Abstract
The nifU and nifS genes encode the components of a cellular machinery dedicated to the assembly of [2Fe-2S] and [4Fe-4S] clusters required for growth under nitrogen-fixing conditions. The NifU and NifS proteins are involved in the production of active forms of the nitrogenase component proteins, NifH and NifDK. Although NifH contains a [4Fe-4S] cluster, the NifDK component carries two complex metalloclusters, the iron-molybdenum cofactor (FeMo-co) and the [8Fe-7S] P-cluster. FeMo-co, located at the active site of NifDK, is composed of 7 iron, 9 sulfur, 1 molybdenum, 1 homocitrate, and 1 unidentified light atom. To investigate whether NifUS are required for FeMo-co biosynthesis and to understand at what level(s) they might participate in this process, we analyzed the effect of nifU and nifS mutations on the formation of active NifB protein and on the accumulation of NifB-co, an isolatable intermediate of the FeMo-co biosynthetic pathway synthesized by the product of the nifB gene. The nifU and nifS genes were required to accumulate NifB-co in a nifN mutant background. This result clearly demonstrates the participation of NifUS in NifB-co synthesis and suggests a specific role of NifUS as the major provider of [Fe-S] clusters that serve as metabolic substrates for the biosynthesis of FeMo-co. Surprisingly, although nifB expression was attenuated in nifUS mutants, the assembly of the [Fe-S] clusters of NifB was compensated by other non-nif machinery for the assembly of [Fe-S] clusters, indicating that NifUS are not essential to synthesize active NifB.
Highlights
K. pneumoniae nifU and nifS Mutants Fail to Accumulate NifB-co—Strain UN1217 carries a mutation in NifE polypeptide of UN1217 (nifN) that impairs FeMo-co synthesis and results in the accumulation of NifB-co activity [20]
The above results can be summarized as follows: (i) NifU and NifS are not required for the synthesis of a functional SAMradical NifB protein, (ii) glutathione S-transferase (GST)-NifB preparations purified from a ⌬nifUS strain carry substantial amounts of FeMo-co precursor, and (iii) nifU and nifS mutants do not synthesize enough NifB-co as to observe its accumulation in a strain unable to process it into FeMo-co
Dean and co-workers first suggested that NifU and NifS are involved in the maturation or stability of both nitrogenase component proteins, because A. vinelandii nifU or nifS deletion mutants exhibit a 15-fold reduction in NifH activity and a 4-fold reduction in NifDK activity [10]
Summary
K. pneumoniae Strains and Growth Conditions—K. pneumoniae strains UN (wild type) and UN1217 (nifN4536::mu) have been previously described [23]. A 1-kb DNA fragment containing the nifA gene, the nifB promoter (PnifB), and a XbaI restriction site at the 5Ј-end, was PCR-amplified from the chromosome of K. pneumoniae UN1217 using oligonucleotides nifA-m1 (5Ј-CCCCTCTAGAATCGCCAACGCCATCCACCATAAT-3Ј) and nifA-c1 (5Ј-GGTCGTACCTTCGTGGTTGGGC-3Ј) as primers. A 2.1-kb DNA fragment containing the gst-nifB gene and a SacI restriction site at the 3Ј-end was PCR-amplified from pRHB233 using oligonucleotides RNF17 (5Ј-ATGTCCCCTATACTAGGTTATTGGAAATTAAG-3Ј) and nifB-c3 (5Ј-CCGAGCTCTCAGGCGACCCCCTTATGCGGCAA-3Ј) as primers Both DNA fragments were ligated into the XbaI and SacI sites of the suicide plasmid pDS132, which carries an sacB gene [30], to generate plasmid pRHB292. Plasmid pRHB235 was transferred to strains UN (wild type), UN1217 (nifN::mu), and UC3 (⌬nifUS nifN::mu) to generate strains UC9, UC10, and UC11, respectively, by a procedure analogous to that described above for gst-nifB replacement. Iron content in purified GST-NifB preparations was determined according to a previous study [40]
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