Discovery of a dual protease mechanism that promotes DNA damage checkpoint recovery

The DNA damage response is a signaling pathway found throughout biology. In many bacteria the DNA damage checkpoint is enforced by inducing expression of a small, membrane bound inhibitor that delays cell division providing time to repair damaged chromosomes. How cells promote checkpoint recovery after sensing successful repair is unknown. By using a high-throughput, forward genetic screen, we identified two unrelated proteases, YlbL and CtpA, that promote DNA damage checkpoint recovery in Bacillus subtilis. Deletion of both proteases leads to accumulation of the checkpoint protein YneA. We show that DNA damage sensitivity and increased cell elongation in protease mutants depends on yneA. Further, expression of YneA in protease mutants was sufficient to inhibit cell proliferation. Finally, we show that both proteases interact with YneA and that one of the two proteases, CtpA, directly cleaves YneA in vitro. With these results, we report the mechanism for DNA damage checkpoint recovery in bacteria that use membrane bound cell division inhibitors.


ylbK disruption results in a polar effect on ylbL
We noticed that ylbK, the gene upstream of ylbL, had a phenotype similar to ylbL in the Tn-seq experiments (Table S2 & S3). Therefore, we tested whether ylbKL functioned together in the DNA damage response. Deletion of ylbK resulted in sensitivity to MMC (Fig S3A). Ectopic expression of ylbK failed to complement the ΔylbK phenotype ( Fig S3A). Given that ylbK is upstream of ylbL we attempted to complement the ΔylbK phenotype using ylbL and found that sensitivity to MMC was rescued ( Fig S3A). Closer examination of the ylbKL locus revealed that a putative ribosome binding site (RBS) for ylbL translation was present within the 3′ end of ylbK ( Fig S3B). Thus, a second deletion of ylbK was made (ΔylbK-2) which included deletion of the codons for all but the first 3 and the last 14 amino acids, leaving the RBS for ylbL intact ( Fig   S3B). This deletion was not sensitive to MMC (Fig S3A). Western blotting revealed that the initial ΔylbK strain did not express YlbL, whereas ΔylbK-2 did (Fig S3C). We conclude that disruption of ylbK results in a polar effect on ylbL, indicating that YlbL functions independently of YlbK.
In order to better understand the prevalence of false positives in Tn-seq experiments we attempted to validate the MMC phenotypes of the forty genes with the lowest relative fitness values in the second growth period of the experiment. Intriguingly, we found that seven additional genes, queA, ylmG, lgt, ylmE, sdaAB, cymR, and ywrC, resulted in no sensitivity to MMC when deleted (Table 1). We also found that the genomic loci of queA, cymR, ylmG, ylmE, and sdaAB, were proximal to genes with validated phenotypes (Table 1). The other two genes, ywrC and lgt have less obvious explanations. For lgt it is possible that the polar effect is on the upstream gene hprK, which codes for the kinase HprK that phosphorylates Crh (1). Deletion of crh resulted in sensitivity to MMC (Table 1), but we did not detect hprK in our Tn-seq experiments. Finally, ywrC does not have a clear explanation. We could not identify validated mutant phenotypes or essential genes proximal to ywrC. It is possible that the transposon resulted in increased expression of neighboring genes that resulted in sensitivity to MMC, and that increased expression is not duplicated in the deletion mutant, though other explanations exist.
Taken together, our results underscore the importance of validating results from forward genetic screens.

Cell wall metabolism genes are sensitive to DNA damage
Our forward genetic screens identified several cell wall metabolism genes as being sensitive to DNA damaging agents, including walH, yycI, walJ, ponA, and brcC (Table 1 and Table S2). We validated that deletion mutants were indeed sensitive to MMC (Table 1). These genes have not previously been implicated in the DNA damage response, though it is possible that they function in regulating cell division. Specifically, the genes walH and yycI are negative regulators of the essential two-component system WalRK (2)(3)(4)(5). A recent publication provided evidence that the WalRK system interacts with components of the divisome (6). Further, a study of walJ found that WalJ likely coordinates cell division with DNA replication (7). As a result, it is tempting to speculate that WalRK and the associated WalHIJ represent one of the connections between DNA replication, the DNA damage response, cell wall metabolism, and cell division.

