Table S1: Strains, Plasmids and Primers

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pBAD30 plasmid with GFP and mCherry tandem reporter with their respective Shine-Dalgarno sequences. This work. pBAD30mw721. pBAD30mw700 with PPP ...
Table S1: Strains, Plasmids and Primers Strain

Genotype or relevant characteristics

Reference

BW25113 MW1014 MW1700 MW1721 MW1723 MW17231 MW17232 MW17233 MW1751 MW1722 MW1731 MW1732 MW1741 MW1753 MW14700 MW14721 MW14723 MW147231 MW147232 MW147233 MW14751 MW14722 MW14731 MW14732 MW14741 MW14753

“wild type” E. coli, parental strain used for the Keio collection. Δefp of Keio collection with kanamycin cassette removed using FLP recombinase. BW25113 with pBAD30mw700. BW25113 with pBAD30mw721 BW25113 with pBAD30mw723 BW25113 with pBAD30mw7231 BW25113 with pBAD30mw7232 BW25113 with pBAD30mw7233 BW25113 with pBAD30mw751 BW25113 with pBAD30mw722 BW25113 with pBAD30mw731 BW25113 with pBAD30mw732 BW25113 with pBAD30mw741 BW25113 with pBAD30mw753 MW1014 with pBAD30mw700 MW1014 with pBAD30mw721 MW1014 with pBAD30mw723 MW1014 with pBAD30mw7231 MW1014 with pBAD30mw7232 MW1014 with pBAD30mw7233 MW1014 with pBAD30mw751 MW1014 with pBAD30mw722 MW1014 with pBAD30mw731 MW1014 with pBAD30mw732 MW1014 with pBAD30mw741 MW1014 with pBAD30mw753

WN1269

ΔargH ΔlysA (Arg Lys Auxotroph) of Salmonella enteric serovar Typhimurium strain SL1344

WN1308 14028s WN1405

WN1269 Δefp (mutation as in Zou, 2012) “wild type” Salmonella enterica serovar Typhimurium strain 14028s 14028s Δefp (kanamycin cassette removed using FLP recombinase)

WN355

14028s ΔpoxA::Km

WN1336

14028s ΔyjeK::Km

WN1421

14028s ΔyfcM::Km

WN1677 WN1617 WN1619 WN1621 WN1623 WN1625 WN1627 WN1629 WN1631 WN1633 WN1635 WN1637 WN1639 WN1653 WN1655 WN1657 WN1659 WN1661 WN1663 WN1665 WN1667 WN1669 WN1671 WN1673 WN1675 WN1700

14028s with pXG10sf-LacZ186 14028s with pXG10sf-PoxB FL 14028s with pXG10sf-PoxB 499 14028s with pXG10sf-PoxB 397 14028s with pXG10sf-PoxB 325 14028s with pXG10sf-PoxB 247 14028s with pXG10sf-PoxB 171 14028s with pXG10sf-PoxB 97 14028s with pXG10sf-PoxB 82 14028s with pXG10sf-PoxB 68 14028s with pXG10sf-PoxB 52 14028s with pXG10sf-PoxB 36 14028s with pXG10sf-PoxB 21 14028s with pXG10sf-PoxB E66L 14028s with pXG10sf-PoxB L67A 14028s with pXG10sf-PoxB C70L 14028s with pXG10sf-PoxB A71L 14028s with pXG10sf-PoxB G72L 14028s with pXG10sf-PoxB S73L 14028s with pXG10sf-PoxB C74L 14028s with pXG10sf-PoxB G75L 14028s with pXG10sf-PoxB P76L 14028s with pXG10sf-PoxB G77L 14028s with pXG10sf-PoxB N78L 14028s with pXG10sf-PoxB H80L 14028s with pXG10sf-PoxB Shifted

Baba, 2006 This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work Auweter, 2011 (1) This work ATCC This work Navarre, 2010 (4) This work Bullwinkle, 2013 (2) This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work

