A-Type Carrier Proteins Are Involved in [4Fe-4S] Cluster Insertion into the Radical S-Adenosylmethionine Protein MoaA for the Synthesis of Active Molybdoenzymes

Abstract

Iron sulfur (Fe-S) clusters are important biological cofactors present in proteins with crucial biological functions, from photosynthesis to DNA repair, gene expression, and bioenergetic processes. For the insertion of Fe-S clusters into proteins, A-type carrier proteins have been identified. So far, three of them have been characterized in detail in Escherichia coli, namely, IscA, SufA, and ErpA, which were shown to partially replace each other in their roles in [4Fe-4S] cluster insertion into specific target proteins. To further expand the knowledge of [4Fe-4S] cluster insertion into proteins, we analyzed the complex Fe-S cluster-dependent network for the synthesis of the molybdenum cofactor (Moco) and the expression of genes encoding nitrate reductase in E. coli. Our studies include the identification of the A-type carrier proteins ErpA and IscA, involved in [4Fe-4S] cluster insertion into the radical S-adenosyl-methionine (SAM) enzyme MoaA. We show that ErpA and IscA can partially replace each other in their role to provide [4Fe-4S] clusters for MoaA. Since most genes expressing molybdoenzymes are regulated by the transcriptional regulator for fumarate and nitrate reduction (FNR) under anaerobic conditions, we also identified the proteins that are crucial to obtain an active FNR under conditions of nitrate respiration. We show that ErpA is essential for the FNR-dependent expression of the narGHJI operon, a role that cannot be compensated by IscA under the growth conditions tested. SufA does not appear to have a role in Fe-S cluster insertion into MoaA or FNR under anaerobic growth employing nitrate respiration, based on the low level of gene expression. IMPORTANCE Understanding the assembly of iron-sulfur (Fe-S) proteins is relevant to many fields, including nitrogen fixation, photosynthesis, bioenergetics, and gene regulation. Remaining critical gaps in our knowledge include how Fe-S clusters are transferred to their target proteins and how the specificity in this process is achieved, since different forms of Fe-S clusters need to be delivered to structurally highly diverse target proteins. Numerous Fe-S carrier proteins have been identified in prokaryotes like Escherichia coli, including ErpA, IscA, SufA, and NfuA. In addition, the diverse Fe-S cluster delivery proteins and their target proteins underlie a complex regulatory network of expression, to ensure that both proteins are synthesized under particular growth conditions.

Keywords: A-type carrier protein; FNR; MoaA; Moco biosynthesis; iron-sulfur clusters; molybdenum cofactor; nitrate reductase.

FIG 1

Expression of a PmoaA-L–lacZ fusion in mutant strains of A-type carrier proteins and nfuA and fnr. The β-galactosidase activities in Miller units were determined for the PmoaA-L–lacZ fusion in wild type (Wt, MG1655 MVA⁺), ΔerpA, ΔiscA, ΔnfuA, ΔsufA, and Δfnr mutant strains. Standard deviations were calculated from three biological replicates. The P values were calculated using an unpaired t test (ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01).

FIG 2

Immunodetection of IscS in mutant strains of A-type carrier proteins and nfuA. Aliquots of 50 μg of the total protein fraction of cell extracts of strains MG1655 MVA⁺ (wild type), ΔmoeB, ΔiscA, ΔerpA, ΔnfuA, and ΔsufA were separated by 12% SDS-PAGE and transferred onto a PVDF membrane. An IscS-specific antiserum (1:5,000 dilution) was used and visualized by enhanced chemiluminescence. The ΔiscS cell extract served as a negative control. The band at 45 kDa corresponds to IscS.

FIG 3

Expression of a PiscR-lacZ (A) and a PsufA-lacZ fusion (B) in mutant strains of A-type carrier proteins and nfuA. The β-galactosidase activities in Miller units were determined for the PiscR-lacZ (A) and the PsufA-lacZ (B) fusion in wild type (Wt, MG1655 MVA⁺), ΔerpA, ΔiscA, ΔnfuA, and ΔsufA mutant strains. Standard deviations were calculated from three biological replicates. The P values were calculated using an unpaired t test (ns, P > 0.05; **, P ≤ 0.01; ***, P ≤ 0.005).

FIG 4

The levels of Moco in different E. coli strains. Quantification of relative amounts of Moco in the indicated mutant strains (A) and the same mutant strains transformed with a moaABCDE expression plasmid under the control of an arabinose-inducible promoter (B). Expression was induced by the addition of 0.2% l-arabinose. Total Moco is oxidized to FormA, which is quantified by its fluorescence (LU) monitored at an excitation of 383 nm and an emission of 450 nm. The integrated areas of the FormA peaks were normalized to the OD₆₀₀. Standard deviations were calculated from three biological replicates. The P values were calculated using an unpaired t test (ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005). n.d., not detectable.

FIG 5

Nitrate reductase activities in different E. coli strains. Nitrate reductase (NR) activities in E. coli BW25113 and ΔnarG, ΔnapA, or ΔnarZ mutant strains were quantified. Nitrate reductase activities (in units) were normalized to the OD₆₀₀ value. Standard deviations were calculated from three biological replicates. n.d., not detectable.

