Plasmid amplification in Escherichia coli after temperature upshift is impaired by induction of recombinant protein synthesis

Production of recombinant proteins often interferes with the physiology of the host organism by causing stress responses. In recombinant Escherichia coli, the cellular content of ColE1-derived plasmids and, consequently, the synthesis of the constitutively synthesized plasmid-encoded proteins generally increases after a temperature upshift. Simultaneous induction of inducible recombinant proteins that are synthesized at high levels and tend to form inclusion bodies, however, attenuates the plasmid amplification. This phenomenon was observed using temperature- as well as IPTG-inducible expression systems. Thus, high-level recombinant gene expression in connection with inclusion body formation does not only interfere with host cell but also with plasmid-related functions.     

A temperature upshift induces initiation of replication at oriC on the Escherichia coli chromosome

A temperature upshift of 10 or more degrees in the growth temperature of a bacterial culture causes induction of extra rounds of chromosome replication. This heat-induced replication (HIR) initiates at oriC , is transitory, requires RNase H1 and RecA proteins and requires neither RNA polymerase activity nor de novo protein synthesis. The number of origins activated by heat is growth rate and temperature differential dependent. An origin activation higher than 20% increases the DNA:mass ratio around twofold, and this value is kept constant for the subsequent generations of growth at 41°C. We have also shown that HIR is neither related to SDR nor induced by the heat shock response. We suggest that a thermodynamic alteration of oriC structure or of membrane fluidity could explain the observed HIR.     

Escherichia coli shows two types of behavioral responses to osmotic upshift.

Behavioral responses to osmotic upshift were characterized by temporal assays of free-swimming cells of Escherichia coli. Small osmotic upshifts (200 to 300 mosM) elicited tumble responses which were chemotaxis dependent, while large osmotic upshifts (400 to 500 mosM) elicited stopping followed by pseudotumbling which was chemotaxis independent.     

Escherichia coli mutant temperature sensitive for group I RNA bacteriophages.

The temperature-sensitive conjugational transfer-deficient mutant Escherichia coli JCFL39, carrying a traD(Ts) mutation, is herein described as also being temperature sensitive for group I RNA phages (MS2, f2, and R17) but not for Q beta. Temperature shift experiments showed that the growth of group I phage MS2 in the mutant could be inhibited by a post-penetration event at high temperature. A possible role for the traD cistron of sex factor F in the intracellular development of MS2 is suggested.     

Adjustment of polyamine contents in Escherichia coli.

Adjustment of polyamine contents in Escherichia coli was studied with strains of Escherichia coli producing normal (DR112) and excessive amounts of ornithine decarboxylase [DR112(pODC)] or S-adenosylmethionine decarboxylase [DR112(pSAMDC)]. Although DR112(pODC) produced approximately 70 times more ornithine decarboxylase than DR112 did, the amounts of polyamines in the cells of both strains did not change significantly. The amounts of polyamines in DR112(pODC) were adjusted by excretion of excessive amounts of putrescine to the medium. When ornithine was deficient in cells, polyamine contents in DR112(pODC) were much higher than those in DR112, although polyamine contents were low in both strains. This indicates that large amounts of ornithine decarboxylase increased the utilization of ornithine for putrescine synthesis. During ornithine deficiency, strain DR112 produced 3.4 times more ornithine decarboxylase. Strain DR112(pSAMDC) produced seven times more S-adenosylmethionine decarboxylase than DR112 did. In DR112(pSAMDC) an increase (40%) in spermidine content, a decrease (35%) in putrescine content, and no significant excretion of putrescine and spermidine were observed. The amount of ornithine decarboxylase in DR112(pSAMDC) was approximately 30% less than that in DR112. In addition, S-adenosylmethionine decarboxylase activity was strongly inhibited by spermidine. A possible regulatory mechanism to maintain polyamine contents in Escherichia coli is discussed based on the results.     

Rapid response to osmotic upshift by osmoregulated genes in Escherichia coli and Salmonella typhimurium.

