Control of autogenous activation of Herbaspirillum seropedicae nifA promoter by the IHF protein

Control of autogenous activation of Herbaspirillum seropedicae nifA promoter by the IHF protein

FEMS Microbiology Letters 212 (2002) 177^182 Control of autogenous activation of Herbaspirillum seropedicae nifA promoter ...

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FEMS Microbiology Letters 212 (2002) 177^182

Control of autogenous activation of Herbaspirillum seropedicae nifA promoter by the IHF protein Roseli Wassem, Fa¤bio O. Pedrosa, Marshall G. Yates, Fabiane G.M. Rego, Leda S. Chubatsu, Liu U. Rigo, Emanuel M. Souza  Departamento de Bioqu|¤mica - UFPR, Caixa Postal 19046, CEP 81531-990, Curitiba, PR Brazil Received in revised form 19 April 2002; accepted 24 April 2002 First published online 29 May 2002

Abstract Analysis of the expression of the Herbaspirillum seropedicae nifA promoter in Escherichia coli and Herbaspirillum seropedicae, showed that nifA expression is primarily dependent on NtrC but also required NifA for maximal expression under nitrogen-fixing conditions. Deletion of the IHF (integration host factor)-binding site produced a promoter with two-fold higher activity than the native promoter in the H. seropedicae wild-type strain but not in a nifA strain, indicating that IHF controls NifA auto-activation. IHF is apparently required to prevent overexpression of the NifA protein via auto-activation under nitrogen-fixing conditions in H. seropedicae. 7 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : NifA; Nitrogen ¢xation; Integration host factor; Herbaspirillum

1. Introduction The NifA protein is the speci¢c activator of nif genes in Proteobacteria. Although, its synthesis and activity are tightly regulated, the mechanisms of such regulation vary greatly among diazotrophs. Oxygen and ammonium levels are usually the main signals to these control mechanisms [1]. In Herbaspirillum seropedicae, an endophytic diazotroph that associates with several grasses [2,3], the nifA promoter region has a complex structure (Fig. 1) which is evidence for a tightly controlled transcription regulation. Three putative 324/312 promoter sequences as well as two putative NtrC- and three NifA-binding sites (BS) were found in the nifA promoter region [4]. A highly conserved 324/ 312 promoter sequence located 12 bp upstream from the mapped transcription start was identi¢ed as the functional nifA promoter, which is activated by NtrC [5]. Although NifA was not essential for promoter activity, the NifABS2 was occupied in vivo under nitrogen-¢xing conditions, suggesting auto-regulation [5]. Furthermore, deletion studies of the H. seropedicae nifA promoter suggested that

both NtrC-BS were functional and that the putative IHF-BS could play a regulatory role in preventing NifAdependent autogenous transcriptional activation [5]. In vitro studies showed that two other low homology NifA-BS were capable of binding NifA [6]. The putative IHF-BS was also shown to bind IHF and one of the 324/312 promoter sequences was con¢rmed as the only one able to bind the cN -RNA polymerase [6]. These results suggested a role for the NifA and IHF proteins in the regulation of nifA expression in H. seropedicae. We now report that the concerted action of NtrC, NifA and IHF on the nifA promoter of H. seropedicae is essential to ¢ne-tune the regulation of the intracellular concentration of the NifA protein in response to ammonium and oxygen levels, con¢rming previous in vitro results [6].

2. Materials and methods 2.1. Bacterial strains The strains used in this work are described in Table 1. 2.2. Construction of deletions in the nifA promoter

* Corresponding author. Fax: +55 (41) 266 2042. E-mail address : [email protected] (E.M. Souza).

An overlapping extension PCR-based method for site-

0378-1097 / 02 / $22.00 7 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 7 1 6 - 4

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Fig. 1. Nucleotide sequence of the promoter region of H. seropedicae nifA gene. Regulatory sequences are in bold; arrow indicates the RNA start; arrowheads indicate point mutations ; deleted sequences are underlined.

speci¢c deletion [10] was used to produce DNA fragments lacking the potential BS sequences. The deleted sequences were multiples of ten residues in an attempt to maintain the remaining upstream sites in the same face of the DNA helix, except in deletions of the NifA-BS2 and both NtrCBSs, where 34 and 54 nucleotides were deleted, respectively, due to primer design constraints.

pended in fresh NFbHP [14] medium (3 ml in 25-ml bottles) to an OD600 of 0.2 with or without 20 mmol/l NH4 Cl and incubated under air or low O2 (1.5%) at 30‡C and 120 rpm for 7 h. L-Galactosidase activity was determined [15] and expressed as nmol of o-nitrophenol produced min31 mg31 protein. Protein in alkali-lysed cells was determined as described [16].