Extraction of PY79 chromosomal DNA
Cell pellets (10 mL OD 600 = 1 equivalent) from stationary phase cultures were re-suspended in lysis buffer (50 mM Tris, pH 8.0, 10 mM EDTA, pH 8.0, 1% (v/v) Triton X-100, 0.5 mg/mL RNase A, 1 mg/mL lysozyme) and incubated at 37°C for 30 minutes. Proteins were digested by addition of 40 μL 10 mg/mL proteinase K (dissolved in TE buffer plus 10% glycerol) and 30 μL of 10% SDS and incubated at 55°C for 30 minutes. 600 μL of PB buffer and 30% (v/v) isopropanol) were added, mixed well by pipetting and added directly to a silica spin column (Epoch life sciences) and centrifuged at 12,000 g for 1 minute at room temperature.
The column was washed with 500 μL PB buffer, then 750 μL PE buffer (10 mM Tris,pH 7.5,and 80% (v/v) ethanol), centrifuging as above to remove buffer. The column was dried by centrifugation as above. Chromosomal DNA was eluted by adding 100 μL ultra-pure water and centrifugation as above.

Purification of Himar1-C9 transposase
Himar1-C9 was purified as described previously (8). E. coli TB1 cells with plasmid pMalC9 (Strain PEB234) were struck out on LB + 100 μg/mL ampicillin and incubated at 37°C overnight. An overnight starter culture was grown in LB + 100 μg/mL ampicillin at 37°C. The starter culture was diluted 1:100 and incubated at 37°C until OD 600 = 0.5 and Himar1-C9 was induced by addition of IPTG to a final concentration of 0.3 mM. The culture was incubated for 2 hours at 37°C. Cells were collected via centrifugation: 5,000 g for 20 minutes at 4°C. Cell pellets were re-suspended in 20 mL ice-cold column buffer (CB; 20 mM Tris, pH 7.5, 200 mM NaCl, 1 mM EDTA, and 1x Roche protease inhibitors). Cells were lysed via French press, and the lysate was clarified via centrifugation: 18,000 rpm (Sorvall SS-34 rotor) for 30 minutes at 4°C. The lysate was loaded onto 1 mL amylose resin (NEB) pre-equilibrated with CB, and placed on a rotator at 4°C for 1 hour. Resin was collected via centrifugation: 3,000 g for 10 minutes at 4°C.
The supernatant was removed and the resin was washed with 4 volumes wash buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 1 mM EDTA, 2 mM DTT, and 10% (v/v) glycerol) five times by resuspending the resin, then collecting via centrifugation and aspirating the wash buffer. The column was eluted by adding 0.8 volume of elution buffer (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM EDTA, 2 mM DTT, 10% (v/v) glycerol, and 10 mM maltose) and incubating on ice for 5 minutes and agitating frequently. The protein was analyzed via SDS-PAGE and concentration was determined using a Bradford assay. The protein was aliquoted, frozen in liquid nitrogen and stored at -80°C.

Transposition reaction
A transposon insertion library was constructed in vitro as described with minor modifications (9). A 50 μL transposition was prepared: 3 μg chromosomal DNA, 1 μg PCR product mariner transposon (The mariner transposon was PCR amplified from pCJ41 using primers oPEB368/369 and Q5 DNA polymerase (NEB)), 10 μL 5x Buffer A (102.5 mM HEPES pH 7.9,47.5% glycerol,467.5 mM NaCl,47.5 mM MgCl 2 ,1.19 mg/mL BSA,9.5 mM DTT), 100 nM Himar1-C9. Reactions were incubated at 30 °C for 16 hours. DNA was precipitated via addition of 0.1 volume sodium acetate, pH 5.2 and 2.5 volume 100% ice-cold ethanol. DNA was collected via centrifugation: 16,000g for 20 minutes at 4°C. DNA pellet was washed with 5 volumes (relative to reaction volume) ice-cold 70% ethanol and pelleted again via centrifugation: 16,000g for 10 minutes at 4°C. The ethanol wash was aspirated and the DNA pellet was dried at room temperature. The transposon junctions were repaired by re-suspending the DNA pellet in the following 50 μL reaction: 1x T 4 DNA ligase buffer (Lucigen), 1 mg/mL BSA (NEB), 0.5 mM dNTPs, 1 mM ATP, 50 mM NaCl, 6 units T 4 DNA polymerase exo-(Lucigen), and 480 units T 4 DNA ligase (Lucigen), and incubating at room temperature for 2 hours. The reactions were mixed by pipetting every 30 minutes over the 2 hour incubation. The reactions were moved to 16°C for 16 hours, and then stored at 4°C until used to transform PY79.

