Phenotype test of metk modification:

Transcription

1

2 Figure S1 Phenotype test of metk modification: Strain with metk replaced by SAM transporter could grow on LB medium with SAM added, and could not grow on LB medium without SAM added. Beginning strain MG1655 could grow on LB with or without SAM. Strain with synonymous mutation in metk showed the same phenotype. Only intermediate strain A10 (yqgc::apr, galp::spc) could grow on medium with spectinomycin or apramycin.

3 A cada(wt) cada::apr ΔcadA Marker B Marker gntt(wt) gntt::spc gntt::gdha 2.4 k 1.9 k 0.9 k 3 k 1 k 0.5 k 2.8 k 2.5 k 1.1 k

4 Figure S2. PCR for cada deletion and gdha integration. A: MG1655 (wild-type cada), 1.9 kb; intermediate strain (cada::apr), 2.4 kb; final strain ( cada), 1.0 kb. B: MG1655 (wild-type gntt site), 1.1 kb; intermediate strain (spectinomycin-resistance gene integration), 2.5 kb; final strain (gdha integration), 2.8 kb.

5 Table S1: Oligonucleotide primers used in this study Primers Sequence (5 3 ) T3_IsceI AAGCGCGCAATTACCCTGTTATCCCTACCAGGCCTCTGCTGGCCAATTAACCC TCACTAA T7_IsceI CAGCGCGCATTACCCTGTTATCCCTAGCCAGCGCTTTGATGGTGTAATACGAC TCACTAT Apr-ISceI-F TCCCCCGGGGCTAGGGATAACAGGGTAATATTCCGGGGATCCGTCGACC Apr-ISceI-R TCCCCCGGGGCATTACCCTGTTATCCCTAGTGTAGGCTGGAGCTGCTTC mdr TTCCCGGCGATCCTCTGG mdf GCATGACGGCAAGTGGACG rharas(ncoi) ACCATGGGGCATGGCGAATTAATCT I-SceIdn(NcoI) TCCATGGTTATTATTTCAGGAAA I-SceIup ATGCATCAAAAAAACC Rhaov GGTTCATTACCTGGTTTTTTTGATGCATAATGTGATCCTGCTGAAT pred-f CGATTTAAATGGTTTAAACATTTATTATGACAACTTGACGGC pred-r CGATTTAAATGGTTTAAACGTAACTGTCAGACCAAGTTTACTC kandn CCAACCAATTAACCAATTCTGATTAG kanup CCTGCAGGGGGGGGGGGAAAGCCACGTTGTGTC IsceIdn_kd GGTTTGTGCCAATACCAGTAGAAACAGACGAAGAATCCATGGTTATTATTTCAG GAAAGT IsceIup_lac GAGCGGATAACAATTTCACACAGGAAACAGCATATGCATCAAAAAAACCAGGTA ATGAAC Placdn_Is GTTCATTACCTGGTTTTTTTGATGCATATGCTGTTTCCTGTGTGAAATTGTTATC CGCTC Placup_kd CTGTTCACCGTTACATATCAAAGGGAAAACTGTCCATACCCATGGAAGGCGAA GCGGCAT BLdn500 AACCCATGGGCATTAAGCGAAAGC BLup400 GCTGGTTGTCGTGATCGTAG BLup_ROL CCATCCGGCAGCAAATCAAATAATTAGTTAAGTCTCCTTGTCGATCACCCGCA BLdn400 AGAGAATTCGGTTTCTGGGCATTGGG BLdn_FOL TGCGGGTGATCGACAAGGAGACTTAACTAATTATTTGATTTGCTGCCGGATGG mdfpepd2 CGCTTCGGTTTTCGCGCCAGCCAGTTTACCCAGCGAATCCAGTTCCCGGCGA TCCTCTGG mdrpepd1 GATCGTGAAGCGGTTCCAGCTGGCTTTGAAACCTTCAAGTTGCATGACGGCA AGTGGACG cada-f ATGAAATCTGATATTTCCATTTCAG cada-r ATAGTGAGCGCCGATTTCACACAG ISceI-cadA-F GCTAGGGATAACAGGGTAATATGAAATCTGATATTTCCATTTCAG ISceI-cadA-R GCATTACCCTGTTATCCCTAATAGTGAGCGCCGATTTCACACAG Apr-ISceI-F0 TCCCCCGGGGCATTACCCTGTTATCCCTAATTCCGGGGATCCGTCGACC Apr-ISceI-R0 TCCCCCGGGGCTAGGGATAACAGGGTAATGTGTAGGCTGGAGCTGCTTC cada-v-f GCTGTGAGGGTGTTTTCATGTG Apr-V-R GTTGATGGCAAAGGTTCCCTATG

