The mechanism of anti-activation at the Pseudomonas sp. ADP σN ...

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The mechanism of anti-activation at the Pseudomonas sp. ADP 2

σN-dependent PatzT promoter 3

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Running title: Anti-activation of a σN-dependent promoter 5

Ana Isabel Platero†, Aroa López-Sánchez, Laura Tomás-Gallardo, 6

Eduardo Santero and Fernando Govantes# 7

Key words: σN-dependent transcription, transcriptional repression, 8

Pseudomonas, atrazine degradation. 9

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Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide/Consejo 11

Superior de Investigaciones Científicas/Junta de Andalucía, and Departamento de 12

Biología Molecular e Ingeniería Bioquímica, Universidad Pablo de Olavide. 13

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†Current address: Abengoa Bioenergy. C/ Energía Solar, nº 1. Palmas Altas. 41014 15

Sevilla, Spain 16

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#Corresponding author. Address: Centro Andaluz de Biología del Desarrollo. Universidad 18

Pablo de Olavide. Carretera de Utrera, Km. 1. 41013 Sevilla, Spain 19

Telephone: +34-954977877. Fax: +34-954349376. E-mail: [email protected] 20

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AEM Accepted Manuscript Posted Online 13 May 2016Appl. Environ. Microbiol. doi:10.1128/AEM.00906-16Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 22

PatzT is an internal promoter of the atzRSTUVW operon directing the synthesis of 23

AtzT, AtzU, AtzV and AtzW, components of an ABC-type cyanuric acid transport system. 24

PatzT is σN-dependent, activated by the general nitrogen control regulator NtrC with the 25

assistance of IHF, and repressed by the LysR-type transcriptional regulator (LTTR) AtzR. 26

We have used a variety of in vivo and in vitro gene expression and protein-DNA interaction 27

assays to assess the mechanisms underlying AtzR-dependent repression of PatzT. Here 28

we show that repression only occurs when AtzR and NtrC interact simultaneously with the 29

PatzT promoter region, indicating that AtzR acts as an anti-activator to antagonize 30

activation by NtrC. Furthermore, repression requires a precise rotational orientation of the 31

AtzR and NtrC binding sites, strongly suggesting protein-protein interaction between both 32

proteins on the promoter region. Further exploration of the anti-activation mechanism 33

showed that although AtzR-dependent repression occurs prior to open complex formation, 34

AtzR does not alter the oligomerization state of NtrC or inhibit NtrC ATPase activity when 35

bound to the PatzT promoter region. Taken together, these results strongly suggest that 36

PatzT-bound AtzR interacts with NtrC to prevent coupling of NtrC-mediated ATP 37

hydrolysis with the remodeling of the interactions between E-σN and PatzT that lead to 38

open complex formation. 39

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IMPORTANCE 41

Here we describe a unique mechanism by which the regulatory protein AtzR 42

prevents activation of the σN-dependent promoter PatzT. Promoters of this family are 43

always positively regulated, but there are a few examples of overlapping negative 44

regulation. The mechanism described here is highly unconventional, and involves 45

interaction between the repressor and activator protein to prevent the action of the former 46

on the RNA polymerase-promoter complex. 47

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INTRODUCTION 49

Promoters transcribed by RNA polymerase bearing the alternative σ factor σN (E-50

σN) are obligately subjected to positive control by enhancer-binding proteins (EBPs) that 51

bind DNA at sites >100 bp upstream from the E-σN binding motifs, designated upstream 52

activation sites (UASs). Activation occurs by a unique mechanism involving 53

oligomerization of the EBP at the UAS, interaction with E-σN by looping out the intervening 54

sequences, and stimulation of closed complex to open complex isomerization concomitant 55

to the hydrolysis of ATP by the conserved central domain of the EBP (1-6). In addition, a 56

handful of σN-dependent promoters are subjected to negative control by proteins other 57

than their EBPs by means of a variety of mechanisms, including (i) interference with DNA 58

loop formation (7-9); (ii) locking RNA polymerase in a form of closed complex insensitive to 59

activation (10); and (iii) competition with the activator for DNA binding (11,12). 60

Interestingly, all of these mechanisms target the activation process, rather than interaction 61

of RNA polymerase with the promoter region, which represents a common form of 62

repression for σ70-dependent promoters in bacteria (13,14). The sole known exception to 63

this rule is AtzR competition with E-σN for DNA binding at the PatzR promoter, a 64

mechanism that was explained by the lack of an UAS in this promoter region, and the 65

concomitant requirement of high promoter occupancy for efficient UAS-independent 66

activation (15). 67

The Pseudomonas sp. ADP 108 kbp plasmid pADP-1 harbors the genes involved in 68

the hydrolytic degradation of the s-triazine atrazine (2-chloro-4-ethylamino-6-69

isopropylamino-1,3,5-triazine)(16). Atrazine degradation requires the products of the 70

constitutively expressed atzA, atzB and atzC genes, involved in atrazine conversion to the 71

central metabolite of s-triazine degradation, cyanuric acid, and the atzDEF operon, 72

encoding the activities required for cyanuric acid conversion to ammonium and carbon 73


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dioxide (17,18). Atrazine and cyanuric acid are primarily used as nitrogen sources, and the 74

atzDEF operon is a target for transcriptional regulation by the general nitrogen control 75

system (19). A regulatory cascade was characterized in which the EBP NtrC activates σN-76

dependent transcription of atzR, encoding the LysR-type transcriptional regulator (LTTR). 77

AtzR in turn activates the divergent atzDEF operon in response to cyanuric acid and a 78

nitrogen limitation signal transduced by the PII protein GlnK. In addition, AtzR represses its 79

own synthesis (17-21). Recently, we showed that atzR is co-transcribed with five additional 80

genes: atzS, encoding a putative outer membrane protein, and the atzTUVW cluster, 81

encoding an ABC-type transport system involved in high affinity uptake of cyanuric acid 82

(22,23). However, an additional σN-dependent promoter, PatzT, located within the atzS 83

coding region, is responsible for most of the transcription of the atzTUVW cluster (22). 84

Similarly to PatzR, PatzT is activated by NtrC in response to nitrogen limitation, and 85

repressed by AtzR in a cyanuric acid-independent fashion. 86

Although PatzR and PatzT share the same regulatory scheme, their architectural 87

features are widely divergent. No UAS for NtrC is present at the PatzR promoter region 88

and NtrC activates from solution or non-specifically bound to DNA, in a UAS-independent 89

fashion (15). UAS-independent activation is known to be fostered by high occupancy of 90

the promoter by E-σN (24-26). Accordingly, PatzR bears an E-σN binding motif with high 91

similarity to the consensus that is strongly bound by E-σN (15). A single AtzR binding site 92

overlaps the PatzR E-σN-binding element and is strictly required for repression (15). In 93

contrast with PatzR, our previous results depicted PatzT as an archetypical σN-dependent 94

promoter, featuring an UAS made out of two NtrC binding sites that is required for high-95

level activation and a binding site for the DNA-bending protein IHF, likely involved in 96

looping out the intervening sequences between the NtrC UAS and the E-σN-binding motif 97

(Fig. 1). Nevertheless, PatzT also displays significant levels of UAS-independent 98


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activation, suggesting a strong interaction between E-σN and its highly conserved binding 99

site (22). AtzR binds the PatzT promoter region at a single site, containing a strong 100

recognition motif centered at position -112 (Fig. 1). This AtzR binding site was also found 101

to be strictly required for repression (22). 102

PatzR and PatzT promoters are two examples of negatively regulated σN-103

dependent transcription. The mechanism of repression has been solved for PatzR, in 104

which AtzR competes with E-σN for interaction with their overlapping binding sites (15). In 105

contrast, the unusual location of the AtzR binding site at the PatzT promoter region, far 106

upstream from the E-σN recognition motif, and the intriguing observation that AtzR cannot 107

repress PatzT transcription when the NtrC binding sites are absent suggest that an 108

unconventional repression mechanism may operate at this promoter. 109

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MATERIALS AND METHODS 111

Bacterial strains and growth conditions 112

Bacterial strains used in this work and their relevant genotypes are summarized in 113