WN1694 WN1696 WN1698 WN1678 WN1618 WN1620 WN1622 WN1624 WN1626 WN1628 WN1630 WN1632 WN1634 WN1636 WN1638 WN1640 WN1654 WN1656 WN1658 WN1660 WN1662 WN1664 WN1666 WN1668 WN1670 WN1672 WN1674 WN1676 WN1701 WN1695 WN1697 WN1699 WN1682 WN1685 WN1688 WN1683 WN1686 WN1689 WN1681 WN1684 WN1687

14028s with pXG10sf-PoxB P76::CCA 14028s with pXG10sf-PoxB P76::CCC 14028s with pXG10sf-PoxB P76::CCT WN1405 with pXG10sf-LacZ186 WN1405 with pXG10sf-PoxB FL WN1405 with pXG10sf-PoxB 499 WN1405 with pXG10sf-PoxB 397 WN1405 with pXG10sf-PoxB 325 WN1405 with pXG10sf-PoxB 247 WN1405 with pXG10sf-PoxB 171 WN1405 with pXG10sf-PoxB 97 WN1405 with pXG10sf-PoxB 82 WN1405 with pXG10sf-PoxB 68 WN1405 with pXG10sf-PoxB 52 WN1405 with pXG10sf-PoxB 36 WN1405 with pXG10sf-PoxB 21 WN1405 with pXG10sf-PoxB E66L WN1405 with pXG10sf-PoxB L67A WN1405 with pXG10sf-PoxB C70L WN1405 with pXG10sf-PoxB A71L WN1405 with pXG10sf-PoxB G72L WN1405 with pXG10sf-PoxB S73L WN1405 with pXG10sf-PoxB C74L WN1405 with pXG10sf-PoxB G75L WN1405 with pXG10sf-PoxB P76L WN1405 with pXG10sf-PoxB G77L WN1405 with pXG10sf-PoxB N78L WN1405 with pXG10sf-PoxB H80L WN1405 with pXG10sf-PoxB Shifted WN1405 with pXG10sf-PoxB P76::CCA WN1405 with pXG10sf-PoxB P76::CCC WN1405 with pXG10sf-PoxB P76::CCT WN355 with pXG10sf-LacZ186 WN355 with pXG10sf-PoxB FL WN355 with pXG10sf-PoxB 21 WN1336 with pXG10sf-LacZ186 WN1336 with pXG10sf-PoxB FL WN1336 with pXG10sf-PoxB 21 WN1421 with pXG10sf-LacZ186 WN1421 with pXG10sf-PoxB FL WN1421 with pXG10sf-PoxB 21

This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work

Plasmids

Description

Reference

pBAD30mw700 pBAD30mw721 pBAD30mw723 pBAD30mw7231 pBAD30mw7232 pBAD30mw7233 pBAD30mw751 pBAD30mw722 pBAD30mw731 pBAD30mw732 pBAD30mw741 pBAD30mw753 pXG10sf

pBAD30 plasmid with GFP and mCherry tandem reporter with their respective Shine-Dalgarno sequences pBAD30mw700 with PPP inserted at 4th codon position of gfp pBAD30mw700 with optimized PPPPPPo (nucleotide sequence: ccg ccg ccg ccg ccg ccg) inserted at 4th codon position of gfp pBAD30mw700 with codon randomized PPPPPP1 (nucleotide sequence: ccg ccg cca ccg ccg cca) inserted at 4th codon position of gfp pBAD30mw700 with codon randomized PPPPPP2 (nucleotide sequence: cca ccg ccc ccc ccg ccc) inserted at 4th codon position of gfp pBAD30mw700 with codon randomized PPPPPP3 (nucleotide sequence: ccc cca ccc cca ccc cca) inserted at 4th codon position of gfp pBAD30mw700 with APP inserted at 4th codon position of gfp pBAD30mw700 with PPG inserted at 4th codon position of gfp pBAD30mw700 with YIR inserted at 4th codon position of gfp pBAD30mw700 with YIRYIR inserted at 4 th codon position of gfp pBAD30mw700 with RME inserted at 4th codon position of gfp pBAD30mw700 with PFF inserted at 4th codon position of gfp An expression vector that places cloned genes under the control of the constitutively active tet01promoter and in frame with ‘super-folder’ GFP