Transformation of PY79
PY79 was struck out on LB agar and incubated at 37°C overnight. A single colony was used to inoculate a 2 mL LM culture (LB + 3 mM MgSO 4 ) in a 14 mL round bottom culture tube. The culture was incubated at 37°C on a rolling rack until OD 600 of about 1.5. Then, 40 μL of the LM culture was transferred to 1.2 mL pre-warmed MD media (1x PC buffer (107 g/L K 2 HPO4, 60 g/L KH 2 PO 4 , 11.8 g/L trisodium citrate dihydrate), 2% glucose, 50 μg/mL phenylalanine, 50 μg/mL tryptophan, 11 μg/mL ferric ammonium citrate, 2.5 mg/mL potassium aspartate, 3 mM MgSO 4 ) and incubated on a rolling rack at 37°C for 6 hours. To each 1.2 mL competent cell culture, 15 μL of the transposase reaction were added, and the cultures were incubated on a rolling rack at 37°C for an additional 1.5 hours. Transformations were plated on LB agar + 100 μg/mL spectinomycin (200 μL per 100 cm plate, and 124 plates in total), and incubated at 37°C overnight. The Library consisted of approximately 900,000 transformants, which were pooled in 1x S7 50 salts + 15% glycerol with a resulting OD 600 of approximately 37.0. The library was distributed into 1 mL aliquots, frozen in liquid nitrogen, and stored at -80°C.

Tn-seq experimental details
Tn-seq experiments were designed with multiple growth periods similar to a prior description (10). The experiment was performed using triplicate samples for each condition, which originated from three aliquots of the transposon insertion library. The experiment was initiated by thawing three aliquots of the transposon insertion library in a beaker of water at 37°C. Each aliquot of the thawed library was used to inoculate 50 mL starter cultures in 500 mL beakers. For the MMC experiment 270 μL were used to inoculate, and 500 μL were used for the MMS and phleomycin experiment. The three starter cultures were incubated with shaking (200 rpm) at 30°C until OD 600 of about 0.8. Starter cultures were used to inoculate paired 25 mL cultures in 250 mL flasks at an OD 600 = 0.05 for control or treatment. For the MMC experiment, MMC was added to a final concentration of 15 ng/mL and an equal volume of the vehicle in which MMC was dissolved (25% v/v DMSO) was added to the control flasks. For MMS, a final concentration of 50 μg/mL was used, and a final concentration of 25 ng/mL was used for phleomycin. An equal volume of water was used for the vehicle control in the MMS and phleomycin experiment. The paired cultures were incubated with shaking (200 rpm) at 30°C until OD 600 of about 1.5 (growth period 1). Then the cultures were back diluted into fresh control or treatment media at an OD 600 = 0.05, and incubated with shaking (200 rpm) at 30°C until OD 600 of about 1.5 (growth period 2).
The cultures were back diluted as above one more time and grown as above (growth period 3).
At all steps of the experiment three samples of OD 600 = 10 were saved as cell pellets, and each sample was serially diluted and plated for viable cells in triplicate to estimate the number of cells at the start and end of the growth periods (Table S1), which were used in the fitness calculations.

Tn-seq sequencing library construction
Sequencing libraries were prepared similar to previous reports (9,11), with some modifications in adaptor sequences to increase compatibility with standard Hi-seq reagents. Genomic DNA was extracted from each sample as described in "Extraction of PY79 genomic DNA." A 200 μL restriction digest using MmeI was assembled for each sample as follows: 6 μg gDNA, 1x Cutsmart buffer (NEB), 64 μM SAM (NEB), 12 units MmeI (NEB). Reactions were incubated at 37°C for 4 hours. CIP (20 units) was added and the reactions were incubated at 37°C for 1 hour.
MmeI was heat inactivated by incubating at 65°C for 30 minutes, and the digested genomic DNA was extracted by addition of 600 μL of PB buffer and binding to a silica spin-column. The column was washed with 500 μL PB, then 750 μL PE buffer, and then eluted with 65 μL ultrapure water. Adaptors (oPEB312/313) were annealed in 1x annealing buffer (10 mM Tris, pH 8.0, 100 mM NaCl, 10 μM EDTA) at a concentration of 25 mM each, by boiling at 100°C for 5 minutes, followed by transferring to a beaker of 100°C water which was allowed to cool slowly to room temperature. The adaptors were ligated to the digested genomic DNA in two 40 μL reactions, each containing 30 μL of the eluate from above, 1x T 4 DNA ligase buffer (NEB), 2.5 μM annealed adaptors, and 800 units T 4 DNA ligase (NEB). Reactions were left at room temperature for 30 minutes, and then transferred to 16°C for 16 hours. The resulting reactions were pooled for each sample, and the DNA was extracted via spin-column as above, but the PB wash was excluded, and the sample was eluted with 100 μL ultra-pure water.
The DNA was extracted by dissolving the gel slice in 400 μL QG buffer (5.5 M guanidine thiocyanate and 20 mM Tris, pH 6.6) by heating at 65°C, and then 250 μL of isopropanol were added and the mixture was added to a silica spin-column. The column was washed with PB and PE buffer as above, and eluted with 120 μL ultra-pure water. The eluate was submitted for sequencing using a Hi-seq 2500 instrument on high output mode with v3 (MMC experiment) or v4 reagents (MMS and phleomycin experiment).