Table 1. Minimal medium containing 25 mM sodium succinate as the sole carbon source 114

was used for in vivo gene expression analysis (27). Nitrogen sources were ammonium 115

chloride or L-serine (1 g l-1). Luria-Bertani (LB) medium was used as rich medium (28). 116

Liquid cultures were grown in culture tubes or flasks with shaking (180 rpm) at 30 or 37ºC 117

(for P. putida or E. coli strains, respectively). For solid media, Bacto-Agar (Difco) was 118

added to a final concentration of 18 g l-1. Antibiotics and other additions were used, when 119

required, at the following concentrations: ampicillin (100 mg l-1), kanamycin (20 mg l-1), 120

carbenicillin (500 mg l-1), rifampicin (10 mg l-1), chloramphenicol (15mg l-1), tetracyclin (5 121

mg l-1) and 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside (X-gal) (25 mg l-1). All 122

reagents were purchased from Sigma-Aldrich. 123

Plasmid construction 124

Plasmids used in this work are summarized in Table 1. All DNA manipulations were 125

performed according to standard procedures (28). Restriction enzymes, DNA polymerases 126

and T4 DNA ligase were purchased from Roche Applied Science. The Klenow fragment or 127

T4 DNA polymerase was routinely used to fill-in recessed 3' ends and trim protruding 3' 128

ends of incompatible restriction sites. Plasmid DNA preparation and DNA purification kits 129

were purchased from Sigma-Aldrich, General Electric Healthcare or Macherey-Nagel and 130

used according to the manufacturers specifications. Plasmid DNA was transferred to E. 131

coli and P. putida strains by transformation (29) or by triparental mating (30). E. coli DH5α 132

was used as a host in all cloning procedures. 133


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Site-directed mutagenesis of the PatzT promoter region by overlap extension PCR 134

was performed essentially as described (31), using mutagenic and external 135

oligonucleotides pairs as primers (sequences available upon request), and pMPO805 as a 136

template. The final PCR products containing PatzT promoter sequences from -218 to +319 137

(wild-type PatzT coordinates, relative to the transcriptional start site) were digested with 138

EcoRI and BamHI and cloned into EcoRI- and BamHI-cleaved pMPO234 to generate the 139

PatzT-lacZ fusion plasmids pMPO835, pMPO836, pMPO837, pMPO849, pMPO850, 140

pMPO851, pMPO863 and pMPO864. The presence of the desired mutations and the 141

absence of unwanted alterations were determined by commercial sequencing (Secugen). 142

The EcoRI-BamHI fragments containing the N+5A, N+6A and N+10A PatzT promoter 143

derivatives were excised from pMPO849, pMPO850 and pMPO851, respectively and 144

cloned into EcoRI- and BamHI-digested pTE103 to yield the in vitro transcription template 145

plasmids pMPO853, pMPO854 and pMPO855. 146

β-Galactosidase assays 147

Steady-state β-galactosidase assays were used to examine the expression of the 148

wild-type and mutant PatzT-lacZ fusions in P. putida KT2442. Preinocula of bacterial 149

strains harboring the relevant plasmids were grown to saturation in minimal medium under 150

nitrogen-sufficient conditions (ammonium chloride 1 gl-1) and cells were then diluted in 151

minimal medium containing the appropriate nitrogen sources (1 gl-1 ammonium chloride for 152

nitrogen excess; 1 gl-1 L-serine for nitrogen limitation). Diluted cultures were shaken for 24 153

hours to mid-exponential phase (A600=0.25-0.5). Growth was then stopped and β-154

galactosidase activity was determined from SDS- and chloroform-permeabilized cells as 155

previously described (32). 156

Protein purification 157


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AtzR-His6 was purified from the overproducing strain NCM631 harboring pMPO135 158

and pIZ227 by nickel affinity chromatography as previously described (21). P. putida 159

NtrCD55E,S161F (33) and σN (34) were kind gifts of A. B. Hervás and V. Shingler. E. coli IHF 160

was purified from the overproducing strain E. coli K5746 by ammonium sulfate 161

fractionation and affinity chromatography on heparin-Sepharose as described previously 162

(35). Core E. coli RNA polymerase was purchased from Epicenter Biotechnologies. 163

Gel mobility shift and DNAse I footprinting assays 164

Probes containing the wild-type or mutant PatzT promoter region derivatives were 165

obtained by PCR using the PatzT-lacZ fusion plasmids as templates (primer sequences 166

available upon request). The PCR products were subsequently digested with EcoRI and 167

BamHI and gel purified. DNA fragments were strand-specifically labeled by filling in 5' 168

overhanging ends using the Klenow fragment in a reaction mixture containing [α-32P]-169

dCTP. Unincorporate nucleotides were removed using the MSB Spin PCRapace kit 170

(Invitek). 171

AtzR-DNA complexes were formed at room temperature in 20 μl reactions 172

containing 10 ng of the radiolabeled probe and increasing amounts of purified AtzR-His6 (0 173

to 100 nM) in binding buffer (35 mM Tris acetate pH 7.9, 70 mM potassium acetate, 20 174

mM ammonium acetate, 2 mM magnesium acetate, 1 mM calcium chloride, 1 mM DTT, 5 175

% glycerol, 100 μg ml-1 salmon sperm DNA, 250 μg ml-1 BSA) for 20 minutes. Reactions 176

were stopped with 4 μl of loading buffer (0.125 % w/v bromophenol blue, 0.125% w/v 177

xylene cyanol, 10 mM Tris HCl (pH 8), 1 mM EDTA, 30% glycerol) and samples were 178

separated on a 5% polyacrylamide native gel in Tris-borate-EDTA buffer at 4ºC. Dried gels 179

were exposed to a phosphoscreen and analyzed using the ImageQuant software 180

(Amersham). For competitive gel mobility shift assays, reactions were incubated for 20 181

minutes in the presence of 400 nM AtzR-His6, and subsequently challenged with 182


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increasing concentrations (0-2 μM) NtrCD55E,S161F for 20 additional minutes, or 183

preincubated with 2 μM NtrCD55E,S161F and challenged with 0-400 nM AtzR-His6, in the 184

same conditions as above. Reactions were stopped with 4 μl of loading buffer (0.125 % 185

w/v bromophenol blue, 0.125% w/v xylene cyanol, 10 mM Tris HCl (pH 8), 1 mM EDTA, 186

30% glycerol) and samples were separated on 6.5% polyacrylamide native gels in Tris-187

borate-EDTA buffer at 4ºC. Dried gels were exposed to a phosphoscreen and analyzed 188

using the ImageQuant software (Amersham). 189

Protein-DNA complexes for DNAse I footprinting assays were formed as above. 190

Partial digestion of the DNA was initiated by the addition of 1 μl of an empirically 191

determined dilution (typically 10-2 to 10-3) of a DNAse I stock solution (10 U ml-1, Roche 192

Diagnostics). Incubation was continued for 30 additional seconds and reactions were 193

stopped by the addition of 5 μl stop buffer (1.5 M sodium acetate, pH 5.2; 130 mM EDTA; 194

1 mg ml-1 salmon sperm DNA; 2.4 mg ml-1 glycogen). DNA was subsequently ethanol 195

precipitated, resuspended in 5 μl loading buffer (0.125% w/v bromophenol blue, 0.125% 196

w/v xylene cyanol, 20 mM EDTA, 95% v/v formamide) and separated by gel 197

electrophoresis on a 6% polyacrylamide-6 M urea denaturing sequencing gel. Sequencing 198

reactions were performed with the Sequenase 2.0 kit (USB). Gels were processed and 199

analyzed as above. 200

In vitro transcription 201

Multiround in vitro transcription reactions were performed essentially as described 202

(22) in a final volume of 20 μl containing 35 mM Tris-acetate, pH 7.9; 70 mM potassium 203

acetate; 20 mM ammonium acetate; 5 mM magnesium acetate; 1 mM DTT; 10% glycerol; 204