pXG10sf-LacZ186

First 186 codons of lacZ in pXG10sf

pXG10sf-PoxB FL

Full-length poxB (excluding stop codon) in pXG10sf

This work This work This work This work This work This work This work This work This work This work This work This work Corcoran, 2012 (3) Corcoran, 2012 (3) This work

pXG10sf-PoxB 499 pXG10sf-PoxB 397 pXG10sf-PoxB 325 pXG10sf-PoxB 247 pXG10sf-PoxB 171 pXG10sf-PoxB 97 pXG10sf-PoxB 82 pXG10sf-PoxB 68 pXG10sf-PoxB 52 pXG10sf-PoxB 36 pXG10sf-PoxB 21 pXG10sf-PoxB E66L pXG10sf-PoxB L67A pXG10sf-PoxB C70L pXG10sf-PoxB A71L pXG10sf-PoxB G72L pXG10sf-PoxB S73L pXG10sf-PoxB C74L pXG10sf-PoxB G75L pXG10sf-PoxB P76L pXG10sf-PoxB G77L pXG10sf-PoxB N78L pXG10sf-PoxB H80L pXG10sf-PoxB Shifted pXG10sf-PoxB P76::CCA pXG10sf-PoxB P76::CCC pXG10sf-PoxB P76::CCT Purpose Confirmation of insertion in pBAD30. Forward primer. Confirmation of insertion in pBAD30. Reverse primer.

First 499 codons of poxB in pXG10sf First 397 codons of poxB in pXG10sf First 325 codons of poxB in pXG10sf First 247 codons of poxB in pXG10sf First 171 codons of poxB in pXG10sf First 97 codons of poxB in pXG10sf First 82 codons of poxB in pXG10sf First 68 codons of poxB in pXG10sf First 52 codons of poxB in pXG10sf First 36 codons of poxB in pXG10sf First 21 codons of poxB in pXG10sf pXG10sf-PoxB FL with E66L mutation pXG10sf-PoxB FL with L67A mutation pXG10sf-PoxB FL with C70L mutation pXG10sf-PoxB FL with A71L mutation pXG10sf-PoxB FL with G72L mutation pXG10sf-PoxB FL with S73L mutation pXG10sf-PoxB FL with C74L mutation pXG10sf-PoxB FL with G75L mutation pXG10sf-PoxB FL with P76L mutation pXG10sf-PoxB FL with G77L mutation pXG10sf-PoxB FL with N78L mutation pXG10sf-PoxB FL with H80L mutation pXG10sf-PoxB with an adenine insertion and cytosine deletion shifting codons 69-82 to the +1 reading frame as outlined in Figure S3

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pXG10sf-PoxB FL with P76 codon (CCG) mutated to CCA

This work

pXG10sf-PoxB FL with P76 codon (CCG) mutated to CCC

This work

pXG10sf-PoxB FL with P76 codon (CCG) mutated to CCT

This work

Primer Name

Sequence

HR700_pBAD30_S

5’_CCTGACGCTTTTTATCGCAAC_3’

HR701_pABD30_AS

5’_TACTGCCGCCAGGCAAATTCTG_3’