6 cada-v-r gntt-iscei-f1 gntt-iscei-r2 gntt-r3 gntt-f3 gdha-f gdha-r gntt-v-r gdha-m-r2 gdha-m-f2 gntt-v-f mdfgalp1 mdrgalp1 mdfyqgc1 mdryqgc2 metkup1k_spea metkdn1k_galp metkdn1.1k metkup1.1k metkmut_f metkmut_r metkr250 metkf1k metkupr metkf1.1k ATAAGATCAAGCAGACCACTGACG GCTAGGGATAACAGGGTAATGATGATCCGCTTCAAAATGAAC GCATTACCCTGTTATCCCTAGTACGGCAGCAATCGACCAGG CCCCCGCAAAATTTCAGCCGTTTATGAGTATTTAACGGATACCGGACCGGCGA GAATCACG GCAAGAAAAAAATGACCCAGGAAAGCAAATTAATAACGAGTTACGCTCGCGTG TTGAAAGG CTCGTTATTAATTTGCTTTCCTGG ATCCGTTAAATACTCATAAACGGC CCAACCGCAATAACCATCAGC GACCATCGCGACTGGCTTTGATTTC GAAATCAAAGCCAGTCGCGATGGTC GATCATGGGTTTTGGCGCAATG CACCTATCTTAATTCACAATAAAAAATAACCATATTGGACCCTTCCCGGCGATC CTCTGG TGTTTGACCGCCCCTGTTTTTTAGCGTCAGGCATGATGCCCGCATGACGGCAA GTGGACG GTGGCAACACGAAAGAAACGTCGTGTGCTTTTTATTTAACCCTTCCCGGCGATC CTCTGG ATTTTCTGCCATTGTGGGGTATAAAACGCGGCGCGCGGCCCGCATGACGGCA AGTGGACG ACACGCTAATGTGGCCCAGCTC GAGCTTACGACCCATTCTTG TTTGCGCCCGAGTTTAAAGGAGAGC TTTCACTAACTGCGCGAGATCGACG GACGCGATCCTCGAACAGGAC... CCGAAAGCG... CGCGTTGCA... TGCGAAACCTAC GTAGGTTTCGCATGC... AACGCGCGC... TTTCGGGTC... CTGTTCGAGGATCGCGTC GATAGCGCTCAGAACCGCACAGGAGTTAG AGCCCGGGCATGCAAAGTGCCTTCTGAACAACTG TTGCCATGGTTAATATCACCTAAAGAGAATTTGGTT AATCTAGATCTTTGGTCGTGAACATTTCCCGTG I-SceI sites are in bound font. Enzyme restriction sites are in italic font. Homologous arms sequences are underlined. Mutation sites are marked by...