250 mg l-1 BSA; 20 nM E-σN and 0.5 μg of supercoiled plasmid template containing wild-205

type PatzT (pMPO831) or its mutant variants (pMPO853, pMPO854 and pMPO855). For 206

PatzT activation assays, E. coli core RNA polymerase (Epicentre) (100 nM), P. putida σN 207


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factor (200 nM), IHF (75 nM) and 4 mM ATP were added and incubated for 10 minutes at 208

30°C. Open complex formation was stimulated by addition of NtrCD55E,S161F (0-400 nM) and 209

further incubation for 10 minutes. In repression experiments, AtzR-His6 (0-240 nM) was 210

added either before or after the formation of open complex and incubated for an additional 211

10 minutes at 30ºC. A mixture of ATP, GTP, CTP (final concentration 0.4 mM each), UTP 212

(0.07 mM) and [α32P]-UTP (0.033 μM, Perkin Elmer) was added to initiate transcription. 213

After a 5 min incubation at 30°C, re-initiation was prevented by the addition of heparin 214

(final concentration 0.1 mg ml-1). The samples were incubated for an additional 5 min at 215

30°C, and the reactions were terminated by the addition of 5 μl of stop buffer (150 mM 216

EDTA, 1.05 M NaCl, 14 M urea, 3% glycerol, 0.075% xylene cyanol, and 0.075% 217

bromophenol blue). Samples were run in 6% polyacrylamide-urea gels in Tris-borate-218

EDTA buffer at room temperature. Gels were processed and analyzed as described 219

above. 220

ATPase activity assays 221

NtrC ATPase activity was assayed by measuring the production of inorganic 222

phosphate (Pi) using the Enzchek Phosphate Assay Kit (Life Technologies). In order to 223

avoid Pi contamination from the AtzR-His6 preparation, the AtzR-His6 buffer was changed 224

to 50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 20 % glycerol, 0.1 mM DTT by discontinuous 225

diafiltration using Nanosep 10K Omega (Pall) centrifugal devices following the 226

manufacturer's instructions. AtzR-His6 DNA binding activity was essentially unchanged as 227

judged from gel motility shift assays. A 110 bp DNA fragment containing the PatzT NtrC 228

and AtzR binding sites was obtained by annealing two complementary DNA 229

oligonucloetides (sequences available upon request). 230

ATPase assay reactions were carried out as specified by the manufacturer, except 231

that the MgCl concentration was increased to 2 mM and 10 mM KCl and 1 mM DTT were 232


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added. Mixtures containing NtrCD55E,S161F (231 nM dimer), AtzR (240 nM tetramer) and/or 233

the PatzT DNA fragment (770 nM) were preincubated for 20 minutes at room temperature. 234

Purine nucleotide phosphorylase was added, followed by 10 min incubation before ATP 235

was added to start the reaction. The kinetics of Pi production was monitored by A360 and 236

used to determine the specific ATPase activity (mol Pi min-1 mol NtrC-1). Phosphate 237

concentrations were derived from standard curves using KH2PO4 as the source of 238

inorganic phosphate. Phosphate release from reactions not containing NtrC was 239

negligible. 240

Blue Native gel electrophoresis 241

Blue Native gel electrophoresis was performed using the Native PAGETM Bis-Tris Gel 242

System (ThermoFisher Scientific). AtzR-His6 and/or NtrCD55E,S161F were diluted in 20 µl in 243

vitro transcription buffer (35 mM Tris-acetate, pH 7.9; 70 mM potassium acetate; 20 mM 244

ammonium acetate; 5 mM magnesium acetate; 1 mM DTT; 10% glycerol), incubated for 245

20 minutes at room temperature, and loaded on a 4-16% Bis-Tris Blue Native gel. 246

Electrophoresis was run and the gel was further processed according to the 247

manufacturer's instructions. 248

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RESULTS 250

AtzR-dependent repression occurs prior to open complex formation 251

We have previously shown that PatzT transcription activation can be replicated in 252

vitro in the presence of pure E-σN, the constitutively active NtrC variant NtrCD55E,S161F and 253

IHF. NtrCD55E,S161F has been shown to activate and repress transcription in vivo and in vitro 254

to levels similar to those obtained with wild-type NtrC in a signal-independent fashion 255

(33,36). In order to characterize AtzR-dependent repression of the PatzT promoter in vitro, 256

multi-round transcription assays were performed using supercoiled plasmid pMPO831, 257

which bears the PatzT promoter region from position -218 to +319, as a template, 258

Escherichia coli RNA polymerase core and IHF protein, Pseudomonas putida σN and 259

NtrCD55E,S161F, and hexahistidine-tagged AtzR (AtzR-His6). To determine the effect of open 260

complex formation on AtzR-dependent repression, two sets of transcription reactions were 261

prepared. In the first set, the template was incubated with E-σN, IHF and different 262

concentrations of AtzR-His6 in the presence of ATP. Isomerization to open complex was 263

subsequently triggered by the addition of NtrCD55E,S161F. In the second set, the DNA 264

template was incubated with NtrCD55E,S161F, E-σN and IHF in the presence of ATP to allow 265

open complex formation, which was subsequently challenged with different concentrations 266

of AtzR-His6 prior to the addition of NTPs (see Experimental Procedures for details)(Fig. 267

2,A). When added prior to open complex formation, a clear AtzR-His6 concentration-268

dependent decrease in PatzT activity was observed, to reach a maximum 90% decrease 269

in transcript levels at the highest concentration used. This result indicates that AtzR can 270

efficiently repress PatzT transcription in vitro. In contrast, addition of AtzR-His6 after open 271

complex formation resulted in low levels of repression, reaching a maximum of 20% at the 272

highest concentration used, suggesting that open complex formation prevents AtzR-273

dependent repression. 274


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The data above may also be explained if the presence of NtrC renders the promoter 275

insensitive to repression due to interactions with E-σN or DNA, rather than by promoter 276

closed complex isomerization. To distinguish between these two possibilities two 277

additional sets of in vitro transcription reactions were performed. In the first set, E-σN was 278

pre-incubated with the PatzT template and NtrCD55E,S161F. The closed complex was 279

challenged with AtzR-His6, and then ATP was added to allow isomerization the the open 280

complex prior to the addition of NTPs. In the second set, ATP was added to the template- 281

NtrCD55E,S161F mixture, thus allowing open complex formation, and the open complex was 282

subsequently challenged with increasing AtzR-His6 concentrations prior to the addition of 283

NTPs. (Fig. 2,B). The results were equivalent to those obtained above, as efficient PatzT 284

repression was only observed when open complex formation was not allowed prior to the 285

addition of AtzR-His6. These results confirm that isomerization to an open complex 286

protects PatzT from AtzR-dependent repression, strongly suggesting that AtzR acts on the 287

early steps of the initiation pathway to prevent either closed complex formation or 288

isomerization to the open complex. 289

NtrC and AtzR bind the PatzT promoter region simultaneously 290

The location of the AtzR binding motif, centered at position -112, far upstream from 291

the E-σN binding motif and in the vicinity of the NtrC binding site NtrC-2 (centered at -130), 292

and the fact that AtzR cannot repress PatzT transcription when it is activated by NtrC in an 293

UAS-independent fashion (22) suggest that, rather than tampering with RNA polymerase 294

function, AtzR may antagonize NtrC interaction with the PatzT promoter region (22). To 295

test this possibility, gel mobility shift assays were performed in which PatzT-bound 296

NtrCD55E,S161F was challenged with increasing concentrations of AtzR-His6 and vice versa 297

(Fig. 3). Interaction of NtrCD55E,S161F with the PatzT promoter region resulted in the 298

replacement of the free probe band by a smear spanning much of the corresponding 299


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PAGE lane (Fig. 3, lane 2). This was previously observed (22), and suggests that the 300

NtrCD55E,S161F-PatzT complex in unstable during gel electrophoresis. In contrast, AtzR-His6 301

retarded the PatzT probe to form a single stable complex (Fig. 3, lane 7). Addition of AtzR-302

