Bacterial strains, growth conditions and mutant construction.
Bacterial strains and plasmids are listed in Supplementary Table S1 and Table S2. Nutrient Luria-Bertani medium (LB, containing 10 g/L Tryptone, 5 g/L Yeast Extract, 10 g/L NaCl) was used for general growth of B. subtilis. Supplements were added as required: erythromycin (ery, 5 µg/mL) and kanamycin (kan, 50 µg/mL). XynA and AmyM were overexpressed in B. subtilis strain BWB09 using plasmid pCS58 and pCS74, respectively. The empty plasmid pBW17 was used as control. For XynA and AmyM secretion profile measurements, 10 mL overnight culture were grown in LB liquid medium supplemented with 50 µg/mL kan in 100 mL flasks at 30°C and 210 rpm to prevent sporulation; the next morning, 1mL overnight was were quickly spin-down and the supernatant was removed and cell pellet was resuspend in 37°C pre-warmed LB and diluted into fresh and prewarmed LB liquid supplemented with 50 µg/mL kan to a start OD600 of 0.05, grown with 210 rpm shaking at 37°C and sampled at desired timepoint for follow-up enzymatic or protein experiments. We used 100mL flask for 10mL liquid culture and 250 mL flask for 25 mL culture to guarantee aerobic growth.
For B. subtilis DNA transformation, the Spizizen-plus and Spizizen-starvation media (SMM, containing 15 mM (NH4)2SO4, 80 mM K2HPO4, 44 mM KH2PO4, 3 mM tri-sodium citrate, 0.5% glucose, 6 mM MgSO4, 0.2 mg/mL tryptophan, 0.02% casamino acids, and 0.000 11% ferric ammonium citrate (NH4)5Fe(C6H4O7)2) were used and then the transformants were selected in LB-agar plate with antibiotic selection [47].
The single mutant strains ∆hrcA and ∆lonA were constructed by transformation of the chromosomal DNA from respective BKE library mutants [48] into competent BWB09 or BWB143 cells, selected via LB + Em agar plates and verified by PCR and sequencing the PCR products.
RNA extraction
RNA extraction was based on the methods described in [49, 50]. Briefly, 2 mL cells were collected from either the logarithmic growth phase (3 h) and stationary growth phase (6 h). Cell pellets were resuspended in 0.4 mL ice-cold growth medium and added to a screw cap tube containing 1.5 g glass beads (0.1 mm), 0.4 mL phenol/chloroform/isoamyl alcohol mixture (25:24:1, Carlroth) and 50 µl 10% SDS, vortexed to mix, and stored by flash freezing in liquid nitrogen. Cell disruption was achieved by bead beating (Precellys 24). After centrifugation, RNA in the upper aqueous phase was ethanol-precipitated, washed twice with 70% ice cold ethanol, dried and dissolved in water. DNA was removed by DNAseI (NEB) treatment. The RNA was then extracted by a second-round of P/C/I extraction, followed by ethanol precipitation and 70% ethanol washing, and dissolving in water.
RNA-seq and sequencing data analysis
Prior to the deep-sequencing, the RNA samples were treated with the MICROBExpress™ Bacterial mRNA Enrichment Kit (Thermo Fisher) to remove most of the 16S and 23S rRNA. Subsequently, the RNA-seq libraries were constructed using the NEBNext® Ultra™ II Directional RNA Library Prep Kit from Illumina® (New England Biolabs) using NEBNext® Multiplex Oligos for Illumina® (New England Biolabs), according to the manufacturer's protocol. Sequencing was performed on an Illumina NextSeg 550 System using NextSeq 500/550 High Output v2.5 kit (75-bp read length), and the raw data were processed using the web-based platform Galaxy (https://usegalaxy.org/). We aimed at a sequencing depth of 5–10 million reads/library [28]. Trimmomatic was used to trim the adaptor sequence and filter bad reads. The trimmed reads were aligned to the Bacillus reference genome (NC_000913) with Bowtie2. After mapping, aligned reads were counted by FeatureCount, referred to the BSU locus_tags. DESeq2 was used to determine differentially expressed features between samples. A customized Excel plugin, GINtool [23], was used to analyse the transcriptome data using prior knowledge on operons, functional categories and regulons.