2.3. Site-directed mutagenesis of the nifA promoter 3. Results and discussion The megaprime procedure of Kammann et al. [11] was employed to create point mutations in the nifA promoter region. 2.4. Construction of nifA: :lacZ fusions A 1.1-kb EcoRI/EcoRV fragment from pEMS301A [4], containing part of the N-terminus of the nifA gene and the whole promoter region was ¢rst cloned into pTZ19R [12] digested with EcoRI/SmaI (pRW3A3), and then subcloned as an EcoRI/PstI fragment into the transcriptional fusion vector pPW452 [13], yielding the plasmid pRW1. The PCR fragments carrying the mutant nifA promoters were restricted with BamHI/NsiI, gel puri¢ed and, ¢nally, cloned into pRW3A3 digested with the same enzymes. The mutant promoters were ¢nally subcloned into pPW452 as EcoRI/PstI fragments. All the constructs were sequenced. 2.5. L-Galactosidase activity Overnight-grown cultures of H. seropedicae strains containing the nifA : :lacZ fusions were centrifuged and resus-

3.1. Expression of native and mutant nifA promoters of H. seropedicae in Escherichia coli The NifA protein can activate the expression of the nifA promoter of H. seropedicae in E. coli [4]. We produced site-directed deletions and point mutations in the nifA promoter region to identify the functional regulatory sequences involved in NifA-dependent activation. Removal of pRWC or double mutation (TGTCTCT and ACACAGA) pRW3 in the NifA-BS2 abolished or reduced promoter activity by ¢ve-fold, respectively, in E. coli N99 (Table 2) and YMC10 (not shown) strains, when Klebsiella pneumoniae NifA (KpNifA) was the activator. However a single mutation in the TGT motif (pRW9) of the NifA-BS2 produced a mutant promoter that still responded to NifA, implying either that this site was only partially active or, alternatively, that NifA-BS1 and NifABS3 also contributed to promoter activity in the N99 strain (Table 2). On the other hand, the zero promoter activity shown by pRWC suggests that NifA-BS1 and NifA-BS3 were silent to NifA activation when the NifABS2 was completely removed (Table 2). These results sug-

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Table 1 Strains used in this work Strain E. coli N99 HN414 YMC10 H. seropedicae SmR1 SmR54 DCP286A



SmR , sup‡, galK3 , F3 SmR , sup‡, galK3 , F3 , himA42 pro, thi, endA, hsdR, Proþ , vlacU169, hutC, NtrCþ

[7] [7] [8]

SmR , Nifþ SmR , KmR , Nif3 , nifA: :Km SmR , KmR , ntrC: :Tn5-B20

[5] [5] [9]

gest that NifA-BS2 is essential for autogenous activation of the nifA promoter by NifA. Removal of the IHF-BS (pRW7) caused a 60% increase in NifA-dependent activation of the H. seropedicae nifA promoter compared to the native promoter in the wildtype E. coli strain N99 (Table 2). In addition, the native promoter (pRW1) had a two-fold higher activity in an ihfA (himA42) mutant of E. coli (HN414), indicating that IHF inhibited NifA-dependent activation of the H. seropedicae nifA promoter (Table 2). 3.2. The nifA promoter of H. seropedicae is a 324/312 promoter primarily dependent on NtrC For L-galactosidase activity measurements, the cultures with high ammonium and oxygen conditions grew much more after 7 h incubation than under other conditions. Since the linear correlation between optical density and protein concentration at high cell density might be lost [17], the L-galactosidase activities were expressed as speci¢c activity (nmol o-nitrophenol produced min31 mg31 protein; Fig. 2) and not as Miller units as in E. coli (Table 2). In the wild-type H. seropedicae strain SMR1 transformants containing pRW1 (native nifA: :lacZ) L-galactosidase activities were ¢ve to eight-fold higher in the absence than in the presence of ammonium ions (Fig. 2A). Mutation at the 325 residues (GCT; pRW22A) produced very low