His6 to a preformed NtrCD55E,S161F-PatzT complex (Fig. 3, lanes 3-6), or NtrCD55E,S161F to a 303

preformed AtzR-His6-PatzT (Figure 2, lanes 8-11) provoked the substitution of the pre-304

existing complexes for a single, stable, slow migrating protein-DNA complex, strongly 305

suggesting that both proteins can bind the PatzT promoter region simultaneously. 306

To characterize the interactions of NtrC and AtzR with the PatzT promoter region 307

further, the effect of challenging a preformed protein-DNA complex with the other protein 308

was also assessed by means of DNAse I footprinting (Fig. 4). NtrCD55E,S161F binding to the 309

PatzT promoter region (Fig. 4, lane 2) resulted in protection of positions -161, -162, -156 to 310

-150 and -136, and hypersensitivity at positions -157, -149, -148, -127 to -125, -119, -118 311

and -116, consistent with interactions at the NtrC1 and NtrC-2 sites (22). AtzR-His6 312

interaction with its site (Fig. 4, lane 7) resulted in a continuously protected region between 313

-109 and -125 with hypersensitive positions at -139, -136, -128, -99, -98, -86 to -84 and -314

64 to -62 (22). Addition of increasing concentrations of AtzR-His6 to a preformed 315

NtrCD55E,S161F-PatzT complex (Fig. 4, lanes 3-6) did not modify the footprinting pattern at 316

the NtrC1 site and the promoter-distal half of the NtrC-2 site. However, the hypersensitivity 317

signature observed at the promoter-proximal half and downstream from the NtrC-2 site 318

(positions -125, -119, -118 and -116) was substituted by the characteristic AtzR 319

footprinting pattern. Challenging a preformed AtzR-His6-PatzT complex with increasing 320

concentrations of NtrCD55E,S161F resulted in the gradual emergence of the NtrC footprinting 321

signature at NtrC1 and promoter-distal half of NtrC2, with no apparent effect on the AtzR 322

footprint. These results are fully consistent with the gel mobility shift results above, 323

indicating that NtrC and AtzR simultaneously bind the PatzT promoter region, although we 324


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cannot rule out that the presence of DNA-bound AtzR may alter the interaction of NtrC with 325

its binding sites, or the oligomeric form of NtrC (dimers or hexamers) bound. 326

PatzT repression requires a precise relative rotational orientation of the NtrC and 327

AtzR sites 328

It has long been known that, although DNA bending or looping acts as a facilitator 329

of EBP-E-σN interactions in many σN-dependent promoters, a misoriented bend may lead 330

to incorrect alignment of the interacting pair, thus preventing activation (37,38). This 331

mechanism has been claimed to operate in several examples of σN-dependent promoter 332

repression (7-9). AtzR is a DNA-bending protein (21), and circular permutation analysis 333

has shown that it causes a 74º bend centered at position -92 of the PatzT promoter region 334

(Supplemental Material, Fig. S1). Thus, interference with DNA loop formation is a feasible 335

mechanism for AtzR repression of the PatzT promoter. To test this hypothesis, we 336

constructed a set of PatzT fusion plasmids in which the rotational orientation and/or the 337

distance between the NtrC and AtzR binding sites, or between both protein binding sites 338

and the downstream elements of the promoter region (IHF and E-σN binding sites) was 339

modified (Fig. 5,A). Plasmids pMPO849, pMPO850, pMPO851, pMPO863 and pMPO864 340

bear 5-, 6-, 10-, 21- or 32-bp insertions between the NtrC2 and AtzR binding sites (these 341

constructs were designated N+5A, N+6A, N+10A, M+21A and N+32 A, respectively). 342

Plasmids pMPO835, pMPO836 and pMPO837 bear 4-, 6- and 10-bp insertions between 343

the AtzR and IHF binding sites (constructs A+4I, A+6I and A10I). To test the effect of these 344

mutations on in vivo PatzT activation and repression, all fusion plasmids were transferred, 345

along with the wild-type PatzT-lacZ fusion pMPO805, to P. putida KT2442, used here and 346

in our previous work as a surrogate host for in vivo gene expression studies (15,21-347

23,39,40), or P. putida KT2442 bearing the AtzR-producing plasmid pMPO109. PatzT 348

expression was assessed from cultures grown in minimal medium containing ammonium 349


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(nitrogen excess) or serine (nitrogen limitation) by means of β-galactosidase assays (Fig. 350

5,B). 351

Analysis of in vivo expression from the wild-type PatzT-lacZ fusion in the absence 352

of AtzR revealed a >100-fold increase in response to nitrogen limitation. This is consistent 353

with our previous observations, and expected for a promoter regulated by the general 354

nitrogen control system (22). None of the mutant promoter regions displayed a defect in 355

activation, and in fact all constructs bearing insertions between the NtrC and AtzR sites 356

(N+5A, N+6A, N+10A, M+21A and N+32A) showed 1.5- to 2-fold increased induction in 357

nitrogen-limited medium. PatzT expression from the wild-type N+5A and N+10A was also 358

assessed in ihf- and ∆ntrC backgrounds (Supplemental Material, Fig. S2). Nitrogen 359

limitation induction was nearly abolished in the absence of NtrC and severely decreased in 360

the absence of IHF in all cases, indicating that expression is similarly dependent on both 361

transcription factors, regardless of the construct. The effect of rotational orientation 362

mutations on PatzT promoter expression was further explored by means of multi-round in 363

vitro transcription assays using supercoiled pMPO831, pMPO853, pMPO854 or 364

pMPO855, bearing wild-type, N+5A, N+6A and N+10A PatzT promoter fragments, 365

respectively, as templates (Fig. 6). In reactions containing E-σN, IHF and NtrCD55E,S161F, but 366

lacking AtzR-His6, the mutant constructs exhibited NtrC concentration-dependent PatzT 367

transcript levels that were similar or greater than those obtained by the wild-type template 368

(Fig. 6,A), consistent with the in vivo observations above. Taken together, our results 369

strongly suggest that NtrC-dependent activation of PatzT is not restricted by the distance 370

or orientation of DNA-bound NtrC relative to promoter-bound E-σN. 371

Analysis of in vivo expression from the wild-type PatzT-lacZ fusion in the presence 372

of AtzR (Fig. 5), showed a 4-fold decrease in activity under nitrogen limitation, consistent 373

with negative control of the PatzT promoter by AtzR (22). Notably, the mutant promoters 374


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bearing insertions of approximately half a helix turn between the NtrC and AtzR sites 375

(N+5A and N+6A) were completely insensitive to AtzR repression, while insertions of 376

approximately one (N+10A), two (N+21A) or three (N+32A) helix turns allowed repression 377

levels similar to those in the wild-type. None of the insertions downstream from the AtzR 378

site (A4I, A6I and A10I) had a significant effect on AtzR-dependent repression of PatzT. In 379

vitro transcription assays performed in the presence of E-σN, IHF, NtrCD55E,S161F and AtzR-380

His6 showed >10-fold AtzR-dependent repression of the wild-type promoter region at both 381

AtzR-His6 concentrations used (Fig. 6,B). A 5 bp insertion (N+5A construct) fully abolished 382

AtzR-dependent repression, while the N+6A template displayed a minor decrease in 383

transcript levels (less than 2-fold). Insertion of a full helix turn (N+10A construct) resulted 384

in repression that was only slightly less efficient than in the wild-type template. Taken 385

together, our results indicate that PatzT repression (i) requires a correct rotational 386

orientation between DNA-bound NtrC and AtzR, (ii) can tolerate an increase in the 387

distance between DNA-bound NtrC and AtzR provided that condition (i) is met, and (iii) 388

does not require correct alignment or a precise distance between the upstream (NtrC and 389

AtzR binding sites) and downstream (IHF and E-σN binding sites) elements of the PatzT 390

promoter region. Considering that NtrC-dependent activation is not sensitive to changes in 391

the distance or orientation of the UAS relative to the E-σN binding site, the evidence 392

obtained does not support the hypothesis that AtzR interference with DNA looping 393

prevents PatzT activation. 394

The rotational orientation of the binding sites does not significantly alter NtrC and 395

