Stationary phase expression of the arginine biosynthetic operon argCBH in Escherichia coli

Abstract

Background: Arginine biosynthesis in Escherichia coli is elevated in response to nutrient limitation, stress or arginine restriction. Though control of the pathway in response to arginine limitation is largely modulated by the ArgR repressor, other factors may be involved in increased stationary phase and stress expression.

Results: In this study, we report that expression of the argCBH operon is induced in stationary phase cultures and is reduced in strains possessing a mutation in rpoS, which encodes an alternative sigma factor. Using strains carrying defined argR, and rpoS mutations, we evaluated the relative contributions of these two regulators to the expression of argH using operon-lacZ fusions. While ArgR was the main factor responsible for modulating expression of argCBH, RpoS was also required for full expression of this biosynthetic operon at low arginine concentrations (below 60 microM L-arginine), a level at which growth of an arginine auxotroph was limited by arginine. When the argCBH operon was fully de-repressed (arginine limited), levels of expression were only one third of those observed in deltaargR mutants, indicating that the argCBH operon is partially repressed by ArgR even in the absence of arginine. In addition, argCBH expression was 30-fold higher in deltaargR mutants relative to levels found in wild type, fully-repressed strains, and this expression was independent of RpoS.

Conclusion: The results of this study indicate that both derepression and positive control by RpoS are required for full control of arginine biosynthesis in stationary phase cultures of E. coli.

Figure 1

Location of operon fusions in strains used in this study. Arrows indicate the direction of transcription of genes.

Figure 2

Expression of argCBH-lacZ in WT, ΔrpoS, ΔargR and ΔrpoSΔargR strains on LB plates containing X-Gal.

Figure 3

Expression of argCBH in isogenic wild type (○) and rpoS mutant (●) strains in glucose minimal media supplemented with exogenous arginine. Expression was tested in exponential phase (OD₆₀₀ = 0.3)

Figure 4

Effect of astCADBE depleted arginine on argCBH expression in wild type, ΔrpoS, Δast ,and ΔrpoS Δast strains. Strains were grown in triplicate in LB rich media. Samples were taken in exponential phase (□, OD₆₀₀ = 0.3) and stationary phase (■, OD₆₀₀ = 1.5), and β-glactosidase activity was assayed as described in Methods.

Figure 5

Effect of exogenous arginine on growth and argCBH expression. Growth rate and argCBH expression were determined using an arginine auxotrophic strain, HS1072 and an arginine prototrophic strain HS1066, respectively, at the indicated arginine concentrations. Overnight cultures were grown with appropriate antibiotics, sub-cultured into minimal media and maintained in exponential phase for at least 8 generations prior to the start of the experiment.

Figure 6

RpoS dependent expression of argH in exponential and stationary phase determined by Northern analysis. RNA was extracted from cultures grown in LB to OD₆₀₀ = 0.3 for exponential phase (E) and OD₆₀₀ = 1.5 for stationary phase (S) using the hot phenol method (as described in Methods). 5 μg of RNA was loaded in each lane. Signal intensity was quantified by densitometry and normalized to an arbitrary value of 10 for expression of the operon in stationary phase in the wild type strain.

Figure 7

The arginine biosynthetic pathway. Note that ArgF and ArgI are ornithine transcarbamylase, while the carAB operon encodes subunits of carbamoylphosphate synthase. Adapted from EcoCyc: Encyclopedia of Escherichia coli Genes and Metabolism .