levels of nifA expression, indicating that this 324/312 promoter-like sequence is indeed the functional promoter of the nifA gene of H. seropedicae (Fig. 2A). Deletion of both NtrC-BSs (pRW2) produced a promoter with a marked decrease (four-fold) in L-galactosidase activity under nitrogen-limiting and high oxygen conditions when compared with the native promoter (Fig. 2A). Deletion of the NtrC-BS1 (pRW11) caused a similar decrease in promoter activity (Fig. 2A). However, deletion of the two NtrC sites did not completely abolish L-galactosidase activity under N-limitation and high oxygen, which may have been due to NtrC binding to a cryptic site located immediately upstream of NtrC-BS1, as seen in vitro [6]. Furthermore, the native nifA promoter failed to derepress in the H. seropedicae ntrC mutant strain DCP286A (Fig. 2C), con¢rming the requirement for NtrC as the primary activator. These results are in agreement with the Nif3 phenotype of mutant DCP286A [9] and con¢rm previous work [5,6]. 3.3. NifA-dependent activation of the nifA promoter of H. seropedicae The presence of three NifA-BSs upstream from the 324/ 312 nifA promoter together with the above results suggested auto-activation by the NifA protein. Previously, we observed that native nifA promoter activity, reported in Miller units, decreased only 25^30% in the H. seropedicae

Table 2 E¡ect of mutation/deletion of regulatory BSs in the nifA promoter activity in di¡erent E. coli backgrounds Plasmid

L-Galactosidase activity E. coli strains (containing pMC71A)


N99 (wt)

HN414 (himA42)

1578 263 1070 0 2503 2000

3312 1230 2197 83 2802 3246

E. coli strains [7] were transformed with pMC71A (KpnifAC ; [18]) plus the indicated nifA: :lacZ fusions and grown aerobically at 30‡C in NFDM medium [19] supplemented with the appropriate antibiotics, glutamine (10Wg ml31 ), and in the presence of 20 mmol l31 NH4 Cl. Bottles (10 ml) containing 4 ml of medium were inoculated with an aliquot (50 Wl) of an overnight culture in Terri¢c Broth. After 18^20 h incubation, the L-galactosidase activity was determined and expressed as Miller units [15]. Figures are activities subtracted from the background levels (500 Miller units) and are average of duplicates from a representative experiment.

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Fig. 2. nifA promoter activity in H. seropedicae. L-Galactosidase activity expressed as nmol of o-nitrophenol min31 mg31 protein in H. seropedicae wild-type SMR1 (A), nifA SMR54 (B) and ntrC DCP286A (C) strains containing the indicated native or mutated nifA: :lacZ fusions. Activities were determined as described in Section 2. Results are the mean of at least three di¡erent experiments. Full crosses indicate deletions; dotted crosses indicate point mutations. The L-galactosidase activities of the strains under low oxygen and 20 mmol/l NH4 Cl varied from 86 to 135 nmol of o-nitrophenol min31 mg31 protein.

nifA mutant [5]. When expressed as L-galactosidase specific activity (Fig. 2A), the native nifA promoter (pRW1) activity in the absence of NH4 Cl was 50% higher under low oxygen than under air in the wild-type H. seropedicae strain SMR1. This increase in promoter activity under low oxygen was not observed in the H. seropedicae nifA mutant strain SMR54, where the native nifA promoter (pRW1) was regulated by ammonium but not a¡ected by oxygen (Fig. 2B). Moreover, deletion of (pRWC) or double mutation in the NifA-BS2 (pRW3) reduced promoter activity under low oxygen in the wild-type strain (Fig. 2A), suggesting that full activation of the nifA promoter requires NifA and that auto-activation requires