AtzR interactions with the PatzT promoter region 396

A trivial explanation to the results above is that the nucleotides inserted in the N+5A 397

and N+6A mutant promoters serendipitously affect AtzR interactions with its binding site, 398

while those inserted in the N+10A, N+21A and N+32 do not, regardless on their effect on 399


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the PatzT promoter architecture. To assess this possibility, AtzR-His6 gel shift assays 400

were performed using wild-type, N+5A, N+6A and N+10A PatzT promoter fragments as 401

probes (Figure 7). AtzR-His6 efficiently retarded all four promoter fragments, indicating that 402

the lack of AtzR-dependent repression of the N+5A and N+6A promoter derivatives is not 403

due to a general defect in AtzR binding. 404

Additionally, we questioned whether the architecture of the NtrC-PatzT promoter 405

complexes formed at the opposite side of the helix might somehow hamper AtzR 406

interaction with its binding site at the mutant N+5A and N+6A promoters. To address this 407

question, we performed DNAse I footprinting analyses in which NtrCD55E,S161F-PatzT 408

complexes preformed on the wild-type, N+5A, and N+10A promoter variants were 409

challenged with increasing concentrations of AtzR (Fig. 8). Addition of NtrCD55E,S161F to the 410

wild-type and mutant probes resulted in similar footprinting patterns around sites NtrC1 411

and NtrC2. Additional hypersensitive bands overlapping the AtzR binding site were also 412

observed, in a variable pattern that may be attributed to differences in sequence due to the 413

insertions present in the mutant promoters. Addition of AtzR-His6 to preformed 414

NtrCD55E,S161F complexes resulted in the replacement of the NtrC-elicited hypersensitive 415

bands overlapping the AtzR binding site with the characteristic AtzR footprint. AtzR-His6 416

binding did not alter the NtrCD55E,S161F footprint upstream from position -126 in the wild-type 417

probe, as discussed above, while the NtrCD55E,S161F footprint was discernible down to 418

position -121 in the N+5A mutant and position -116 in the N+10A mutant. This effect is 419

clearly attributable to a decrease in the overlap of the NtrC and AtzR footprinting patterns 420

as the NtrC2 and AtzR sites are moved apart and correlates very well with the 5- and 10-421

bp insertions present in the mutant promoters. No observable feature in the footprinting 422

patterns could be correlated with the repression proficiency of the promoter variants. 423

Taking together, our results suggest that NtrC and AtzR interact with all the tested PatzT 424

promoter region variants in an independent fashion, and therefore the ability of AtzR to 425


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exert repression cannot be correlated with competition or interference with NtrC for DNA 426

binding. 427

AtzR does not alter NtrC ATPase activity or oligomerization state. 428

The E-σN-dependent promoter activation pathway involves oligomerization of the 429

EBP upon UAS binding to a hexameric conformation that promotes the EBP ATPase 430

activity. Further contacts with the E-σN-promoter closed complex couple ATP hydrolysis to 431

the remodeling of the closed complex into a transcriptionally active open complex (41,42). 432

In order to discern the particular step of this pathway at which AtzR inhibits NtrC activation 433

of PatzT, the possible ability of AtzR to inhibit NtrC oligomerization, AtzR-His6 and 434

NtrCD55E,S161F were incubated separately and simultaneously in in vitro transcription buffer 435

and the protein complexes were resolved in Blue Native gel electrophoresis (Supplemental 436

Material, Fig. S3). Surprisingly, native AtzR-His6 did not migrate as a discrete band and 437

could not be detected in this electrophoresis system. Also, heat-denatured NtrCD55E,S161F 438

did not migrate to its expected size (~53 kDa), but as a single band with an apparent 439

molecular weight of ~90kDa. Both proteins were previously shown to migrate according to 440

their monomer molecular weights in SDS-PAGE (21,33). The native form of NtrCD55E,S161F 441

migrated as three bands, with apparent molecular weights of ~90, ~180 and ~540 kDa, 442

suggesting that this constitutive form is a mixture of monomers, dimers and hexamers in 443

solution. This migration pattern was unchanged in the presence of AtzR-His6, suggesting 444

that the presence of AtzR does not alter the equilibrium between the different oligomeric 445

forms of NtrC. 446

In addition, ATPase activity assays were performed using pure NtrCD55E,S161F in the 447

absence or in the presence of AtzR-His6 (Fig. 9). Addition of AtzR-His6 did not result in a 448

significant decrease in specific ATPase activity (1.3-fold change, p-value=0.26). This 449

assay was also performed in the presence of a ~3-fold molar excess of a 110-bp PatzT 450


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promoter region fragment spanning the NtrC and AtzR binding sites. In the presence of 451

promoter DNA a 2-fold increase (p-value<0.05) in ATPase activity was observed in an 452

AtzR-independent fashion, consistent with the notion that DNA binding stimulates P. 453

putida NtrC oligomerization and ATPase activity, as previously shown for its 454

enterobacterial counterpart and other proteins in this family (41). Significantly, addition of 455

AtzR-His6 did not result in decreased ATP hydrolysis in these conditions. 456

Taken together, these results strongly suggest that AtzR does not inhibit ATP hydrolysis or 457

any of the steps leading to it. As our results above (Fig. 2) indicate that AtzR must operate 458

prior to open complex formation, we propose that PatzT-bound AtzR prevents coupling of 459

ATP hydrolysis to oligomerization of the closed complex into the open complex. 460

461


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DISCUSSION 462

Promoters dependent on the alternative σ factor σN have been widely studied as 463

unique examples of a eukaryotic-like transcriptional activation mechanism in prokaryotic 464

systems (1,24,43). In addition to positive control, a small subset of σN-dependent 465

promoters is subjected to negative regulation. The work presented here demonstrates that 466

a new, unconventional mechanism by which AtzR prevents coupling of NtrC-catalyzed 467

ATP hydrolysis to the isomerization of the E-σN-promoter closed complex to the open 468

complex is accountable for the repression of the Pseudomonas sp. ADP PatzT promoter. 469

Analysis of the few examples of negatively regulated σN-dependent promoters has 470

revealed a fairly diverse array of regulatory mechanisms, generally involving interference 471

of the repressor protein with the activation process (7-12,15,22). The location of the AtzR 472

binding site, adjacent to the NtrC UAS and far upstream from the E-σN recognition 473

element, as well as the early observation that AtzR cannot repress the relatively high 474

levels of UAS-independent, NtrC-activated PatzT transcription observed when the NtrC 475

UAS was inactivated by mutation (22), prompted us to hypothesize that AtzR antagonizes 476

NtrC function when bound to the PatzT promoter region, and therefore performs the role of 477

an anti-activator. To this end, the simplest mechanism would be activator exclusion, 478

involving competition between repressor and activator for interaction with the promoter 479

region, as previously shown for repression of the XylR-activated P. putida Pu promoter by 480

TurA and PprA (11,12). However, gel mobility shift assays showed that AtzR and NtrC 481

simultaneously interact with the PatzT promoter region (Fig. 3), and DNAse I footprinting 482

revealed the simultaneous occupancy of the NtrC1, NtrC2 and AtzR sites (Fig. 4), strongly 483

suggesting that AtzR does not compete with NtrC for DNA binding. It may be argued that, 484

even though AtzR does not exclude NtrC binding the NtrC2 site, the vicinity of DNA-bound 485

AtzR may alter the mode of interaction with this site, a notion supported by the altered 486


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NtrC-induced DNAse I hypersensitivity pattern around NtrC2 in the presence of AtzR (Fig. 487

4). However, promoter derivatives containing insertions of up to 32 bp between the NtrC2 488

and AtzR sites, in which the sites are moved up to three helix turns apart, still show a 489

normal response to AtzR (Fig. 5), indicating that interference with normal NtrC binding is 490

not likely involved in the mechanism of repression. Similarly, the observation that promoter 491

derivatives bearing 4, 6 or 10 bp insertions between the AtzR and IHF binding sites still 492

support wild-type levels of activation and repression (Fig. 5) rules out the possibility that 493

