Table S3. Primers used in this work Primer Sequence - PLOS

Table S3. Primers used in this work Primer

Sequence (5`-3`)a

Locationb

ACrnaF ACrnaR ADrnaF ADrnaR AErnaF AErnaR creDrnaF creDrnaR ADF ADR ADH2F ADH2R ADH3F ADH3R AEF AER ARF

GGGCTGGCCTCGAAAGAGGAC GCACCGAGTCGGGGAACTGCA CGCGCATTTCCTCATCGAACGC TCGCAGTGGCCCTGGATGCG CCGCTGCACCTTCTGGTGGTG GCCACCGGGCCGAGCAAGGCA CGGCGTGCTGCAGGATATCGC TGTCGACGTGGTACAGGCGCG GTACGCCTGCTGGACGATG GAGGGCAGATCCTCGACCAG GGGCAGCGCGGTCAGGC TCAGGAAGTCGGCACCGCC TTGGCCGGCCCCTGAAC GCGACGACCTGAGCGACG GCCTGGACCCGAACGAAC TCAGAGGAACAGCGCGCAG GTCGACCCAGTGCCTTCAGG

208 to 228 434 to 454 225 to 246 457 to 476 245 to 265 488 to 508 114 to 134 344 to 364 -233 to -253 707 to 726 -19 to -39 762 to 780 -81 to -97 845 to 862 -93 to -111 1013 to 1032 1138 to 1156

PCR product size (bp) 246

Use

Reference or source

Quantification of ampC mRNA

Juan et al (2006)

251

Quantification of ampD mRNA

Juan et al (2006)

246

Quantification of ampE mRNA

This work

251

Quantification of creD mRNA

This work

916

ampD amplification and sequencing Juan et al (2005)

815

ampDh2 amplification and Juan et al (2006) sequencing ampDh3 amplification and Juan et al (2006) sequencing ampE amplification and sequencing This work

960 1231 1391

ampR and ampC-ampR intergenic Juan et al (2005) region amplification and sequencing

ARR CreBF CreBR CreBIR CreCF CreCR CreCIF CreDF CreDR CreDIF2 CreDIR CreDIF DACB-F DACB-R DACB-I-R DACB-I-F DACB-I-F2 AD-F0ERI AD-R3HDIII AD-F2HDIII AD-RBHI DACB-F-ERI DACB-I-R-HD3 DACB-I-F-HD3 DACB-R-BHI AmpC-F-ERI AmpC-I-R-HDIII AmpC-I-F-HDIII AmpC-R-BHI AEXBI-F1 AEHDIII-R1 AEHDIII-F2 AEERI-R2

CTCGAGAGCGAGATCGTTGC CACCAAGGGCTTGCGCA TCGAGCATCTCCGGCAGG CTGGAACGGCCCGCTC CAGCCCGGACCACGCCTG CCAGCGTGCGGTTCATG CCTGCCGGAGATGCTCG GGCTGCCGCGCGGCTGA TCAGGCCCTGGCGGGTAC GTCGACAACAAGGTTGCCGG GATGCCATAGTGCGCAGGC GCGACGGCTTCCAGGCGC CGACCATTCGGCGATATGAC CGCGTAATCCGAAGATCCATC GTCGCGCATCAGCAGCCAG GCCAGGGCAGCGTACCGC GTGCTCAACGGCAACCTCTAC TCGAATTCGTCCTGACTTCGCCGGAC TCAAGCTTCACGGACCCAGCCGGTAAC TCAAGCTTGCCTGGACCCGAACGAAC TCGGATCCGAGGGCAGATCCTCGACCAG TCGAATTCCGACCATTCGGCGATATGAC TCAAGCTTGTCGCGCATCAGCAGCCAG TCAAGCTTGCCAGGGCAGCGTACCGC TCGGATCCCGCGTAATCCGAAGATCCATC TCGAATTCGCGCGCAGGGCGTTCAG TCAAGCTTCGTCCTCTTACGAGGCCAG TCAAGCTTCAGGGCAGCCGCTTCGAC TCGGATCCCAGGTTGGCATCGACGAAG GCTCTAGACGCATCCAGGGCCACTGCG TCAAGCTTCGTTCGGCGACATGGAAGGC TCAAGCTTGCACGGCCATCTGCTCTGGC TCGAATTCTTGACGCACGGAGTCCGCTC

-239 to -220 -147 to -166 887 to 903 393 to 408 -18 to -1 1526 to 1542 198 to 214 -118 to -102 1342 to 1359 358 to 377 389 to 408 842 to 859 -178 to -159 1526 to 1546 378 to 396 854 to 871 316 to 336 -478 to -496 16 to 34 170 to 187 663 to 682 -175 to -156 378 to 396 854 to 871 1526 to 1546 -186 to -169 210 to 229 366 to 384 779 to 798 -93 to -111 358 to 377 430 to 449 1016 to 1035

1066

creB amplification and sequencing

This work

1560

creB sequencing creC amplification and sequencing

This work This work

1477

creC sequencing creD amplification and sequencing

This Work This work

creD sequencing

This work

dacB amplification and sequencing

This work

dacB sequencing

This work

AmpD inactivation

Juan et al (2006)

DacB inactivation

This work

AmpC inactivation

Moya et al (2008)

AmpE inactivation

This work

1721

523 512 571 693 415 432 488 606

TCGAATTCCACCAGGTGAAGAGCCTCG AmpR-F-ERI 123 to 141 AmpR-I-R-HDIII TCAAGCTTGACTGTGCAACTGGGCGG 552 to 572 728 to 744 AmpR-I-F-HDIII TCAAGCTTCAGGGTGTCGGCGTGGC TCGGATCCGCCTATGCCGCCAGCCTG 1140 to 1157 AmpR-R-BHI TCGAATTCCACCAAGGGCTTGCGCA -147 to -166 CreB-F-ERI TCAAGCTTCTGGAACGGCCCGCTC CreB-I-R-HD3 393 to 408 TCAAGCTTCCTGCCGGAGATGCTCG 198 to 214 CreC-I-F-HD3 TCGGATCCGCTGACCGCCTGGGCG 1337 to 1352 CreC-Rb-BHI TCGGATCCGGCTGCCGCGCGGCTGA -118 to -102 creD-F-BHI TCAAGCTTGATGCCATAGTGCGCAGGC creD-I-R-HD3 390 to 408 TCAAGCTTGATCACCGCCCAGGGCTTC 747 to 765 creD-I-F-HD3 TCGAATTCTCAGGCCCTGGCGGGTAC creD-R-ERI 1342 to 1359 a Sites for restriction endonucleases are underlined. b Location of the primers respect to the start codon of the corresponding genes.

424

AmpR inactivation

This work

CreBC inactivation

This work

CreD inactivation

This work

409 571 466 526 613

References Juan C, Macia MD, Gutierrez O, Vidal C, Perez JL, et al. (2005) Molecular mechanisms of β-lactam resistance mediated by AmpC hyperproduction in Pseudomonas aeruginosa clinical strains. Antimicrob Agents Chemother 49: 4733-4738. Juan C, Moya B, Perez JL, Oliver A (2006) Stepwise upregulation of the Pseudomonas aeruginosa chromosomal cephalosporinase conferring high level beta-lactam resistance involves three AmpD homologues. Antimicrob Agents Chemother 50: 1780-1787. Moya B, Juan C, Alberti S, Perez JL, Oliver A (2008) Benefit of having multiple ampD genes for acquiring β-lactam resistance without losing fitness and virulence in Pseudomonas aeruginosa. Antimicrob Agents Chemother 52: 3694-3700.