Tn-seq data analysis
The 50 bp sequencing reads were trimmed to 43 bp using Fastx Trimmer, because the sequencing reaction went outside of the inverted repeat, and those seven base pairs could not be aligned to our reference database. The trimmed reads were aligned to a reference database in which every TA site found in the PY79 genome was placed adjacent to the transposon sequence (reference database fasta file and sequencing data accession number GSE109366) using bwa (12). The reads were imported into the R statistical software package RStudio (RStudio 13) for further analysis. Each transposon insertion site was provided a coverage value where each read was equal to a coverage of one. Each transposon insertion site was indexed to its position in the genome and the gene or intergenic region in which it resides. Fitness was calculated for each insertion for the control and the treatment using the equation: where N 0 and N f are the number of bacteria at the start and end of the growth period, respectively, and F 0 and F f are the transposon frequency in the population as measured by Illumina sequencing (insertion coverage divided by total reads in the sample), respectively (11; also see Fig 1C). The ratio of treatment to control was calculated and defined as the relative fitness (see Fig 1C). For each gene (or intergenic region), the insertions containing less than 10 reads were removed and any gene without at least 12 insertions from the combined triplicate data were also removed, thus requiring that each gene have at least 4 insertions in each replicate to be included in the analysis. Each gene's average relative fitness was then calculated by determining the mean relative fitness of all insertion sites within each gene. We trimmed insertions in our fitness calculation that were in the upper or lower five percent of the data for that gene, reasoning that not all insertions will be a true representation of a null allele. A t-test was used to determine if the gene relative fitness differed significantly from one. P-values were adjusted for multiple comparisons using the method of Benjamini and Hochberg (14).

