Regulation of the Rhodobacter sphaeroides 2.4.1 hemA gene by

Regulation of the Rhodobacter sphaeroides 2.4.1 hemA gene by PrrA and FnrL 1

2

3

Britton Ranson-Olson1#

and Jill H. Zeilstra-Ryalls2* 4

1Department of Biological Sciences, Oakland University, Rochester, Michigan 48309 and 5

2Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 6

43403 7

8

9

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*Corresponding author. Mailing address: Department of Biological Sciences, 217 Life Sciences 12

Building, Bowling Green State University, Bowling Green, OH 43403. Phone: (419) 372-2872. 13

Fax: (419) 372-2024. E-mail: [email protected] 14

15

#Permanent address: Department of Biological Sciences, Lake Superior State University, 615 W. 16

Easterday Avenue, Sault Ste. Marie, MI 49783. 17

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Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.00828-08 JB Accepts, published online ahead of print on 8 August 2008

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ABSTRACT 1

Part of the oxygen responsiveness of Rhodobacter sphaeroides 2.4.1 tetrapyrrole 2

production is through changes in transcription of the hemA gene, which codes for one of two 3

isoenzymes catalyzing 5-aminolevulinic acid synthesis. Regulation of hemA transcription from 4

its two promoters is mediated by the DNA binding proteins FnrL and PrrA. The two PrrA 5

binding sites, I and II, located upstream of the more 5' hemA promoter (P1), are of equal 6

importance to transcription under aerobic conditions while anaerobically binding site II is of 7

greater importance. By phosphoprotein affinity chromatography and immunoblot analyses we 8

have now shown that phosphorylated PrrA levels in the cell increase with decreasing oxygen 9

tensions. Then, using both in vivo and in vitro methods we demonstrated that the relative 10

affinities of phosphorylated versus unphosphorylated PrrA towards the two binding sites differ, 11

with phosphorylated PrrA having greater affinity for site II. We also showed that PrrA 12

regulation is directed towards the P1 promoter. We propose that the PrrA component of 13

anaerobic induction of P1 transcription is attributable to higher affinity of phosphorylated versus 14

unphosphorylated PrrA towards binding site II. Anaerobic activation of the more 3' hemA 15

promoter (P2) is thought to involve FnrL binding to an FNR consensus-like sequence located 16

upstream of the P2 promoter, but the contribution of FnrL to P1 induction may be indirect since 17

the P1 transcription start is within the putative FnrL binding site. We present evidence 18

suggesting that the indirect action of FnrL works through PrrA, and discuss possible 19

mechanisms. 20

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INTRODUCTION 1

The hemA gene of the α-Proteobacterium Rhodobacter sphaeroides 2.4.1 codes for one 2

of two ALA synthase isoenzymes responsible for 5-aminolevulinic acid (ALA) production. 3

ALA is the precursor to all tetrapyrroles, and in keeping with the ability of this bacterium to 4

generate energy from respiratory chains and from anoxygenic photosynthesis, R. sphaeroides 5

synthesizes both heme and bacteriochlorophyll (Bchl) as well as vitamin B12, a necessary 6

cofactor in Bchl formation (14) and also methionine and cysteine biosynthesis (3). Both the 7

absolute and relative amounts of these tetrapyrroles change dramatically in this bacterium as part 8

of its metabolic response to changes in its environment, and oxygen is a key player in controlling 9

those events. Certainly one of the most notable responses is the over 100-fold increase in Bchl 10

production when oxygen tensions are reduced (23), which is necessary in preparation for 11

photosynthesis. At the same time, appropriate levels of heme and vitamin B12 must also be 12

maintained. This radical change in tetrapyrrole biosynthesis is enabled in part by an increase in 13

ALA formation (23), and with respect to oxygen control we have found that among the ALA 14

synthase genes present in this organism, alterations in hemA expression are responsible (38, 40). 15

Therefore, investigations of hemA regulation should provide insights into the mechanisms of 16

oxygen control in the cell. Our studies to date have shown that hemA is transcribed from two 17

promoters and that two global regulatory proteins, PrrA and FnrL, regulate hemA transcription in 18

response to changes in oxygen tensions (11, 30). 19

FnrL is a homolog of the Escherichia coli anaerobic regulatory protein Fnr, an oxygen-20

labile DNA binding protein that is active anaerobically (20). The hemA upstream sequences 21

contain a perfect FNR consensus sequence (35). FnrL has been shown to activate transcription 22

from the downstream P2 promoter in response to lowering oxygen tensions (11), and this is 23

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consistent with the -45 position of the FNR consensus sequence relative to the transcription start 1

site of P2. However, as the transcription start site of the upstream P1 promoter is centrally 2

positioned within the FNR consensus sequence an activator role towards P1 involving FnrL 3

binding to the consensus would not be expected. Nevertheless, we have demonstrated that an 4

intact fnrL gene is also required for increased expression from the P1 promoter in response to 5

lowering oxygen tensions (11, 38). Therefore, we have proposed that FnrL has an indirect role 6

with respect to transcription from P1, which predicts the existence of another factor that would 7

act directly. 8

PrrA is the DNA binding regulator protein of the PrrBA two-component redox-9

responsive regulatory system in R. sphaeroides. From a combination of microarray and other 10

investigations, the PrrA regulon has been shown to include tetrapyrrole biosynthesis genes (10, 11

