Polyadenylation can regulate ColE1 type plasmid copy number independently of any effect on RNAI decay by decreasing the interaction of antisense RNAI with its RNAII target

Replication of ColE1-type plasmids is regulated by RNAI, an antisense RNA that interacts with the replication pre-primer, RNAII. Exonucleolytic attack at the 3' end of RNAI is impeded in pcnB mutant bacteria, which lack poly(A) polymerase I-the principal RNA polyadenylase of E. coli; this leads to accumulation of an RNAI decay intermediate (RNAI(-5)) and dramatic reduction of the plasmid copy number. Here, we report that polyadenylation can also affect RNAI-mediated control of plasmid DNA replication by inhibiting interaction of RNAI(-5) with RNAII. We show that mutation of the host pcnB gene profoundly affects the plasmid copy number, even under experimental conditions that limit the effects of polyadenylation on RNAI(-5) decay. Moreover, poly(A) tails interfere with RNAI/RNAII interaction in vitro without producing any detectable alteration of RNAI secondary structure. Our results establish the existence of a previously undetected mechanism by which RNA polyadenylation can control plasmid copy number.     

Maintenance of pBR322-derived plasmids without functional RNAI

pBR322-derived plasmids that lack the bla gene and 40% of the gene for the replication inhibitor, RNAI, have been constructed. Since the RNAI gene totally overlaps with the gene for the replication primer, RNAII, this primer is similarly defective and also lacks its normal promoter. The primer is presumed to by synthesized either from the counter-tet promoter (plasmid pCL59) or from an inserted lacUV5 promoter (plasmid pCL59-65). Based mainly on the observation that the plasmid Rom protein, which normally assists in the RNAI/RNAII interaction, has no effect on the replication of the RNAI/RNAII-defective plasmids, we suggest that the defective RNAI is not functional while the defective RNAII primer, although less efficient, still allows plasmid replication. The defective plasmids are fully compatible with the intact parent plasmid, indicating that they do not share a common control of replication. In the absence of antibiotics, the bacteria lose the defective plasmid, beginning after 80 generations; under the same conditions, the parent plasmid is retained even after 140 generations. During exponential growth of their host, the number of defective plasmids in a culture increases exponentially with a doubling time either smaller or greater than that of the host cell growth, depending on the growth medium and, in the case of pCL59-65, on the presence or absence of lac inducer IPTG. As a result of these differences in host cell growth and plasmid replication, the plasmids are either gradually diluted out or their copy number continually increases. This shows that, without RNAI, plasmid replication is uncoupled from the host cell growth and not, as usual, adjusted to it. It also implies that the RNAI mechanism is the only means of replication control for ColE1-type plasmids that senses and adjusts the copy number; limiting host factors cannot provide a back-up control to stabilize copy numbers.     

PcnB is required for the rapid degradation of RNAI, the antisense RNA that controls the copy number of CoIE1-related plasmids

The replication of CoIE1-related plasmids is controlled by an unstable antisense RNA, RNAI, which can interfere with the successful processing of the RNAII primer of replication. We show here that a host protein, PcnB, supports replication by promoting the decay of RNAI. In bacterial strains deleted for pcnB a stable, active form of RNAI, RNAI*, which appears to be identical to the product of 5′-end processing by RNAse E, accumulates. This leads to a reduction in plasmid copy number. We show, using a GST- PcnB fusion protein, that PcnB does not interfere with RNAI/RNAII binding in vitro. The fusion protein, like PcnB, has polyadenylating activity and is able to polyadenylate RNAI (and also another antisense RNA, CopA) in vitro.     

Inheritance of the replication complex by one of two daughter copies during lambda plasmid replication in Escherichia coli.

Direct measurement of DNA synthesis confirmed that lambda plasmid replication proceeds for several hours in an amino acid-starved relA mutant of Escherichia coli, leading to plasmid amplification; this replication is lambda cro-independent, but requires the function of lambda O initiator in the absence of its synthesis. This suggests that after the assembly of the replication complex (RC) at ori lambda the lambda O protein remains in this structure and the affinity of lambda O to ori lambda is alleviated in the assembled RC allowing its movement along the DNA. During amino acid starvation the lambda plasmid DNA synthesis per bacterial mass occurs at a constant level, as would be expected if the number of functioning RCs remained constant. This favors the idea that under these conditions the next replication round operates due to the activity of the RC inherited from the preceding round. Density shift experiments reveal indeed that, from two daughter plasmid copies synthesized after the onset of amino acid starvation only one is able to enter into the next round of replication. We infer that this is the plasmid copy that inherits the lambda O-enclosing RC from the previous replication round. Moreover, the same results of density shift experiments were obtained for plasmids synthesized before the onset of amino acid starvation. Therefore, we presume that in lambda plasmid-harboring bacteria growing in nutrient medium, every second plasmid circle bears an RC that originates from the preceding round of replication. This structure has to be assembled de novo only on the daughter plasmid copy that does not inherit the parental RC. In the absence of lambda O initiator synthesis in amino acid-starved relA cells this process cannot occur, leaving as the only replication pathway that driven by the parental RC. Our results are discussed in relation to the model of regulation of lambda plasmid replication.     