Proteomics experimental details
Ms Bioworks processed samples as described below. Submitted samples were washed three times with PBS. The washed pellets were suspended in modified RIPA buffer (2% SDS, 150 mM NaCl, 50 mM Tris HCl pH 8) and lysed using mechanical disruption in a Next Advance Bullet Blender using 1.0mm silica beads, setting 8 for 3 minutes. The lysate was centrifuged at 10,000 g for 10 minutes. Protein concentrations were determined by Qubit fluorometry. 20 μg of each sample was processed by SDS-PAGE using a 10% Bis-Tris NuPAGE gel (Invitrogen) with the MES buffer system; the gel was electrophoresed approximately 5 cm. The mobility region was excised into 20 equal sized segments for further processing by in-gel digestion. In-gel digestion was performed on each submitted sample using a robot (ProGest, DigiLab) with the following protocol: 1) Washed with 25mM ammonium bicarbonate followed by acetonitrile . 2) Reduced with 10mM dithiothreitol at 60°C followed by alkylation with 50mM iodoacetamide at room temperature. 3) Digested with trypsin (Promega) at 37°C for 4h. 4) Quenched with formic acid and the supernatant was analyzed directly without further processing.
Each gel digest was analyzed by nano LC-MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive. Peptides were loaded on a trapping column and eluted over a 75μm analytical column at 350 nL/min; both columns were packed with Luna C18 resin
Cells were lysed via sonication and lysates were cleared via centrifugation: 18,000 rpm (Sorvall SS-34 rotor) for 45 minutes at 4°C. The supernatant was removed and incubated with Ni 2+ -NTAagarose (Qiagen) for 1 hour at 4°C. The lysate/bead slurry was loaded onto a gravity flow column and the beads were allowed to settle for 5-10 minutes. The lysate was collected as the flow-through. The column was washed with 50 column volumes wash buffer (50 mM potassium phosphate pH 8.0, 300 mM NaCl, 5% (v/v) glycerol, and 30 mM imidazole). YlbL was eluted from the column via digestion with 6xHis-Ulp1 in digestion buffer (50 mM potassium phosphate pH 8.0, 150 mM NaCl, 5% (v/v) glycerol, 1 mM DTT, and 10 mM imidazole) on a rotator at room temperature for 2 hours, yielding untagged YlbL (a.a. 36-341). The digestion buffer was collected as the flow-through and concentrated to approximately 5 mL using a 10 kDa Amicon centrifugal filter. YlbL was then loaded onto a HiLoad superdex 200-PG 16/60 column preequilibrated with SEC buffer (50 mM potassium phosphate pH 8.0, 150 mM NaCl, and 5% (v/v) glycerol) and the column was washed using SEC buffer at a flow rate of 1 mL/min. The peak fractions were pooled, glycerol was added to a final concentration of 20%, and concentrated using a 10 kDa Amicon centrifugal filter. Aliquots were frozen in liquid nitrogen, and stored at 80°C.
YlbL and YlbL-S234A were purified for in vitro assays as follows. 10xHis-Smt3-YlbL (a.a 36-341) and 10His-Smt3-YlbL-S234A (a.a. 36-341) were expressed from plasmids pPB157 and pPB181, respectively, as described above for antibody production. Cell pellets from a one liter culture were re-suspended in 30 mL lysis buffer (50 mM Tris pH 7.5, 250 mM NaCl, 10% sucrose, and 20 mM imidazole) and lysed via sonication. Cell lysates were clarified via centrifugation: 18,000 rpm (Sorvall SS-34 rotor) for 30 minutes at 4°C. Clarified lysates were applied to Ni 2+ -NTA-agarose pre-equilibrated with lysis buffer. The column was washed with 20 column volumes wash buffer (25 mM Tris pH 8.0, 100 mM NaCl, 30 mM imidazole, and 5% glycerol). The Ni 2+ column was eluted in four fractions of 1.2 column volumes using elution buffer (25 mM Tris pH 8.0, 100 mM NaCl, 10% glycerol, and 250 mM imidazole). Fractions 1-3 were pooled, DTT was added to 2.5 mM and 0.3 mg of Ulp1 was added. The elution was digested at 4°C overnight yielding untagged YlbL (a.a. 36-341) or untagged YlbL-S234A (a.a 36-341). The digest was desalted using a Zeba spin column into equilibration buffer (25 mM Tris pH 8.0, 50 mM NaCl, 5% glycerol, and 10 mM imidazole). The desalted digest was applied to a second column of Ni 2+ -NTA-agarose and the flow-through was collected. The column was washed with one column volume of equilibration buffer. The flow-through and wash fractions were pooled and concentrated using a 10 kDa Amicon centrifugal filter. The concentrated protein was loaded onto a Sephacryl S-200 size exclusion column pre-equilibrated with SEC buffer (25 mM Tris pH 7.5, 5% glycerol, and 25 mM NaCl) and eluted over 1 column volume with SEC buffer at a flow rate of 1 ml/min. Peak fractions were pooled, glycerol was added to 20% and the final protein was concentrated using a 10 kDa Amicon centrifugal filter. The concentrated protein was aliquoted, frozen in liquid nitrogen, and stored at -80°C.

