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At least 19 recordsLinked to original sources
PubMed2006

Staphylococcus aureus mutants with increased lysostaphin resistance.

Staphylococcus simulans secretes lysostaphin, a bacteriolytic enzyme that specifically binds to the cell wall envelope of Staphylococcus aureus and cleaves the pentaglycine cross bridges of peptidoglycan, thereby killing staphylococci. The study of S. aureus mutants with resistance to lysostaphin-mediated killing has revealed biosynthetic pathways for cell wall assembly. To identify additional genes involved in cell wall envelope biosynthesis, we have screened a collection of S. aureus strain Newman transposon mutants for lysostaphin resistance. Bursa aurealis insertion in SAV2335, encoding a polytopic membrane protein with predicted protease domain, caused a high degree of lysostaphin resistance, similar to the case for a previously described femAB promoter mutant. In contrast to the case for this femAB mutant, transposon insertion in SAV2335, herein named lyrA (lysostaphin resistance A), did not cause gross alterations of cell wall cross bridges such as truncations of pentaglycine to tri- or monoglycine. Also, inactivation of LyrA in a methicillin-resistant S. aureus strain did not precipitate a decrease in beta-lactam resistance as observed for fem (factor essential for methicillin resistance) mutants. Lysostaphin bound to the cell wall envelopes of lyrA mutants in a manner similar to that for wild-type staphylococci. Lysostaphin resistance of lyrA mutants is attributable to altered cell wall envelope properties and may in part be due to increased abundance of altered cross bridges. Other lyr mutants with intermediate lysostaphin resistance carried bursa aurealis insertions in genes specifying GTP pyrophosphokinase or enzymes of the purine biosynthetic pathway.

Amino Acid Sequence↗
PubMed1985

Regulation of the expression of the tufB operon: DNA sequences directly involved in the stringent control.

We have located the DNA sequence involved in the stringent control of the Escherichia coli tufB operon. Various deletion and insertion mutants of the promoter locus were constructed by in vitro mutagenesis, and their response to guanosine-5'-diphosphate-3'-diphosphate (ppGpp) was examined in a cell-free transcription system consisting of purified RNA polymerase holoenzyme. The nucleotide sequence (GpCpGpC) from positions -7 to -4 (designating the initiation site of mRNA as position +1) is responsible for the selective inhibition by ppGpp of tufB transcription. Point mutations were then constructed in which each one of the above four nucleotides was replaced by an A or T residue and tested for their response to ppGpp in the in vitro transcription system. The results indicated that the alteration of any nucleotide in the GpCpGpC sequence leads to the loss of the stringent response.

Base Sequence↗
PubMed1980

Functional homology between E. coli ribosomal protein L11 and B. megaterium protein BM-L11.

Ribosomes from the thiostrepton-resistant mutant MJ1 of Bacillus megaterium completely lack a protein designated BM-L11. When assayed in vitro, such ribosomes show an impaired ability to hydrolyse GTP in the presence of the elongation factor EF-G and are unable to support the synthesis of (p)ppGpp in response to the stringent factor. Restoration of both these activities can be achieved by re-addition of either protein BM-L11 or its serological homologue from Escherichia coli, protein L11, implying that these two proteins are related functionally as well as immunologically.

Bacillus megaterium↗
PubMed1980

Genetics and physiology of the rel system of Bacillus subtilis.

Stringent factor (ATP:GTP-3' pyrophosphotransferase) has been purified from wild type Bacillus subtilis and it has been shown that guanosine tetra- and pentaphosphate (ppGpp and pppGpp) are synthesized in vitro in the presence of ribosomes, unacylated tRNA and its specific codon, as has been demonstrated in Escherichia coli. relA, the genetic determinant for the stringent factor, has been mapped on the B. subtilis chromosome by transduction and is found between aroD and leu. The relC locus, defined by mutations which were originally selected by resistance to thiostrepton, has been mapped adjacent to spoOH in the order cysA, spoOH, relC, rif. Sringent factor and ribosomes are functional for the in vitro synthesis of (p)ppGpp in early stages of sporulation (up to at least 4 h). This contradicts the findings of other laboratories.

Bacillus subtilis↗
PubMed1980

Uncharged tRNA inhibits guanosine 3',5'-bis (diphosphate) 3'-pyrophosphohydrolase [ppGppase], the spoT gene product, from Escherichia coli.

