This test was therefore used for two purposes: (i) to distinguish relA+ from relA− strains, and (ii) as a qualitative measure of transcriptional polarity relief at the ilv locus. Growth in the presence of amino acids Serine, Methionine, and Glycine (SMG) was scored on glucose-minimal A plates supplemented with each of the amino acids at 100 μg/ml and compared with the growth on non-supplemented glucose-minimal A plates to score for SMG phenotype

The E. coli relA mutants exhibit SMG-sensitive (SMGS) phenotype i.e. growth-inhibition in the presence of Serine, Methionine and Glycine at 1 mM concentration each (Uzan and Danchin, 1978) and is proposed to be a consequence of transcriptional polarity exerted by a frameshift mutation in the ilvG gene on the expression of downstream genes of the ilvGMEDA operon (Lopes et al., 1989). It was observed in another study that the rho and nusG mutants that are defective for transcription termination conferred SMG-resistant (SMGR) phenotype in a relA1 strain (Harinarayanan and Gowrishankar, 2003)

SMG resistance

Genomic mapping of disrupted locusin Tn7insertion mutants was carried out as describedpreviously(Kaur et al., 2004).C. glabratamutants carrying Tn7insertionswere grown in YPD-liquid medium and genomic DNA was isolated fromovernight cultures. 10 μg genomic DNA was digested either with restriction enzyme MfeIor SpeI.Restriction enzyme-digestedDNA was precipitated with 1 ml ethanol and 1/10thvolume of sodiumacetate (3 M,pH 5.2). DNA pellet was washed twice with ice-cold 70% ethanol, air driedand was resuspended in sterilewater. DNA was recircularized with T4 DNA ligase.Resultant circular DNA carriedTn7cassette flanked on bothsidesby the disrupted locus oftheC. glabratagenome. CircularDNA wastransformed in E. coliBW23473 strainwhich contains protein Π (the product of the pirgene) required by R6Kγorifor replication.Twoverified transformants were grown overnight in LB-kanamycin medium and plasmids were extracted. Purified plasmids were sequenced withprimers reading outwards (OgRK 183 and OgRK 184) from both ends ofTn7cassette.Sequences obtained were compared,usingBLAST,against C. glabratagenome sequence database and regionsof Tn7insertions in C. glabratawere mapped

Mutant rescue