Role of peptide chain elongation factor G in guanosine 5'-diphosphate 3'-diphosphate synthesis.

In a wild-type strain (relA+) of Escherichia coli, starvation of amino acid led to an immediate cessation of the synthesis of stable ribonucleic acids, together with the accumulation of an unusual nucleotide, guanosine 5'-diphosphate 3'-diphosphate, commonly known as ppGpp. This compound also accumulated during heat shock. When temperature-sensitive protein synthesis elongation factor G (EF-G) was introduced into E. coli NF859, a relA+ strain, the synthesis of ppGpp was reduced to approximately one-half that of wild-type EF-G+ cells at a nonpermissive temperature of 40 degrees C. Furthermore, fusidic acid, an inhibitor of protein synthesis which specifically inactivates EF-G, prevented any accumulation of ppGpp during the heat shock. We suggest that a functional EF-G protein is necessary for ppGpp accumulation under temperature shift conditions, possibly by mediating changes in the function of another protein, the relA gene product. However, EF-G is probably not required for the synthesis of ppGpp during the stringent response, since its inactivation did not prevent ppGpp accumulation during amino acid starvation.     

Mode of initiation of constitutive stable DNA replication in RNase H-defective mutants of Escherichia coli K-12.

The alternative pathway of DNA replication in rnh mutants of Escherichia coli can be continuously initiated in the presence of chloramphenicol, giving rise to constitutive stable DNA replication (cSDR). We conducted a physiological analysis of cSDR in rnh-224 mutants in the presence or absence of the normal DNA replication system. The following results were obtained. cSDR allowed the cells to grow in the absence of the normal replication system at a 30 to 40% reduced growth rate and with an approximately twofold-decreased DNA content. cSDR initiation was random with respect to time in the cell cycle as well as choice of origins. cSDR initiation continued to increase exponentially for more than one doubling time when protein synthesis was inhibited by chloramphenicol. cSDR initiation was inhibited during amino acid starvation in stringent (relA+) but not in relaxed (relA1) strains, indicating its sensitivity to ppGpp. cSDR initiation was rifampin sensitive, demonstrating that RNA polymerase was involved. cSDR functioned in dnaA+ rnh-224 strains parallel to the normal oriC+ dnaA+-dependent chromosome replication system.     

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.     

Role of DnaA protein during replication of plasmid pBR322 in Escherichia coli

Summary The in vivo role of the Escherichia coli protein DnaA in the replication of plasmid pBR322 was investigated, using a plasmid derivative carrying an inducible dnaA ⁺ gene. In LB medium without inducer, the replication of this plasmid, like that of pBR322, was inhibited by heat inactivation of chromosomal DnaA46 protein so that plasmid accumulation ceased 1 to 2 h after the temperature shift. This inhibition did not occur when the plasmid dnaA ⁺ gene was expressed in the presence of the inducer isopropyl-1-thin--d-galactopyranoside (IPTG). Inhibition was also not observed in glycerol minimal medium or in the presence of low concentrations of rifampicin or chloramphenicol. Deletion of the DnaA binding site and the primosome assembly sites (pas, rri) downstream of the replication origin did not affect the plasmid copy number during exponential growth at 30° C, or after inactivation of DnaA by a shift to 42° C in a dnaA46 host, or after oversupply of DnaA, indicating that these sites are not involved in a rate-limiting step for replication in vivo. The accumulation of the replication inhibitor, RNAI, was independent of DnaA activity, ruling out the possibility that DnaA acts as a repressor of RNAI synthesis, as has been suggested in the literature. Changes in the rate of plasmid replication in response to changes in DnaA activity (in LB medium) could be resolved into an early, rom-dependent, and a late, rom-independent component. Rom ^– plasmids show only the late effect. After heat inactivation of DnaC, plasmid replication ceased immediately. These results, together with previously published reports, suggest that DnaA plays no specific role during in vivo replication of ColE1 plasmids and that the gradual cessation of plasmid replication after heat inactivation of DnaA in LB medium results from indirect effects of the inhibition of chromosome replication and the ensuing saturation of promoters with RNA polymerase under nonpermissive growth conditions.     

