dRNA-Seq Reveals Genomewide TSSs and Noncoding RNAs of Plant Beneficial Rhizobacterium Bacillus amyloliquefaciens FZB42

Abstract

Bacillus amyloliquefaciens subsp. plantarum FZB42 is a representative of Gram-positive plant-growth-promoting rhizobacteria (PGPR) that inhabit plant root environments. In order to better understand the molecular mechanisms of bacteria-plant symbiosis, we have systematically analyzed the primary transcriptome of strain FZB42 grown under rhizosphere-mimicking conditions using differential RNA sequencing (dRNA-seq). Our analysis revealed 4,877 transcription start sites for protein-coding genes, identified genes differentially expressed under different growth conditions, and corrected many previously mis-annotated genes. We also identified a large number of riboswitches and cis-encoded antisense RNAs, as well as trans-encoded small noncoding RNAs that may play important roles in the gene regulation of Bacillus. Overall, our analyses provided a landscape of Bacillus primary transcriptome and improved the knowledge of rhizobacteria-host interactions.

Fig 1. Growth conditions of Bacillus amyloliquefaciens FZB42.

(A) Schematic diagram of the dRNA-seq workflow of B. amyloliquefaciens FZB42. FZB42 was cultured in four media: i) 1CM medium; ii) 1CM medium supplemented with maize root exudates (+RE); iii) 1CM medium supplemented with soil extract (+SE); iv) 1CM medium supplemented with both maize root exudates and soil (+RS). (B) Growth curves of FZB42 growing in the four media. The cultures were collected for total RNA preparation at different time points (#6h & #9h) as indicated by the arrows. TEX: terminator exonuclease; ESP: early stationary phase; MSP: middle stationary phase.

Fig 2. General profile of the sequencing data.

Distribution of the mapped reads of various RNAs from each library varying in different culture conditions or terminator exonuclease treatment. 1CM: 1CM medium; +RE: 1CM medium supplemented with maize root exudates (RE); +SE: 1CM medium supplemented with soil extract (RE); +RS: 1CM medium supplemented with both maize root exudates and soil (RS); TEX: terminator exonuclease; ESP: early stationary phase; MSP: middle stationary phase. (B) Venn diagram showing overlaps among various TSS categories. The TSS were designated into the categories as defined in [12]. (C) Length distribution of 5’leader sequence in B. amyloliquefaciens FZB42. (D) Motif analysis among upstream of TSSs of B. amyloliquefaciens FZB42. Sequences upstream of TSSs were extracted (positions -1 to -50) and common sequence motifs were searched using MEME software. The coordinates give positions relative to TSSs.

Fig 3. A subset of operons antisense to known genes.

Two operons identified antisense to a known operon (A) or to known genes (B). cDNA reads in each library without (-TEX) or with (+TEX) terminator exonuclease treatment were mapped to B. amyloliquefaciens FZB42 chromosome and visualized by IGB 8.1. Vertical bars indicate cDNA recovery detected in each library and were adjusted to the same scale of 20. The genes indicated above the horizontal coordinates are located on the forward strand while the genes indicated below the horizontal coordinates are located on the reverse strand. Bacterial cultures were sampled for total RNA extraction at two time points (#6h&#9h) from four conditions: i) in 1CM medium; ii) 1CM medium supplemented with the maize root exudates (+RE); iii) 1CM medium supplemented with soil extract (+SE); iv) 1CM medium supplemented with both maize root exudates and soil extract (+RS). The time point #6h corresponded to early stationary phase while #9h corresponded to middle stationary phase.

Fig 4. Verification of sRNAs in B. amyloliquefaciens FZB42.

sRNAs identified by sequencing data were validated by Northern blot. One representative lane was displayed for each sRNA identified. The names of sRNAs and the molecular marker were indicated on the top and on the left respectively. For more details of each sRNA see S6 Fig.

Fig 5. Genomic Visualization of various RNAs identified.

The 1st ring from inside depicts genomic size maker; 2nd ring in black indicates DNA GC content; 3rd ring in purple and green indicates GC skew (+/-); 4th ring in grey indicates housekeeping rRNA and tRNAs; 5th and 6th ring indicates primary TSS (in red) and genes located on the leading strand (black arrows clockwise), 7th ring with oliver arrows indicates the re-annotated genes on the leading strand, including corrected CDS, leaderless mRNAs and proposed operons; 8th and 9th ring indicates primary TSS(in red) and genes (black arrows counterclockwise) located on the lagging strand; 10th ring with Olive green arrows indicates re-annotated genes on the lagging strand; 11th-12th ring show nucleotide conservation patterns with genomes of B. amyloliquefaciens DSM7 (NC_014551.1), B. amyloliquefaciens subsp. plantarum CAU B946 (NC_016784.1) and B. subtilis 168 (NC_000964.3), respectively; 13th ring in orange depicts all the cis-encoded RNAs annotated in this study, including riboswitches and all the antisense RNAs; 14th ring (the most outer-ring in red) depicts experimentally validated sRNAs in this study and their names are labeled.