Ribosome profiling
Ribosome profiling was based on the methods described in [16, 30] and a workflow diagram can be found in Figure S4. Briefly, 100 mL cells at the logarithmic growth phase (3 h) or stationary growth phase (6 h) were mixed with 0.4 mL 250 mM chloramphenicol (final concentration 1 mM) and 100 mL crushed ice made of 1mM chloramphenicol in PBS pH 7.4 and centrifuged for 5 min at 9000 xg at 4°C. Cell pellet was subjected to flash freeze in liquid nitrogen and stored at -80°C. The pellet was resuspended in 2 mL lysis buffer (100 mM NH4Cl, 10 mM MgCl2, 20 mM Tris pH 8.0, 0.4% Triton X-100, 0.1% NP-40, 5 mM CaCl2, 1 mM chloramphenicol) and pulverized cryogenically in a 25 mL stainless steel grinding jar with a 12 mm ball (Retsch) using the mixer mill MM400 (Retsch). The pulverization was achieved by 8 cycles of 2 min milling at a frequency of 20 1/s and 1 min cooling in liquid nitrogen between the cycles. The pulverized cells were thawed on ice and added with 50 µL DNase I (NEB) and 10 mL lysis buffer. The lysate was centrifuged for 10 min at 15,000 xg at 4°C. 1 mL of the clarified supernatant was used for total RNA isolation and 9 mL of the clarified supernatant was subjected to ultracentrifugation over a 8 mL sucrose cushion (20% sucrose, 100 mM NH4Cl, 10 mM MgCl2, 20 mM Tris pH 8.0, 0.5 mM EDTA, 0.4% Triton X-100, 0.1% NP-40, 1 mM chloramphenicol) to collect ribosome pellets. The ultracentrifugation was performed using OptiSeal polypropylene tubes (Beckman) in a Ti-60 rotor at 50,000 rpm and 4°C for 2 h. Ribosome pellets were resuspended in 200 µL resuspension buffer (100 mM NH4Cl, 10 mM MgCl2, 20 mM Tris pH 8.0) and the RNA concentration was measured using Nanodrop (Thermo Scientific). To generate monosomes from polysomes, 1 mg of ribosome RNA was digested with 8 µl micrococcal nuclease (NEB, 2000 U/ µL) at 37°C and after 30 min 8 µL 0.25 M EGTA was added to quench the reaction. The digested sample was subjected to ultracentrifugation over a sucrose gradient solution (10% − 50% sucrose in 100 mM NH4Cl, 10 mM MgCl2, 20 mM Tris pH 8.0, 2 mM DTT) to isolate monosomes from polysomes. The ultracentrifugation was performed using Open-Top thin-wall polypropylene tubes (Beckman) with 0.9 mL of 10–50% sucrose solutions (4.5 mL in total) from the bottom in an SWTi-55 rotor at 42,000 rpm and 4°C for 2.5 h. The 0.9 mL of the 30%, 40% and 50% sucrose fractions were collected and subjected to RNA extraction using P/C/I (Carlroth), followed by isopropanol precipitation and 70% ethanol washing, and resuspended in 8 µL nuclease free water. The RNA sample was separated by electrophoresis on a 15% TBE 7M Urea PAGE gel at 60 V for 20 min and then at 180 V for 1 h. The ribosome protected fragments (RPFs) of size between 22 nt and 34 nt were excised from the gel, purified and subjected to sequencing library construction using NEBNext® Small RNA Library Prep Set for Illumina (NEB) according to the manufacturer's instructions.
The total RNA from clarified lysate supernatant was purified using P/C/I method and the mRNA was enriched via the RNaseH-mediated rRNA depletion. The enriched mRNA was fragmented using Magnesium RNA Fragmentation Module (NEB) and subjected to electrophoresis separation. Similarly, the fragmented mRNAs of size between 22 nt and 34 nt were excised from the gel, purified and subjected to sequencing library construction, as vehicle control. Sequencing was also performed on an Illumina NextSeg 550 System using NextSeq 500/550 High Output v2.5 kit.
Ribosome profiling data analysis
The raw sequencing data were processed using the Galaxy platform and the RiboGalaxy platform (https://ribogalaxy.ucc.ie/). On the Galaxy platform, Cutadapt was used to remove 3’ adapter sequences and select reads of size between 18 and 32 nucleotide [51]. FastQC was used to check the read quality [52], the Bowtie2 was used to align reads to the B. subtilis genome sequence NC_000964.3 to generate SAM files [53], which link reads to their genomic position. The SAM files were then converted to BAM format using Filter SAM or BAM (MAPQ > = 3) [54]. The aligned reads were counted by FeatureCount [55]. We processed the BAM files on the RiboGalaxy platform for further analysis.
Xylanase and Amylase enzyme activity Assays
100 µl cells were taken from the culture transferred into a 1.5 mL Eppendorf tube and centrifuged at 20,000 RCF for 1 min at 4°C, and then 70 µl supernatant was transferred to a new tube and stored by flash freezing in liquid nitrogen and storage at -80°C. Xylanase enzyme activity in the supernatant was determined using the fluorescence-based assay EnzChek® Ultra Xylanase Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Amylase enzyme activity were tested using α-Amylase Assay Kit (Megazyme, Ceralpha Method #K-CERA) in a optimized and scaled-down system based on the manufacturer’s instructions. Briefly, 50 µL substrate was reacted with 50 µL AmyM samples at 25°C for 20 min in a 96-well plate, and then 100 µL Tris-Stop solution (20g/L Tris in pH 10.0) was added to stop the reaction. The absorbance (405 nm) was measured using a Multiskan FC microplate photometer (Thermo Fischer Scientific, #51119000). All supernatant samples were only thawed on ice-water prior to the test. The commercial Xylanase (Sigma, X2753) and α-Amylase from Bacillus subtilis (Sigma, 10069) were used for construction of standard curve for the detection of xylanase and amylase enzyme activity respectively.