Inserting PPPPPP0

MW.ins.PPPPPP_fwd

Inserting PPPPPP1

MW.ins.PPPPPP.rand(1)_fwd

Inserting PPPPPP2

MW.ins.PPPPPP.rand(2)_fwd

Inserting PPPPPP3

MW.ins.PPPPPP.rand(3)_fwd

Inserting PPP

MW.ins.PPP_fwd

Inserting PPG

MW.ins.PPG_fwd

Inserting APP

MW.ins.APP_fwd

Inserting RME

MW.ins.RME_fwd

Inserting YIR

MW.ins.YIR_fwd

Inserting YIRYIR

MW.ins.YIRYIR_fwd

Inserting PFF

MW.ins.PFF_fwd

GFP forward

MW.716_GFP_F

This work

5’_CTAGCGAATTCAGGAGGAATTTACCATGCCGCCGCCGCCGCCGCC GATGAGTAAAAGAGAAGAACTTTT_3’ 5’_CTAGCGAATTCAGGAGGAATTTACCATGAGTAAACCACCGCCCCC CCCGCCCAGAGAAGAACTTTTCACTG_3’ 5’_CTAGCGAATTCAGGAGGAATTTACCATGAGTAAACCGCCGCCACC ACCGCCAAGAGAAGAACTTTTCACTG_3’ 5’_CTAGCGAATTCAGGAGGAATTTACCATGAGTAAACCCCCACCCCC ACCCCCAAGAGAAGAACTTTTCACTG_3’ 5’_CTAGCGAATTCAGGAGGAATTTACCATGCCGCCGCCGATGAGTAA AAGAGAAGAACTTTT_3’ 5’_CTAGCGAATTCAGGAGGAATTTACCATGAGTAAACCGCCGGGCAG AGAAGAACTTTTCACTG_3’ 5’_CTAGCGAATTCAGGAGGAATTTACCATGAGTAAAGCGCCGCCGAG AGAAGAACTTTTCACTG_3’ 5’_CTAGCGAATTCAGGAGGAATTTACCATGAGTAAACGCGAAATGA GAGAAGAACTTTTCACTG_3’ 5’_CTAGCGAATTCAGGAGGAATTTACCATGAGTAAATATATTCGCAG AGAAGAACTTTTCACTG_3’ 5’_CTAGCGAATTCAGGAGGAATTTACCATGAGTAAATATATTCGCTA TATTCGC AGA GAAGAACTTTTCACTGGAGT_3’ 5’_CTAGCGAATTCAGGAGGAATTTACCATGAGTAAACCGTTTTTTAG AGAAGAACTTTTCACTG_3’ 5’_CGAATTCAGGAGGAATTTACCATGAGTAAAAGAGAAGAACTTTTC ACTGGAG_3’

GFP reverse

MW.717_GFP_R

5’_CATGGTAAATTCCTCCTAGCTTGTTATTTGTAGAGCTCATCCATGC C_3’

mCherry forward

MW.718_Cherry_F

5’_CAAGCTAGGAGGAATTTACCATGGTTTCCAAGGGCGAGGAGG_3’

mCherry reverse

MW.719_Cherry_R

5’_CGGGTACCCGGATCCCCGGAATCATCTA_3’

Confirmation of efp deletions 16s rRNA-specific qPCR standard

WNp582 WNp583 WNp555 WNp556 new sfGFP qPCR F new sfGFP qPCR R see 'PoxB F NsiI' see 'PoxB -536 NheI R' PoxB F NsiI

5’_TACAACGACGGTGAGTTCTGGCAT_3’ 5’_AAAGTTCGGCGGAGTGACAGAGAT_3’ 5’_CGGGGAGGAAGGTGTTGTG_3’ 5’_GAGCCCGGGGATTTCACATC_3’ 5’_GTTCCATGGCCAACACTTGTCACT_3’ 5’_TACATAACCTTCGGGCATGGCACT_3’

poxB FL R NheI poxB -73c R NheI poxB -175c R NheI poxB -247c R NheI poxB -325c R NheI poxB -401c R NheI poxB -475c R NheI PoxB -490c NheI R PoxB -504c NheI R PoxB -520c NheI R PoxB -536c NheI R PoxB R NheI PoxB E66L F PoxB E66L R PoxB L67A F PoxB L67A R PoxB C70L F PoxB C70L R PoxB A71L F PoxB A71L R PoxB G72L F PoxB G72L R PoxB S73L F PoxB S73L R PoxB C74L F PoxB C74L R PoxB G75L F PoxB G75L R PoxB P76L F PoxB P76L R PoxB G77L F PoxB G77L R PoxB N78L F PoxB N78L R PoxB H80L F PoxB H80L R PoxB +1 frame F

