Controlled expression of nif and isc iron-sulfur protein maturation components reveals target specificity and limited functional replacement between the two systems

Abstract

The nitrogen-fixing organism Azotobacter vinelandii contains at least two systems that catalyze formation of [Fe-S] clusters. One of these systems is encoded by nif genes, whose products supply [Fe-S] clusters required for maturation of nitrogenase. The other system is encoded by isc genes, whose products are required for maturation of [Fe-S] proteins that participate in general metabolic processes. The two systems are similar in that they include an enzyme for the mobilization of sulfur (NifS or IscS) and an assembly scaffold (NifU or IscU) upon which [Fe-S] clusters are formed. Normal cellular levels of the Nif system, which supplies [Fe-S] clusters for the maturation of nitrogenase, cannot also supply [Fe-S] clusters for the maturation of other cellular [Fe-S] proteins. Conversely, when produced at the normal physiological levels, the Isc system cannot supply [Fe-S] clusters for the maturation of nitrogenase. In the present work we found that such target specificity for IscU can be overcome by elevated production of NifU. We also found that NifU, when expressed at normal levels, is able to partially replace the function of IscU if cells are cultured under low-oxygen-availability conditions. In contrast to the situation with IscU, we could not establish conditions in which the function of IscS could be replaced by NifS. We also found that elevated expression of the Isc components, as a result of deletion of the regulatory iscR gene, improved the capacity for nitrogen-fixing growth of strains deficient in either NifU or NifS.

FIG. 1.

(A) A. vinelandii [Fe-S] cluster biosynthetic gene regions. Genes whose products are thought to have similar functions are indicated by the same color. (B) Schematic diagram of the incorporation of a gene into the A. vinelandii genome through homologous recombination. The red lines indicate locations of reciprocal recombination events. The approximate position of the araBAD promoter is indicated by bent arrows.

FIG. 2.

Analysis of nif-, scr- and ara-regulated expression of NifUS. (A) Schematic diagrams of the genetic organizations of strains DJ, DJ1475, and DJ1626. The complete genotypes of these strains are shown in Table 1. The cross-hatched regions indicate deletions. Expression of genes regulated by the nif control elements is induced in the absence of a fixed nitrogen source and is repressed in the presence of a fixed nitrogen source. Expression of genes regulated by the scr control elements and expression of genes regulated by the ara control elements are induced by inclusion of sucrose and arabinose, respectively, in the growth medium. (B) Coomassie brilliant blue-stained 15% SDS-PAGE gel containing crude extracts of strains DJ, DJ1475, and DJ1626 grown under different culture conditions. Lane 1, crude extract of DJ grown in the absence of a fixed nitrogen source with sucrose as the carbon source; lane 2, crude extract of DJ1475 grown in the absence of a fixed nitrogen source with sucrose as the carbon source; lane 3, crude extract of DJ1626 grown in the absence of a fixed nitrogen source with sucrose as the carbon source and arabinose added to the growth medium; lane 4, crude extract of DJ1626 grown in the presence of a fixed nitrogen source with sucrose as the carbon source and arabinose added to the growth medium; lane 5, crude extract of DJ1626 grown in the presence of a fixed nitrogen source with sucrose as the carbon source and no arabinose added to the growth medium. Unlabeled lanes contained M_r standards (phosphorylase b, bovine serum albumin, ovalbuminn, carbonic anhydrase, and soybean trypsin inhibitor). The positions of nitrogenase catalytic components are indicated as follows: D, nitrogenase MoFe protein α-subunit; K, nitrogenase MoFe protein β-subunit; and H, nitrogenase Fe protein. Bands corresponding to these components are apparent in lanes 1, 2 and 3 but not in lane 4 or 5. The positions of NifU and NifS are indicated by U and S, respectively. The accumulation of NifS could not be evaluated on stained SDS-PAGE gels because of the presence of another band at the same location. (C) Western blot analysis of NifS accumulation. A duplicate of the SDS-PAGE gel shown in panel B was used for blotting and analysis of NifS accumulation using anti-NifS antibodies. High levels of NifS in lanes 3 and 4 relative to the levels in lanes 1, 2, and 5 are apparent.

FIG. 3.

Rescue of the null growth phenotype associated with the functional loss of IscU by elevated ara-directed expression of NifUS. (A) Schematic diagrams of the genetic organizations of DJ1626, DJ1639, and DJ1640. See Table 1 for complete genotypes of these strains. (B) Growth of strains DJ1626, DJ1639, and DJ1640 when they were cultured in the absence (▴) or in the presence (▪) of arabinose. Cells were precultured in media containing arabinose and were switched to the growth conditions indicated above at zero time. OD (600 nm), optical density at 600 nm.

FIG. 4.

Effect of ara-dependent NifU or NifS expression on growth of cells with IscU or IscS depleted. (Top) Schematic diagrams of the genetic organizations of DJ1421, DJ1680, and DJ1713. See Table 1 for complete genotypes of these strains. (Bottom) Growth phenotypes of DJ1421, DJ1680, and DJ1713 when they were cultured under the conditions indicated. Genes whose expression was under control of the nif regulatory elements were induced when N₂ was used as the nitrogen source and were repressed when NH₃ was used as the nitrogen source. Genes whose expression was under control of the scr regulatory elements were induced when sucrose was used as the carbon source and were repressed when glucose was used as the carbon source. Genes whose expression was under control of the ara regulatory elements were induced when arabinose was added to the growth medium.

FIG. 5.

Effect of IscS depletion on aconitase activity in the presence or absence of arabinose-induced NifS expression. (A) Schematic diagrams of the genetic organizations of strains DJ1454, DJ1450, and DJ1726. (B) Ratio of aconitase activity to isocitrate dehydrogenase activity for cells with IscS depleted (DJ1450 and DJ1726) normalized to the ratio for control cells (DJ1454) (WT) in which IscS was not depleted. IscS was depleted from DJ1450 and DJ1726 by a carbon source shift (sucrose to glucose) as described in Materials and Methods. Arabinose was also added at the time of the carbon source shift for DJ1726 in order to induce a high level of NifS expression. The values are the averages of at least two independent experiments.

FIG. 6.

IscU is not essential under nitrogen-fixing conditions when cells are cultured under an atmosphere containing 5% oxygen. (A) Schematic diagrams of the genetic organizations of DJ1454, DJ1450, and DJ1445. (B) Growth of strains under nitrogen-fixing conditions with an atmosphere containing either 20 or 5% oxygen using glucose as the carbon source, when either IscU (DJ1445) or IscS (DJ1450) was depleted. Growth of DJ1454 was eliminated when this strain was cultured under an atmosphere containing 5% oxygen if a fixed source of nitrogen was added to the growth medium (data not shown).

FIG. 7.

Elevated expression of Isc components improves the diazotrophic growth capacity of strains with nifU deleted. (A) Schematic diagrams of the genetic organizations of DJ1421, DJ1616, DJ1646, and DJ1734. (B) Diazotrophic growth of DJ1421 (•), DJ1616 (▪), and DJ1646 (⧫). Strain DJ1734 exhibited the same very slow diazotrophic growth exhibited by DJ1616 (data not shown). OD (600 nm), optical density at 600 nm.