A model for regulation of ColE1-like plasmid replication by uncharged tRNAs in amino acid-starved Escherichia coli cells

It has been previously observed that various ColE1-like plasmids replicate differentially in Escherichia coli cells during the relaxed response to amino acid starvation. Here we develop a kinetic model to explain these observations based on the possibility of interaction of the 3' CCA-OH sequence with the UGG triplets in loops of RNA I and RNA II encoded by ColE1-like plasmids. According to our model, when the interaction of uncharged CCA with RNA I is possible, the replication of the ColE1-like plasmid is affected by differences in the concentration of various tRNAs in the starved cell, but it is not affected by the tRNA concentration if the hypothetical pairing occurs between the CCA-OH and RNA II. Using the previously determined parameters for the pBR322 plasmid, the concentration of uncharged tRNAs in the amino acid starved relaxed strains and the assumed efficiency of binding of tRNA and RNA I, we show that our model explains the differences in pBR322 copy number in the relaxed strain starved for several amino acids.     

Suppression of ColE1 high-copy-number mutants by mutations in the polA gene of Escherichia coli.

We isolated three Escherichia coli suppressor strains that reduce the copy number of a mutant ColE1 high-copy-number plasmid. These mutations lower the copy number of the mutant plasmid in vivo up to 15-fold; the wild-type plasmid copy number is reduced by two- to threefold. The suppressor strains do not affect the copy numbers of non-ColE1-type plasmids tested, suggesting that their effects are specific for ColE1-type plasmids. Two of the suppressor strains show ColE1 allele-specific suppression; i.e., certain plasmid copy number mutations are suppressed more efficiently than others, suggesting specificity in the interaction between the suppressor gene product and plasmid replication component(s). All of the mutations were genetically mapped to the chromosomal polA gene, which encodes DNA polymerase I. The suppressor mutational changes were identified by DNA sequencing and found to alter single nucleotides in the region encoding the Klenow fragment of DNA polymerase I. Two mutations map in the DNA-binding cleft of the polymerase region and are suggested to affect specific interactions of the enzyme with the replication primer RNA encoded by the plasmid. The third suppressor alters a residue in the 3'-5' exonuclease domain of the enzyme. Implications for the interaction of DNA polymerase I with the ColE1 primer RNA are discussed.     

RelA mutation and pBR322 plasmid amplification in amino acid-starved cells of Escherichia coli

Plasmid pBR322 is amplified following amino-acid limitation in Escherichia coli relA hosts. In relA+ hosts there was no significant amplification or a much smaller one. Plasmid amplification is due to the relA mutation; when the relA+ allele is transferred into the relA mutant CP79 this strain no longer amplifies plasmid DNA during amino acid starvation. It is concluded that ppGpp is a negative effector of plasmid replication. Amplification is temperature dependent, being maximal at 32 degrees C and negligible at 37 degrees C.     

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.     

pMS76, a plasmid capable of amplification by treatment with chloramphenicol

pMS76 is a nonconjugative, 5.54-megadalton plasmid. This plasmid is present in Escherichia coli K12 cells at about 20 copies per chromosome. In addition, the pMS76 plasmid can be mobilized by conjugative plasmids and it shares a number of other properties with the amplifiable ColE1 plasmid, including the ability to amplify copy number in the presence of chloramphenicol. However, pMS76 is compatible with ColE1-like replicons, pBR313, and with other multicopy plasmids, RSF1030 and pACYC184. Also the replication of pMS76 is rifampicin sensitive and requires DNA polymerase 1.     

Characterization of a mini-ColC1 plasmid.

An in vitro constructed plasmid, pVH15, consisting of the entire genome of the plasmid ColE1, the tryptophan operon of Escherichia coli, and regions of the bacteriophage PHI80pt190, spontaneously gave rise in E. coli to a mini-ColE1 plasmid consisting of approximately one-half of the ColE1 genome and a small segment of phi80pt190 DNA. This mini-ColE1 plasmid, designated pVH51, has a molecular weight of approximately 2.1 X 10(6) and possesses a single EcoRI restriction site. Heteroduplex analyses showed that about 90% of the pVH51 plasmid hybridizes to about 50% of the ColE1 plasmid. Phenotypically, pVH51 did not produce colicin E1 but conferred immunity to this colicin. The number of mini-ColE1 plasmid molecules per cell was maintained at a four- to fivefold higher level than normal ColE1. A mini-ColE1 hybrid plasmid, designated pML21 and consisting of pVH51 and the kan fragment of plasmid pSC105 inserted at the EcoRI restriction site of mini-ColE1, was maintained at a lower copy number level than pVH51. As in the case of normal ColE1, both pVH51 and pML21 continued to replicate in the presence of chloramphenicol. The promotion of conjugal transfer of pVH51 and pML21 by a self-transmissible plasmid was greatly reduced compared with normal ColE1.     

