Stringency without ppGpp accumulation

(7) have proposed that the coincident restrictions in biosynthetic activity observed following such a light transition (7,11,16,23) are mediated by ppGpp. This proposal is especially compelling with regard to cellular phospholipid synthesis, because both the percent inhibition of the rate of phospholipid synthesis and the extent of accumulation of cellular ppGpp were found to be directly related to the magnitude of the light transition. However, a direct involvement of ppGpp in the control of phospholipid synthesis or other cellular biosynthetic activities was not established, and the mechanism responsible for the light-mediated formation of ppGpp was not identified.To evaluate the potential effector role of ppGpp in the coordination of metabolic processes in R. sphaeroides, we found it of interest to determine whether R. sphaeroides possessed a typical ribosome-dependent, Rel+ mechanism for the production of ppGpp and, if present, whether this mechanism was involved in the light-mediated formation of ppGpp. Cells of R. sphaeroides M29-5 (Leu-Met-) were grown in t succinic acid minimal medium as described by Lueking et al. (24) and were deprived of leucine by being washed and suspended in medium lacking this amino acid. Cells used for nucleotide measurements were adapted and grown in the low.phosphate minimal medium described by Campbell and Lueking (8). Phototrophic growth was conducted under an atmosphere of 95% N2-5% CO2 at 31°C and with saturrating illumination (5,380 Ix). Chemotrophic growth was conducted aerobically in the dark with constant sparging

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confidence: 92%

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confidence: 99%

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confidence: 99%

See 1 more Smart Citation

Stringency in the absence of ppGpp accumulation in Rhodobacter sphaeroides

Acosta¹,

Lueking²

1987

“…Because the function of so many cellular tRNA species is influenced by PSUI-mediated pseudouridine modification, mutations in hisT have strong pleiotropic effects on cellular metabolism and regulation (see references in reference 13). For this reason, hisT mutants have been particularly valuable in studies of the effects of undermodification on translation (17), attenuation (7,11), and cellular physiology (11,18,19).…”

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confidence: 99%

Structural analysis of the Escherichia coli K-12 hisT operon by using a kanamycin resistance cassette

Arps¹,

Winkler²

1987

We constructed a series of recombinant plasmids containing a kanamycin resistance (Kmr) cassette upstream from, within, and downstream from hisT, which encodes the tRNA modification enzyme pseudouridine synthase I. These Kmr insertions were then crossed directly into the bacterial chromosome. We determined growth characteristics, assayed in vivo hisT expression, and mapped in vivo hisT operon transcripts for the Kmr insertion mutants. We also analyzed polypeptides synthesized in minicells from plasmids containing Kmr cassettes. The combined results from these experiments demonstrate new features concerning the structure and expression of the complex operon that contains hisT. We show that the minimum size of the operon is approximately 3,500 base pairs and that it contains at least four genes, which are arranged in the order usg-2 (pdxB), usg-1, hisT, and dsg-l and encode polypeptides with apparent molecular masses of 42,000, 45,000, 31,000, and 17,000 daltons, respectively. Of these genes, only the functions of usg-2 (pdrB) and hisT are known, and genetic evidence suggests that these two genes do not require usg-l or dsg-l for function. usg-2 (pdxB) is required for growth of bacteria on minimal medium at 37°C. In contrast, the three genes at the end of the hisT operon are dispensable and form a transcription unit that is expressed from a relatively strong internal promoter. The phenotypes of the Kmr insertion mutants and results from gene expression experiments further confirm the position of the internal promoter and locate additional genetic signals in the DNA sequence around hisT. The experiments reported here also indicate several interesting properties of the Kmr cassette as a tool for probing complex operons.

“…Mutations in hisT and miaA, which cause deficiencies in pseudouridine (38,39,40) and ms2io6A37, respectively, influence the synthesis (7,8,19,50,66) and degradation (29) of many amino acids through tRNAmediated attenuation. However, several regulatory features of such modification-deficient strains are not easily reconciled with regulation through attenuation (3,6,8,46,52,57,65). The nuvC gene in E. coli encodes an enzyme that is needed both for 4-thiouridine synthesis in tRNA and for thiamine biosynthesis (53).…”

Section: Discussionmentioning

confidence: 99%

Isolation of the gene (miaE) encoding the hydroxylase involved in the synthesis of 2-methylthio-cis-ribozeatin in tRNA of Salmonella typhimurium and characterization of mutants

Persson

Björk

1993

The modified nucleoside 2-methylthio-N-6-isopentenyl adenosine (ms2i6A) is present at position 37 (3' of the anticodon) of tRNAs that read codons beginning with U except tRNASer in Escherichia coli. Salmonella typhimurium 2-methylthio-cis-ribozeatin (ms2io6A) is found in tRNA, probably in the corresponding species that have ms2i6A in E. coli. The gene (miaE) for the tRNA(ms2io6A)hydroxylase of S. typhimurium was isolated by complementation in E. coli. The miaE gene was localized close to the argI gene at min 99 of the S. typhimurium chromosomal map. Its DNA sequence and transcription pattern together with complementation studies revealed that the miaE gene is the second gene of a dicistronic operon. Southern blot analysis showed that the miaE gene is absent in E. coli, a finding consistent with the absence of the hydroxylated derivative of ms2i6A in this species. Mutants of S. typhimurium which have MudJ inserted in the miaE gene and which, consequently, are blocked in the ms2i6A hydroxylation reaction were isolated. Unexpectedly, such mutants cannot utilize the citric acid cycle intermediates malate, fumarate, and succinate as carbon sources.