Identification of Critical Staphylococcal Genes Using Conditional Phenotypes Generated by Antisense RNA Y. Ji, B. Zhang, S. F. Van Horn, P. Warren, G. Woodnutt, M. K. R. Burnham, M. Rosenberg

Supplemental Figure 1. (A) Construction of the inducible antisense S. aureus library. S. aureus WCUH29 chromosomal DNA was sheared, treated with Bal31 to convert all ends to 3'OH and 5'P termini and to create a mixture of blunt and frayed ends. The Bal31-digested DNA fragments in the range of 200bp to 800bp were purified and ligated into the EcoRV site of downstream Pxyl/tet o in ATc inducible vector pYJ335. The ligated DNA was electroporated into E. coli DH10B using Amp as selection. The transformants were pooled and incubated in LB-Amp (100g/ml) for purification of library plasmid DNA, which was electroporated into S. aureus laboratory strain RN4220 and subsequently after passage into S. aureus clinical strain WCUH29 (29). (B) Screening for essential genes. The electrotransformants, WCHU29 carrying library plasmid DNA, were selected on TSA-Erm (5g/ml) and duplicated onto TSA-Erm plates with ATc and without ATc. Growth defect and lethal colonies on TSA-Erm with ATc were selected after overnight incubation. Medium version | Full size version

Reference:
29. Plasmid library DNA prepared from the collection of E. coli clones was subsequently electroporated into 50 l of S. aureus laboratory strain RN4220 competent cells at 1.8 KV, 100 resistance, and 25 FD capacitance, then the electrotransformants were spread for single colonies on TSA-Erm (5 g/ml) plates. The colonies were collected and inoculated into 50 ml TBS-Erm and incubated with shaking at 37°C overnight. Plasmid library DNA was purified from this culture using QIAprep Miniprep Kit (Qiagen) and electroporated into S. aureus WCUH29.

Supplemental Table 1. Additional S. aureus antisense mutants
Isolate	Gene	Function	Phenotype on TSA-Erm with ATc	Isolate	Gene	Proposed function	Phenotypes on TSA-Erm with Atc
JSB373	dnaX	DNA polymerase	defect	JSB325	lepA	GTP-bining protein	lethal
JSB309	addA	ATP-dependent nuclease	defect	JY476	pfoD	oxidoreductase	lethal
JSB434	addB	ATP-dependent nuclease	defect	JSB24	yqiY	transporter	defect
JSB428	deaD	ATP-dependent RNA helicase	defect	JSB83	yhjX	transporter	lethal
JSB520	rnpA	ribonuclease	defect	JSB128	yurY	transporter	lethal
YJ49-8	uvrC	exinuclease	defect	JSB298	cbrB	transporter	lethal
JSB375	rpsQ	30s ribosomal protein	defect	JSB303	yhcL	transporter	defect
JSB953	rpsE	30s ribosomal protein	lethal	JSB427	bcaT	transporter	defect
JSB964	rpsK	30s ribosomal protein	lethal	JY245	ykaB	transporter	defect
JSB948	rpsH	30s ribosomal protein	lethal	JY271	yckK	ABC transporter	defect
JSB933	rpsS	30s ribosomal protein	lethal	JY311	_	dehydrogenase	defect
JSB932	rplB	50s ribosomal protein	lethal	YJ55-9	glyD	dehydrogenase	defect
JSB930	rplD	50s ribosomal protein	lethal	YJ41-3	_	unknown	defect
JSB945	rplE	50s ribosomal protein	lethal	JSB94	yaaO	unknown	defect
JSB950	rplF	50s ribosomal protein	defect	JSB166	yvlA	unknown	defect
JSB967	rplQ	50s ribosomal protein	lethal	JSB105	ylbC	unknown	lethal
JSB250	lysS	tRNA synthetase	lethal	JSB170	ykrU	unknown	defect
JSB441	ileS	tRNA synthetase	defect	JSB175	ydiL	unknown	defect
JSB765	tyrS	tRNA synthetase	defect	JSB398	yeeF	unknown	lethal
JSB618	proS	tRNA synthetase	defect	JSB411	yxdM	unknown	lethal
JSB902	thrS	tRNA synthetase	defect	JSB564	yjjP	unknown	lethal
JSB21	glrS	tRNA synthetase	lethal	JY19	_	unknown	defect
YJ59-2	cbk	cabonate kinase	defect	JY91	_	unknown	defect
JSB379	sporel	GTP-pyrophosphokinase	lethal	JY345	_	unknown	lethal
JY163	purB	adenlosuccinate lyase	defect	JSB52	_	unknown	lethal
JY202	purC	saicar synthase	defect	JSB62	_	unknown	lethal
JSB116	pgaM	phosphoglyceromutase	defect	JSB79	_	unknown	defect
JSB256	glcT	transcription antiterminator	defect	JSB80	_	unknown	defect
JY261	trkA	transketolase	lethal	JSB86	_	unknown	lethal
YJ59-11	pdhE1	dihydrolipoamide dehydrogenase	defect	JSB125	_	unknown	lethal
YJ45-8	odP2	suE2 of pyruvate dehydrogenase	defect	JSB138	_	unknown	lethal
YJ59-9	ypcA	glutamate dehydrogenase	defect	JSB165	_	unknown	defect
JSB77	adh1	alchole dehydrogenase	defect	JSB186	_	unknown	defect
YJ60-2	fmhA	FmhA cell wall biosynthesis	defect	JSB371	_	unknown	lethal
JY262	ykqkI	Na+/H+ antiporter	defect
JY154	glpP	glyceral uptake operon	defect
JSB444	kdpA	ATPase	defect
Note: gene = closest Bacillus subtilis homologue; "_" designates no Bacillus subtilis.

Supplemental Table 2. Identification number of unknown genes without name
Isolate		Accession top hit
JSB125		BAB44552.1
JSB138		BAB45583.1
JSB165		BAB45224.1
JSB174		BAB46465.1
JSB186		BAB44593.1
JSB187		BAB46062.1
JSB196		BAB45084.1
JSB291		BAB44970.1
JSB313		BAB44881.1
JSB316		BAB45982.1
JSB371		BAB46035.1
JSB399		BAB45209.1
JSB414		BAB46027.1
JSB433		BAB44249.1
JSB459		BAB45878.1
JSB52		BAB46149.1
JSB59		BAB46802.1
JSB60		BAB44590.1
JSB62		BAB46063.1
JSB79		BAB46069.1
JSB80		BAB46638.1
JSB86		BAB44595.1
JSB91		BAB45356.1
JY156		BAB44466.1
JY188		BAB44269.1
JY19		BAB46708.1
JY311		BAB45087.1
JY345		BAB44449.1
JY349		BAB45095.1
JY367		P42424
JY511		BAB45207.1
JY91		BAB46759.1
YJ47-6		Q57060
YJ49-11		BAB44703.1
YJ65-2		BAB45784.1
YJ67-4		BAB45356.1
YJ68-17		BAB45879.1
YJ68-4		P80544
YJ69-15		BAB44722.1
YJ69-6		BAB46464.1
YJ7-7		P33025