The serine/threonine kinase Stk and the phosphatase Stp regulate cell wall synthesis in Staphylococcus aureus


6-10 transformants were grown in LB with antibiotics (100 µg/ml streptomycin and ampicillin + 50 µg/ml kanamycin) and 0.5 mM IPTG for 3 days at 18 °C. 20 µl culture was diluted in 80 µl Z-buffer and OD 600 was measured. 25 µl 1 mg/ml MUG in DMSO was added. After 15 min incubation at room temperature 30 µl 1 M NaC0 3 was added and the fluorescence was measured (Ex. 355 nm, Em. 460 nm). Arbitrary units of β-gal activity (MUG units) were calculated as follows: F 355/460nm / (t x OD 600 ). 
Significance values were calculated using a two-tailed unpaired student t-test (* p < 0.05, ** p < 0.01, *** p < 0.001). Horizontal dotted line indicate threshold of 90 MUG units. This threshold is based on the lowest value of a significant protein interaction (FemA-FemA). 

Figure S1 .

 S1 Figure S1. Cell wall phenotype of S. aureus NewmanHG wt, Δstk, Δstp and ΔstkΔstp strains at logarithmic growth phase. (a) Analysis of the cell morphology and the cell wall thickness of S. aureus wt and mutant at the same stage in the cell cycle by TEM. The mean cell diameter (CD) and the cell wall thickness (CWT) was assessed based on the diameter of the 20 largest cells out of 100 cells per strain. The CWT was measured at five different points on the cell wall. (b) Muropeptide profile of S. aureus wt and mutant strains obtained by UPLC-MS. Highlighted muropeptide peaks were identified by MS and their structures are shown (c). Masses and retention times of indicated peaks are shown in Fig. S2a. (d) Lysostaphin susceptibility of S. aureus wt and mutant strains. Data represent percentages of the initial optical density (OD 600 ) over three hours after treatment with 0.5 µg/ml lysostaphin. The graphs show the mean and standard deviation of three independent experiments. Significance values of CD, CWT and lysostaphin susceptibility were calculated using a two-tailed unpaired student t-test (* p < 0.05, ** p < 0.01, *** p < 0.001). 


Figure S3 .Figure S4 .FemXFemXFigure S5d .

 S3S4S5d Figure S3. PGN precursor concentration in S. aureus wild type and mutant strains.Relative PGN precursor concentration of S. aureus strains at logaritmic-(a) and stationary-growth phase (b) adjusted to an internal standard (5 nmol camphorsulfonic acid). Graph (c) and (d) show the same data represented as the log 2 -fold changes of Δstk, Δstp and ΔstkΔstp mutants compared to wild-type cells at logaritmic-and stationary-growth phase. Significant differences (n=4; nonparametric unpaired Mann-Whitney test; * p < 0.05) are marked with asterisks. UDP-MurNAc-A-E-K was not detectable (n.d.) at stationary-growth phase. A alanine, E glutamic acid, K lysine 




  Figure S7. Stk/Stp in vitro kinase/phosphatase assay.One µg recombinant protein (FemX, FemA or FemB) was incubated with 1 µg Stk kinase domain (Stk KD ) in kinase buffer with or without 20 mM ATP for 1 h at 37 °C. For the dephosphorlyation reaction, 1 µg Stp was added to the reaction mixture. Each reaction was stopped by addition of 5 x SDS sample buffer and heating for 5 min at 95 °C. The phosphorylated proteins were detected by Western blot using an anti-phosphothreonine antibody (αP-Thr #9381S, Cell Signaling, Germany) and were visualized using the ImageQuant LAS 4000 imaging system (GE Healtcare). The upper panel shows the loading control with Coomassie-blue staining; the bottom panel shows phosphorylated proteins detected by αP-Thr Western blot. M: Marker PageRuler™ Prestained Protein Ladder (Thermo Fisher Scientific Inc, USA) 


Figure S13 .

 S13 Figure S13. Quantitative measurement of selected protein interactions.Measurement of β-galactosidase activity using fluorogenic substrate 4-methyllumbelliferyl β-Dgalactopyranoside (MUG) 4 to quantify the protein interactions based on the spot assay (figure4a). 