FIG 6

Nitrate reductase activities in different E. coli strains. Nitrate reductase activities in different E. coli mutant strains (A) and the same mutant strains transformed with a narGHJI expression plasmid under the control of an IPTG-inducible promoter (B). Expression was induced by the addition of 20 μM IPTG. Nitrate reductase activities (in units) were normalized to the OD₆₀₀ value. Standard deviations were calculated from three biological replicates. The P values were calculated using an unpaired t test (ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005).

FIG 7

Relative expression levels of erpA, iscA, and sufA in different E. coli mutant strains. Expression analysis was carried out using qRT-PCR. The expression levels of erpA (A), iscA (B), and sufA (C) in different E. coli mutant strains are shown. The y axis denotes the relative expression values in log of fold changes relative to the wild type, which was set to 1. Data represent the means from three biological replicates (±standard deviation [SD]). The P values were calculated using an unpaired t test (ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01). n.d., no expression detectable.

FIG 8

Levels of Moco and the activity of nitrate reductase in different E. coli strains. (A, C, and E) Quantification of relative amounts of Moco in the indicated mutant strains. Total Moco is oxidized to FormA and monitored at an excitation of 383 nm and an emission of 450 nm. The integrated areas of the FormA peaks (in LU per second) were normalized to the OD₆₀₀ value. Standard deviations were calculated from three biological replicates. (B, D, and F) Nitrate reductase activities in different E. coli mutant strains. Nitrate reductase activities (in units) were normalized to the OD₆₀₀ value. Standard deviations were calculated from three biological replicates. The P values were calculated using an unpaired t test (ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005). n.d., not detectable. Black bars correspond to the indicated mutant strains, and white bars correspond to the same mutant strains containing either a plasmid expressing erpA (A and B), iscA (C and D), or sufA (E and F). Protein expression was induced by the addition of 0.2% l-arabinose.

FIG 9

Expression levels of narG in different E. coli mutant strains. Expression analysis was carried out using qRT-PCR. The expression levels of narG in different E. coli mutants were quantified. The y axis denotes the relative expression values in log of fold changes relative to the wild type, which was set to 1. Data represent the means of three biological replicates (± SD). The P values were calculated using an unpaired t test (ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01). n.d., no expression detectable.

FIG 10

Expression of a PpepT-lacZ fusion in different E. coli mutant strains. The β-galactosidase activities in Miller units were determined for the PpepT-lacZ fusion in MG1655, ΔerpA, ΔiscA, ΔsufA, ΔnfuA, Δfnr, ΔiscA ΔerpA, ΔiscA ΔsufA, and ΔsufA ΔerpA mutant strains. The standard deviations were calculated from three biological replicates. The P values were calculated using an unpaired t test (ns, P > 0.05; **, P ≤ 0.01; ***, P ≤ 0.005). n.d., not detectable.

FIG 11

Expression of a Pfnr-lacZ fusion in different E. coli mutant strains. The β-galactosidase activities in Miller units were determined for the Pfnr-lacZ fusion in MG1655, ΔerpA, ΔiscA, ΔnfuA, ΔsufA, and Δfnr mutant strains. Standard deviations were calculated from three biological replicates. The P values were calculated using an unpaired t test (ns, P > 0.05; **, P ≤ 0.01; ***, P ≤ 0.005).

FIG 12

Expression of a PpepT-lacZ fusion in different E. coli mutant strains with coexpression of plasmids containing erpA, iscA, or sufA. The β-galactosidase activities in Miller units were determined for the PpepT-lacZ fusion in MG1655, ΔerpA, ΔiscA, and ΔiscA/ΔerpA mutant strains. Standard deviations were calculated from three biological replicates. The P values were calculated using an unpaired t test (ns, P > 0.05; **, P ≤ 0.01). n.d., not detectable. Black bars correspond to the indicated mutant strains, and white bars correspond to the same mutant strains containing either a plasmid expressing erpA (A), iscA (B), or sufA (C). Expression was induced by the addition of 0.2% l-arabinose.

FIG 13

Model for the insertion of Fe-S clusters into MoaA and FNR under anaerobic respiration with nitrate. The ISC system is the major system for assembling Fe-S clusters under anaerobic conditions, since the SUF system is generally expressed under aerobic conditions of oxidative stress and iron limitation. The [2Fe-2S] clusters assembled on IscU with help of IscS are passed to IscA and further to ErpA, which delivers it to the target protein (the dotted line shows that IscU is also able to directly supply the [4Fe-4S] clusters to ErpA). Both IscA and ErpA can insert Fe-S clusters into MoaA. SufA is unable to substitute the roles of both proteins under these conditions, based on a low gene expression. For FNR, ErpA is the major A-type carrier protein, a role that is not substituted by IscA and SufA under cellular conditions of nitrate respiration. FNR activates the transcription of the narGHJI operon and the moaABCDE operon. Moco, in general, is inserted into apo-molybdoenzymes, like NarG, after the insertion of Fe-S clusters into the enzyme. The proteins that insert Fe-S clusters inti NarGHI were not investigated in this study (more details are given in the text).