5’_GTTTTTGCTAGCCCTTAGCCAGTTCGTTTTCGC_3’ 5’_GTTTTTGCTAGCAATACCGGTAATGCCGCAGG_3’ 5’_GTTTTTGCTAGCGCGCTTGCCGTTCATTTTTAGA_3’ 5’_GTTTTTGCTAGCTTCTTCCACCAGCGGCAG_3’ 5’_GTTTTTGCTAGCATCATAAGGGTTATCGTACTCAACGTG_3’ 5’_GTTTTTGCTAGCGTTTTCAGGCGCGGGTTTC_3’ 5’_GTTTTTGCTAGCCGCCAGAACAGGGACGTG_3’ 5’_GTTTTTGCTAGCGATCAGGTGCAGGTTGCCCG_3’ 5’_GTTTTTGCTAGCTGCCAGCTCGCCGGTAAG _3’ 5’_GTTTTTGCTAGCGACCTCTTCGTGGCGGGT_3’ 5’_GTTTTTGCTAGCAAGACTATCGCTCAGGCCGTTCA_3’ 5’_GTTTTTGCTAGCGATTTGTTTTACGCCAGCCTG_3’ 5’_CACAAGCGCAGCTTACCGGCCTGCTGGCAGTGTGCGCCGGTT_3’ 5’_AACCGGCGCACACTGCCAGCAGGCCGGTAAGCTGCGCTTGTG_3’ 5’_AAGCGCAGCTTACCGGCGAGGCGGCAGTGTGCGCCGGTTCAT_3’ 5’_ATGAACCGGCGCACACTGCCGCCTCGCCGGTAAGCTGCGCTT_3’ 5’_TTACCGGCGAGCTGGCAGTGCTCGCCGGTTCATGTGGACCGG_3’ 5’_CCGGTCCACATGAACCGGCGAGCACTGCCAGCTCGCCGGTAA_3’ 5’_CCGGCGAGCTGGCAGTGTGCCTCGGTTCATGTGGACCGGGCA_3’ 5’_TGCCCGGTCCACATGAACCGAGGCACACTGCCAGCTCGCCGG_3’ 5’_GCGAGCTGGCAGTGTGCGCCCTTTCATGTGGACCGGGCAACC_3’ 5’_GGTTGCCCGGTCCACATGAAAGGGCGCACACTGCCAGCTCGC_3’ 5’_GCTGGCAGTGTGCGCCGGTTTATGTGGACCGGGCAACCTGC_3’ 5’_GCAGGTTGCCCGGTCCACATAAACCGGCGCACACTGCCAGC_3’ 5’_TGGCAGTGTGCGCCGGTTCACTTGGACCGGGCAACCTGCACC_3’ 5’_GGTGCAGGTTGCCCGGTCCAAGTGAACCGGCGCACACTGCCA_3’ 5’_CAGTGTGCGCCGGTTCATGTTTACCGGGCAACCTGCACCTGA_3’ 5’_CAGTGTGCGCCGGTTCATGTTTACCGGGCAACCTGCACCTGA_3’ 5’_GTGCGCCGGTTCATGTGGACTGGGCAACCTGCACCTGATCA_3’ 5’_TGATCAGGTGCAGGTTGCCCAGTCCACATGAACCGGCGCAC_3’ 5’_GCGCCGGTTCATGTGGACCGCTCAACCTGCACCTGATCAATG_3’ 5’_CATTGATCAGGTGCAGGTTGAGCGGTCCACATGAACCGGCGC_3’ 5’_CCGGTTCATGTGGACCGGGCCTCCTGCACCTGATCAATGGCC_3’ 5’_GGCCATTGATCAGGTGCAGGAGGCCCGGTCCACATGAACCGG_3’ 5’_ATGTGGACCGGGCAACCTGCTCCTGATCAATGGCCTGTTTG_3’ 5’_CAAACAGGCCATTGATCAGGAGCAGGTTGCCCGGTCCACAT_3’ 5’_CGGTTCATGTGGACCGGGCAACCTGCACCTGATAATGGCCTGTTT GATTGCCACCG_3’ 5’_AGGTTGCCCGGTCCACATGAACCGGCGCACACTTGCCAGCTCGCC GGTAAG_3’ 5’_TGCGCCGGTTCATGTGGACCAGGCAACCTGCACCTGATCAA_3’ 5’_TTGATCAGGTGCAGGTTGCCTGGTCCACATGAACCGGCGCA_3’ 5’_TGCGCCGGTTCATGTGGACCCGGCAACCTGCACCTGATCAA_3’ 5’_TTGATCAGGTGCAGGTTGCCGGGTCCACATGAACCGGCGCA_3’ 5’_TGCGCCGGTTCATGTGGACCTGGCAACCTGCACCTGATCAA_3’

