Transcription of ribosomal protein genes carried on F'' plasmids of Escherichia coli.

Summary Spontaneous mutants of the petite-negative yeast Kluyveromyces lactis, resistant to the antibiotics chloramphenicol and oligomycin, were isolated and genetically characterized.Three chloramphenicol-resistant mutants showed non-Mendelian inheritance when crossed to sensitive parents.Of 5 oligomycin-resistant strains studied, three exhibited resistance due to the presence of an extrachromosomal mutation. The resistance of the other two deriving from a nuclear and recessive mutation.When two factor crosses in trans configuration were performed between two of the chloramphenicol and the five oligomycin-resistant mutants a polarity in recombination was observed with a predominance of sensitive (O^SC^S) over resistant (O^RC^R) reciprocal recombinants.Allelism tests carried out among the oligomycin-resistant mutants indicated the presence of at least two distinct extrachromosomal regions responsible for the resistance.     

Influence of low-temperature storage and glucose starvation on growth recovery in Escherichia coli relA and relA+ strains.

To study the influence of microgravity on bacterial growth behavior during a space mission, the special experimental conditions and the hardware environment necessitate storage of cells at low temperature, and permit a relatively short experimental period. Before this experimental period, cells have to recover their condition of steady-state growth, because it is only in this condition that the growth behavior of the flight and ground populations can be adequately compared. To meet these requirements and to obtain cells which recover rapidly their steady-state growth, we analyzed the size and shape of Escherichia coli cells during storage at 4 degrees C, with and without previous glucose starvation of the cells. It appeared that cells stored at low temperature in the presence of glucose continued to increase in average mass and assumed ovoid shapes. In addition, upon restoration of maximal growth rate at 37 degrees C, they continued to increase in size and showed a transient overshoot of their final steady-state value, which was reached after about 5 h. Cells previously starved for glucose, however, maintained their average size and rod-shape during low-temperature storage. Recovery of the starved cells was most rapid in the relA+ strain which, contrary to the isogenic relA strain, showed no overshoot and reached its final steady-state size within 2 h.     

Characterization of the relA1 mutation and a comparison of relA1 with new relA null alleles in Escherichia coli

The most widely studied "relaxed" mutant of the relA locus, the relA1 allele, is shown here to consist of an IS2 insertion between the 85th and 86th codons of the otherwise wild-type relA structural gene, which normally encodes a 743-amino acid (84 kDa) protein. The RelA protein is a ribosome-dependent ATP:GTP (GDP) pyrophosphoryltransferase that is activated during the stringent response to amino acid starvation and thereby occasions the accumulation of guanosine 3',5'-bispyrophosphate (ppGpp). We propose that the IS2 insertion functionally splits the RelA protein into two (alpha and beta) peptide fragments which can complement each other in trans to yield residual ppGpp synthetic activity; neither fragment shows this activity when expressed alone. Cell strains with a single copy relA null allele show physiological behavior that is much the same as relA1 mutant strains. Both relA1 and relA null strains accumulate ppGpp during glucose starvation and do not accumulate ppGpp during the stringent response. The presence of ppGpp in verifiable relA null strains is interpreted as unequivocal evidence for an alternate route of ppGpp synthesis that exists in addition to the relA-dependent reaction.     

Escherichia coli relA strains as hosts for amplification of pBR322 plasmid DNA

Abstract We have proposed that guanosine tetraphosphate produced in Escherichia coli cells subjected to an isoleucine limitation inhibits pBR322 DNA replication [1]. In E. coli relA which cannot synthesize guanosine tetraphosphate (ppGpp) upon amino acid limitation pBR322 DNA is amplified after arginine starvation. The yield of plasmid DNA amplified either by chloramphenicol (Cm) or by arginine limitation is compared. The plasmid yield per cell is equal in amino acid-starved cells and in cells treated with Cm. To increase the plasmid content per ml of cell suspension the growth medium was supplemented with increasing amounts of nutrients. Plasmid DNA can be isolated in large quantities by this procedure. This simple method can be used for the enrichment of pBR325 DNA which cannot be amplified by Cm treatment. Our results indicate that E. coli relA strains might be suitable hosts for the amplification of pBR322 and related plasmids in E. coli .     

