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Supplementary Data for

Properties of a novel PBP2A protein homologue from Staphylococcus aureus strain LGA251 and its

contribution to the β-lactam resistant phenotype

Choonkeun Kim1,‡ , Catarina Milheiriço2,‡ , Susana Gardete1,2, Mark A. Holmes3,

Matt T. G. Holden4, Hermínia de Lencastre1,2, and Alexander Tomasz1,§

1Laboratory of Microbiology and Infectious Diseases, The Rockefeller University, New York, USA

2Laboratory of Molecular Genetics, Instituto de Tecnologia Química e Biológica da Universidade Nova

de Lisboa, 2780 Oeiras, Portugal 3Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, UK

4Pathogen Genomics, The Welcome Trust Sanger Institute, Hinxton, Cambridge, UK

§To whom correspondence should be addressed: Alexander Tomasz, The Rockefeller University,

1230 York Avenue, New York, NY 10065, USA

Tel.: (212) 327-8277; Fax: (212) 327-8688; E-mail: tomasz@rockefeller.edu

‡Both authors contributed equally to this work

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Table S1. Bacterial strains and plasmid vectors. Strain or plasmid Description Reference or source

E. coli strains DC10B Δdcm in the DH10B background; Dam methylation only (1)

S. aureus strains COL-S COL with SCCmec excised; Oxs Ems Cms Tcs (2)

LGA251 S. aureus veterinary strain (3)

COL-SLGAmecA COL-S expressing LGA251 mecA gene in trans from the inducible Pcad promoter, Kanr Neor

This study

COL-Sspac::pbpB COL-S derivate in which pbpB gene is under the control of spac promoter/pPBP2i, Emr (4)

COL-Sspac::pbpB/LGAmecA COL-Sspac::pbpB carrying the plasmid-borne S. aureus LGA251 mecA region/ pBCB8::mecA_LGA, Emr Kanr Neor

This study

COL-Sspac::pbpB/SAmecA COL-Sspac::pbpB carrying the plasmid-borne S. aureus COL mecA region/pSTSW2C, Emr

Cmr (4,5)

Plasmids

pBCB8 pCN51 derivate with the ermC cassette replaced with the aphA-3 cassette, Pcad inducible promotor, Apr Kanr Neor

(6), Sobral, R. andPinho, M.; unpublished data,

pBCB8::mecALGA251 pBCB8 cloning vector with the LGA251 mecA gene (including RBS site) fused to Pcad promoter, Apr Kanr Neor

This study

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Table S2. Oligonucleotide primers used for cloning of mecA and mecALGA251 in E. coli and S. aureus.

Name Sequence (5´ 3´)

PCR

CHismecAF ACTACCATGGCTTCAAAAGATAAAGAAATTAATA

CHismecAR ATTTCTCGAGTTCATCTATATCGTATTTTTTATTACC

CHismecALGAF ATTACCATGGAAGATGACGATATTGAGAAAACAATTA

CHismecALGAR ATTCCTCGAGCTGATCTATATCAAATTGAGTTTTTCC

mecALGAF5 TATAGAGCTCTTGTAGTACAAAAGGAGG

mecALGAR6 TCTCGAATTCCAAGCACTTAATATCAACGC

Sequencing

mecASF CTATTAGGTTATGTTGGTCCC

mecASR TTGCCAACCTTTACCATCGAT

mecALGAF3 ACACCTTTTAGGTTATGTGG

mecALGAF4 AATATCCCGAGTGATTATCC

mecALGAR2 TTTACTAGTATCTCGCCTTGG

mecALGAR3 TTAATTGGACCCACATAACC

mecALGAR7 CTACAATTTCTTCTTCTCC

pCad-F GCACTTATTCAAGTGTATTT

Restriction sites are underlined: Nco I (CCATGG), Xho I (CTCGAG), Sac I (GAGCTC),

and EcoR I (GAATTC).

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FIGURE S1. Susceptibility of strain LGA251 to the β-lactam antibiotics oxacillin and cefoxitin. Minimal

inhibitory concentrations (MIC values) were determined at two temperatures using the Etest. Overnight

cultures were diluted to 0.08 of OD620 and spread on Müller-Hinton agar plates, and then the Etest strips

were placed. The MIC values were evaluated after 24 h incubation. This is stated in the material and

methods section.

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FIGURE S2. Western blotting of the membrane preparations by an anti-PBP2A antibody. The anti-

PBP2A antibody can partially recognize an epitope on PBP2ALGA since PBP2A and PBP2ALGA share only

9 out of 20 amino acids composing of the antigenic determinant. PBP2ALGA is barely detected as LGA251

is grown in the presence of cefoxitin (lane 5).

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Score Coverage # Unique Peptides

# PSMs

Area # AAs MW

[kDa] calc. pI

25105.91 98.93 169 778 8.23 ×

108 654 74.7 8.16

Score Coverage # Unique Peptides

# PSMs

Area # AAs MW

[kDa] calc. pI

27505.46 98.77 166 952 1.68 ×

109 652 74.5 9.19

FIGURE S3. Sequence coverage of C-terminal His-tagged PBP2A (A) and PBP2ALGA (B) - by mass

spectrometry (MS). MS covered 99% of peptide sequences for both proteins (green highlights),

confirming their full length and lack of post-translational modification. Oxidation of methionine residues

is a common phenomenon during MS procedure. The number of PSMs represents the total number of

identified peptide sequences.

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FIGURE S4. Preference of PBP2A and PBP2ALGA for two β-lactam antibiotics. PBP2A barely has

preference for cefoxitin compared with oxacillin (A), while PBP2ALGA has much lower affinity to

cefoxitin than oxacillin (B).

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