Niels P. Fiil

Accumulation and turnover of guanosine tetraphosphate in Escherichia coli

Abstract A strain ofEscherichia coli B possessing a temperature-labile valyl-transfer RNA synthetase (strain NF 342) has been used to study guanosine tetraphosphate metabolism. Culturing this strain at a variety of temperatures intermediate between the permissive and non-permissive (30 to 42 °C) yielded a range of growth rates, presumably as a result of varying degrees of limitation for charged valyl-tRNA. The rate of RNA accumulation and the level of guanosine tetraphosphate were measured at these different growth rates. These two parameters were found to be related to one another in a hyperbolic manner, suggesting inhibition of stable RNA synthesis by ppGpp ‡ the concentration corresponding to half-maximal inhibition was approximately 0.1 to 0.2 mM. We suggest a model whereby the transcription initiation frequency of ribosomal RNA and other stringently regulated genes is dependent upon the concentration of a positive control protein, whose transcription-stimulating activity is modulated by the ppGpp concentration. At intermediate temperatures RNA synthesis and growth continue at a reduced rate and a substantial pool of ppGpp accumulates in the cell. This permits entry of exogenous precursors into the intracellular nucleotide pools and enables a study to be made of the interrelation between GTP and ppGpp. Upon amino-acid starvation ppGpp accumulates rapidly, reaching a maximal concentration after 3 to 5 minutes and then declines slightly to a plateau level which is maintained for at least one hour. When [3H]guanosine is added 7 minutes after the onset of valyl-tRNA starvation, the specific activity of ppGpp lags about 3 minutes after that of GTP. The kinetics of labelling with [3H]guanosine are consistent with a rather direct precursor-product relationship between GTP and ppGpp. After prolonged limitation for charged valyl-tRNA, the steady-state rate of ppGpp formation is about half the rate of ppGpp accumulation occurring immediately after the onset of starvation. Release of amino-acid limitation increases the rate of ppGpp degradation at least fourfold. From this it is inferred that not only the rate of ppGpp synthesis, but the rate of its degradation also, is subject to regulation, presumably involving translational processes.

Interaction of alleles of therelA, relC andspoT genes inEscherichia coli: Analysis of the interconversion of GTP, ppGpp and pppGpp

SummaryMutants in thespoT gene have been isolated as stringent second site revertants of therelC mutation. These show varying degrees of the characteristics associated with thespoT1 gene,viz relative amount and absolute levels of both pppGpp and ppGpp and the decay rate of the latter. The entry of3H-guanosine into GTP and ppGpp pools inspoT+ andspoT1 cells either growing exponentially or during amino acid starvation was determined, and the rate of ppGpp synthesis and its decay constant calculated. During exponential growth the ppGpp pool is 2-fold higher, its decay constant 10-fold lower, and its synthesis rate 5-fold lower inspoT- than inspoT+ cells; during amino acid starvation the ppGpp pool is 2-fold higher, its decay constant 20-fold lower, and its synthesis rate 10-fold lower inspoT than inspoT+ cells. In one of the “intermediate”spoT mutants the rate of entry of3H-guanosine into GTP, ppGpp and pppGpp was measured during amino acid starvation. The data form the basis of a model for the interconversion of the guanosine nucleotides in which the flow is:GDP→GTP→pppGpp→ppGpp→Y. Calculations of the rates of synthesis and conversion of pppGpp and ppGpp under various conditions in variousspoT+ andspoT- strains indicate that the ppGpp concentration indirectly controls the rate of pppGpp synthesis.ThespoT1 allele was introduced into various relaxed mutants. It was shown that many phenomena associated with the relaxed response ofrelC and “intermediate”relA mutants were phenotypically suppressed when thespoT1 allele was introduced into these mutants. These double mutants exhibit ppGpp accumulation, rate of RNA accumulation, rate of β-galactosidase synthesis, and heat lability of β-galactosidase synthesized during amino acid starvation similar to the stringent wild-type. It is concluded that the relaxed response is due directly to the lack of ppGpp and that the stringest response is due directly to ppGpp.

A previously unidentified gene in the spc operon of Escherichia coli K12 specifies a component of the protein export machinery

The gene prlA codes for a factor that appears to function in the export of proteins in Escherichia coli. This conclusion is based on the finding that mutations altering the prlA gene product restore export of envelope proteins with defective signal sequences. Previous results showed that the prlA gene lies in an operon (spc) known to code for ten different ribosomal proteins. Our studies show that the prlA gene lies promoter-distal to the last known ribosomal protein gene in this operon. Evidence from gene fusions constructed in vitro suggests that prlA codes for a protein containing at least 300 amino acids. Thus a heretofore unidentified protein specified by a gene within the spc operon appears to be a component of the cellular protein export machinery.