7 Supplementary Methods Construction of the modular plasmids for donor sequences and templates A 1.5-kb NotI fragment containing the kanmx gene from pug6 (EUROSCARF) was cloned into pbluescript II KS (-) (Stratagene) at the NotI site, yielding pks-k. A 1.7-kb DNA fragment containing the kanmx gene was amplified from pks-k using the primer pair T3_IsceI/T7_IsceI and digested with BssHII. The resulting fragment was used to replace the 0.2-kb BssHII region containing the multiple cloning sites (MCS) of the cloing plasmids pbluescript II KS (-). The resulting ampicillin and kanamycin resistance plasmids were named pkski-1 and pkski-2, which differ in the direction of insertion. pkski-2 was then digested with NotI to excise kanmx and the backbone fragment of the plasmid was re-circularized by T4 DNA ligase, yielding pksi kb DNA fragments containing the apramycin or spectinomycin resistance genes were amplified from pij773 and pij778, using the primer pair Apr-ISceI-F / Apr-ISceI-R and T/A cloned into pmd18-t simple (Takara). The resulting plasmids were named pmdiai and pmdisi. Construction of the helper plasmids A 2-kb DNA fragment containing the rhab promoter was amplified from the E. coli W3110 genome and a 0.7-kb fragment containing the I-SceI encoding region was amplified puc19rp12 (Posfai at al, Nucleic Acids Res. 27 (22), ), using the primers pairs rharas(ncoi)/rhaov and I-SceIdn(NcoI)/ I-SceIup. The fragments were joined together by overlap PCR and cloned into pkd46 at the NcoI site. The end product was named predia, which is similar to the previously reported predi plasmid. The only distinctions are some differences in the I-SceI conding region. The ampicillin resistance gene of pkd46 and predia were replaced by a kanamycin resistance gene. PCR fragments obtained with the primer pair pred-f/predr were digested with PmeI and ligated with a 0.9-kb kanamycin resistance gene fragment from ppic3.5k (Invitrogen) (obtained by PCR with kandn/kanup), yielding pkd-k and predki. A 2-kb DNA fragment containing the trc promoter was amplified from ptrc99a and a 0.7-kb fragment containing the I-SceI encoding region was amplified from predia, with the primers pairs Placup_kd / Placdn_Is and IsceIup_lac / IsceIdn_kd. The fragments were joined by overlap PCR and cloned into pkd46 and predki at the NcoI site, yielding predtai and predtki. Disruption of the cada gene and marker regeneration To create a plasmid for the disruption of the cada gene, a 1.3-kb fragment of the cada gene was amplified from E. coli MG1655 genomic DNA using the primer pair cada-f/ cada-r, then subcloned into pmd18-t Simple (Takara) to create pmdcada.

8 The apramycin resistance gene contained on a 1.5-kb PCR fragment from pij773 (obtained using primers Apr-ISceI-F0/Apr-ISceI-R0) was flanked by two I-SceI recognition sites. It was inserted into pmdcada at the EcoRI site (by Klenow fill-in after EcoRI digestion), to generate pmd-cada-iscei-apr. A 2-kb PCR fragment was amplified from pmdcada-iscei-apr, using the primer pair cada-f/ cada-r. This fragment was used to transforme E. coli MG1655 carrying the helper plasmid predia by electroporation. Transformants were analyzed by colony PCR, and MG1655 ( cada::iscei-apra-iscei) was obtained. pmdcada was digested with EcoRI to evict a 1-kb fragment from the cada ORF, and the plasmid was re-circularized by T4 DNA ligase, yielding pmdcadal. A 0.7-kb DNA fragment containing the truncated cada gene was amplified from pmdcadal using the primer pair ISceI-cadA-F / ISceI-cadA-R and cloned into pbackzero-t (Takara). The resulting plasmid, pbackzero-iscei-cada has two I-SceI sites in tandem, flanking the truncated cada gene. The sequence of the plasmid was confirmed by I-SceI endonuclease analysis and sequencing. To regenerate the marker, donor plasmid pbackzero-iscei-cada was used to transform into MG1655 ( cada::iscei-apra-iscei) carrying the helper plasmid predia. Several resulting colonies were first incubated in LB liquid medium with 0.5% glucose. Forty microliters of overnight seed culture were inoculated in a test tube containing 4 ml LB medium containing 50µg/mL ampicillin and 10 mm L-arabinose and the test tube was incubated at 30 ºC and 200 rpm for ~2 h. L-rhamnose was added to the mixture to a final concentration of 20 mm and the test tube was incubated for another 6~8 h. Then L-rhamnose and L-arabinose were further added to concentrations of 10 mm and 5 mm (final concentration 30mM and 15mM, respectively), and the test tube was incubated overnight. The next day, the overnight culture was harvested, suspended in sterile water, diluted and spread on LB plates supplemented with 20mM L-rhamnose, 10mM L-arabinose and 50µg/mL ampicillin. Colonies appeared after overnight incubation at 30 ºC. Then the colonies were analyzed for apramycin sensitivity, and the final clones were confirmed by PCR with primer pair cada-v-f/cada-v-r. All procedures using predtai as helper plasmid were the same as described above for predia, except that L-rhamnose was substituted with Isopropyl β-d-1-thiogalactopyranoside (IPTG). Seamless deletion of the pepd gene Seamless deletion of the pepd gene in BL21 (DE3) was performed by a method similar to that for the disruption of the cada gene. A 1.6-kb PCR fragment was amplified from pmdiai, using the primers pair mdfpepd2/mdrpepd1. This fragment was introduced into BL21 (DE3) carrying the helper plasmid predtki by electroporation. The transformants were tested by colony PCR with the primer pair BLdn500/BLdn400. The apramycin resistance gene with I-SceI sites was inserted into the pepd gene and disrupted it. To construct the donor plasmid for seamless deletion of the pepd gene, DNA fragments containing the upstream and downsteam regions of the pepd gene were amplified from the BL21

