals
This article does not contain any studies with human participants or animals performed by any of these authors.
Informed consent is not required in this work.
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