The spot gene product from Escherichia coli, the guanosine 3',5'-bis(diphosphate) 3'-pyrophos phohydrolase [ppGppase] catalyzes the specific release of pyrophosphate from the 3'-position of guanosine 3',5'-bis(diphosphate) [ppGpp]; this reaction is significantly inhibited in the presence of uncharged tRNAPhe yeast. Little or no inhibition is observed with Phe-tRNAPhe, tRNAPhe-CpCpAoxi-red or ribosomal RNA (16S and 23S).

Depression, Chemical↗
PubMed1982

The regulation of the ammonia assimilatory enzymes in Rel+ and Rel- strains of Salmonella typhimurium.

The influence of the relA1 mutation on the regulation of the ammonia assimilatory enzymes, glutamate dehydrogenase (EC 1.4.1.4), glutamine synthetase (EC 6.3.1.2), and glutamate synthase (EC 1.4.1.3), was examined. When cells grown in rich media (either Luria broth or glucose-ammonia plus casamino acids) were transferred to a glucose-ammonia medium, the relA mutant failed to resume growth and did not have the same increase in any of the assimilatory enzyme activities as the rel+ strain. This effect was particularly dramatic for glutamate dehydrogenase, which increased 6-fold in the rel+ strain. Measurements of the guanosine nucleotide concentrations showed that the rel+ strain had a ppGpp concentration about 9 times that of the relA mutant 5 min after the shift to minimal medium. These results are consistent with those for other biosynthetic enzymes and show that the ammonia assimilatory enzymes require a relA product for their synthesis during shift from rich to minimal media. In addition, we examined the response of these strains to a change in nitrogen source. The relA mutant again failed to resume growth after a shift from glucose-ammonia to glucose-arginine medium. Even though the ppGpp concentration did not increase, the rel+ strain grew and increased glutamine synthetase activities about 2-fold. These changes the absence of increased ppGpp levels suggest that some other relA-mediated function is important during this change in nitrogen source.

Ammonia↗
PubMed1979

The stimulation of Escherichia coli stringent factor-dependent synthesis of guanosine 3',5'-polyphosphate [(p)ppGpp] by rat liver ribosomal proteins.

The effect of groups of proteins from rat liver ribosomes on the Escherichia coli stringent factor-catalyzed synthesis of (p)ppGpp was tested. Most groups were capable of supporting (p)ppGpp synthesis; the exceptions were A40, B140, B240 and B160 which contain proteins which are relatively less basic than those in the active groups. The capacity of 30 individual rat liver ribosomal proteins to activate stringent factor was assessed; most sustained the synthesis of (p)ppGpp. Proteins S12, S21, L12, P1, and P2 (which are acidic or relatively acid) had no activity; proteins S6, S8, and L3 were the most active: the others had moderate activity.

Animals↗
PubMed1980

The relA locus and the regulation of lysine biosynthesis in Escherichia coli.

The allelic state of relA influences the phenotype of Escherichia coli strains carrying the lysA22 mutation:lysA22 relA strains are Lys- where lysA22 relA+ strains grow (slowly) in the absence of lysine. This physiological effect has been related to an effect of the expression of the relA locus on the regulation of lysine biosynthesis. The fully derepressed levels of some lysine enzymes (aspartokinase III, aspartic semialdehyde dehydrogenase, didhydrodipicolinate reductase) are observed under lysine limitation only in rel+ strains. And the induction of DAP-decarboxylase by DAP is much higher in rel+ than in rel- strains when an amino acid limitation of growth is also realised. These results are in agreement with the hypothesis of Stephens et al. (1975) on a possible role of the stringent regulation as a general signal for amino acid deficiency.

Aspartate Kinase↗
PubMed1985

Cloning of the Escherichia coli gene for the stringent starvation protein.

In order to clone the Escherichia coli gene for the stringent starvation protein (SSP), we determined its N-terminal sequence as well as the sequence of two peptide fragments obtained by cyanogen bromide cleavage of the protein. We then chemically synthesized four sets of oligodeoxyribonucleotide mixtures that represented possible codon combinations for parts of these amino acid sequences. The synthetic oligonucleotides were labelled with 32P at their 5'-termini and used as hybridization probes to detect DNA fragments containing the complementary sequences. Genomic Southern hybridization of E. coli chromosomal DNA gave up to ten DNA fragments hybridizing with each probe but only a few hybridized with two or more of the probes. The latter fragments were cloned in pBR322. By determining partial base sequences with a rapid method and examining proteins encoded by the DNA fragments, we were able to show that we had isolated a clone containing the complete SSP structural gene.

