tRNA2Thr complements temperature sensitivity caused by null mutations in the htrB gene in Escherichia coli

Abstract

According to the wobble rule, tRNA2Thr is nonessential for protein synthesis, because the codon (ACG) that is recognized by tRNA2Thr is also recognized by tRNA4Thr. In order to investigate the reason that this nonessential tRNA nevertheless exists in Escherichia coli, we attempted to isolate tRNA2Thr-requiring mutants. Using strain JM101F(-), which lacks the gene for tRNA2Thr, we succeeded in isolating two temperature-sensitive mutants whose temperature sensitivity was complemented by introduction of the gene for tRNA2Thr. These mutants had a mutation in the htrB gene, whose product is an enzyme involved in lipid A biosynthesis. Although it is known that some null mutations in the htrB gene give a temperature-sensitive phenotype, our mutants exhibited tighter temperature sensitivity. We discuss a possible mechanism for the requirement for tRNA2Thr.

FIG. 1.

Complementation of temperature-sensitive mutants (ts8 and ts124) derived from strain JM101F⁻ by cross-streaking with Kohara's phage clone 128 (16). An overnight culture of each temperature-sensitive mutant grown in lambda broth at 32°C was streaked left to right horizontally over Kohara's phage clone 128, which had been streaked vertically on a lambda plate (11). The plate was incubated at 42°C overnight and photographed. JM101F⁻, which is a derivative of JM101 (22) that cures F′ (proAB-lac) spontaneously, was screened by testing pro and lac markers. Temperature-sensitive mutants were isolated after mutagenesis with N-methyl-N′-nitrosoguanidine (18). The isolation procedure was as described by Nakayashiki and Inokuchi (20).

FIG. 2.

Growth curves of the temperature-sensitive mutants. Overnight cultures of the bacteria grown at 32°C in Luria-Bertani medium were diluted to an optical density at 600 nm (OD₆₀₀) of 0.005. The diluted cultures were shaken at 42°C. Aliquots were withdrawn at every 2 h and the OD₆₀₀ was measured by using a model UV-160A Shimadzu spectrophotometer (Shimadzu Co. Ltd., Kyoto, Japan). Symbols: ×, JM101F⁻; ▵, ts8; □, ts124.

FIG. 3.

Schematic presentation of the mutations in the temperature-sensitive mutants, ts8 and ts124. PCR was carried out by using genomic DNA from the mutant bacteria as a template and two primers, 5′-GCACACGAATTCTGCGCCCGACTTC-3′ and 5′-GATATAGGAATTCTCGATAATTAAC-3′. The resultant fragments (1.2 kb) were cloned into the EcoRI site of pUC18. Plasmids were isolated by the alkaline lysis method (4). The methods used for digestion with restriction enzyme, agarose gel electrophoresis and DNA ligation, were those described by Sambrook et al. (21). DNA was sequenced by the chain termination method by using materials and protocols from a DYEnamic ET terminator cycle sequencing kit. Eight sequencing primers for the 1.2-kb insert were synthesized by KURABO Co. (Osaka, Japan). To avoid PCR errors, three independent clones were used for sequencing. Shadowed letters in the figure indicate mutated bases.

FIG. 4.

Growth comparisons of the deletion mutants in the various tRNA genes and in the htrB gene of the isogenic strain. W3110 wild-type and mutant bacteria were cultured overnight in Luria-Bertani medium at 32°C, diluted, and spotted on a Luria-Bertani plate. The plate was incubated at 42°C overnight and photographed. The ΔleuX deletion mutant was described previously (21). Other deletion mutants used in this experiment were constructed by following the same procedures described in reference : leuX and thrW were replaced by the kanamycin resistance (Km^r) gene from pUC4KAPA (Pharmacia, Uppsala, Sweden), and serU was replaced by the chloramphenicol resistance (Cm^r) gene from pHSG399 (Takara Shuzo, Kyoto, Japan). The htrB1::Tn10 marker (tetracycline resistance [Tet^r] gene) in strain MLK53 (13) and other drug resistance markers were moved with phage P1-mediated transduction into the wild-type strain W3110 in our laboratory stocks. The replacement of drug resistance genes in the mutant strains was verified by DNA sequencing of PCR products amplified from the regions including the junction between the drug resistance gene and host chromosome.