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 .     

Role of protein synthesis in the survival of carbon-starved Escherichia coli K-12.

In a typical Escherichia coli K-12 culture starved for glucose, 50% of the cells lose viability in ca. 6 days (Reeve et al., J. Bacteriol. 157:758-763, 1984). Inhibition of protein synthesis by chloramphenicol resulted in a more rapid loss of viability in glucose-starved E. coli K-12 cultures. The more chloramphenicol added (i.e., the more protein synthesis was inhibited) and the earlier during starvation it was added, the greater was its effect on culture viability. Chloramphenicol was found to have the same effect on a relA strain as on an isogenic relA+ strain of E. coli. Addition of the amino acid analogs S-2-aminoethylcysteine, 7-azatryptophan, and p-fluorophenylalanine to carbon-starved cultures to induce synthesis of abnormal proteins had an effect on viability similar to that observed when 50 micrograms of chloramphenicol per ml was added at zero time for starvation. Both chloramphenicol and the amino acid analogs had delayed effects on viability, compared with their effects on synthesis of normal proteins. The need for protein synthesis did not arise from cryptic growth, since no cryptic growth of the starving cells was observed under the conditions used. From these and previous results obtained from work with peptidase-deficient mutants of E. coli K-12 and Salmonella typhimurium LT2 (Reeve et al., J. Bacteriol. 157:758-763, 1984), we concluded that a number of survival-related proteins are synthesized by E. coli K-12 cells as a response to carbon starvation. These proteins are largely synthesized during the early hours of starvation, but their continued activity is required for long-term survival.     

Effects of starvation for potassium and other inorganic ions on protein degradation and ribonucleic acid synthesis in Escherichia coli.

Starvation of Escherichia coli for potassium, phosphate, or magnesium ions leads to a reversible increase in the rate of protein degradation and an inhibition of ribonucleic acid (RNA) synthesis. In cells deprived of potassium, the breakdown of the more stable cell proteins increased two- to threefold, whereas the hydrolysis of short-lived proteins, both normal ones and analog-containing polypeptides, did not change. The mechanisms initiating the enhancement of proteolysis during starvation for these ions were examined. Upon starvation for amino acids or amino acyl-transfer RNA (tRNA), protein breakdown increases in relA+ (but not relA) cells as a result of the rapid synthesis of guanosine-5'-diphosphate-3'-diphosphate (ppGpp). However, a lack of amino acyl-tRNA does not appear to be responsible for the increased protein breakdown in cells starved for inorganic ions, since protein breakdown increased in the absence of these ions in both relA+ and relA cultures, and since a large excess of amino acids did not affect this response. In bacteria in which energy production is restricted, ppGpp levels also rise, and protein breakdown increases. The ion-deprived cultures did show a 40 to 75% reduction in adenosine-5'-triphosphate levels,l similar to that seen upon glucose starvation. However, this decrease in ATP content does not appear to cause the increase in protein breakdown or lead to an accumulation of ppGpp. No consistent change in intracellular ppGpp levels was found in relA+ or relA cells starved for these ions. In addition, in relX mutants, removal of these ions led to accelerated protein degradation even though relX cells are unable to increase ppGpp levels or proteolysis when deprived of a carbon source. In the potassium-, phosphate-, and magnesium-deprived cultures, the addition of choramphenicol or tetracycline caused a reduction in protein breakdown toward basal levels. Such findings, however, do not indicate that protein synthesis is essential for the enhancement of protein degradation, since blockage of protein synthesis by inactivation of a temperature-sensitive valyl-tRNA synthetase did not restore protein catabolism to basal levels. These various results and related studies suggest that the mechanism for increased protein catabolism on starvation for inorganic ions differs from that occurring upon amino acid or arbon deprivation and probably involves an enhanced susceptibility of various cell proteins (especially ribosomal proteins) to proteolysis.     

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.     