The rapid in vivo response of both Escherichia coli and Salmonella typhimurium osmoregulated genes to an osmotic upshift was analyzed in detail by using chromosomal operon fusions. Within 10 min after the addition of 0.3 M NaCl to the culture medium, the differential rates of expression of both an S. typhimurium proU-lac fusion and a proP-lac fusion increased by 180- and 17-fold respectively, while an E. coli ompC-lac fusion increased by 3.4-fold. For all three stimulated promoters, the increased rate of expression was maintained until about 40 min after the osmotic upshift. Thereafter, proU expression continued at a steady-state rate that was 27-fold higher than that of the control, while proP and ompC expression fell to 1.4- and 2-fold of the control rates, respectively. In contrast, expression of an E. coli ompF-lac fusion decreased twofold within 2.5 min. For proU, the length of the lag phase, which preceded the onset of the rapid response, increased with the degree of osmotic upshift, above a threshold of 0.2 M NaCl; the onset of the rapid proU response also preceded the resumption of growth. The rapid response phase, which was first quantitated for proU, proP, ompC, and ompF in this study, is an important component of the osmoregulation of these promoters. The addition of the osmoprotectant glycine betaine at the time of osmotic upshift decreased both the length of the rapid response and the subsequent steady-state of expression of proU.     

RNA synthesis in Escherichia coli during K + -depletion

During K⁺ depletion of a mutant of $\text{Escherichia}$$\text{coli}$ which cannot concentrate this cation, protein synthesis is inhibited but RNA formation continues. The RNA produced during K⁺ depletion was analyzed by gel electrophoresis. It was found that 4S, 5S and 23S RNA were synthesized by K⁺-depleted cells whether uninfected or infected with phage T4. In addition, an RNA species moving close to 16S (presumably 17S) and material of about 6–10S were made during K⁺ depletion. These species of RNA were not evident in growing cells. Methylation of RNA is severely inhibited during K⁺ depletion.     

Control of RNA synthesis in Escherichia coli after a shift to higher temperature.

Parameters of RNA synthesis were measured after a temperature upshift in a pair of Escherichia coli B/r strains that are isogenic except for having relA and relA+ loci, to examine the cause for a reported anomaly in the correlation between guanosine tetraphosphate (ppGpp) and stable RNA (rRNA, tRNA) synthesis under such conditions. Two main results were: (i) the specific stable RNA gene activity (stable RNA per total RNA synthesis) correlated in the conventionally expected fashion with the level of ppGpp but was obscured by a nonspecific increase in the RNA chain elongation rate due to the higher temperature; (ii) the temperature upshift caused a transient reduction in the RNA polymerase activity (transcribing per total enzyme) that accounts for the previously observed oscillating RNA synthesis rate after a temperature shift.     

The role of antioxidant enzymes in response of Escherichia coli to osmotic upshift

Aerobic growth of Escherichia coli sodAsodB and katE mutants lacking cytosolic superoxide dismutases and catalase hydroperoxidase II was inhibited by osmotic upshift to a greater extent than of their wild-type parent strains. The fur mutation leading to an intracellular overload of iron also increased sensitivity of growing E. coli cells to osmotic upshift. Using lacZ fusions, it was shown that expression of antioxidant genes soxS and katE was stimulated by an increase in osmolarity. These data suggest that in aerobically growing E. coli cells, moderate osmotic upshift causes activation of certain antioxidant systems.     

Regulation of membrane phospholipid syntheesis in Escherichia coli during temperature up-shift.

The synthesis of membrane phospholipids and that of stable ribonucleic acid were inhibited during temperature up-shift of both rel+ and relA strains of Escherichia coli. The kinetics of the inhibition of the synthesis of both molecules were correlated with the kinetics of guanosine 5'-diphosphate-3'-diphosphate synthesis. Metabolic down-shift experiments gave similar results.     

Recognition of messenger RNA during translational initiation in Escherichia coli

The structural aspects of recognition by E. coli ribosomes of translational initiation regions on homologous messenger RNAs have been reviewed. Also discussed is the location of initiation region on mRNA, its confines, typical nucleotide sequences responsible for initiation signal, and the influence of RNA macrostructure on protein synthesis initiation. Most of the published DNA nucleotide sequences surrounding the start of various E. coli genes and those of its phages have been collected.