Amino Acid Sequence↗
PubMed1981

Isolation and characterization of prototrophic relaxed mutants of Klebsiella pneumoniae.

A new selection procedure has been developed for isolating prototrophic relaxed mutants of Klebsiella pneumoniae. Two mutants were isolated. One of them showed a fully relaxed phenotype, while the other one behaved in a semi-relaxed way. The wild-type strain, as well as the rel mutants exerted similar patterns to their E. coli counterparts in RNA, protein, ppGpp and pppGpp accumulation during amino starvation, carbon source shift-down and nitrogen starvation. Both mutants became stringent after introducing an F'-factor carrying the relA+ allele from Escherichia coli. The relaxed phenotype could be recovered by curing the F'-factor. Some of the pleiotropic consequences of rel mutations found in E. coli are present in the Klebsiella mutants also while some of them are absent. The mutants are defective in dinitrogen fixation after the exhaustion of limiting ammonium from the culture medium. However, their merodiploid derivatives, carrying the E. coli relA+ allele, showed the wild-type level of nitrogenase activity under the same conditions.

Carbon↗
PubMed2006

The flexible N-terminal domain of ribosomal protein L11 from Escherichia coli is necessary for the activation of stringent factor.

The stringent response is activated by the binding of stringent factor to stalled ribosomes that have an unacylated tRNA in the ribosomal aminoacyl-site. Ribosomes lacking ribosomal protein L11 are deficient in stimulating stringent factor. L11 consists of a dynamic N-terminal domain (amino acid residues 1-72) connected to an RNA-binding C-terminal domain (amino acid residues 76-142) by a flexible linker (amino acid residues 73-75). In vivo data show that mutation of proline 22 in the N-terminal domain is important for initiation of the stringent response. Here, six different L11 point and deletion-mutants have been constructed to determine which regions of L11 are necessary for the activation of stringent factor. The different mutants were reconstituted with programmed 70 S(DeltaL11) ribosomes and tested for their ability to stimulate stringent factor in a sensitive in vitro pppGpp synthesis assay. It was found that a single-site mutation at proline 74 in the linker region between the two domains did not affect the stimulatory activity of the reconstituted ribosomes, whereas the single-site mutation at proline 22 reduced the activity of SF to 33% compared to ribosomes reconstituted with wild-type L11. Removal of the entire linker between the N and C-terminal domains or removal of the entire proline-rich helix beginning at proline 22 in L11 resulted in an L11 protein, which was unable to stimulate stringent factor in the ribosome-dependent assay. Surprisingly, the N-terminal domain of L11 on its own activated stringent factor in a ribosome-dependent manner without restoring the L11 footprint in 23 S rRNA in the 50 S subunit. This suggests that the N-terminal domain can activate stringent factor in trans. It is also shown that this activation is dependent on unacylated tRNA.

Alanine↗
PubMed1992

Drosophila glutathione S-transferases have sequence homology to the stringent starvation protein of Escherichia coli.

The Drosophila glutathione S-transferase D genes encode a family of isozymes. We have determined the amino acid sequence of a new member of this family by nucleotide sequence analysis of a genomic DNA clone. The open reading frame of this intronless gene should encode an isozyme subunit of 211 amino acids. This sequence has significant homology to the E. coli stringent starvation protein, SSP, which is also a protein of two identical 211 amino acid subunits. The two proteins have very similar overall amino acid composition as well. It is possible that SSP may be a glutathione S-transferase(s) in E. coli or is evolutionarily related to glutathione S-transferases. Because SSP is known to be tightly associated with the RNA polymerase holoenzyme during purification, it is conceivable that Drosophila glutathione S-transferase(s) may potentially interact with the transcription machinery in a fashion similar to SSP's interaction with E. coli RNA polymerase holoenzyme.

Amino Acid Sequence↗
PubMed2004

Conformational antagonism between opposing active sites in a bifunctional RelA/SpoT homolog modulates (p)ppGpp metabolism during the stringent response [corrected].