Involvement of the lac regulatory genes in catabolite repression in Escherichia coli

1. Acute transient catabolite repression of beta-galactosidase synthesis, observed when glucose is added to glycerol-grown cells of Escherichia coli (Moses & Prevost, 1966), requires the presence of a functional operator gene (o) in the lactose operon. Total deletion of the operator gene abolished acute transient repression, even in the presence of a functional regulator gene (i). 2. Regulator constitutives (i(-)) also show transient repression provided that the operator gene is functional. Regulator deletion mutants (i(del)), with which to test specifically the role of the i gene, have not so far been available. 3. The above mutants, showing various changes in the lactose operon, show no alteration in the effect of glucose on induced tryptophanase synthesis. Glucose metabolism, as measured in terms of the release of (14)CO(2) from [1-(14)C]glucose and [6-(14)C]glucose, also showed no differences between strains exhibiting or not exhibiting transient repression. This suggests no change in the operation of the pentose phosphate cycle, a metabolic activity known to be of paramount importance for glucose repression of beta-galactosidase synthesis (Prevost & Moses, 1967). 4. Chronic permanent repression by glucose of beta-galactosidase synthesis (less severe in degree than acute transient repression) persists in strains in which transient repression has been genetically abolished. Constitutive alkaline-phosphatase synthesis, which shows no transient repression, also demonstrates chronic permanent repression by glucose. 5. Chloramphenicol repression also persists in mutants with no transient repression, and also affects alkaline phosphatase. It is suggested that chronic permanent repression and chloramphenicol repression are non-specific, and that they do not influence beta-galactosidase synthesis via the regulatory system of the lactose operon.     

Synthesis of ribosomal proteins in merodiploid strains of Escherichia coli

Summary The regulation of the synthesis of r-proteins in Escherichia coli was investigated by increasing the dosage of the genes for a limited number of ribosomal proteins (r-proteins) using either transducing phage fus 3 (Lindahl et al. 1977) or rif ^d18 (Kirschbaum and Konrad 1973). During exponential growth the presence in the cell of either lysogenised transducing phage did not increase the rate of synthesis or degradation of any of the 31 r-proteins whose genes are duplicated. Experiments were also performed to determine whether r-protein synthesis during the period of unbalanced r-protein synthesis that follows nutritional enrichment was sensitive to an increase in gene dosage. Duplication of the 27 r-protein genes on fus 3 did not alter the rate of synthesis of any of the r-proteins after enrichment. However, gene dosage effects were detected for at least 3 of the r-proteins whose genes were duplicated of rif ^d18.     

Escherichia coli K12 relA strains as safe hosts for expression of recombinant DNA

Most Escherichia coli K12 strains survive for a relatively long time outside the laboratory. Under the same conditions the isoallelic E. coli K12 relA mutants die faster because they lack the stringent response. The killing rate is increased by using a plasmid-encoded suicide system consisting of the phage T7 lysozyme gene driven by the E. coli alkaline phosphatase gene promoter (phoA). Cells containing this system were rapidly and effectively killed as soon as phosphate was made limiting. The combination of the chromosomal relA mutation and a conditional suicide system of this type provides an effective means of biological containment for recombinant E. coli strains.     

Salt tolerance of lactose-grown Vibrio parahaemolyticus carrying Escherichia coli lac genes

Lac- strains of Vibrio parahaemolyticus were converted to Lac+ on receiving a hybrid plasmid containing the lactose utilization genes of Escherichia coli K-12. A V. parahaemolyticus strain containing this hybrid plasmid exhibited optimal growth rates on glucose and other carbon sources in the presence of 0.2 to 0.4 M NaCl. Growth of the same strain on lactose was inhibited at similar concentrations of NaCl. The altered growth rate responses in lactose medium appeared to be attributable to effects of NaCl on the activity of lactose permease, and possibly on that of beta-galactosidase, rather than on the levels of these enzymes in V. parahaemolyticus cells.     

Genetic studies of the ribosomal proteins in Escherichia coli. 3. Compositions of ribosomal proteins in various strains of Escherichia coli

Summary The comparative chromatographic investigations into the ribosomal proteins of various strains of E. coli have demonstrated that most of the strains including three strains of E. coli subsp. communior had ribosomes with the same protein compositions (C-type). The ribosomes from strain B are different from the C-type ribosomes in having the specific 30-4 (B) component in place of 30-4 (B-type), while those from strains K 12 may be distinguished from the type-C ribosomes by the presence of the specific 30-7 (K) component in place of 30-7 (K-type) or, in addition to 30-7 (K), the presence of 30-9 (W3637) in place of 30-9 (K-3637 type). Two strains, IAM 1132 and IAM 1182, have R-type ribosomes, in which at least six 50s proteins and four 30s protein components are distinct from the corresponding protein components in the C-type ribosomes.     

Protein sequences encoded by the relA and the spoT genes of Escherichia coli are interrelated

relA and spoT are designations for two unlinked Escherichia coli genes whose products function in the synthesis and degradation of guanosine 3',5'-bispyrophosphate during the stringent regulatory response to amino acid deprivation. The RelA protein catalyzes an ATP:GTP 3'-pyrophosphoryl group transfer reaction, and the SpoT protein has a guanosine 3',5'-bispyrophosphate 3'-pyrophosphohydrolyase activity. Both genes have been sequenced recently; the relA gene produces an 84-kDa protein, and the spoT gene is deduced to encode a 79-kDa protein. We report here that the protein sequences of the relA and spoT genes are extensively interrelated.