Characterization of Salmonella enterica serovar Typhimurium aconitase A

Abstract

Aconitases (Acn) are iron-sulfur proteins that catalyse the reversible isomerization of citrate and isocitrate via the intermediate cis-aconitate in the Krebs cycle. Some Acn proteins are bifunctional and under conditions of iron starvation and oxidative stress lose their iron-sulfur clusters and become post-transcriptional regulators by binding specific mRNA targets. Many bacterial species possess two genetically distinct aconitase proteins, AcnA and AcnB. Current understanding of the regulation and functions of AcnA and AcnB in dual Acn bacteria is based on a model developed in Escherichia coli. Thus, AcnB is the major Krebs cycle enzyme expressed during exponential growth, whereas AcnA is a more stable, stationary phase and stress-induced enzyme, and both E. coli Acns are bi-functional. Here a second dual Acn bacterium, Salmonella enterica serovar Typhimurium (S. Typhimurium), has been analysed. Phenotypic traits of S. Typhimurium acn mutants were consistent with AcnB acting as the major Acn protein. Promoter fusion experiments indicated that acnB transcription was~10-fold greater than that of acnA and that acnA expression was regulated by the cyclic-AMP receptor protein (CRP, glucose starvation), the fumarate nitrate reduction regulator (FNR, oxygen starvation), the ferric uptake regulator (Fur, iron starvation) and the superoxide response protein (SoxR, oxidative stress). In contrast to E. coli, S. Typhimurium acnA was not induced in the stationary phase. Furthermore, acnA expression was enhanced in an acnB mutant, presumably to partially compensate for the lack of AcnB activity. Isolated S. Typhimurium AcnA protein had kinetic and mRNA-binding properties similar to those described for E. coli AcnA. Thus, the work reported here provides a second example of the regulation and function of AcnA and AcnB proteins in a dual Acn bacterium.

Coupling of Peptidoglycan Synthesis to Central Metabolism in Mycobacteria: Post-transcriptional Control of CwlM by Aconitase

“…Recombinant AcnA protein had an iron content (5.7 ± 0.6 iron atoms per AcnA; n = 7) and a UV-visible spectrum consistent with the presence of a [4Fe-4S] cluster ( Figure 1 D). The catalytic parameters of M. tuberculosis AcnA were typical of an Acn enzyme, with V max of 1.6 ± 0.1 U mg −1 (where 1 U is equal to 1 μmol of citrate converted to isocitrate per min) and K M of 3.3 ± 0.2 mM, similar to those reported previously for bacterial AcnA proteins ( Figure S1 A; Jordan et al., 1999 ; Baothman et al., 2013 ). Acn activity decreased when AcnA was exposed to superoxide (ROS), nitric oxide (NO), or BIP, and the inhibition was associated with disassembly of the iron-sulfur cluster, as indicated by the lower iron contents of the treated AcnA protein ( Figure 1 E).…”

Impact of ROS-Induced Damage of TCA Cycle Enzymes on Metabolism and Virulence of Salmonella enterica serovar Typhimurium

“…Further qPCR experiments revealed that not only AcnA amounts, but also expression of acnA is reduced in STM Δ sodAB compared to WT at all tested time points (Figure 5A). This result is contradictive to studies which revealed enhanced expression of acnA upon short-time exposure to methyl viologen (MV) (Baothman et al, 2013; Seo et al, 2015). STM WT grown for 3.5 h in LB was treated with 10 mM MV for 30 min (Figure 5B).…”

“…A STM strain defective in SOD is known to be sensitive toward oxidative stress and exhibits a reduced virulence in macrophages (Tsolis et al, 1995). Furthermore, STM with deleted aconitases was reported to be sensitive toward oxidative stress (Baothman et al, 2013). Motivated by the strong similarities between the metabolic profiles, we set out to elucidate if metabolic perturbations of the TCA cycle affect tolerance of STM toward oxidative burst of phagocytes.…”

The TCA Pathway is an Important Player in the Regulatory Network Governing Vibrio alginolyticus Adhesion Under Adversity

“…pdhB is a pivotal metabolic gene, and the pdhB deletion mutant displayed less efficient adhesion than the wild type strain to FN ( Rajab et al, 2010 ; Wallwiener et al, 2012 ; Vastano et al, 2014 ). acnA is an essential component of the TCA cycle ( Gruer et al, 1997 ; Baothman et al, 2013 ). Research on Salmonella enterica has shown that acnA might be involved in the regulation of flagellar assembly and adhesion to the surface of J774 macrophage-like cells ( Tang et al, 2004 ).…”