Enzymes of the Rel/Spo family enable bacteria to survive prolonged periods of nutrient limitation by producing an intracellular signaling alarmone, (p)ppGpp, which triggers the so-called stringent response. Both the synthesis of (p)ppGpp from ATP and GDP(GTP), and its hydrolysis to GDP(GTP) and pyrophosphate, are catalyzed by Rel/Spo proteins. The 2.1 A crystal structure of the bifunctional catalytic fragment of the Rel/Spo homolog from Streptococcus dysgalactiae subsp. equisimilis, Rel(Seq), reveals two conformations of the enzyme corresponding to known reciprocal activity states: (p)ppGpp-hydrolase-OFF/(p)ppGpp-synthetase-ON and hydrolase-ON/synthetase-OFF. The hydrolase and synthetase domains bear remarkable similarities to the catalytic domains of the cyclic phosphodiesterase and nucleotidyltransferase superfamilies, respectively. The active sites, separated by more than 30 A, contain bound nucleotides including an unusual (p)ppGpp derivative, GDP-2':3'-cyclic monophosphate. Reciprocal regulation of the antagonistic catalytic activities, suggested by the structure, is supported by mutagenesis experiments and appears to involve ligand-induced signal transmission between the two active sites.

Amino Acid Sequence↗
PubMed1986

The physiology of stringent factor (ATP:GTP 3'-diphosphotransferase) in Escherichia coli.

The enzyme ATP:GTP 3'-diphosphotransferase catalyzes the transfer of the beta, gamma-pyrophosphate of ATP to the 3' position of GTP or GDP. The amounts of enzyme were measured in cell extracts of a relA+ strain of E. coli grown at different growth rates between 0.4 and 1.9 generations per hour, using precipitation with specific antibodies to purify the enzyme. The amount of enzyme was found to be a constant fraction of total protein at all growth rates corresponding to about 45 molecules of enzyme per genome equivalent of DNA. The purified enzyme has little catalytic activity by itself but has to be activated either by a complex of 70S ribosomes, mRNA and uncharged tRNA or by a solvent like ethanol at a concentration of about 20%. The kinetic constants of the enzyme for the transfer pyrophosphate from ATP to GTP in the ribosome-activated state were determined. The Vmax was estimated to be 140 mumol/min X mg at 37 degrees C and the S0.5 values for GTP and ATP were 0.35 and 0.53 mM, respectively. The reaction was estimated to have an equilibrium constant of about 300. In the pyrophosphate transfer from ATP to GDP the Vmax was estimated to be 90 mumol/min X mg at 37 degrees C and the S0.5 for GDP as 0.3 mM. During amino acid starvation of a relA+ strain of E. coli the amounts of enzyme and the catalytic capacity of the enzyme are sufficient to maintain the observed ppGpp levels in the cells at all growth rates.

Adenosine Triphosphate↗
PubMed1980

Escherichia coli elongation factor G blocks stringent factor.

The relationship between the binding domains of elongation factor G(EF-G) and stringent factor (SF) on ribosomes was studied. The binding of highly purified, radioactively labeled, protein factors to ribosomes was monitored with a column system. The data show that binding of EF-G to ribosomes in the presence of fusidic acid and GDP or of the noncleavable analogue GDPCP prevents subsequent binding of SF to ribosomes. In addition, stabilization of the EF-G-ribosome complex by fusidic acid inhibits SF's enzymatic activities. Removal of protein L7/L12 from ribosomes leads to weaker binding of EF-G, while SF's binding and activity are unaffected. In the absence of L7/L12, EF-G-dependent inhibition of SF binding and function is reduced. The data presented in this report suggest that these two factors bind at overlapping, or at least interacting, ribosomal domains.

Escherichia coli↗
PubMed1987

Structure of the gene for the stringent starvation protein of Escherichia coli.

The nucleotide sequence of the gene for the stringent starvation protein (SSP) of E. coli was determined. The deduced amino acid sequences shows that the SSP is composed of 212 amino acid residues, rich in both positively and negatively charged amino acids and has a molecular weight of 24,305. Primer extension experiments and nuclease S1 mapping analysis showed a site on the chromosome DNA corresponding to the 5' end of the transcript of the SSP gene. However, the consensus promoter sequences were not found at the upstream region. In the 3' flanking region a long coding frame was found immediately following the SSP gene, suggesting that the SSP gene is a member of a multicistronic operon.

Amino Acid Sequence↗