Isolation of chromosomal mutations that affect carotenoid production in Escherichia coli: mutations alter copy number of ColE1-type plasmids

Chromosomal mutants were isolated in Escherichia coli that altered carotenoid production from transformed carotenoid biosynthesis genes on a pACYC-derived plasmid (pPCB15). The mutations were mapped by sequencing. One group of mutations appeared to affect the cell metabolism without changing the copy number of the carotenoid synthesis plasmid. The other group of mutations either increased or decreased the copy number of the pPCB15 plasmid as determined by real-time PCR. The copy number change in most mutants was likely specific for ColE1-type plasmids for which copy number is controlled by a small antisense RNA. This collection of host strains would be useful for fine tuning expression of proteins and adjusting production of desired molecules without recloning to different vectors.     

Cloning and characterization of a relA/spoT homologue from Bacillus subtilis

A PCR-amplified DNA fragment of the relA gene from genomic Bacillus subtilis DNA was used to isolate the entire relA / spoT homologue and two adjacent open reading frames (ORFs) from a λ ZAP Express library. The relA gene, which encodes a protein of 734 amino acid residues (aa), is flanked by an ORF (170 aa) that shares high similarity to adenine phosphoribosyltransferase genes ( apt ), and downstream by an ORF (131 aa) of unknown function. This genetic organization is similar to that in Streptomyces coelicolor A3(2) and Streptococcus equismilis H46A. relA shows significant similarity to the Escherichia coli relA and spoT genes, which are responsible for the synthesis and degradation of the highly phosphorylated guanosine nucleotides (p)ppGpp, triggering the stringent response. Deletion of the relA gene generated a (p)ppGpp⁰ phenotype that demonstrated its essential role in the response to amino acid deprivation and resulted in impaired/lowered induction of proteins involved in stress response as well as amino acid biosynthesis, as judged by two-dimensional gel electrophoresis. The same effects of impaired induction of some σ^B-independent proteins could also be shown in a sigB/relA double mutant, supporting the role of relA in derepression/induction of catabolic and anabolic genes during stringent response.     

Stringent and growth control of rRNA synthesis in Escherichia coli are both mediated by ppGpp

Weak stringent or relaxed responses were induced in Escherichia coli (relA+), using mild amino acid starvation or treatment with chloramphenicol at low concentrations, respectively, such that the growth rate was barely reduced. In this manner, the intracellular concentration of the nucleotide guanosine tetraphosphate, ppGpp, could be varied in any desired range between 0 and 1000 pmol of ppGpp per OD460 unit of culture mass. At the same time, the rate of synthesis of stable RNA (rs; rRNA and tRNA) was measured, relative to the total instantaneous rate of RNA synthesis (rt). The correlation between the cytoplasmic concentration of ppGpp and stable RNA gene activity (rs/rt) was the same as that observed previously with relA+ and relA strains growing exponentially at different rates in different media. This suggests that the distinction between growth control and stringent control of stable RNA synthesis is arbitrary, and that both kinds of control reflect the same ppGpp-dependent phenomenon. By increasing the stable RNA gene dosage, using high copy number plasmids carrying an rrn gene, we have tested the idea that ppGpp partitions the bacterial RNA polymerase into two forms with different probabilities to initiate at stable RNA and mRNA promoters. The relaxed response was not significantly altered, but the extent of the stringent response was reduced by the presence of extra rrn genes. The results agree with quantitative predictions derived from the RNA polymerase partitioning hypothesis.     

Suppressive effect of l-phenylalanine on lignin peroxidase in the white-rot fungus Phanerochaete chrysosporium

Abstract Guanosine-5'-diphosphate-3'-diphosphate (ppGpp), an effector for many metabolic pathways, is synthesized by the relA gene product after amino acid limitation. Studies of stringent controlled Escherichia coli CP78 (relA⁺) and relaxed controlled E. coli CP79 (relA⁻) were carried out to test whether these strains differ in the appearance of their cytoplasmic membranes after induction of stringent and relaxed response. Cytoplasmic membrane structures of the cells were investigated by freeze-fracture electron microscopy after cooling the cells. The obtained micrographs showed a net-like distribution of the particles in the cytoplasmic membranes of relaxed controlled cells whereas such a pattern was not detectable in the stringent controlled counterparts.