Analysis of the proteins synthesized in ultraviolet light-irradiated Escherichia coli following infection with the bacteriophages ?drif d 18 and ?dfus-3

SummaryThe presence of EF-Tu, RNA polymerase subunit α, and EF-G on the λdfus-3 genome and EF-Tu, ribosomal proteins L7/L12, and RNA polymerase subunit β on the λdrifd 18 genome has been confirmed using a two-dimensional gel electrophoresis technique sensitive to changes in isoelectric point and molecular weight. In this system two EF-Tu gene products could not be resolved. Following infection of ultraviolet light-irradiated Escherichia coli with either λdfus-3 or λdrifd18, the EF-Tu gene, tufA, near 65 minutes on the genetic map is expressed as 3–4 copies per EF-G molecule. The EF-Tu gene, tufB, near 79 minutes on the genetic map, is expressed at about one-third of this rate. α is expressed as 1 copy per EF-G molecule, β as 0.14 per EF-G molecule and L7/L12 as 2.5 per EF-G. These figures compare well with the relative amounts found in exponentially-growing cells, in which the ratio of EF-Tu to EF-G is approximately 5. Almost 90% of the total number of proteins (calculated on a molecular weight basis) which theoretically can be encoded on the λdrifd18 have been identified on the two-dimensional gel.

A relaxed mutant with an altered ribosomal protein L11

SummaryRelaxed mutants of Escherichia coli have been isolated which have an altered electrophoretic mobility of ribosomal protein L11. It can be shown that reversion to stringency in one of these mutants occurs simultaneously with a reversion of the L11 protein to its normal mobility. The L11 structural gene, rplK, maping near rif, is carried by the bacteriophage λcI857S7drifd18, and is most likely identical with relC.

Expression of Escherichia coli Ribosomal Protein and RNA Polymerase Genes Cloned on Plasmids

SummaryFragments of λdrifd 18 DNA with different end-points within the set of structural genes of ribosomal proteins L11 (rplK), L1 (rplA), L10 (rplJ) and L12 (rplL) as well as the β (rpoB) and β′ (rpoC) subunits of RNA polymerase have been cloned on plasmids. These plasmids were transformed in host cells which were mutant for each of the genes, enabling expression of both wild-type (plasmid-borne) and mutant (chromosomal) genes to be differentiated. On the basis of these results we propose the following genetic structure for the region: rplK and rplA are in one operon; rplL, rpoB and rpoC are in a second. Our data suggest the possibility that rplJ is by itself in an operon situated between the other two.

A functional analysis of the rel gene in Escherichia coli

Abstract An F-prime episome carrying the rel † gene was isolated from an Escherichia coli Hfr strain which transfers this gene as an early marker. Strains heterozygotic for the rel gene were constructed and rel + was shown to be dominant over rel − . The kinetics of expression of the stringent phenotype were determined after transfer of the F′ rel + episome into a rel − strain. The rel + allele is expressed immediately after transfer and the recombinants become fully stringent after 40 minutes.

Isolation of a transducing phage carrying rpsT, the structural gene for ribosomal protein S20

Summaryλ transducing phages carrying segments of the Escherichia coli chromosome in the dapB region have been isolated and their in vivo gene products analyzed by two-dimensional gel electrophoresis. One of these phages, λddapB-2, carries the structural genes for ribosomal protein S20 (rpsT) and isoleucyltransfer RNA synthetase (ileS). The most likely gene order is thr-rpsT-ileS-dapB-pyrA.

Nonsense and insertion mutants in the relA gene of E. coli: Cloning relA

We have made use of lysogens of a specialized transducing bacteriophage, lambdapyrG+ relA+, to select nonsense (relAnon) and insertion (relAins) mutations in the relA gene. Three independent relAnon mutants were isolated on the phage. In all three, the relaxed phenotype was suppressed by supD, supE, supF or sup6. Three independent relAins mutants were isolated, all containing an insertion element (probably IS2) in an apparently identical location in the relA gene. Polyacrylamide gel electrophoretic analysis of peptides synthesized by the phages in ultraviolet lightkilled host cells revealed that no stringent factor was coded for by either the relAins or relAnon phages (the latter in a sup+ cell); stringent factor was detected when the relAnon phages were used in a similar experiment with supD or supE host cells. The relAnon and relAins mutations could be crossed in haploid form in the E. coli chromosome. These recombinants grew with a normal doubling time, had a ppGpp pool which was between 70 and 100% compared with the classical relA strain, and underwent a normal carbon source shift-down. A restriction endonuclease map of the pyrG relA region of the specialized transducing phage is presented in which the position of the insertion element (recognized by a novel Hind III-cut site) defines the position of the relA gene. This position was verified by an analysis of the structure of five plasmids formed by cloning portions of the region in the pBR322 cloning vehicle. Our results indicate that the relA gene is not an essential cellular function, that there might be a second mechanism for the synthesis of basal level ppGpp in the cell and that the sole function of the relA gene is apparently the high level ppGpp synthesis triggered in response to deacylated tRNA.

A transducing bacteriophage λ carrying the structural gene for elongation factor Ts

SummaryA specialized transducing bacteriophage λdpolCdapD-9 has been isolated that carries the structural gene for EF-Ts1 (tsf). The presence of EF-Ts among the proteins synthesized under the direction of this phage in UVL-inactivated cells has been detected by two-dimensional gel electrophoresis and has been verified by antibody precipitation. In an induced lysogen of this phage the relative rate of synthesis of EF-Ts is increased 4-fold. Evidence is presented which suggests that the structural genes for ribosomal protein S2 (rpsB) and RNA polymerase σ factor (sit) also lies on λdpolCdapD-9.