9 (DE3) genome using the primer pairs BLup400/ BLup_ROL and BLdn400/ BLdn_FOL. The fragments were joined by over-lap PCR and cloned into pksi-1 at the EcoRI/SphI sites, yielding pksipepd0.7k. The donor plasmid pksipepd0.7k was introduced into BL21 (DE3) pepd::apr /predtki. Then, the deletion of the apramycin resistance gene was performed as descried above, with IPTG as the inducer and kanamycin to maintain the helper plasmid predtki. The final strains were analyzed by colony PCR with the primer pairs BLdn500/BLup400. Integration of the gdha gene into the gntt locus A 1.3-kb fragment of the gntt gene was amplified from MG1655 genomic DNA, using the primer pair gntt-iscei-f1/gntt-iscei-r2. Then this fragment was subcloned into pmd19-t Simple (Takara) to create pmd19-gntt. A 1.5-kb PCR fragment containing the spectinomycin resistance gene flanked by two I-SceI recognition sites from pmdisi, which was obtained using the primers mdf/mdr containing spectinomycin resistance gene flanked by two I-SceI recognition sites was blunt cloned into pmd19-gntt at the ClaI site (by Klenow fill-in after ClaI digestion), to yield pmd19-gntt-spc. A 2.5-kb PCR fragment was amplified from pmd19-gntt-spc, using the primers pair M13F / M13R. This fragment was introduced into MG1655(pREDTKI) by electroporation to yield MG1655gntT::spc(pREDTKI). A 1.6-kb gdha fragment was amplified from the MG1655 genome and insert into pmd19-gntt by Gibson Assembly using primers gdha-f / gdha-r and gntt-r3 / gntt-f3. The donor plasmid obtained was named pmd19-gntt-gdha. The donor plasmid pmd19-gntt-gdha was introduced into MG1655gntT::spc carrying predtki. Maker eviction and gdha gene integration were carried out as described above, using IPTG as the inducer. The final strains were tested by colony PCR with the primer pair gntt-v-r / gntt-v-f. Site-directed mutagenesis of the metk gene at the native locus Approximate 1.6-kb PCR fragments were amplified from pmdiai and pmdisi, using the primers pairs mdfyqgc1/mdryqgc2 and mdfgalp1 / mdrgalp1. The PCR fragments were introduced into MG1655(pKD46) by successive electroporation. The resulting strain, with IsceI-aprR-IsceI cassette inserted into the yqgc site and IsceI-spcR-IsceI cassette inserted into the galp site, was named A10. yqgc and galp are the non-essential genes located next to the essential gene metk. The helper plasmid pkd46 was cured via growth at 42 o C. A 2.5-kb PCR fragments, containing part of the spea gene, the full-length yqgb-yqgc-metk genes and part of the galp gene, was amplified from the MG1655 genome, using the primer pairs metkup1k_spea /metkdn1k_galp and cloned into pksi-1 at the EcoRI / SacI sites to obtain pksi-metk. Site-directed mutagenesis of the metk gene was performed using the primers pair metkmut_f/ metkmut_r, following the method of the QuikChange Site-Directed Mutagenesis Kit (Agilent) to obtain the donor plasmid pksi-metkmut. The donor plasmid pksi-metkmut and the helper plasmid predtki were co-transformed into A10. Maker eviction and native locus site-directed mutagenesis were carried out as described

