Severity of the Streptomycin Resistance and Streptomycin Dependence Phenotypes of Ribosomal Protein S12 of Thermus thermophilus Depends on the Identity of Highly Conserved Amino Acid Residues

DOI: 10.1128/JB.187.10.3548-3550.2005

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FIG. 1.
Construction of T. thermophilus rpsL mutants by gene replacement. The P90R Str^d strain was transformed with plasmid pUC18 containing the rpsL gene with the desired mutation (here, K42T). Plasmid pUC18 does not replicate in T. thermophilus; therefore, replacement of chromosomal rpsL with the plasmid-borne rpsL gene occurs via homologous recombination with selection on antibiotic-free medium.
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FIG. 2.
Three-dimensional structure of S12 (blue) showing the location of K42 and P90 (Protein Data Bank accession no. 1FJG ). K42 and P90 lie in two highly conserved loops (2), shown in green. K42 contacts streptomycin (strep; red) and 16S RNA (grey) via the phosphate of A913 (13). This figure was illustrated with PyMol (6).

TABLE 1.

T. thermophilus mutants and their streptomycin phenotypes

Wild type or mutation(s)	Codon change	Phenotype	Streptomycin MIC (μg/ml)	Paromomycin MIC (μg/ml)
IB21 (wild type)		Str^s	25	5
rpsL R37C	CGC→TGC	Str^r	500
rpsL K42T	AAG→ACG	Str^r	2,000
rpsL K42C	AAG→TGC	Str^r	2,000
rpsL P90R	CCG→CGG	Str^d Par^d	10^a	1^a
rpsL P90L	CCG→CTG	Str^d Par^d	10^a	1^a
rpsL P90E	CCG→GAG	Str^d	10^a
rpsL P90M	CCG→ATG	Str^d	10^a
rpsL P90W	CCG→TGG	Str^d	10^a
rpsL P90A	CCG→GCC	Str^r	500	5
rpsL P90C	CCG→TGC	Str^r	500	5
rpsL P90G	CCG→GGG	Str^r	500	5
rpsL K42T, P90R		Str^r	>2,000
rpsL K42C, P90C		Lethal
rpsL R37C, P90L		Par^d	ND	1^a
rpsL P90R, rpsD E201K	GAG→AAG^b	Str^r	ND
rpsL P90R, rpsE G103R	GGG→AGG^c	Str^r	ND