Z

  -buffer: 8 g of Na 2 HPO 4 x12H 2 O, 3.125 g of NaH 2 PO 4 xH 2 O, 0.375 g KCl, 0.123 g MgSO 4 x7H 2 O dissolved in 500 ml distilled Water) 




  




  




  




  




  




  




  




  




  


Table S6 . Minimal inhibitory concentration (MIC) of cell wall-active antibiotics for S. aureus NewmanHG wt and mutant strains.

 S6 
			MICs [µg/ml] of S. aureus
		wt	Δstk	Δstp	ΔstkΔstp
	fosfomycin	8	16	4	4
	D-cycloserine	32	32	16	32
	tunicamycin	32	4	16	4
	ramoplanin	0.06	0.03	0.06	0.03
	nisin	128	128	256	128
	methicillin	2	1	2	1
	penicillin G	0.25	0.13	0.25	0.13
	vancomycin	2	2	2	2
	daptomycin	2	2	2	2
	bacitracin	32	32	32	32

Table S8 . Bacterial strains used in this study Table S9. E. coli bacterial two-hybrid strains used in this study

 S8 
	Stk KD Stp ATP strains strains S. aureus NewmanHG + ---+ + -+ strains B04 E.coli DH5α p25N (pKNT25), Kan R + + + -+ + + + + ---FemA characteristic + + -+ + + + -characteristic + + + + FemB characteristic S. aureus Newman, saeS + B131 E.coli DH5α p18C-GlmS (NWMN_2056) S. aureus NewmanHG Δstk stk deletion, stk::ermB, Erm R B05 E.coli DH5α p25C (pKT25) Kan R B133 E.coli DH5α p18C-GlmM (NWMN_2062) S. aureus NewmanHG Δstp stp deletion, stp::ermB, Erm R B06 E.coli DH5α p18C (pUT18C), Amp R B135 E.coli DC10B p18C-GlmU (NWMN_0462, gcaD) S. aureus NewmanHG ΔstkΔstp stk/stp deletion, stk/stp::ermB, Erm R + ---B07 E.coli DH5α p18N (pUT18), Amp R B173 E.coli DC10B p18C-FemX (NWMN_2163, fmhB) S. aureus RN4220 NCTC 8325-4-r; 11 bp Deletion in rsbU B08 E.coli DH5α pUT18C-zip, Amp R B175 E.coli DC10B p18C-FemA (NWMN_1286) E. coli DH5α F -endA1 hsdR17 supE44 thi-1 recA1 B09 E.coli DH5α pKT25-zip, Kan R B177 E.coli DC10B p18C-FemB (NWMN_1287) B15 E.coli DH5α p25C-Stk (NWMN_1130) B203 E.coli DC10B p18C-Pbp4 (NWMN_0612) gyrA96 relA1(A) Δ(argF-lac)U169 B17 E.coli DH5α p18C-Stk (NWMN_1130) B207 E.coli DC10B p18C-Ddl (NWMN_1987) Φ80dlacZΔM15 E.coli DC10B mcrA Δ(mrr-hsdRMS-mcrBC) B18 E.coli DH5α p25N-Stp (NWMN_1129) B209 E.coli DC10B p18C-OatA (NWMN_2467) B19 E.coli DH5α p25C-Stp (NWMN_1129) B213 E.coli DC10B p18C-MurI (NWMN_1063) φ80lacZΔM15 ΔlacX74 recA1 araD139 B20 E.coli DH5α p18N-Stp (NWMN_1129) B215 E.coli DC10B p18C-MurT (NWMN_1830) Δ(ara-leu)7697 galU galK rpsL endA1 B21 E.coli DH5α p18C-Stp (NWMN_1129) B217 E.coli DC10B p18C-Sle1 (NWMN_0429) nupG Δdcm E.coli BL21(DE3) F -ompT hsdSB (rS_ mS_) gal dcm (DE3) B37 E.coli DH5α p18C-MurC (NWMN_1633) B219 E.coli DC10B p18C-LytH (NWMN_1534) E. coli BTH101 F-, cya-99, araD139, galE15, galK16, B63 E.coli DH5α p18C-DivIB (pGL544) B221 E.coli DC10B p18C-SsaA (NWMN_2443) B64 E.coli DH5α p18C-FtsA (pGL545) B223 E.coli DC10B p18C-Mgt (NWMN_ 1766) rpsL1 (Str r), hsdR2, mcrA1, mcrB1 B65 E.coli DH5α p18C-FtsL (pGL546) B225 E.coli DC10B p18C-GatD (NWMN_1829)	+ + -+	+ + + -source or reference + + + + source or reference source or reference Euromedex This work Maniero et al. 1 Euromedex This work This work Euromedex This work This work Euromedex This work This work Euromedex This work Novick, R. Euromedex This work MBI-Fermentas This work This work This work This work This work This work Monk, et al. 2 This work This work This work This work This work This work This work This work Novagen Steele et al, 2011 3 This work Euromedex Steele et al, 2011 3 This work Steele et al, 2011 3 This work
	B66 B226	E.coli DH5α p18C-Pbp2 (pGL547) E.coli DC10B p25C-FemX (NWMN_2163, fmhB)		Steele et al, 2011 3 This work
	B67 B227	E.coli DH5α p18C-FtsZ (pGL562) E.coli DC10B p25C-FemA (NWMN_1286)		Steele et al, 2011 3 This work
	B68 B228	E.coli DH5α p18C-DivIC (pGL554) E.coli DC10B p25C-FemB (NWMN_1287)		Steele et al, 2011 3 This work
	B69 B233	E.coli DH5α p18C-GpsB (pGL570) E.coli DC10B p25C-MurG (NWMN_1330)		Steele et al, 2011 3 This work
	B70	E.coli DH5α p18C-SepF (pGL572)		Steele et al, 2011 3
	B71	E.coli DH5α p18C-FtsW (pALB6)		Steele et al, 2011 3
	B72	E.coli DH5α p18C-RodA (pALB14)		Steele et al, 2011 3
	B73	E.coli DH5α p18C-EzrA (pVF32)		Steele et al, 2011 3
	B92	E.coli DH5α p18C-Pbp2		D. Lopez
	B93	E.coli DH5α p18C-Pbp3		D. Lopez
	B117	E.coli DH5α p18C-MurA (NWMN_2004)		This work
	B119	E.coli DH5α p18C-MurB (NWMN_0707)		This work
	B121	E.coli DH5α p18C-MurD (NWMN_1093)		This work
	B123	E.coli DH5α p18C-MurE (NWMN_0888)		This work
	B125	E.coli DH5α p18C-MurF (NWMN_1986)		This work
	B127	E.coli DH5α p18C-MraY (NWMN_1092)		This work
	B129	E.coli DH5α p18C-MurG (NWMN_1330)		This work