AtzR may interfere with IHF binding to the PatzT promoter region. 494

A second mechanism of anti-activation of σN-dependent promoters exploits the 495

requirement of a DNA loop to facilitate interaction between the DNA-bound EBP and E-σN. 496

Several σN-dependent promoter repressors have been proposed to alter the orientation of 497

the DNA loop hence preventing activation, including the Klebsiella pneumoniae LTTR Nac 498

at its own promoter (7), CcpA at the Bacillus subtilis levanase operon (8) and CRP at the 499

E. coli glnHp2 promoter (9). As AtzR bends DNA upon binding at the PatzT promoter 500

region (Supplemental Material, Fig. S1), AtzR-induced bending may alter the orientation of 501

the DNA loop at the PatzT promoter, thus impairing NtrC interaction with E-σN. However, 502

insertions designed to rotate the NtrC UAS to the opposite side of the helix or to separate 503

it up to three full helix turns from the E-σN binding site failed to diminish PatzT activation 504

(Figs. 5 and 6), indicating that flexibility of the DNA loop is sufficient to accommodate NtrC 505

interaction with RNA polymerase from various orientations and distances. This result is 506

expected for IHF-independent σN-dependent promoters, in which strong binding of E-σN 507

facilitates interaction with the EBP in a position- and orientation-independent fashion 508

(44,45). However, for IHF-dependent promoters, strict dependence on the correct 509

rotational orientation for activation has been documented (38). In vivo expression of the 510

wild-type and mutant PatzT promoters was dependent on both NtrC and IHF 511

(Supplemental Material, Fig. S2). On the other hand, the PatzT promoter displays very 512


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high similarity to the E-σN consensus [TGGCCC-N5-TTGC vs. TGGCAC-N5-TTGC 513

(conserved positions are underlined)]. We propose that, despite the fact that PatzT 514

activation is aided by IHF, strong interaction of E-σN with its highly conserved binding motif 515

may compensate for suboptimal orientation of the NtrC UAS, enabling orientation-516

independent activation. The presence of substantial levels of UAS-independent activation 517

of PatzT (22), a phenomenon associated with promoters with high E-σN occupancy (15,24-518

26) lend further support to this notion. Strikingly, despite the lack of rotational orientation 519

dependency of PatzT activation, AtzR repression of PatzT was impaired when a half-helix 520

turn was inserted between the NtrC UAS and the AtzR binding site, but not when one, two 521

or three full helix turns were inserted at this location (Figs. 5 and 6), indicative of a 522

rotational orientation-dependent effect of AtzR on PatzT activation. However, this pattern 523

was not reproduced when half or a full helix turn was inserted downstream from the AtzR 524

binding site, indicating that a precise rotational orientation of AtzR relative to the 525

downstream elements of the promoter is not required for repression (Fig. 5). If the role of 526

AtzR were to alter the orientation of the DNA bend, a similar dependence on rotational 527

orientation would be expected on both sides of the AtzR binding site (37,38). Taken 528

together, the insensitivity of NtrC-dependent activation to changes in position and 529

rotational orientation, along with the inconsistent effect of altered rotational orientation of 530

the AtzR binding site relative to the upstream and downstream promoter elements fail to 531

support an anti-activation model based solely on a distortion exerted by AtzR on the DNA 532

loop formed at the PatzT promoter. 533

Activation of σN-dependent promoter is a complex process. NtrC can bind its UAS 534

elements both in its unphosphorylated (inactive) and phosphorylated (active) forms. 535

However, phosphorylation promotes oligomerization to a hexameric conformation that 536

enhances ATPase activity. DNA loop-mediated interaction with E-σN bound to DNA in a 537

closed complex couples ATP hydrolysis with the remodeling of the E-σN-promoter closed 538


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complex structure, leading to isomerization to the open complex (6,41,42). Our 539

experimental results show that (i) AtzR does not interfere with NtrC binding to the PatzT 540

UAS, as documented in competitive gel mobility shift and DNAse I footprinting assays 541

(Figs. 3, 4 and 8); (ii) AtzR does not alter NtrC oligomerization state, as assessed by Blue 542

Native gel electrophoresis (Supplemental Material, Fig. S3); (iii) AtzR does not interfere 543

with NtrC phosphorylation, as repression was observed in vitro with an NtrC variant 544

(NtrCD55E,S161F), that does not require phosphorylation, in the absence of a phosphate 545

donor (Figs. 2 and 6); (iv) AtzR does not have a relevant effect on DNA loop formation, as 546

evidenced by the in vivo and in vitro phenotypes of our mutants bearing rotationally altered 547

variants of the PatzT promoter region (Figs. 5 and 6); (v) AtzR does not interfere with NtrC 548

ATPase activity or the events (DNA binding and oligomerization), as shown by the fact that 549

AtzR does not decrease the ATPase activity of NtrC, even in the presence of PatzT 550

promoter region DNA (Fig. 9), and (vi) finally, our in vitro transcription assays indicate that 551

AtzR repression occurs prior to open complex formation (Fig. 2). Taken together, our 552

experimental evidence leads to the conclusion that AtzR prevents activation of the PatzT 553

promoter by preventing the productive interactions between NtrC and E-σN that result in 554

the isomerization of the stalled transcriptional initiation closed complex to the initiation-555

proficient closed complex. 556

The observation that AtzR failed to repress PatzT transcription in vivo when bound 557

at the opposite side of the DNA helix, but did so when full helix turn insertions restores the 558

relative orientation of the AtzR and NtrC binding sites (Figs. 5 and 6) indicates that precise 559

alignment between both DNA-bound proteins is critical for repression. Two different 560

mechanisms of anti-activation are compatible with these observations. Firstly, DNA-bound 561

AtzR may interact specifically with NtrC to form a complex that is not proficient in coupling 562

ATP hydrolysis with closed complex remodeling. Should this be the case, the effects of 563

positive or negative cooperativity between both proteins may be too subtle to detect with 564


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the sensitivity of our DNA-binding assays. Additional experimentation in search for further 565

evidence of specific protein-protein interaction between NtrC and AtzR has unfortunately 566

been inconclusive: in vivo interaction was apparent with the bacterial adenylate cyclase-567

based two-hybrid (BACTH) assay, but attempts to detect interaction between the purified 568

proteins in vitro have been so far unsuccessful (data not shown). Alternatively, AtzR may 569

act as a "roadblock" to sterically hinder productive interactions between NtrC and E-σN. 570

This mechanism would not require specific interactions between AtzR and NtrC, and may 571

be mediated either by the bulk of the AtzR tetramer bound next to the NtrC binding site, or 572

by the loss of flexibility of the intervening DNA between the NtrC and E-σN binding sites 573

due to AtzR-mediated DNA bending. While neither of these mechanisms has been 574

previously documented for the repression of a σN-dependent promoter, they are 575

reminiscent of the repression mechanism at the E. coli CRP-dependent deo promoter, in 576

which CytR binds in the vicinity of the CRP binding site and interacts with CRP to prevent 577

contacts with the carboxyl terminal domain of the RNA polymerase α subunit (46,47). We 578

hope that our future research will help clarify these specific details of the repression 579

mechanism at this unique bacterial promoter. 580

581


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ACKNOWLEDGMENTS 582

We wish to thank Ana B. Hervás (CABD, Universidad Pablo de Olavide), Linda U. 583

M. Johansson, Lisandro M.D. Bernardo, Eleonore Skärfstad and Victoria Shingler (Umeå 584

University) for purified proteins; Guadalupe Martín and Nuria Pérez for technical help; and 585

all members of the Govantes and Santero laboratories at CABD for their insights and 586

helpful suggestions. 587

588


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Regulatory Network for Nitrogen Assimilation in Pseudomonas putida. J Bacteriol 681

