Construction and characterization of a set of E. coli strains deficient in all known loci affecting the proteolytic stability of secreted recombinant proteins

Even though secretion offers numerous advantages for the production of proteins in Escherichia coli, the expression of many heterologous proteins is severely limited by degradation in the periplasmic space. We found that mutations in rpoH, the RNA polymerase sigma factor responsible for heat shock protein synthesis, affect the stability of heterologous secreted proteins. A particularly dramatic increase in expression was further observed in rpoH degP double mutants. To minimize proteolytic degradation, we constructed a family of 25 isogenic strains deficient in all known cell envelope proteases (DegP, Protease III, Tsp(Prc), and OmpT), as well as the rpoH15 mutant allele, and characterized their growth in both shake flasks and fermentors. The availability of this set of strains permits the selection of a suitable host based on the optimal combination between the optimum reduction in protease activity and acceptable growth properties.     

Is the insect glutathione S-transferase I gene family intronless?

Salmonella requires its alternative sigma factor sigma S (RpoS) for virulence in mice. rpoS mutants can be frequently isolated from highly passaged Salmonella laboratory strains. In particular, the live typhoid oral vaccine Salmonella typhi Ty21a and its parental strain Ty2, a 'wild-type' strain widely used for vaccine development, are rpoS mutants. Here, we show that the nucleotide sequence of the rpoS mutant allele of Ty2 is identical to that of the rpoS mutant allele of Ty21a. This demonstrates that the rpoS mutation arose in Ty2 before the isolation of Ty21a in 1975, an observation that may have implications for vaccine research.     

Trehalose is not relevant for in vivo activity of sigmaS-containing RNA polymerase in Escherichia coli

The sigmaS- and sigma70-associated forms of RNA polymerase core enzyme (E) of Escherichia coli have very similar promoter recognition specificities in vitro. Nevertheless, the in vivo expression of many stress response genes is strongly dependent on sigmaS. Based on in vitro assays, it has recently been proposed that the disaccharide trehalose specifically stimulates the formation and activity of EsigmaS and thereby contributes to promoter selectivity (S. Kusano and A. Ishihama, J. Bacteriol. 179:3649-3654, 1997). However, we demonstrate here that a trehalose-free otsA mutant exhibits growth phase-related and osmotic induction of various sigmaS-dependent genes which is indistinguishable from that of an otherwise isogenic wild-type strain and that stationary-phase cells do not accumulate trehalose (even though the trehalose-synthesizing enzymes are induced). We conclude that in vivo trehalose does not play a role in the expression of sigmaS-dependent genes and therefore also not in sigma factor selectivity at the promoters of these genes.     

Cloning and characterization of the rpoH gene of Rhodobacter capsulatus

By using an oligonucleotide mixture corresponding to a region highly conserved among alternative sigma factors we identified a new sigma factor gene (rpoH) from Rhodobacter capsulatus. This gene encodes a protein of 34 kDa with strong similarity to the RpoH (sigma32) factors from other bacterial species. It was not possible to inactivate the R. capsulatus rpoH gene by introducing a resistance cassette, implying that it is essential for growth. The 5' ends of the mRNAs were mapped to two sequences with similarity to an rpoH- and an rpoD-dependent promoter, respectively. The amounts of both these mRNAs increased after heat shock, but were unaffected by a decrease in oxygen tension. Western analysis using a sigma factor-specific antibody revealed the accumulation of a protein of about 34 kDa after heat shock, and an increase in the amounts of a protein with the same size after reduction of oxygen tension in R. capsulatus cultures.     

Role of rpoS in stress survival and virulence of Vibrio cholerae

Vibrio cholerae is known to persist in aquatic environments under nutrient-limiting conditions. To analyze the possible involvement of the alternative sigma factor encoded by rpoS, which is shown to be important for survival during nutrient deprivation in several other bacterial species, a V. cholerae rpoS homolog was cloned by functional complementation of an Escherichia coli mutant by using a wild-type genomic library. Sequence analysis of the complementing clone revealed an 1.008-bp open reading frame which is predicted to encode a 336-amino-acid protein with 71 to 63% overall identity to other reported rpoS gene products. To determine the functional role of rpoS in V. cholerae, we inactivated rpoS by homologous recombination. V. cholerae strains lacking rpoS are impaired in the ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and carbon starvation. These results suggest that rpoS may be required for the persistence of V. cholerae in aquatic habitats. In addition, the rpoS mutation led to reduced production or secretion of hemagglutinin/protease. However, rpoS is not critical for in vivo survival, as determined by an infant mouse intestinal competition assay.     

Identification of protein synthesis elongation factor G as a 4.5 S RNA-binding protein in Escherichia coli

Escherichia coli 4.5 S RNA is metabolically stable and abundant. It consists of 114 nucleotides, and it is structurally homologous to domain IV of mammalian signal recognition particle (SRP) RNA. In this study, we found two 4.5 S RNA-binding proteins in cell extracts by means of a gel mobility shift assay. One protein was identified as Ffh, which has been characterized as 4.5 S RNA-binding protein. The other protein was separated from Ffh by two consecutive column chromatographic elutions and by monitoring the 4.5 S RNA binding activity. After the second chromatography, a dominant protein with an approximate molecular weight of 78,000 was associated with 4.5 S RNA binding activity. A sequence of the NH2-terminal 19 residues of the 78-kDa protein was completely identical to that of the protein elongation factor G (EF-G) of E. coli, and further it cross-reacted with antiserum against E. coli EF-G. The results obtained using a synthetic oligo RNA corresponding to the 23 S rRNA defining the EF-G binding site indicated that 4.5 S RNA and 23 S rRNA are competitive in 4.5 S RNA binding and that a decanucleotide sequence conserved between them serves as a binding site for EF-G. Conservation of the SRP RNA binding activity of EF-G from Bacillus subtilis suggests that the binding of EF-G to SRP RNA is essential for its function.     

The response regulator SprE controls the stability of RpoS

In Escherichia coli, the sigma factor, RpoS, is a central regulator in stationary-phase cells. We have identified a gene, sprE (stationary-phase regulator), as essential for the negative regulation of rpoS expression. SprE negatively regulates the rpoS gene product at the level of protein stability, perhaps in response to nutrient availability. The ability of SprE to destabilize RpoS is dependent on the ClpX/ClpP protease. Based on homology, SprE is a member of the response regulator family of proteins. SprE is the first response regulator identified that is implicated in the control of protein stability. Moreover, SprE is the first reported protein that appears to regulate rpoS in response to a specific environmental parameter.