NifA-BS2. Moreover, in vitro and in vivo footprinting showed that NifA-BS2 binds to NifA at physiologically signi¢cant concentrations (20^50 nM) and nitrogen-¢xing conditions, respectively [6,5]. Since the H. seropedicae NifA protein is oxygen-sensitive [20,21], NifA-dependent auto-activation of the nifA promoter is expected to occur only under oxygen limitation. In agreement with these results, the wild-type H. seropedicae strain SMR1 carrying the NtrC-BS-deleted nifA : : lacZ fusions (pRW2 and pRW11) had activities three to four-fold higher under low oxygen than under air and in the absence of ammonium, probably activated by endogenous NifA protein, which is sensitive to both NHþ 4 and

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oxygen (Fig. 2A). In the H. seropedicae nifA strain SMR54 the NtrC-BS deleted promoters showed very low activity in relation to the wild-type promoter, and similar activities in the presence or absence of oxygen. 3.4. The IHF-BS inhibits NifA auto-activation in H. seropedicae The L-galactosidase activity of H. seropedicae SMR1 carrying plasmid pRW7 (IHF-BS deleted) was two-fold higher than that of the native nifA promoter in the absence of ammonium ions and under limiting oxygen (Fig. 2A). However, this increase in L-galactosidase activity was not observed in the nifA strain SMR54 under low oxygen, where the IHF-BS-deleted fusion had activity similar to that of the native promoter under nitrogen-limiting conditions at both low and high oxygen levels (Fig. 2B). Similar results were obtained with a promoter carrying a mutated IHF-BS (AACCC, positions 3182/3181 pRW2B). These results indicate that the IHF-BS 36 bases upstream from the NifA-BS2 inhibits NifA-dependent transcription in vivo. 3.5. The IHF protein as a transcription regulator of the nifA promoter of H. seropedicae Binding of the IHF protein to several promoters leads to di¡erent e¡ects on promoter regulation. It can either stimulate or inhibit transcription by di¡erent mechanisms (reviewed in [22,23]). In cN -dependent promoters, IHF increases speci¢city of activation by favoring interaction of the activator bound to the upstream BS and the cN RNA polymerase and/or preventing contact of non-specific activators in solution or bound to cryptic sites [24]. However, in the case of the nifA promoter of H. seropedicae, IHF apparently controls the extent of auto-activation by NifA bound to a functional enhancer sequence. Three NifA-BSs are also present in the nifU promoter of K. pneumoniae [25]. In this promoter, where the IHF-BS partially overlaps NifA-BS3, IHF inhibited NifA binding to this site by competition and stimulated nifU promoter transcription by facilitating NifA activation bound to NifA-BS1 and NifA-BS2 [25]. In the nifA promoter of H. seropedicae, however, the IHF-BS is located 36 bp upstream from the NifA-BS2, indicating that direct contact between the two proteins is less likely to occur. In vitro DNaseI footprinting showed that NifA and IHF did not compete for binding to the H. seropedicae nifA promoter [6]. These results indicate that, while IHF binding did not prevent NifA interaction with the nifA promoter, it could induce changes in the DNA structure upon binding, and modulate the activity of the bound activator (NifA), which may constitute a novel way of ¢ne-tuning transcription activation by an enhancer-binding protein. In conclusion, activation of the native nifA promoter of H. seropedicae was shown to be primarily dependent on


the 324/312 promoter sequence and on the NtrC protein. Under nitrogen-¢xing conditions (limiting oxygen and NHþ 4 ), the nifA promoter is initially activated by NtrC and the increasing NifA concentrations stimulate its own transcription. Therefore, the NifA protein, while not essential, is required for full promoter activity. The role of the IHF protein is probably to control the extent of NifA autogenous activation by a mechanism that does not prevent NifA binding. This control is presumed necessary to avoid a potential deleterious e¡ect of NifA overexpression under nitrogen-¢xing conditions. On the other hand the presence of three NifA-BSs in the nifA promoter of H. seropedicae points to the requirement for and the involvement of the NifA protein in its own overexpression in order to ful¢l diverse physiological roles.

Acknowledgements We thank CNPq/PRONEX/FINEP for ¢nancial support. R.W. was a recipient of CNPq studentship. We also want to thank Darlene C. Persuhn for the gift of the ntrC strain DCP286A and Roseli Prado, Valter A. de Baura and Julieta Pie for technical assistance.

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