YneA
10xHis-Smt3-YneA (a.a. 28-103) was expressed from pPB204 and harvested as described for YlbL above. Cell pellets were re-suspended in lysis buffer (50 mM Tris pH 7.5, 250 mM NaCl, 10% (w/v) sucrose, 20 mM imidazole). Cells were lysed via sonication and the lysate was clarified as described for YlbL above. The clarified lysate was incubated with Ni 2+ -NTA-agarose beads, pre-equilibrated with lysis buffer, and incubated on rotator at 4°C for 1 hour. The lysate/bead slurry was loaded into a gravity flow column and the beads were allowed to settle for mM NaCl, and 5% (v/v) glycerol) and washed at a flow rate of 2 mL/min. The peak fractions were pooled, and glycerol was added to 20%. The protein was loaded onto a HiTrap Q column (GE life sciences), pre-equilibrated with 7.5% Q-finish buffer (25 mM Tris pH 8.0, 5% (v/v) glycerol, and 1 M NaCl) and 92.5% Q-start buffer (25 mM Tris pH 8.0, and 5% (v/v) glycerol).
The column was washed with 5 column volumes 7.5% Q-finish buffer. The column was eluted with 30 column volumes over a linear gradient from 7.5% to 50% Q-finish buffer at a flow rate of 2 mL/min. Peak fractions were pooled, glycerol was added to a final concentration of 20%, and the protein was concentrated using a 3 kDa Amicon centrifugal filter. Aliquots were frozen in liquid nitrogen and stored at -80°C.
Glucose was added to 0.2% and expression was induced by addition of IPTG to 1 mM and incubating at 37°C for 45 minutes. Cells were harvested via centrifugation: 4,000 g for 20 minutes at 4°C. Cell pellets were re-suspended in 30 mL lysis buffer (50 mM Tris pH 7.5, 250 mM NaCl, 10% (w/v) sucrose) per one liter of culture and lysed via sonication. Lysates were clarified via centrifugation: 12,500 rpm (Sorvall SS-34 rotor) for 45 minutes at 4°C. Clarified lysates were mixed with pre-equilibrated 2.5 mL amylose resin (NEB) on a rotator at 4°C. The lysate/resin mixture was loaded into a gravity flow column and the resin was allowed to settle for 5-10 minutes. The lysate was allowed to flow over the packed resin. The column was washed with 20 column volumes of wash buffer (25 mM Tris pH 8.0, 100 mM NaCl, 10%(v/v) glycerol).
The column was eluted with 4 fractions of 3 mL each in elution buffer (25 mM Tris pH 8.0, 100 mM NaCl, 10%(v/v) glycerol, and 50 mM maltose). Fractions 1-3 were pooled, DTT was added to 2 mM, and digested with MBP-Ulp1 overnight at 4°C. The resulting digest, yielding untagged CtpA (or CtpA-S297A), was diluted two-fold with Q-start buffer (25 mM Tris, pH 8.0, 5% glycerol) to bring the NaCl concentration to 50 mM, and loaded onto a HiTrap Q-column pre-equilibrated with 5% Q-finish buffer (25 mM Tris, pH 8.0, 1 M NaCl, 5% glycerol). The column was washed with 5 column volumes 5% Q-finish buffer. The column was eluted with 25 column volumes over a linear gradient from 5 to 50% Q finish buffer. The peak fractions were pooled, concentrated using a 30 kDa Amicon centrifugal filter, and loaded onto a sephacryl 16/60 S-200 column pre-equilibrated with SEC buffer (25 mM Tris pH 7.5, 25 mM NaCl, 5% glycerol). The column was washed with SEC buffer at a flow rate of 1 mL/min. Peak fractions were pooled, glycerol was added to 20%, and concentrated using a 30 kDa Amicon centrifugal filter. Aliquots were frozen in liquid nitrogen, and stored at -80°C.

MBP-Ulp1
MBP-Ulp1 was expressed from pPB200 in E. coli BL21 DE3 cells. One liter LB + 50 μg/mL kanamycin cultures were inoculated with an overnight culture at 1:100. Cultures were grown at 37°C until OD 600 of about 0.5, glucose was added to 0.2%, and then protein expression was induced by addition of 0.25 mM IPTG. Cultures were incubated at 20°C overnight. Cells were pelleted via centrifugation: 4,000 g for 20 minutes at 4°C. Cell pellets were re-suspended in lysis buffer (50 mM Tris pH 7.5, 250 mM NaCl, 10% (w/v) sucrose, and 1 mM DTT), and lysed via sonication. Lysates were cleared via centrifugation and MBP-Ulp1 was purified on an amylose column as described for CtpA above. Peak fractions from the amylose column were loaded onto a sephacryl S200 26/60 column, pre-equilibrated with SEC buffer (25 mM Tris pH 7.5, 200 mM NaCl, 2% glycerol). The column was washed with SEC buffer at a flow rate of 2 mL/min. Peak fractions were pooled, glycerol was added to 20%, and DTT was added to 1 mM. MBP-Ulp1 was concentrated to about 1.7 mg/mL, aliquots were frozen in liquid nitrogen and stored at -80°C.

