Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae

Abstract

In Klebsiella pneumoniae, products of the nitrogen fixation nifLA operon regulate transcription of the other nif operons. NifA activates transcription by 54 -holoenzyme. In vivo, NifL antagonizes the action of NifA under aerobic conditions or in the presence of combined nitrogen. In contrast to a previous report, we show that depletion of iron (Fe) from the growth medium with the chelating agent o-phenanthroline (20 M) mimics aerobiosis or combined nitrogen in giving rise to inhibition of NifA activity even under anaerobic, nitrogenlimiting conditions. Adding back Fe in only twofold molar excess over phenanthroline restores NifA activity, whereas adding other metals fails to do so. By using strains that lack NifL, we showed that NifA activity itself does not require Fe and is not directly affected by phenanthroline. In the free-living diazotroph Klebsiella pneumoniae, expression of nitrogen fixation (nif) genes is regulated by the products of the nifLA operon (3,8,14,32). NifA activates transcription of all nif genes (except nifLA) by the alternative holoenzyme form of RNA polymerase 54 -holoenzyme. NifA binds to an upstream activation sequence (34) and contacts promoterbound 54 -holoenzyme by means of a DNA loop (9). It catalyzes the ATP-dependent isomerization of closed complexes between 54 -holoenzyme and a nif promoter to transcriptionally productive open complexes (25,35). NifL, which is a negative regulator, inhibits NifA activity in vivo in response to molecular oxygen and/or combined nitrogen (22,32).NifL from K. pneumoniae is composed of two domains separated by a hydrophilic interdomain linker (Q-linker [12]). We showed recently that the C-terminal domain is sufficient to inhibit transcriptional activation by NifA in vitro and in vivo (37). Thus, the inhibitory function of NifL appears to lie in its C-terminal domain, which presumably interacts with NifA. The N-terminal domain of NifL contains a region of homology (ϳ30% amino acid identity over 130 residues) to the product of the bat gene from Halobacterium halobium (19,47,50). Since Bat is an oxygen-responsive activator of the synthesis of bacterio-opsin, it has been proposed that the region of homology may be involved in oxygen sensing. The mechanism(s) by which NifL senses oxygen and/or combined nitrogen is not understood.The N-terminal domain of NifL contains one CysXXCys motif (Cys-184-Ala-Asp-Cys-187). The similarity of this motif to sequences involved in binding metal clusters in proteins such as ferrodoxins and rubredoxins (1, 4, 24) suggested a possible metal-binding role of this region in NifL (21). On the basis of this prediction, Henderson et al. (21) examined the influence of metal ion deficiency on NifL activity. They concluded that iron (Fe) deficiency reduced the inhibitory activity of NifL expressed at high levels in Escherichia coli.To further study the function of a possible metal ion or iron-sulfur (Fe-S) cluster in oxygen-sensing by NifL, we examined the effect of Fe deficiency on NifL inhibition of NifA activity in vivo in a stra...

Fnr Is Required for NifL-Dependent Oxygen Control of nif Gene Expression in Klebsiella pneumoniae

“…We recently showed that in vivo iron is specifically required for nif induction in K. pneumoniae, and additionally, that iron is not present in NifL (31,32). In order to examine whether oxygen is detected by a more general system rather than by NifL directly, we chose to examine the possible influence of Fnr on the nif induction in a heterologous E. coli system.…”

“…NifA-mediated activation of transcription from the nifHDK promoter in K. pneumoniae UN4495 and E. coli strains was monitored by measuring the differential rate of ␤-galactosidase synthesis during exponential growth (units per milliliter per optical density unit at 600 nm [OD 600 ]) (32). Inhibitory effects of NifL on NifA activity were assessed by virtue of a decrease in nifH expression.…”

“…Samples for monitoring growth at 600 nm and determining ␤-galactosidase activity were taken anaerobically. In E. coli strains carrying a plasmid encoding NifL and NifA (pNH3) (11) or NifL C184S/C187S and NifA (pRS107) or a plasmid encoding NifA alone (pJES851) (32), expression of nifLA, nifL C184S/C187S nifA, or nifA was induced from the tac promoter with 10 M IPTG (isopropyl-␤-D-thiogalactopyranoside).…”

“…This indicates that NifL apparently acts as a redox switch in response to the environmental oxygen status and allows NifA activity, only under oxygen-limiting conditions. We recently showed that in vivo, the presence of iron is required to relieve inhibitory effects of NifL on transcriptional activation by NifA and, additionally, that iron is not present in NifL (31,32). Therefore, we have postulated that an unidentified iron-containing protein may be the physiological reductant for NifL.…”

Mechanism of coordinated synthesis of the antagonistic regulatory proteins NifL and NifA of Klebsiella pneumoniae

“…The N-terminal domain of NifL shows homology to the bat gene product of Halobacterium halobium, which is an oxygen-responsive activator, and contains one CysXXCys motif, similar to sequences involved in the binding of metal clusters in ferrodoxins and rubredoxins (3,15), which suggests the involvement of metals in the function of NifL. In fact, the involvement of iron in the function of NifL has been described (12,15,37). Recently, NifL from Azotobacter vinelandii has been reported as a flavoprotein redox sensor (13).…”