Inhibition of uridine 5'-diphosphate-N-acetylmuramyl-L-alanine synthetase by beta-chloro-L-alanine in Escherichia coli

The synthesis of the nucleotide precursors for peptidoglycan is regulated by the relA gene in Escherichia coli. Thus, nucleotide precursors labeled with [3H]diaminopimelic acid accumulated in a relA strain but not in an isogenic relA+ strain during amino acid deprivation. Furthermore, nucleotide precursor synthesis was relaxed in the amino acid deprived relA+ strain by treatment with chloramphenicol. Uridine diphosphate-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) was the major component accumulated during the relaxed synthesis of nucleotide precursors in both relA+ and relA strains. The effect of beta-chloro-L-alanine (CLA) on the relaxed synthesis of nucleotide precursors for peptidoglycan was determined. At a low concentration (0.0625 mM) CLA inhibited the synthesis of UDP-MurNAc-pentapeptide and caused the accumulation of UDP-MurNAc-tripeptide. Thus, low concentrations of CLA probably inhibited alanine racemase, as reported previously. Higher concentrations of CLA also inhibited an earlier step in nucleotide precursor synthesis. This was shown to be due to the inhibition of UDP-MurNAc-L-alanine synthetase by CLA. CLA inhibited the activity of this enzyme in cell-free extracts as well as in intact cells.     

Accumulation of ppGpp in a relA mutant of Escherichia coli during amino acid starvation

In Escherichia coli, amino acid starvation triggers the rapid synthesis of two guanosine polyphosphates, pppGpp and ppGpp (the 3'-pyrophosphates of GTP and GDP, respectively). Determination of the turnover rate of the ppGpp pool indicated that during serine deprivation, as opposed to other amino acid starvations, the rate of ppGpp degradation is dramatically decreased. This results in a slow but significant accumulation of this regulatory nucleotide in a relA mutant during serine starvation. Similar ppGpp accumulation can be seen during serine starvation in different serine auxotrophic mutants carrying different relA alleles. On the other hand, no ppGpp accumulation is induced in various relaxed strains by serine hydroxamate treatment.     

Induction of cat-86 by chloramphenicol and amino acid starvation in relaxed mutants of Bacillus subtilis

The chloramphenicol acetyltransferase gene cat-86 is induced through a mechanism that is a variation of classical attenuation. Induction results from the destabilization of an RNA stem-loop that normally sequesters the cat-86 ribosome-binding site. Destabilization of the stem-loop is due to the stalling of a ribosome in the leader region of cat-86 mRNA at a position that places the A site of the stalled ribosome at leader codon 6. Two events can stall ribosomes at the correct location to induce cat-86 translation: addition of chloramphenicol to cells and starvation of cells for the amino acid specified by leader codon 6. Induction by amino acid starvation is an anomaly because translation of the cat-86 coding sequence requires all 20 amino acids. To explain this apparent contradiction we postulated that amino acid starvation triggers intracellular proteolysis, thereby providing levels of the deprived amino acid sufficient for cat-86 translation. Here we show that a mutation in relA, the structural gene for stringent factor, blocks intracellular proteolysis that is normally triggered by amino acid starvation. The relA mutation also blocks induction of cat-86 by amino acid starvation, but the mutation does not interfere with chloramphenicol induction. Induction by amino acid starvation can be demonstrated in relA mutant cells if the depleted amino acid is restored at very low levels (e.g., 2 micrograms/ml). A mutation in relC, which may be the gene for ribosomal protein L11, blocks induction of cat-86 by either chloramphenicol or amino acid starvation. We believe this effect is due to a structural alteration of the ribosome resulting from the relC mutation and not to the relaxed phenotype of the cells.     

Real time analysis of the RNAI-RNAII-Rop complex by surface plasmon resonance: from a decaying surface to a standard kinetic analysis

RNA loop-loop complexes are motifs that regulate biological functions in both prokaryotic and eukaryotic organisms. In E. coli, RNAI, an antisense RNA encoded by the ColE1 plasmid, regulates the plasmid replication by recognizing through loop-loop interactions RNAII, the RNA primer that binds to the plasmidic DNA to initiate the replication. Rop, a plasmid-encoded homodimeric protein interacts with this transient RNAI-RNAII kissing complex. A surface plasmon resonance (SPR)-based biosensor was used to investigate this protein-nucleic acid ternary complex, at 5 degrees C, in experimental conditions such as the protein binds either to the loop-loop complex while it dissociates or to a saturated stable RNAI-RNAII surface. The results show that RNAI hairpin dissociates from the RNAII surface 110 times slower in the presence of Rop than in its absence. Rop binds to RNAI-RNAII with an on-rate of 3.6 x 10(6) M(-1) s(-1) and an off-rate of 0.11 s(-1), resulting in a binding equilibrium constant equal to 31 nM. A Scatchard-plot analysis of the interaction monitored by SPR confirms a 1:1 complex of Rop and RNAI-RNAII as observed for non-natural Rop-loop-loop complexes.