191:6123-6135. 682

34. Johansson LU, Solera D, Bernardo LM, Moscoso JA, Shingle, V. 2008. Sigma(54)-683

RNA polymerase controls sigma(70)-dependent transcription from a non-overlapping 684

divergent promoter. Mol Microbiol 70:709-723. 685

35. Nash HA, Robertson CA, Flamm E, Weisberg RA, Miller HI. 1987. Overproduction 686

of Escherichia coli integration host factor, a protein with nonidentical subunits. J 687

Bacteriol 169:4124-4127. 688

36. Hervás AB, Canosa I, Santero E. 2010. Regulation of glutamate dehydrogenase 689

expression in Pseudomonas putida results from its direct repression by NtrC under 690

nitrogen-limiting conditions. Mol Microbiol 78:305-319. 691

37. Claverie-Martin F, Magasanik B. 1992. Positive and negative effects of DNA bending 692

on activation of transcription from a distant site. J Mol Biol 227:996-1008. 693

38. Molina-Lopez JA, Govantes F, Santero E. 1994. Geometry of the process of 694

transcription activation at the σ54-dependent nifH promoter of Kebsiella pneumoniae. J 695

Biol Chem 269:25419-25425. 696

39. Porrúa O, Platero AI, Santero E, Del Solar G, Govantes F. 2010. Complex interplay 697

between the LysR-type regulator AtzR and its binding site mediates atzDEF activation 698

in response to two distinct signals. Mol Microbiol 76:331-347. 699

40. Porrúa O, López-Sánchez A, Platero AI, Santero E, Shingler V, Govantes F. 2013. 700

An A-tract at the AtzR binding site assists DNA binding, inducer-dependent 701

repositioning and transcriptional activation of the PatzDEF promoter. Mol Microbiol 702

90:72-87. 703


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41. Bush M, Dixon R. 2012. The role of bacterial enhancer binding proteins as specialized 704

activators of σ54-dependent transcription. Microbiol Mol Biol Rev 76:497-529. 705

42. Joly N, Zhang N, Buck M, Zhang Z. 2012. Coupling AAA protein function to regulated 706

gene expression. Biochim Biophys Acta 1823:108-116. 707

43. Kustu S, Santero E, Keener J, Popham D, and Weiss D. 1989. Expression of sigma-708

54 (ntrA)-dependent genes is probably united by a common mechanism. Microbiol Rev 709

53:367-376. 710

44. Ninfa A, Reitzer L, Magasanik B. 1987. Initiation of transcription at the bacterial 711

glnAp2 promoter by purified E. coli components is facilitated by enhancers. Cell 712

60:1039-1046 713

45. Reitzer L, Magasanik B. 1986. Transcription of glnA in E. coli is stimulated by 714

activator bound to sites far from the promoter. Cell 45:785-787 715

46. Valentin-Hansen P, Sogaard-Andersen L, Pedersen H. 1996. A flexible partnership: 716

the CytR anti-activator and the cAMP-CRP activator protein, comrades in transcription 717

control. Mol Microbiol 20:461-466. 718

47. Kristensen HH, Valentin-Hansen P, Sogaard-Andersen L. 1997. Design of CytR-719

regulated, cAMP-CRP dependent class II promoters in Escherichia coli: RNA 720

polymerase-promoter interactions modulate the efficiency of CytR repression. J Mol 721

Biol 266:866-876. 722

48. Hanahan D. 1983. Studies on transformation of Escherichia coli with plasmids. J Mol 723

Biol 166:557-580. 724


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49. Govantes F, Molina-Lopez JA, Santero E. 1996. Mechanism of coordinated 725

synthesis of the antagonistic regulatory proteins NifL and NifA of Klebsiella 726

pneumoniae. J Bacteriol 178:6817-6823. 727

50. Franklin FC, Bagdasarian M, Bagdasarian MM, Timmis KN. 1981. Molecular and 728

functional analysis of the TOL plasmid pWWO from Pseudomonas putida and cloning 729

of genes for the entire regulated aromatic ring meta cleavage pathway. Proc Natl Acad 730

Sci USA 78:7458-7462. 731

51. Marqués S, Gallegos MT, Manzanera M, Holtel A, Timmis KN, Ramos JL. 1998. 732

Activation and repression of transcription at the double tandem divergent promoters for 733

the xylR and xylS genes of the TOL plasmid of Pseudomonas putida. J Bacteriol 734

180:2889-2894. 735

52. Kim J, Zwieb C, Wu C, Adhya S. 1989. Bending of DNA by gene-regulatory proteins: 736

construction and use of a DNA bending vector. Gene 85:15-23. 737

53. Figurski DH, Helinski DR. 1979. Replication of an origin-containing derivative of 738

plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci 739

USA 76:1648-1652. 740

54. Elliott T, Geiduschek EP. 1984. Defining a bacteriophage T4 late promoter: absence 741

of a "-35" region. Cell 36:211-219. 742

743


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LEGENDS TO FIGURES 744

Figure 1. Comparison of the PatzR and PatzT promoter regions. Cartoon depicting the 745

identified cis-acting elements at the PatzR (top) and PatzT (bottom) promoter regions. 746

Promoters are indicated as closed arrows, NtrC binding sites as closed boxes, the 747

symmetrical AtzR-binding sites as two open boxes, and the IHF binding site as a shaded 748

box. The scale indicates coordinates relative to the transcription start point. 749

Figure 2. In vitro repression of the PatzT promoter. In vitro transcription assays using 750

the PatzT promoter region as a template. Isomerization to the open complex was triggered 751

by addition of NtrCD55E,S161F to an ATP-containing mixture (A), or addition of ATP to an 752

NtrCD55E,S161F-containing reaction mixture (B). Each panel shows an autoradiograph of the 753

representative PAGE gel (top), and a plot of the quantified relative transcript abundance 754

(bottom). AtzR-His6 concentrations were 0 (lanes 1), 40 nM (lanes 2), 80 nM (lanes 3), 160 755

nM (lanes 4) or 240 nM (lanes 5). Legends denote the order of AtzR-His6 addition, before 756

(AtzR-NtrC or AtzR-ATP), or after (NtrC-AtzR or ATP-AtzR) open complex formation. 757

Symbols and error bars represent the averages and standard deviations of at least three 758

independent assays. Significance of the differences between the two data sets for each 759

experiment was assessed by the t-test for unpaired samples not assuming equal 760

variances. *: p<0.05. **: p<0.01. ***: p<0.001. Differences were not significant when not 761

indicated. 762

Figure 3. Gel mobility shift assay of AtzR and NtrC on the PatzT promoter region. 763

Autoradiograph of a representative PAGE gel. Lane 1: free PatzT probe. Lanes 2-6: A pre-764

formed complex containing 2 µM NtrCD55E,S161F challenged with 0, (lane 2), 50 nM (lane 3), 765

100 nM (lane 4), 200 nM (lane 5), or 400 nM AtzR-His6. Lanes 7-11: A pre-formed 766

complex containing 400 nM AtzR-His6, challenged with 0 (lane 7), 250 nM (lane 8), 500 767

nM (lane 9), 1 µM (lane 10), or 2 µM (lane 11) NtrCD55E,S161F. AtzR-DNA, NtrC-DNA or 768


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AtzR-NtrC-DNA complexes are denoted by closed, shaded, and open arrows, respectively. 769

Figure 4. DNAse I footprinting assay of AtzR and NtrC on the PatzT promoter region. 770

Autoradiograph of a representative PAGE gel. Lane 1: free PatzT probe. Lanes 2-6: A pre-771

formed complex containing 2 µM NtrCD55E,S161F challenged with 0, (lane 2), 50 nM (lane 3), 772

100 nM (lane 4), 200 nM (lane 5), or 400 nM AtzR-His6. Lanes 7-11: A pre-formed 773

complex containing 400 nM AtzR-His6, challenged with 0 (lane 7), 250 nM (lane 8), 500 774

nM (lane 9), 1 µM (lane 10), or 2 µM (lane 11) NtrCD55E,S161F. Coordinates relative to PatzT 775

transcriptional start are indicated on the right. The approximate locations of the NtrC and 776

