Expression of biosynthetic genes from Pseudomonas aeruginosa and Escherichia coli in the heterologous host

Abstract

We examine the expression of constitutive or repressible, monocistronic genes from Pseudomonas aeruginosa and Escherichia coli after their transfer to the heterologous host. To this end, chromosomal DNA from P. aeruginosa was cloned into the mobilizable broad-host-range vector pKT240; recombinant plasmids carrying the argA, argF, or proC genes were identified by complementation of the corresponding auxotrophic mutations. The isofunctional E. coli genes and the E. coli proB gene were subcloned into pKT240 from existing recombinant plasmids. The enzyme expression specified by the Pseudomonas genes in E. coli, calculated per gene copy, ranged from 0.3%-5% of the levels observed in Pseudomonas. Fusion of the P. aeruginosa proC gene to the E. coli consensus tac promoter resulted in very high proC enzyme production in E. coli, indicating that, at least in this case, the expression barrier is essentially at the level of transcriptional initiation. The E. coli argA and argF enzymes, which are controlled by repression in their native host, were synthesized constitutively in P. aeruginosa at 5% of the levels measured in E. coli under derepressed conditions. The constitutive E. coli proB and proC genes were expressed at high levels (ca. 50%) in the heterologous host. These results support the idea that P. aeruginosa may be a more permissive host than E. coli for the heterologous expression of genes from gram-negative bacteria.

Transcription initiation at multiple promoters of the pfl gene by E sigma 70-dependent transcription in vitro and heterologous expression in Pseudomonas putida in vivo

“…Little is known about the expression of E. coli genes in Pseudomonas species, but the general conclusion that could be drawn from the studies performed is that Pseudomonas species are much more permissive hosts for expression of E. coli genes than is E. coli for expression of genes from Pseudomonas species (26). Nonregulated E. coli genes are well expressed in P. putida, while regulated genes are less well expressed.…”

“…Previous analyses have determined that when Pseudomonas genes are transcribed from inducible E. coli promoters, there is also only limited restriction to translation in E. coli (26). Synthesis of PFL in P. putida yields a suitable substrate for biochemical analysis since the enzyme cannot be activated in the heterologous host because of the absence of the gene encoding the PFL-activating enzyme.…”

Positive FNR-like control of anaerobic arginine degradation and nitrate respiration in Pseudomonas aeruginosa

“…Recently we realized that anaerobic arginine utilization was defective in strain S1239 (19) (25,32), and the E. coli consensus promoter, which is recognized by the RNA polymerase holoenzyme (EaJ70), functions well in P. aeruginosa (28,50,58). Previously, it has been shown that FNR-dependent genes of E. coli are transcribed and expressed in Pseudomonas species (28,46), but regulation by oxygen limitation in the heterologous host has not been demonstrated.…”

“…Plasmid purification, restriction, DNA fragment isolation from low-melting-point agarose, ligation, transformation, and DNA sequencing by the chain termination method were all carried out by standard techniques described in our previous publications (6,25,42,43) or by Sambrook et al (45). Recombinant plasmids (in E. coli ED8767) were mobilized by pRK2013 (in E. coli HB101) to 20 3 4, 27 …”

The homologous regulators ANR of Pseudornonas aeruginosa and FNR of Escherichia coli have overlapping but distinct specificities for anaerobically inducible promoters

“…aeruginosa (Jeenes et al, 1986). It could be argued that under these conditions the amount of ANR present in the cell might be sub-optimal and thus limit the expression.…”