General strain construction methods
Generation of competent B. subtilis cultures for generating new genotypes was performed as described below in "Transposon insertion mutant library construction," or as previously reported (16).
subtilis strains were transformed with the indicated plasmid (prepared from E. coli MC1061), plated on LB agar + 100 μg/mL spectinomycin and incubated at 30°C overnight. Isolates were colony purified by restreaking on LB agar + 100 μg/mL spectinomycin and incubating at 30°C overnight. The editing plasmid was evicted by restreaking isolates on LB agar and incubating at 45°C for 8-12 hours or overnight. Loss of the editing plasmid was verified by restreaking LB agar + 100 μg/mL spectinomycin and on LB agar. Isolates that were unable to grow in the presence of spectinomycin were used for PCR genotyping.
Gene deletions using the B. subtilis knockout library were performed as described (18).
Chromosomal DNA, extracted as described below (see extraction of PY79 chromosomal DNA), was used to transform PY79 or the indicated strain. Incorporation of the erm cassette at the appropriate locus was verified via PCR genotyping. Removal of the erm cassette was performed following transformation with pDR244, which contains cre recombinase. Eviction of pDR244 was performed as described for a CRISPR/Cas9 genome editing plasmid. Loss of the erm cassette was verified by sensitivity to erythromycin and PCR genotyping.
Integration of inducible constructs at the amyE locus was achieved via double crossover recombination. For constructs containing a xylose inducible promoter (P xyl ), strains were transformed with plasmids that had been digested with two unique restriction enzymes (KpnI-HF and ScaI-HF) or with genomic DNA of a strain already generated (see detailed strain construction) and transformants were selected using LB agar + 5 μg/mL chloramphenicol.
Incorporation via double cross-over at amyE was determined by screening for an inability to utilize starch and by testing for the absence of a spectinomycin resistance cassette that is present on part of the plasmid that is not integrated. For constructs containing an IPTG inducible promoter (P hyp ), strains were transformed with plasmids that had been digested with two unique restriction enzymes (SpeI and SacI-HF) and transformants were selected using LB agar + 100 μg/mL spectinomycin. Isolates were colony purified by restreaking on LB agar + 100 μg/mL spectinomycin. Incorporation via double cross-over at amyE was determined by screening for an inability to utilize starch.

General cloning techniques
Plasmids were assembled using Gibson assembly (19). Gibson  Plasmids that were intermediate products of editing plasmids containing only the targeting spacer were generated by ligation of the proto-spacer into pPB41 as described previously (16,17). Briefly, pPB41 was digested with BsaI-HF (NEB), and then treated with CIP (NEB). The digestion product was purified by gel extraction from an agarose gel. Proto-spacers were annealed in 1x annealing buffer (10 mM Tris, pH 8.0, 100 mM NaCl, 10 μM EDTA) at a concentration of 10 μM each by incubation in a 100°C heat block for 5 minutes, followed by transferring to a beaker of water pre-heated to 100°C. The annealing reactions were allowed to cool slowly to room temperature in the beaker of water. The annealed proto-spacers were phosphorylated using T 4 PNK (NEB). The phosphorylated proto-spacers were ligated to digested pPB41 using T 4 DNA ligase (NEB). The resulting ligations were used to transform Top10 or MC1061 E. coli. Plasmids sequences were verified by Sanger sequencing using oPEB253.