10 above, using IPTG as the inducer. The final strains were tested by colonies PCR with primers metkup1.1k / metkdn1k. The PCR fragments were sequenced to confirm the mutation sites. Replacement of the metk gene with the Rickettsia SAM transporter gene A 1-kb fragment encoding the Rickettsia SAM transporter was synthesized with BspHI / HindIII sites added to the ends. The synthesized fragment was subcloned into plasmid puc57, yielding puc-samt. The metk gene in pksi-metk was replaced by the SAM transporter gene, via PCR with primer pair metkupr / metkf1.1k, digestion with NcoI and BglII, ligation with the 0.9-kb BspHI/BamHI fragment from puc-samt (BamHI site from puc57) containing the SAM transporter gene, yielding pksimetksamt. The process for replacing the metk gene with the Rickettsia SAM transporter gene were the same as for site-directed mutagenesis of the metk gene, with 1mM SAM added into LB medium. Clones were analyzed for SAM auxotrophy. The sequence of the metk locus was confirmed following PCR with primer pair metkup1.1k / metkdn1k. The PCR fragments were sequenced to confirm the gene replacement.

11 Protocols for this method: Note: The protocols are using bifunction(red + I-SceI) helper plasmid predtki (kan R ) and ampicillin resistance donor plasmid. When using other bifunction helper plasmid, the antibiotics should be used depending on the resistance of the helper plasmid. When using bifunction helper plasmid with rhab promoter, L-rhamnose (instead of IPTG) should be used for inducing I-SceI. Note: The cultivation time should be suitable for MG1655, W3110, BL21(DE3) and other derivative strains. For other strains, the time needs to be adjusted. Initial work: 1. Lambda-Red PCR targeting: integration of the spectinomycin or apramycin resistance gene(s) cassette flanked by I-SceI sites in or beside the modification site. Note: Both bifunctional (Red + I-SceI) helper plasmid or conventional Red helper plasmid (such as pkd46) could be used in this step. If conventional Red helper plasmid pkd46 is used, it should be subsequently cured. If the bifunctional helper plasmid is used, it could be either retained or cured. If the intermediate strain is expected to survive long-term preservation, curing the bifunctional helper plasmid is recommended. 2. Donor plasmid construction. Any method can be used to construct the donor plasmid, including traditional restriction cloning, site-specific mutagenesis, Gibson Assembly (Nature Methods 6 (5): ) or artificial whole gene synthesis, etc. Preparation: (1) Culture the intermediate strain overnight for competent cell preparation. (2) Extract donor plasmid (and bifuctional helper plasmid, if needed) Note: Donor plasmid and bifunctional plasmid should harbor different resistance markers. (3) Materials for preparation of competent cell (according to the corresponding protocol.) (4) Test tubes with 4mL LB liquid. (5) LB agar (6) Stock solutions: ampicillin, kanamycin, spectinomycin, apramycin, glucose, L-arabinose, L-rhamnose, IPTG. (7) Sterile water.