AtzR binding sites are denoted by closed and open boxes, respectively. Closed bars and 777

circles indicate positions rendered protected or hypersensitive, respectively, by NtrC 778

binding. Shaded bars and circles indicate positions rendered protected or hypersensitive, 779

respectively, by AtzR binding. 780

Figure 5. β-galactosidase activity of PatzT-lacZ fusions. A. Schematic of the PatzT 781

promoter derivatives used for gene fusion analysis. Sequences of the NtrC2, AtzR and IHF 782

binding sites are shown. Binding sites are coded as in Fig. 1. Vertical arrows indicate the 783

location of the insertions. Plasmid name, construct designation and the number and 784

identity of the nucleotides inserted is shown for each construct. B. Results of the β-785

galactosidase assays performed with the PatzT promoter derivatives above fused to lacZ 786

in pMPO234. Columns and error bars represent the averages and standard deviations of 787

at least three independent assays. Significance of AtzR regulation (-AtzR vs. +AtzR) was 788

assessed for each promoter variant under both nitrogen sufficiency and nitrogen limitation 789

by the t-test for unpaired samples not assuming equal variances. *: p<0.05. **: p<0.01. ***: 790

p<0.001. Differences were not significant when not indicated. Significance of nitrogen 791

regulation (Ammonium vs. serine) was also assessed and differences were found to be 792

significant (0.05>p>0.00002) in all cases (not shown). 793


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Figure 6. In vitro activation and repression of PatzT promoter derivatives. In vitro 794

transcription assays using PatzT promoter region derivatives as templates. A. Activation of 795

PatzT promoter derivatives in the presence of 75 mM IHF and 0 (lanes 1), 200 nM (lanes 796

2) or 400 nM (lanes 3) NtrCD55E,S161F. B. Repression of PatzT promoter region derivatives 797

in the presence of 400 nM NtrCD55E,S161F, 75 nM IHF and 0 (lanes 1), 75 nM (lanes 2) or 798

150 nM (lanes 3) AtzR-His6. Each panel shows an autoradiograph of a representative 799

PAGE gel (top), and a plot of the quantified relative transcript abundance (bottom). Bars 800

represent the averages and standard deviations of at least three independent assays. 801

Significance of NtrC-dependent activation (panel A) and AtzR-dependent repression 802

(panel B) was assessed for each promoter variant by the t-test for unpaired samples not 803

assuming equal variances. *: p<0.05. **: p<0.01. ***: p<0.001. Differences were not 804

significant when not indicated. 805

Figure 7. Gel mobility shift assay of AtzR on PatzT promoter region derivatives. 806

Autoradiograph of a representative PAGE gel, containing the indicated PatzT promoter 807

derivative probes, and 0 (lanes 1), 50 nM (lanes 2) or 100 nM (lanes 3) AtzR-His6. 808

Figure 8. DNAse I footprinting assay of AtzR and NtrC on PatzT promoter region 809

derivatives. Autoradiograph of a representative PAGE gels containing the wild-type (A), 810

N+5A (B), or N+10A (C) probe. Lane 1: free PatzT probe. Lanes 2-6: A pre-formed 811

complex containing 2 µM NtrCD55E,S161F challenged with 0, (lane 2), 50 nM (lane 3), 100 nM 812

(lane 4), 200 nM (lane 5), or 400 nM AtzR-His6. The approximate locations of the NtrC and 813

AtzR binding sites are denoted by closed and open boxes, respectively. Closed bars and 814

circles indicate positions rendered protected or hypersensitive, respectively, by NtrC 815

binding. Shaded bars and circles indicate positions rendered protected or hypersensitive, 816

respectively, by AtzR binding. 817

Figure 9. Effect of AtzR and PatzT promoter DNA on NtrC ATPase activity. ATPase 818


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activity assay of NtrCD55E,S161F (231 nM), in the presence or in the absence of AtzR-His6 819

(240 nM) and/or a PatzT promoter region DNA (770 nM). Data are specific molar activity, 820

assuming that the NtrC active form is a hexamer. Bars represent the averages and 821

standard deviations of at least three independent assays. Significance was assessed by 822

the t-test for unpaired samples not assuming equal variances. *: p<0.05. Differences were 823

not significant when not indicated. 824


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TABLES

Table 1. Bacterial strains and plasmids used in the present work Bacterial strain Genotype/phenotype Reference/source E. coli

DH5α φ80dlacZ∆M15 ∆(lacZYA-argF)U169 recA1 endA1 hsdR17 (rk- mk

+) supE44 thi-1 gyrA relA1 48 KT5746 N5271 [galK ilv his (λ cIts

5857 N7N53 ΔBamΔHI)]/ pPLhimhimA-5. Apr 35 NCM631 hsdS gal λDE3:lacI lacUV5:gen1(T7 RNA polymerase) ∆lac linked to Tn10 49 P. putida KT2440 mt-2 hsdR1 (r- m+) 50 KT2440-IHF3 mt-2 hsdR1 (r- m+) ∆ihfA::Tcr 51 KT2442 mt-2 hsdR1 (r- m+) Rifr 50 MPO201 mt-2 hsdR1 (r- m+) ∆ntrC::Tcr 19 Plasmid Genotype/phenotype Reference/source pBEND2 Vector for circular permutation analysis. Apr 52 pIZ227 pACYC184-derived plasmid containing lacIq and the T7 lysozyme gene; Cmr 49 pMPO103 1.45 Kb fragment containing atzR and the 5´end of atzD, cloned in pBluescript II SK (+). Apr 19 pMPO109 atzR coding sequence and promoter region cloned in pKT230. Kmr 19 pMPO135 pET23b plasmid derivative overexpressing AtzR-His6. Apr 21 pMPO234 Broad-host range lacZ translational fusion vector, based on pBBR1MCS-4. Apr 15 pMPO310 NtrCD55E,S161F expressed from PlacUV5 in a pACYC184-derived plasmid, Cmr 33 pMPO805 PatzT -lacZ transcriptional fusion in pMPO234 carrying the wild-type PatzT promoter. Apr 22 pMPO820 pBEND2 derivative containing the PatzT promoter region This work pMPO831 PatzT wild type template plasmid for in vitro transcription, based on pTE103. Apr 22 pMPO835 PatzT -lacZ transcriptional fusion in pMPO234 bearing a 4 bp insertion between the AtzR and IHF sites. Apr This work pMPO836 PatzT -lacZ transcriptional fusion in pMPO234 bearing a 5 bp insertion between the AtzR and IHF sites. Apr This work pMPO837 PatzT -lacZ transcriptional fusion in pMPO234 bearing a 10 bp insertion between the AtzR and IHF sites. Apr This work pMPO849 PatzT -lacZ transcriptional fusion in pMPO234 bearing a 5 bp insertion between the NtrC2 and AtzR sites. Apr This work pMPO850 PatzT -lacZ transcriptional fusion in pMPO234 bearing a 6 bp insertion between the NtrC2 and AtzR sites. Apr This work pMPO851 PatzT -lacZ transcriptional fusion in pMPO234 bearing a 10 bp insertion between the NtrC2 and AtzR sites. Apr This work pMPO853 PatzT template for in vitro transcription, bearing a 5 bp insertion between the NtrC2 and AtzR sites, based on pTE103. Apr This work pMPO854 PatzT template for in vitro transcription, bearing a 6 bp insertion between the NtrC2 and AtzR sites, based on pTE103. Apr This work pMPO855 PatzT template for in vitro transcription, bearing a 10 bp insertion between the NtrC2 and AtzR sites, based on pTE103. Apr This work pMPO863 PatzT -lacZ transcriptional fusion in pMPO234 bearing a 21 bp insertion between the NtrC2 and AtzR sites. Apr This work pMPO864 PatzT -lacZ transcriptional fusion in pMPO234 bearing a 32 bp insertion between the NtrC2 and AtzR sites. Apr This work pPLhiphimA-5 IHF overproduction plasmid. Apr 35 pRK2013 Helper plasmid used in conjugation. Kmr Tra+ 53 pTE103 Vector for in vitro transcription assays; Apr 54


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