Table S9 . E. coli bacterial two-hybrid strains used in this study Table S10. Plasmids used in this study

 S9 
	plasmid	characteristic	source or reference
	pGEM-T	Amp r	Promega
	pEC1	pUC18 ermB, Amp r , Em r	Brückner, 1992
	pBT2	shuttle vector, Amp r , Cm r	Brückner, 1997
	pET-28a(+)	His 6 expression vector, Kan r	Novagen
	pET-28a-Stk KD	His 6 -Stk 1-291 , cloned in pET28a(+), Kan r	This study
	pET-28a-Stp	His 6 -Stp, cloned in pET28a(+), Kan r	This study
	pET-28a-FemX	His 6 -FemX, cloned in pET28a(+), Kan r	This study
	pET-28a-FemA	His 6 -FemA, cloned in pET28a(+), Kan r	This study
	pET-28a-FemB	His 6 -FemB, cloned in pET28a(+), Kan r	This study

Table S11 . Primer used in this study

 S11 
	Primer	sequence	restriction enzyme
	ermBEcoRI	CGGAATTCGGTGACATCTCTCTATTGTG	EcoRI
	ermBPstI	AACTGCAGGGAAGCTGTCAGTAGTATACC	PstI
	stp-up_EcoRI	CGGAATTCTGGCTCGTTGAACAAGGTC	EcoRI
	stp-up-BamHI	CGGGATCCACAAGCTGCGTCAATATCC	BamHI
	stp-down-PstI	TACCTGCAGTACTCGCGGCTATTGAAGGTG	PstI
	stp-down-EcoRV	CGGATATCAAGGGTGGTTCACCAACAAGC	EcoRV
	stp1	CAACATGATGCGACAAAACC	
	stp2	CTTGCGTCGTTTCACGTTTA	
	pET_Stk KD _fw	ATATTGGATCCataggtaaaataataaatgaacg	BamHI
	pET_ Stk KD _rev	ATATTCTCGAGTTAtaccgctatcgttttc	EcoRI
	pET_Stp_fw	CATATGCTAGAGGCACAATTTTTTACTG	NdeI
	pET_Stp_rev	CGCCCTAGGTCATACTTTATCACCTTCAATAGGCCG	BamHI
	pET FemX_fw	ATATGAATTCgaaaagatgcatatcactaatca	EcoRI
	pET FemX_rev	TATACTCGAGctattttcgttttaatttacgagatat	XhoI
	pET FemA_fw	ATATGGATCCaagtttacaaatttaacagctaaagag	BamHI
	pET FemA_rev	TATACTCGAGctaaaaaattctgtctttaacttttttaa	XhoI
	pET FemB_fw	ATATGGATCCaaatttacagagttaactgttaccg	BamHI
	pET FemB_rev	TATACTCGAGctatttctttaattttttacgtaatttatc	XhoI

