Mutations in Escherichia coli pmp and htpR genes stabilize the products of foreign gene expression

The half lives of mRNA for Escherichia coli chloramphenicol-acetyltransferase, Bacillus amyloliquefaciens alpha-amylase and human leucocyte interferon were measured in E. coli cells by molecular RNA.DNA hybridization. The effect of mutation in pnp gene, coding polynucleotide phosphorylase, on the stability of these mRNA was studied. The half life of interferon mRNA increases from 25 to 90 s in the pnp mutant, resulting in an increase of interferon accumulation. The stability of interferon in E. coli cells depends on the htpR gene, controlling the heat shock response. The yields of leucocyte interferons alpha-2, alpha I-1 and fibroblast interferon beta increase ten times in htpR mutants. Thus, by using pnp and htpR mutants it is possible to enhance considerably the eukaryotic gene expression in bacterial cells.     

The psychrotrophic bacterium Yersinia enterocolitica requires expression of pnp, the gene for polynucleotide phosphorylase, for growth at low temperature (5°C)

The psychrotrophic bacterium Yersinia enterocolitica is characterized by temperature-dependent adaptations. To investigate Y . enterocolitica genes involved in cold adaptation, a mutant restricted in its ability to grow at 5°C was isolated from a transposon mutant library. The transposon insertion site in this psychrotrophy-defective (PD) mutant mapped 16 bp upstream of an open reading frame whose predicted amino acid sequence showed 93% similarity with the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase), encoded by pnp . Expression of this gene was blocked in the PD mutant. However, the introduction of a second copy of pnp , including 0.33 kbp sequences upstream of its coding region, into the chromosome of the PD mutant restored pnp expression as well as the ability to grow at 5°C. Furthermore, the expression of pnp appeared to be temperature dependent: in the parental Y . enterocolitica strain, the levels of both pnp mRNA and PNPase were 1.6-fold higher at 5°C compared with 30°C. A similarly enhanced level of PNPase at 5°C was observed in the merodiploid recombinant strain, which indicates that the 0.33 kbp region upstream of pnp harboured a cold-inducible promoter. A putative cold shock promoter motif (ATTGG) was observed in this region.     

Conserved domains in polynucleotide phosphorylase among eubacteria

Polynucleotide phosphorylase (PNPase) is a polynucleotide nucleotidyl transferase (E. C. 2.7.7.8) that is involved in mRNA degradation in prokaryotes. PNPase structure analysis has been performed in Streptomyces antibioticus; this revealed the presence of five domains: two ribonuclease PH (RPH)-like (pnp1 and pnp2), one alpha helical, one KH, and one S1 domains. The trimeric nature of this enzyme was also confirmed. In this work, we have investigated conserved domains or subdomains in bacterial PNPases (55), for this structure-based sequence homology analysis between predicted amino acid sequences from bacterial PNPases and that of S. antibioticus was performed. Our findings indicate that while pnp2 (% similarity average S = 84/% identity average I = 22), KH (S = 74.3%/I = 5.3%), S1 (S = 71.3%/I = 1.2%); and pnp1 (S = 52.8%/I = 0.3%) domain; structure and sequence are well conserved among different bacteria, alpha helical domain (S = 39.5%/I = 0) although conservation of the structure is somewhat maintained, the sequence is not conserved at all. Implications of such findings in PNPase activity will be discussed.     

Isolation and characterization of a new temperature-sensitive polynucleotide phosphorylase mutation in Escherichia coli K-12

Polynucleotide phosphorylase (PNPase) has been studied in detail since its discovery in 1955 [1]. In an attempt to determine what role, if any, it has in mRNA decay in Escherichia coli, we have isolated and characterized a temperature-sensitive mutation, pnp-200, in the pnp gene. In vitro phosphorolysis, polymerization and exchange activities of the partially purified Pnp-200 enzyme are all reduced to 30-40% of wild-type activity at 50 degrees C compared to 32 degrees C. The pnp-200 mutation alone does not affect cell growth or mRNA stability. A triple mutant strain containing pnp-200 in combination with other temperature-sensitive mutations in genes known to affect mRNA metabolism (rnb-500 and ams-1) is conditionally lethal and shows increased mRNA stability after shift to the non-permissive temperature.     

Cloning and orientation of the gene encoding polynucleotide phosphorylase in Escherichia coli.

Mutations which affect the activity of polynucleotide phosphorylase (PNPase) map near 69 min on the bacterial chromosome. This region of the chromosome has been cloned by inserting the kanamycin-resistant transposon Tn5 near the argG and mtr loci at 68.5 min. Large SalI fragments of chromosomal DNA containing the Tn5 element were inserted into pBR322, and selection was made for kanamycin-resistant recombinant plasmids. Two of these plasmids were found to produce high levels of PNPase activity in both wild-type and host strains lacking PNPase activity. The pnp gene was further localized and subcloned on a 4.8 kilobase HindIII-EcoRI fragment. This fragment was shown to encode an 84,000-molecular weight protein which comigrated with purified PNPase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The orientation of the pnp gene was determined by insertion of Tn5 into the 4.8 kilobase fragment cloned in pBR322. Some of the insertions had lost the ability to elevate the level of PNPase activity in the host bacterium. Restriction mapping of the positions of the Tn5 insertions and analysis of plasmid-encoded polypeptides in UV-irradiated maxi-cells indicated that the pnp gene is oriented in the counterclockwise direction on the bacterial chromosome.     

The C-terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo

RNase E is an essential Escherichia coli endonuclease, which controls both 5S rRNA maturation and bulk mRNA decay. While the C-terminal half of this 1061-residue protein associates with polynucleotide phosphorylase (PNPase) and several other enzymes into a 'degradosome', only the N-terminal half, which carries the catalytic activity, is required for growth. We characterize here a mutation (rne131 ) that yields a metabolically stable polypeptide lacking the last 477 residues of RNAse E. This mutation resembles the N-terminal conditional mutation rne1 in stabilizing mRNAs, both in bulk and individually, but differs from it in leaving rRNA processing and cell growth unaffected. Another mutation (rne105 ) removing the last 469 residues behaves similarly. Thus, the C-terminal half of RNase E is instrumental in degrading mRNAs, but dispensable for processing rRNA. A plausible interpretation is that the former activity requires that RNase E associates with other degradosome proteins; however, PNPase is not essential, as RNase E remains fully active towards mRNAs in rne+pnp mutants. All mRNAs are not stabilized equally by the rne131 mutation: the greater their susceptibility to RNase E, the larger the stabilization. Artificial mRNAs generated by E. coli expression systems based on T7 RNA polymerase can be genuinely unstable, and we show that the mutation can improve the yield of such systems without compromising cell growth.