Individual plasmid construction
pPB44: A proto-spacer targeting recR (oPEB241/242) was ligated to pPB41. pPB103: The upstream and downstream portions of the ΔsodA editing template were PCR amplified using oPEB506/507 and oPEB508/509, respectively. CRISPR/Cas9 was PCR amplified using oPEB232/234 and pPB81 as the template. The pPB41 vector was PCR amplified using oPEB217/218. These four PCR products were used in a Gibson assembly reaction to generate a ΔsodA editing plasmid. Clones were verified by Sanger sequencing with oPEB227, oPEB253, and oPEB512.
pPB107: The upstream portion of amyE and the P xyl promoter were PCR amplified using oPEB370/383. The chloramphenicol resistance cassette and the downstream portion of amyE were amplified using oPEB557/377. pPB47 was PCR amplified using oPEB116/117. The open reading frame (ORF) of ylbK was PCR amplified using oPEB558/559. These four PCR products were used in a Gibson assembly reaction to generate a plasmid to integrate ylbK under the control of P xyl at the amyE locus. Clones were verified by Sanger sequencing with oPEB345 and oPEB348.
pPB108: The upstream portion of amyE and the P xyl promoter were PCR amplified using oPEB370/383. The chloramphenicol resistance cassette and the downstream portion of amyE were amplified using oPEB557/377. pPB47 was PCR amplified using oPEB116/117. The ORF of ylbL was PCR amplified using oPEB560/561. These four PCR products were used in a Gibson assembly reaction to generate a plasmid to integrate ylbL under the control of P xyl at the amyE locus. Clones were verified by Sanger sequencing with oPEB345 and oPEB348. pPB111: The upstream portion of amyE and the P xyl promoter were PCR amplified using oPEB370/383. The chloramphenicol resistance cassette and the downstream portion of amyE were amplified using oPEB557/377. Plasmid pPB47 was PCR amplified using oPEB116/117.
The upstream portion of the ylbL ORF containing the S234A mutation was PCR amplified using oPEB560/567. The downstream portion of the ylbL ORF containing the S234A mutation was PCR amplified using oPEB566/561. These five PCR products were used in a Gibson assembly reaction to generate a plasmid to integrate ylbL-S234A under the control of P xyl at the amyE locus. Clones were verified by Sanger sequencing with oPEB345 and oPEB348.
pPB118: A proto-spacer targeting ylmG (oPEB618/619) was ligated to pPB41. pPB120: The upstream and downstream portions of the ΔqueA editing template were PCR amplified using oPEB579/580 and oPEB581/582, respectively. CRISPR/Cas9 was PCR amplified using oPEB232/234 and pPB113 as the template. The pPB41 vector was PCR amplified using oPEB217/218. These four PCR products were used in a Gibson assembly reaction to generate a ΔqueA editing plasmid. Clones were verified by Sanger sequencing with oPEB227, oPEB253, and oPEB585. pPB121: The upstream and downstream portions of the ΔctpA editing template were PCR amplified using oPEB588/589 and oPEB590/591, respectively. CRISPR/Cas9 was PCR amplified using oPEB232/234 and pPB114 as the template. The pPB41 vector was PCR amplified using oPEB217/218. These four PCR products were used in a Gibson assembly reaction to generate a ΔctpA editing plasmid. Clones were verified by Sanger sequencing with oPEB227, oPEB253, and oPEB594.
pPB122: The upstream and downstream portions of the ΔysoA editing template were PCR amplified using oPEB597/598 and oPEB599/600, respectively. CRISPR/Cas9 was PCR amplified using oPEB232/234 and pPB115 as the template. The pPB41 vector was PCR amplified using oPEB217/218. These four PCR products were used in a Gibson assembly reaction to generate a ΔysoA editing plasmid. Clones were verified by Sanger sequencing with oPEB227, oPEB253, and oPEB603. by Sanger sequencing using oPEB527, oPEB58, oPEB833, and oPEB837.
pPB204: The ORF coding for a.a. 28-103 of YneA was PCR amplified using oPEB842/843. The plasmid pPB12 was PCR amplified using oPEB56/57. These two PCR products were used in a Gibson assembly to generate a plasmid for overexpression of a 10xHis-Smt3-YneA(28-103) fusion protein in E. coli. Clones were verified via Sanger sequencing using oPEB527 and oPEB58.
pPB214: The pET28b-MBP-Smt3 vector was PCR amplified using oPEB56/57 using pPB203 as a template. The ctpA-S297A ORF (coding for a.a. 38-466) was PCR amplified using oPEB831/832 using pPB185 as a template. These two PCR products were used in a Gibson assembly reaction to generate a plasmid for overexpression of the fusion protein MBP-Smt3-CtpA-S297A (38-466) in E. coli. Clones were verified by Sanger sequencing using oPEB527, oPEB58, and oPEB833.
pPB237: The upstream and downstream portions of the ΔfhuG editing template were PCR amplified using oPEB914/915 and oPEB916/917, respectively. CRISPR/Cas9 was PCR amplified using oPEB232/234 and pPB227 as the template. The pPB41 vector was PCR amplified using oPEB217/218. These four PCR products were used in a Gibson assembly reaction to generate a ΔfhuG editing plasmid. Clones were verified by Sanger sequencing with oPEB227, oPEB253, and oPEB920. pPB267: YneA was amplified with primers oPEB1034/1035 and the plasmid pUT18C was amplified using primers oPEB1017/1018. These two PCR products were used in a Gibson