12 Step 1: 1.1 Inoculate the overnight seed culture into fresh LB medium containing kanamycin (depending on the resistance of the helper plasmid) and apramycin or spectinomycin (depending on the marker used for introducing the I-SceI site into genome). Prepare competent cells for electroporation or heat shock transformation. Prepare plates for the next step: LB plates containing glucose(5g/l), ampicillin, kanamycin and apramycin / spectinomycin. (antibiotics concentration: 50 or 100 mg/l) 1.2 Transform donor plasmid into intermediate strain with bifunctional helper plasmid or Co-transform donor plasmid and bifunctional helper plasmid into intermediate strain without bifunction helper plasmid Spread E. coli cells onto LB plates containing glucose, ampicillin, kanamycin and apramycin / spectinomycin

13 Step 2: 2.1 Inoculate several resulting colonies into test tubes containing 4 ml LB medium containing 0.5% glucose and kanamycin Cultivated at 30 ºC * 200 rpm for 6~8 h. (OD600>=1 ) 2.2 Inoculate 40 microliters of seed culture into a test tube containing 4 ml LB medium containing 10mM L-arabinose and 50µg/mL kanamycin (depending on the resistance of the helper plasmid). Cultivate under 30 ºC * 200 rpm for about 2 h. (OD600 about 0.1 ~ 0.5) Add IPTG to a final concentration of 20 mm. Cultivate 30 ºC * 200 rpm overnight. Note: The cultivation time should be suitable for MG1655, W3110, BL21(DE3) and other derivative strains. For other strains, the time needs to be adjusted.

14 Step 3: 3.1 Inoculate 40 microliters of culture (OD600>= 1) into another test tube containing 4 ml LB medium and 20 mm IPTG, 10mM L-arabinose and 50µg/mL kanamycin (depending on the resistance marker of the helper plasmid). Note: Alternatively, in this step and the following step, arabinose-free medium with only L-rhamnose or IPTG for inducing I-SceI and antibiotic for maintaining the helper plasmid could be used, to avoid the potential deleterious effects caused by the prolonged expression of the lambda Red functions. But a slightly decreased efficiency would be obtained. Cultivated the test tube was 30 ºC * 200 rpm for another 6~8h. (to OD600>= 0.5) Note: This step (6~8h cultivation) could be skipped, but it's recommended NOT to skip it. Skipping this step might result in a lower marker eviction rate. Prepare plates for the next step: Plate A: LB with 20mM L-rhamnose or IPTG, 10mM L-arabinose and 50µg/mL ampicillin or kanamycin (depending on the resistance of helper plasmid) Plate B : Plate A plus apramycin or spectinomycin (depending on the marker used for introducing the I-SceI site into the genome) For modifications in essential genes, two Plate B (B1 and B2) should be used. Plate B1: Plate A plus apramycin Plate B2: Plate A plus spectinomycin 3.2 Harvest the culture was ed, suspend in sterile water and dilute (10-1 to 10-4 ). Spread the diluted culture on Plate A and Plate B, respectively. (or Plate A, Plate B1 and Plate B2) Incubate 30 ºC overnight. Prepare plates for the next step: LB plates with kanamycin LB plates with ampicillin LB plates with apramycin or spectinomycin.

15 Step Expected result after overnight incubation: Colonies appear on plate A. Significantly fewer colonies appear on Plate B (or B1 and B2). Analyze colonies appear from plate A : (1) Ampicillin, apramycin / spectinomycin sensitive phenotype. Streak on 3 or 4 plates: LB plates with kanamycin, LB plates with ampicillin, LB plates with apramycin or spectinomycin. (2) Colony PCR. 4.2 Strains with the desired modification should have the following phenotype (1) spc S, apr S, amp s, and kan R and (2) Have the expected colony PCR bands

16 Follow-up work: Colony PCR reconfirm (if necessary) Sequencing the PCR fragments (if necessary) Cure the bifunction helper plasmid via 42 C growth or Retain the bifunctional helper plasmid for further modifications