Back to log phase: σS as a global regulator in the osmotic control of gene expression in Escherichia coli

It is now well established that the σ^S subunit of RNA polymerase is a master regulator in a complex regulatory network that governs the expression of many stationary-phase-inducible genes in Escherichiacoli. In this review, more recent findings will be summarized that demonstrate that σ^S also acts as a global regulator for the osmotic control of gene expression, and actually does so in exponentially growing cells. Thus, many σ^S-dependent genes are induced during entry into stationary phase as well as in response to osmotic upshift. K⁺ glutamate, which accumulates in hyperosmotically stressed cells, seems to specifically stimulate the activity of σ^S-containing RNA polymerase at σ^S-dependent promoters. Moreover, osmotic upshift results in an elevated cellular σ^S level similar to that observed in stationary-phase cells. This increase is the result of a stimulation of rpoS translation as well as an inhibition of the turnover of σ^S, which in exponentially growing non-stressed cells is a highly unstable protein. Whereas the RNA-binding protein HF-I, previously known as a host factor for the replication of phage Qβ RNA, is essential for rpoS translation, the recently discovered response regulator RssB, and ClpXP protease, have been shown to be required for σ^S degradation. The finding that the histone-like protein H-NS is also involved in the control of rpoS translation and σ^S turnover, sheds new light on the function of this protein in osmoregulation. Finally, preliminary evidence suggests that additional stresses, such as heat shock and acid shock, also result in increased cellular σ^S levels in exponentially growing cells. Taken together, σ^S function is clearly not confined to stationary phase. Rather, σ^S may be regarded as a sigma factor associated with general stress conditions.     

Gene <Emphasis Type="Italic">yddG</Emphasis> of <Emphasis Type="Italic">Escherichia coli</Emphasis> encoding the putative exporter of aromatic amino acids: Constitutive transcription and dependence of the expression on the cell growth rate

Gene yddG of Escherichia coli encodes a protein of the inner membrane. Data obtained earlier demonstrated that under conditions of aromatic amino acids overproduction YddG promotes their export from E. coli cells. In this work, a method of primer extension was used to localize the P _yddG promoter, which corresponds to E. coli promoters recognized by RNA polymerase in complex with σ⁷⁰ or σ^S subunits. By constructing a gene of the hybrid protein YddG’-LacZ at the intrinsic site of gene yddG location in the E. coli chromosome and analyzing the activity of β-galactosidase in cells growing on laboratory media LB and M9, the constitutive type of yddG expression at a low level was demonstrated (the activity was about 3 to 4% of the LacZ level under induction of the lac operon in E. coli wild-type cells). The expression of yddG had a twofold increase under conditions of retarded cell growth upon the stress caused by the high NaCl content (0.6 M) or by the presence of phenylalanine excess quantities (>1 mM) in the culture medium.     

Suggested interrelationships of RNA-polymerase sigma S subunit and nitrogen control system in <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis>

The effect of mutation in rpoS gene encoding sigma S subunit of RNA-polymerase on the capacity of Pseudomonas chlororaphis 449 to assimilate nitrogen was investigated. It has been shown that mutant cells with knocked-out rpoS gene had significantly lower capacity to utilize the nitrogen sources such as alanine, proline, histidine, arginine, urea, and ammonium and glutamine synthetase was downregulated in their cell free extracts. Both defects were abolished by glutamine supplementation to the medium. It is suggested that in Pseudomonas chlororaphis the association of the nitrogen control system and the system of gene expression is regulated by RNA-polymerase sigma S subunit, which can be responsible for cell adaptation at nitrogen supply limitation.     

ClpXP affects the cell metabolism of <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> partially in an RpoS-dependent manner

Introduction

ClpXP protease is an important proteolytic system in Salmonella enterica serovar typhimurium (S. typhimurium). Inactivation of ClpXP by deletion of clpP resulted in overproduction of RpoS and a growth defect phenotype. Only one report has indicated that deleting rpoS can restore the growth of a S. typhimurium clpP mutant to the wild-type level. Whether overproduction of RpoS is responsible for the growth deficiency resulting from clpP disruption and how ClpXP affects the cell metabolism of S. typhimurium remain to be elucidated.

Objectives

The aim of this study is to investigate the effect of ClpXP on cell metabolism of S. typhimurium and explore the possible co-effect of RpoS associated with ClpXP in cell metabolism.

Method

We constructed a clpP rpoS double deletion mutant TT-19 (ΔclpP ΔrpoS TT-1) using a two-step phage transduction technique. We then compared the metabolite fingerprints of Salmonella rpoS deletion mutant TT-14 (ΔrpoS TT-1), clpP deletion mutant TT-16 (ΔclpP TT-1), and clpP rpoS double deletion mutant TT-19 (ΔclpP ΔrpoS TT-1) with those of the wild-type strain TT-1 by using gas chromatography coupled with mass spectrometry (GC–MS).

