TABLE 2.

Phenotypes of EMS and site-directed AdhE mutants^a

Mutant type and AdhE clone	Position 267		Position 568		Other mutation		No. of occurrences	Phenotype
	Amino acid	Codon	Amino acid	Codon	Amino acid	Mutation
EMS
pCRADH1 (wt)	Ala	GCA	Glu	GAG				White
pCRADH2 (adhE*)	Thr	ACA	Lys	AAG				Red
pMUT1	Thr	ACA	Glu	GAG				White
pMUT2	Ala	GCA	Lys	AAG				Red
pEMS1	Ala	GCA	Lys	AAG				Red
pEMS2	Ala	GCA	Lys	AAG				Red
pEMS3	Ala	GCA	Lys	AAG				Red
pEMS4	Ala	GCA	Lys	AAG	Ala470Ala	GCG→GCA		Red
pEMS5	Ala	GCA	Lys	AAG	Arg463Arg	CGC→CGG		Red
Site directed
Hydrophobic	Ala	GCA	Ala	GCG			1	Red
	Ala	GCA	Leu	CTG			1	Red
Polar	Ala	GCA	Asn	AAC			1	Red
	Ala	GCA	Gly	GGC			4	Red
	Ala	GCA	Gly	GGG			12	Red
	Ala	GCA	Gly	GGT			1	Red
	Ala	GCA	Gly	GGA			5	Red
	Ala	GCA	Ser	TCT			1	Red
Basic	Ala	GCA	Arg	AGG			7	Red
	Ala	GCA	Arg	CGG			2	Red
	Ala	GCA	Arg	AGA			1	Red
	Ala	GCA	His	CAT			1b	Red
	Ala	GCA	Lys	AAG			1	Red
Acidic	Ala	GCA	Asp	GAC			2	White
	Ala	GCA	Asp	GAT			1	White
	Ala	GCA	Glu	GAG			16	White
	Ala	GCA	Glu	GAA			5	White
Nonsense	Ala	GCA	Stop	TGA			11	White

^aAdhE phenotype and genotype of EMS mutants and site-directed mutants selected for ADH activity under aerobic conditions on alcohol indicator medium as described in the text. Red colonies on this medium indicate oxidation of ethanol by ADH and the coupled reduction of 2,3,5-triphenyl tetrazolium chloride. Mutated nucleotides for the EMS-mutagenized strains are underlined. All mutants derived from site-directed mutagenesis except the one containing His were isolated by random PCR mutagenesis of all three of the Glu568 codon nucleotides. Occurrence is the number of random mutants with the indicated codon. wt, wild type. 

^bThe His mutant was constructed by site-directed mutagenesis.