sfGFP-specific qPCR poxB-specific qPCR pXG10sf-PoxB truncation plasmids: pXG10sf-PoxB FL pXG10sf-PoxB 499 pXG10sf-PoxB 397 pXG10sf-PoxB 325 pXG10sf-PoxB 247 pXG10sf-PoxB 171 pXG10sf-PoxB 97 pXG10sf-PoxB 82 pXG10sf-PoxB 68 pXG10sf-PoxB 52 pXG10sf-PoxB 36 pXG10sf-PoxB 21 pXG10sf-PoxB E66L pXG10sf-PoxB L67A pXG10sf-PoxB C70L pXG10sf-PoxB A71L pXG10sf-PoxB G72L pXG10sf-PoxB S73L pXG10sf-PoxB C74L pXG10sf-PoxB G75L pXG10sf-PoxB P76L pXG10sf-PoxB G77L pXG10sf-PoxB N78L pXG10sf-PoxB H80L

pXG10sf-PoxB Shifted

pXG10sf-PoxB P76::CCA pXG10sf-PoxB P76::CCC pXG10sf-PoxB P76::CCT

PoxB +1 frame R PoxB P76 CCG:CCA F PoxB P76 CCG:CCA R PoxB P76 CCG:CCC F PoxB P76 CCG:CCC R PoxB P76 CCG:CCT F

5’_GTTTTTATGCATGTTATCACATTTAAGGAGATGGAGAACC_3’

REFERENCES FOR RELEVANT STRAINS IN SUPPLEMENTARY TABLE S1 1.

2.

3.

4.

Auweter, S. D., A. P. Bhavsar, C. L. de Hoog, Y. Li, Y. A. Chan, J. van der Heijden, M. J. Lowden, B. K. Coombes, L. D. Rogers, N. Stoynov, L. J. Foster, and B. B. Finlay. 2011. Quantitative mass spectrometry catalogues Salmonella pathogenicity island-2 effectors and identifies their cognate host binding partners. J Biol Chem 286:24023-24035. Bullwinkle, T. J., S. B. Zou, A. Rajkovic, S. J. Hersch, S. Elgamal, N. Robinson, D. Smil, Y. Bolshan, W. W. Navarre, and M. Ibba. 2013. (R)-beta-Lysine-modified Elongation Factor P Functions in Translation Elongation. The Journal of biological chemistry 288:4416-4423. Corcoran, C. P., D. Podkaminski, K. Papenfort, J. H. Urban, J. C. Hinton, and J. Vogel. 2012. Superfolder GFP reporters validate diverse new mRNA targets of the classic porin regulator, MicF RNA. Molecular microbiology 84:428-445. Navarre, W. W., S. B. Zou, H. Roy, J. L. Xie, A. Savchenko, A. Singer, E. Edvokimova, L. R. Prost, R. Kumar, M. Ibba, and F. C. Fang. 2010. PoxA, YjeK, and elongation factor P coordinately modulate virulence and drug resistance in Salmonella enterica. Mol Cell 39:209-221.