TABLE 2.

NreA variants tested in A. fabrum

Variant	Mutagenesis plasmid	Test plasmid	Sequencea	Mutagenesis oligonucleotidesb
C089B NreA (wild type)	pKJ213	pKJ257	MNERPHVHETHPAIIKRLKRADGHLRGIVEMIEAGRPCLDIAQQLHAVEKAIAQAKKTLIQDHLNHCLEDVVGPLALEQRRSIDEFKDITKYL	oKJ608/609
R17A	pKJ216	pKJ260	MNERPHVHETHPAIIKaLKRADGHLRGIVEMIEAGRPCLDIAQQLHAVEKAIAQAKKTLIQDHLNHCLEDVVGPLALEQRRSIDEFKDITKYL	oKJ585/586
R20A	pKJ255	pKJ267	MNERPHVHETHPAIIKRLKaADGHLRGIVEMIEAGRPCLDIAQQLHAVEKAIAQAKKTLIQDHLNHCLEDVVGPLALEQRRSIDEFKDITKYL	oKJ610/611
C38A	pKJ270	pKJ258	MNERPHVHETHPAIIKRLKRADGHLRGIVEMIEAGRPaLDIAQQLHAVEKAIAQAKKTLIQDHLNHCLEDVVGPLALEQRRSIDEFKDITKYL	oKJ581/582
Q44A	pKJ252	pKJ264	MNERPHVHETHPAIIKRLKRADGHLRGIVEMIEAGRPCLDIAQaLHAVEKAIAQAKKTLIQDHLNHCLEDVVGPLALEQRRSIDEFKDITKYL	oKJ604/605
H63A	pKJ215	pKJ259	MNERPHVHETHPAIIKRLKRADGHLRGIVEMIEAGRPCLDIAQQLHAVEKAIAQAKKTLIQDaLNHCLEDVVGPLALEQRRSIDEFKDITKYL	oKJ583/584
H66A	pKJ247	pKJ261	MNERPHVHETHPAIIKRLKRADGHLRGIVEMIEAGRPCLDIAQQLHAVEKAIAQAKKTLIQDHLNaCLEDVVGPLALEQRRSIDEFKDITKYL	oKJ596/597
C67A	pKJ253	pKJ265	MNERPHVHETHPAIIKRLKRADGHLRGIVEMIEAGRPCLDIAQQLHAVEKAIAQAKKTLIQDHLNHaLEDVVGPLALEQRRSIDEFKDITKYL	oKJ606/607
K91A	pKJ256	pKJ268	MNERPHVHETHPAIIKRLKRADGHLRGIVEMIEAGRPCLDIAQQLHAVEKAIAQAKKTLIQDHLNHCLEDVVGPLALEQRRSIDEFKDITaYL	oKJ612/613

^a Sequence includes the entire NreA amino acid sequence for NreA variants used. Bold lowercase letters represent alanine substitutions compared to the C089B NreA (wild type).

^b Mutagenesis oligonucleotides were used to incorporate mutations in nreA via inverse-PCR amplification of pKJ213, followed by circularization, followed by transfer of the variant gene to pKJ227, as described in Materials and Methods. A. fabrum strains harboring these plasmids are listed in Table S1.