Table S12 . Primer for constructing bacterial two-hybrid vectors

 S12 
	primer	sequence	restriction enzyme
	B007 C-Stk (s)	ATTGGATCCGATAGGTAAAATAATAAATGAACGATATAAAAtTG	BamHI
	B008 C-Stk (a)	TAAGAATTCTTATACATCATCATAGCTGACTTCTTTTTC	EcoRI
	B012 C-Stp (s)	ATTGGATCCGCTAGAGGCACAATTTTTTACTGATACTGGA	BamHI
	B013 C-Stp (a)	TAAGAATTCTCATACTTTATCACCTTCAATAGCCGC	EcoRI
	B020 C-MurC (s)	ATTGGATCCGACACACTATCATTTTGTCGGAAT	BamHI
	B021 C-MurC (a)	TAAGAATTCCTAAAACGCATTTTTCATGCCTAATTTA	EcoRI
	B026 N-Stk (s)	ATTGGATCCATGATAGGTAAAATAATAAATGAACGATATAAAATG	BamHI
	B027 N-Stk (a)	TAAGAATTCGCTACATCATCATAGCTGACTTCTTTTTC	EcoRI
	B031 N-Stp (s)	ATTGGATCCGATGCTAGAGGCACAATTTTTTACTGATACTGGA	BamHI
	B032 N-Stp (a)	TAAGAATTCGCTACTTTATCACCTTCAATAGCCGC	EcoRI
	B045 p25C (s)	CGGCGGATATCGACAT	sequencing
	B046 p25C (a)	CTGGCGAAAGGGGGAT	sequencing
	B047 p25/18N (s)	CACTTTATGCTTCCGGCTC	sequencing
	B048 p25N (a)	CTTGATGCCATCGAGTACG	sequencing
	B049 p18C (s)	GAGCGGACGTTCGAAGTT	sequencing
	B050 p18C (a)	GTTGGCGGGTGTCGG	sequencing
	B051 p18N (a)	TCCACAACAAGTCGATGC	sequencing
	B045 p25C (s)	CGGCGGATATCGACAT	sequencing
	B046 p25C (a)	CTGGCGAAAGGGGGAT	sequencing
	B070 C-MurA_fw	ATATTGGATCCGgataaaatagtaatcaaaggtggaa	BamHI
	B071 C-MurA_rev	TATTAGAATTCttaatcgttaatacgttcaatgtc	EcoRI
	B074 C-MurB_fw	ATATTGGATCCGataaataaagacatctatcaagctttac	BamHI
	B075 C-MurB_rev	TATAAGAATTCttacgattcctttggatgtt	EcoRI
	B078 C-MurD_fw	ATATTGGATCCGcttaattatacagggttagaaaataaaaatg	BamHI
	B079 C-MurD_rev	TATAAGAATTCttaataagatggtaaatgggca	EcoRI
	B082 C-MurE_fw	ATATTGGATCCGgatgcaagtacgttgtttaaga	BamHI
	B083 C-MurE_rev	TATAAGAATTCttaatcaacagggccacc	EcoRI
	B086 C-MurF_fw	ATATTGGATCCGattaatgttacattaaagcaaattcaa	BamHI
	B087 C-MurF_rev	TATAAGAATTCctatgaaattaaagcatttaccact	EcoRI
	B090 C-MraY_fw	ATATTGGATCCGatttttgtatatgcgttattagcg	BamHI
	B091 C-MraY_rev	TATAAGAATTCttaatgcactccaatccataa	EcoRI
	B094 C-MurG_fw	ATATTGGATCCGacgaaaatcgcatttacc	BamHI
	B095 C-MurG_rev	TATAAGAATTCttaattcaatgcgtctttaatcat	EcoRI

