Controlled expression and functional analysis of iron-sulfur cluster biosynthetic components within Azotobacter vinelandii

Abstract

A system for the controlled expression of genes in Azotobacter vinelandii by using genomic fusions to the sucrose catabolic regulon was developed. This system was used for the functional analysis of the A. vinelandii isc genes, whose products are involved in the maturation of [Fe-S] proteins. For this analysis, the scrX gene, contained within the sucrose catabolic regulon, was replaced by the contiguous A. vinelandii iscS, iscU, iscA, hscB, hscA, fdx, and iscX genes, resulting in duplicate genomic copies of these genes: one whose expression is directed by the normal isc regulatory elements (Pisc) and the other whose expression is directed by the scrX promoter (PscrX). Functional analysis of [Fe-S] protein maturation components was achieved by placing a mutation within a particular Pisc-controlled gene with subsequent repression of the corresponding PscrX-controlled component by growth on glucose as the carbon source. This experimental strategy was used to show that IscS, IscU, HscBA, and Fdx are essential in A. vinelandii and that their depletion results in a deficiency in the maturation of aconitase, an enzyme that requires a [4Fe-4S] cluster for its catalytic activity. Depletion of IscA results in a null growth phenotype only when cells are cultured under conditions of elevated oxygen, marking the first null phenotype associated with the loss of a bacterial IscA-type protein. Furthermore, the null growth phenotype of cells depleted of HscBA could be partially reversed by culturing cells under conditions of low oxygen. Conserved amino acid residues within IscS, IscU, and IscA that are essential for their respective functions and/or whose replacement results in a partial or complete dominant-negative growth phenotype were also identified using this system.

FIG. 1.

Schematic representation of the relevant genetic organization of key strains used in this work. (A) Organization of the A. vinelandii sucrose catabolic regulon. DJ refers to the wild-type strain. DJ1418 has the scrX gene replaced by lacZ. DJ1476 was derived from DJ1418 and carries an insertion mutation within the scrR gene. Levels of lacZ expression in response to carbon source or as a result of scrR inactivation are shown for strains DJ1418 and DJ1476. (B) Organization of isc genes in strains that have various isc genes duplicated and placed under the sucrose catabolic regulatory elements. Black boxes indicate deleted regions for a particular strain. β-Gal, β-galactosidase.

FIG. 2.

Effect of depletion of Isc components in A. vinelandii. Shown are growth phenotypes, using sucrose or glucose as the carbon source, of strains that carry a deletion in an IscR-regulated copy of an isc gene but that contain an intact copy of the corresponding ScrR-regulated copy. A schematic representation of these strains is shown in Fig. 1B.

FIG. 3.

HscBA are essential Isc components, while IscA is essential only under conditions of elevated oxygen. Strains were cultured in medium using glucose as the carbon source under ambient (∼20%) oxygen or 40% oxygen. Strain DJ1714 has a deletion in the IscR-regulated copy of iscA and an intact duplicated copy of iscA whose expression is directly controlled by Scr regulatory elements. Strain DJ1694 has a deletion in the IscR-controlled hscBA copy and a duplicated copy of hscB-hscA-fdx-iscX whose expression is controlled by PscrX. A schematic representation of the strains used is shown in Fig. 1B. All strains showed normal growth when cultured using sucrose as the carbon source under ambient (∼20%) oxygen.

FIG. 4.

Effect of low oxygen on growth in cells depleted of Isc components. Strains used for the depletion of IscS (DJ1450), IscU (DJ1445), or IscA (DJ1559) also encode a duplicated copy of iscS-iscU-iscA-hscB-hscA-fdx-iscX whose expression is controlled by the Scr regulatory elements. Strains used for the depletion of HscBA (DJ1694) or Fdx (DJ1695) also have a duplicated copy of hscB-hscA-fdx-iscX whose expression is controlled by the Scr regulatory elements (Table 2). A schematic representation of these strains is shown in Fig. 1B. All strains show normal growth when cultured using sucrose as the carbon source under either ambient (∼20%) or 5% oxygen.

FIG. 5.

Depletion of Isc components in A. vinelandii has a detrimental effect on aconitase activity. (A) Effect on growth in liquid culture that occurs upon a carbon source shift for strains DJ1450, DJ1445, DJ1447, and DJ1463. Each strain was diluted at time zero in liquid medium that contains either sucrose (•) or glucose (▪) as the carbon source. The wild-type strain grows at the same rate in liquid culture when either sucrose or glucose is used as the carbon source. (B) Effect of depletion of Isc components on aconitase activity. Strain DJ1450, DJ1445, DJ1559, DJ1447, or DJ1463 was shifted from growth in liquid culture using sucrose as the carbon source to growth using glucose as the carbon source. Cells were harvested once an effect on growth was observed (see panel A) and assayed for aconitase and isocitrate dehydrogenase activities. The ratio of aconitase to isocitrate dehydrogenase (acn/idh) activity for each sample depleted of an Isc component was individually normalized to values obtained from the wild-type strain (DJ1454 [WT]) cultured under identical conditions. All data shown in the figure represent the average of two or three independent experiments. (C) Western analysis showing depletion of IscU in glucose-grown DJ1445 cells. The left panel shows Coomassie brilliant blue staining of proteins, separated by 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), from crude extracts of DJ1454 (wild type) and DJ1445 (ΔiscU) grown in glucose for 14 h. A duplicate of this gel was used for immunostaining with antibody to IscU (right panel). Lane 1, M_r standards (carbonic anhydrase, soybean trypsin inhibitor, and lysozyme); lane 2, crude extract of proteins (26 μg) from DJ1454 (wild type); lane 3, crude extract of proteins (26 μg) from DJ1445 (ΔiscU); lane 4, detection of IscU in DJ1454 (wild type); lane 5, detection of IscU in DJ1445 (ΔiscU).

FIG. 6.

Growth phenotypes exhibited by strains with selected residues of the IscR-regulated copy of IscU replaced by alanine. Cells were cultured using sucrose or glucose as the carbon source.