A New Relaxed Mutant of Escherichia coli with an Altered 50S Ribosomal Subunit

Abstract

Cultures were grown with reciprocal shaking at 370 in either phosphate-buffered medium M9 (10) or in Tris medium (11) with 0.2% glucose (w/v) as energy source. These growth media contained 20 pg/ml of -proline, 20 pg/ml of zAeucine, 10 pg/ml of uracil, and 50 pg/ml of all other growth requirements. To achieve rapid amino-acid starvation, cultures were shifted by membrane filtration to growth medium lacking amino acids. For shift-down experiments, cultures were grown in medium containing 0.01% glucose and 0.2%o sodium acetate. Large cultures for ribosome preparation (5 liters or more) were grown at 370 in L-broth (12) plus 1% glucose with vigorous aeration to A4W = 2.0 (about 5 X 108 cells per ml). The cultures were cooled rapidly with distilled-water ice and harvested in a cooled Sharples continuous-flow centrifuge. Transductions were carried out in Lbroth as described by Lennox (12).Radioactive Labeling. Protein and RNA synthesis were measured by adding ["4Cjproline (0.5 p&Ci/ml) and [5-8H]uracil (5 pCi/ml) (New England Nuclear Corp., Boston, Mass.) to a growing culture at the appropriate time. Samples (100 pl each) were pipetted into 2 ml of ice-cold 5% trichloroacetic acid; samples were prepared and the radioactivity was counted as described (11). Guanosine tetraphosphate measurements were made on cultures growing in Tris medium (0.35 mM phosphate) to which 20-50 ;pCi/ml of ['2PJortho-phosphate had been added at least one-half generation before the first sampling. Samples of 50 pul were pipetted at intervals into ice-cold tubes containing 5 ;pl of 13 M formic acid and were chromatographed as described (11). Tests for stringency or relaxedness in suspected mutants or in recombinants were most reliably done as follows: colonies were picked from agar plates, resuspended in separate tubes containing 0.25 ml of the appropriate selective (minimal) growth medium containing 20,ug/ml of jAeucine and 10 pg/ml'of uracil, and grown overnight with shaking. Next day each culture was diluted about 50-fold into 0.5 ml of the same growth medium and incubated with shaking at 370 for 2-4 hr. Then 1 drop of a mixture containing 500 pg/ml of 5-methyl tryptophan (or

Regulation of Escherichia coli RelA Requires Oligomerization of the C-Terminal Domain

“…Furthermore, the full-length RelA proteins with mutations in the CTD (RelA-C612G, RelA-D637R, and RelA-C638F) were still found to be ribosome associated. Our findings are supported by the fact that in relC mutants, while the endogenous RelA remained ribosome bound, it maintained only 10% of its normal activity (9). Thus, it seems that even though in these cells the ribosomes contain a mutant form of ribosomal protein L11, they can still bind RelA.…”

“…In vitro, ribosomes from the relC strain have only 10% of the (p)ppGpp synthetic activity of ribosomes from the wild-type strain (9). Previously in our laboratory, it was shown that when they were overexpressed separately in the relC strain, the synthetic activity of the full-length RelA was severely impaired, while the truncated N-terminal RelA fragment was active (29 sensitivity test.…”

A relaxed (rel) mutant of Streptomyces coelicolor A3(2) with a missing ribosomal protein lacks the ability to accumulate ppGpp, A-factor and prodigiosin

“…Ochi seen on the gel, although it might have been hidden by another protein spot. Strain 63 can thus be classified as a relC mutant, and the missing protein was tentatively designated ST-L1 1 by analogy with E. coli and B. subtilis mutants which lack ribosomal proteins L11 and BS-L1 1, respectively (Friesen et al, 1974;Smith et al, 1978Smith et al, , 1980. Antibiotic resistance.…”

Involvement of the N Terminus of Ribosomal Protein L11 in Regulation of the RelA Protein of Escherichia coli

“…Such mutations map in either the relA (1) or the relC (11,20) genes. The relC gene was later shown to be identical to rplK, the gene encoding ribosomal protein L11.…”