Results

Deletion of rpoS recovered only a part of the growth of Salmonella clpP mutant. Further metabolome analysis indicated that clpP disruption changed the levels of 16 extra- and 19 intracellular substances, while the extracellular concentrations of 4 compounds (serine, l-5-oxoproline, l-glutamic acid, and l-tryptophan) and intracellular concentrations of 10 compounds (l-isoleucine, glycine, serine, l-methionine, l-phenylalanine, malic acid, citric acid, urea, putrescine, and 6-hydroxypurine) returned to their wild-type levels when rpoS was also deleted.

Conclusion

ClpXP affects the cell metabolism of S. typhimurium partially in an RpoS-dependent manner.

     

Influence of <Emphasis Type="Italic">UPF</Emphasis> genes on severity of <Emphasis Type="Italic">SUP45</Emphasis> mutations

Eukaryotic cells possess a special mechanism for the degradation of mRNAs containing premature termination codons (PTCs), referred to as NMD (nonsense-mediated mRNA decay). The strength of this pathway depends on the recognition of the PTCs by translational machinery and the interaction of translation termination factors eRF1 and eRF3 with Upf1, Upf2 and Upf3 proteins in Sachromyces cerevisiae yeast. Previously, we have shown that the decrease of eRF1 protein amounts in sup45 nonsense mutants leads to the impairment of NMD. Here we show that the deletion of UPF1 or UPF2 genes leads to an increase in the viability of sup45 mutants, while the effect of UPF3 gene deletion is allele-specific. Two-hybrid data have shown that amino acid residues 1–555 of Upf1 protein interact with eRF1. Any UPF gene deletion leads to allosupression of the adel1-14 mutation without a change in eRF1 content. The Upf1 depletion does not influence the synthetic lethality of sup45 mutations and the [PSI ⁺] prion. It is possible that the absence of Upf1 (or its activator Upf2) leads to a more effective formation of the translation termination complex and consequently to the increased viability of the cells containing mutant termination factors.     

Adaptive functions of <Emphasis Type="Italic">Escherichia coli</Emphasis> polyamines in response to sublethal concentrations of antibiotics

Escherichia coli exposure to sublethal antibiotic concentrations induced an increase in cell polyamine contents. Maximum accumulation of putrescine and spermidine in response to antibiotics-induced oxidative stress preceded the increment of cadaverine, the content of which was dependent on the rpoS expression level and reached the maximum in response to fluoroquinolones. The polyamine positive modulating effects on rpoS expression increased in the following order: cadaverine-putrescine-spermidine. The reason for cadaverine accumulation was the increase in activities of lysine decarboxylases CadA and Ldc. High cadaverine accumulation in the cells exposed to fluoroquinolones and cephalosporins resulted in the reduction of porin permeability; so it was considered as a response aimed at cell protection against antibiotic penetration into the cell. Netilmycin, unlike other antibiotics, did not substantially affect the lysine decarboxylase activity and cellular polyamine pools.     

Improved Tolerance of <Emphasis Type="Italic">Escherichia coli</Emphasis> to Propionic Acid by Overexpression of Sigma Factor RpoS

Propionic acid (PA) is an economically important compound, but large-scale microbial production of PA confronts obstacle such as acid stress on microbial cells. Here, we show that overexpressing sigma factor RpoS improves the acid tolerance of Escherichia coli. Four genes including rpoS, fur, pgi and dnaK (encoding RNA polymerase sigma factor, ferric uptake regulator, phosphoglucoisomerase, and chaperone, respectively) were independently overexpressed in E. coli. The recombinant E. coli overexpressing rpoS showed the highest PA tolerance. This strain could grow in M9 medium at pH 4.62, whereas wild type E. coli survived only at pHs above 5.12. Moreover, in the shake-flask cultivation, the E. coli strain overexpressing rpoS grew faster than wild type. Notably, the minimum inhibitory concentration of PA for this recombinant strain was 7.81 mg/mL, which was 2-fold higher in comparison with wild type. Overall these results indicated that overexpression of sigma factor rpoS significantly enhanced E. coli tolerance to PA.     

Identification and Classification of Two-component Systems That Affect rpoS Expression in Escherichia coli

The rpoS-encoded σ^S subunit of RNA polymerase regulates the expression of stationary phase and stress response genes in Escherichia coli. Recent study of our DNA microarray analysis suggested that the rpoS expression is affected by multiple two-component systems. In this study, we identified two-component-system mutants in which the rpoS expression increased. The regulatory manner of the systems on rpoS expression is suggested.