Table S12 . Primer for constructing bacterial two-hybrid vectors

 S12 
	primer	sequence	restriction enzyme
	B098 C-GlmS_fw	ATATTGGATCCGtgtggaattgttggttatattg	BamHI
	B099 C-GlmS_ rev	TATAAGAATTCttattccacagtaactgatttagc	EcoRI
	B111 C-GlmM_fw	ATATTGGATCCGggaaaatattttggtacagacg	BamHI
	B112 C-GlmM_rev	TATAAGAATTCttatttatctaatcccattttatcttga	EcoRI
	B115 C-GlmU_fw	ATATTGGATCCGttcatgcgaagacacg	BamHI
	B116 C-GlmU_rev	TATAAGAATTCttatttcctatatccttcttttgttg	EcoRI
	B119 C-FemX_fw	TATAATCTAGAGgaaaagatgcatatcactaatc	XbaI
	B120 C-FemX_rev	TATAAGAATTCctattttcgttttaatttacga	EcoRI
	B123 C-FemA_fw	ATATTGGATCCGaagtttacaaatttaacag	BamHI
	B124 C-FemA_rev	TATATGAATTCctaaaaaattctgtctttaactt	EcoRI
	B127 C-FemB_fw	ATATTGGATCCGaaatttacagagttaactgt	BamHI
	B128 C-FemB_rev	TATAAGAATTCctatttctttaattttttacgtaat	EcoRI
	B175 C-Pbp4_fw	ATTGGATCCGaaaaatttaatatctattatcatcatt	BamHI
	B176 C-Pbp4_ rev	TAAGAATTCttattttctttttctaaataaacg	EcoRI
	B183 C-Ddl_fw	ATTGGATCCGacaaaagaaaatatttgtatcg	BamHI
	B184 C-Ddl_rev	ATTGAATTCttagtcaattttgtatttatttttc	EcoRI
	B187 C-OatA_fw	ATTGGATCCGgatacaaaagactttaaacgtt	BamHI
	B188 C-OatA_rev	ATTGAATTCttatttcttatttgtagcatgt	EcoRI
	B194 C-MurI_fw	ATTGGATCCGaataaaccaataggtgtaatagac	EcoRI
	B195 C-MurI_rev	ATTGAATTCctagtcattcactgatatacgtt	BamHI
	B198 C-MurT_fw	ATTGGATCCGagacagtggacggca	EcoRI
	B199 C-MurT_rev	TAAGAATTCttatgattgacctccttca	BamHI
	B202 C Sle1_fw	ATTGGATCCGcaaaaaaaagtaattgcag	EcoRI
	B203 C-Sle1_rev	TAAGAATTCttagtgaatatatctataattatttacttg	BamHI
	B206 C-LytH_fw	ATTGGATCCGaaaaaaatagaggcatgg	EcoRI
	B207 C-LytH_rev	TAAGAATTCctacgcagaaaaataaattt	BamHI
	B210 C-SsaA_fw	ATTGGATCCGgaatataaaaagatactaattcgtt	EcoRI
	B211 C-SsaA_rev	TAAGAATTCttagtaaatatagttaaagttacgtg	BamHI
	B214 C-Mgt_fw	ATTGGATCCGaaaagaagcgataggtact	EcoRI
	B215 C-Mgt_rev	TAAGAATTCttaacgatttaattgtgacata	BamHI
	B218 C-GatD_fw	ATTGGATCCGcatgaattgactatttatcatt	EcoRI
	B219 C-GatD_rev	TAAGAATTCttaacgagatttcttctgtc	BamHI

863 +/-32 nm 31 +/-7 nm 931 +/-45 nm 24 +/-4 nm 922 +/-41 nm 39 +/-8 nm 1003 +/-45 nm 30 +/-6 nm CD CWT 


Supplementary Information
Marcel Jarick, Ute Bertsche, Mark Stahl, Daniel Schultz, Karen Methling, Michael Lalk, Christian Stigloher, Mirco Steger, Andreas Schlosser, Knut Ohlsen, Dr Knut Pd, Ohlsen
The serine/threonine kinase Stk and the phosphatase Stp regulate cell wall synthesis in Staphylococcus aureus