11, 27, 30, 41), as well as genes with roles in many other biological processes, including 12

photosynthesis, carbon and nitrogen fixation, denitrification, formaldehyde dehydrogenase 13

activity, and aerotaxis (2, 13, 18, 31). With respect to hemA, two PrrA binding sites that are 14

centered -163 and -67 bp relative to the P1 transcription start site have been identified. In vitro 15

studies indicated that PrrA activates P1 transcription, and while the in vivo data are consistent 16

with an activator role for PrrA they also reveal that although both PrrA binding sites are of equal 17

importance in transcription under aerobic conditions, anaerobically, one binding site 18

predominates (30). 19

From these studies, two central questions arise: How is the differential effect of PrrA 20

towards the two binding sites achieved, and how do PrrA and FnrL work together to achieve the 21

correct levels of hemA expression? Our investigations of these questions not only provide new 22

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insights into hemA gene expression, but they may also contribute to understanding the actions of 1

these DNA binding proteins towards other members of both regulons. 2

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MATERIALS AND METHODS 1

Bacterial strains, plasmids and growth conditions. The bacterial strains and plasmids 2

used in this study are listed in Table 1. The E. coli strains were grown at 37°C in Luria-Bertani 3

media (32). Antibiotics at final concentrations of 100 µg/ml of ampicillin (Ap), 15 µg/ml of 4

tetracycline (Tc), and 50 µg/ml of kanamycin (Kn) or spectinomycin and (Sp) streptomycin (St) 5

were used for strain selection or plasmid maintenance. R. sphaeroides strains were cultured at 6

28°C in Sistrom’s succinate minimal medium A (5, 7, 34). The following growth conditions 7

were applied: Highly aerobic conditions were achieved by sparging liquid cultures with a 8

mixture of 30% oxygen, 2% carbon dioxide, and 68% nitrogen; semi-aerobic conditions were 9

achieved by sparging liquid cultures with a mixture of 2% oxygen, 2% carbon dioxide, and 96% 10

nitrogen; anaerobic conditions were achieved by either sparging liquid cultures with a mixture of 11

2% carbon dioxide and 98% nitrogen or by growing the cells in completely filled screw-capped 12

tubes. When solid media were used for anaerobic incubations, the plates were placed in jars and 13

an anaerobic environment was generated using BD BBL GasPak Anaerobic System Envelopes 14

(Becton, Dickinson and Company, Sparks, MD). For anaerobic dark growth, liquid and solid 15

media were supplemented with yeast extract (final concentration of 1%, w/v) and dimethyl 16

sulfoxide (DMSO) as an alternate electron acceptor (final concentration of 0.06 M), and for 17

photosynthetic growth anaerobic cultures were placed in front of banks of incandescent lights 18

(ca. 10 W/m2). Final concentrations of 50 µg/ml of Sp and St or Kn, and 0.5 µg/ml of Tc were 19

used when appropriate. Reagent-grade antibiotics and chemicals were purchased from Sigma 20

Chemical Co. (St. Louis, MO). 21

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Conjugations and transformations. Plasmids were mobilized into R. sphaeroides by 1

triparental mating as described previously by Davis et al. (5), with the HB101(pRK2013) helper 2

strain (6, 12). Transformation of E. coli was carried out using CaCl2-treated cells (32). 3

DNA manipulations and DNA sequence analysis. Standard protocols (32) or 4

manufacturers' instructions were followed for general DNA and plasmid manipulations, 5

including purification, isolation, restriction endonuclease treatment, and other enzymatic 6

treatment. Plasmid DNA was purified using the FastPlasmid Mini kit (Qiagen, Valencia, CA), 7

and the Zymoclean purification kit (Zymo Research Co., Orange, CA) was used for isolation of 8

DNA from agarose. Restriction enzymes were purchased from New England BioLabs, Inc. 9

(Beverly, MA), Gibco-BRL/Life Technologies, Inc. (Gaithersburg, MD), and Promega

(Madison, 10

WI). DNA sequencing was performed on an ABI Prism 310 Genetic Analyzer with the ABI 11

Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Inc., 12

Foster City, CA), and sequencing reactions were prepared according to manufacturers' 13

instructions. To improve primer extension reactions dimethyl sulfoxide (DMSO) was added at a 14

final concentration of 5% due to the high G+C content of R. sphaeroides templates. 15

Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). 16

Construction of ββββ-galactosidase reporter plasmids. The Quikchange Site-Directed 17

Mutagenesis Kit (Stratagene, La Jolla, CA) was used to carry out oligonucleotide-directed 18

mutagenesis. In all cases, the integrity of the relevant sequences were confirmed by DNA 19

sequencing. The mutations introduced into the hemA sequences are indicated in Fig. 1. Plasmid 20

templates with 316 bp of hemA sequences upstream of the translation initiation site that were 21

either unaltered, altered in PrrA binding site I, binding site II, or both binding sites (30), were 22

used as templates in oligonucleotide-directed mutagenesis reactions to create the P2 promoter 23

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deletions with the primers HemAP2DelUP and its complement, HemAP2DelDOWN (11). Each 1

of the altered hemA upstream sequences (Fig. 1) were excised from the vector using the unique 2

PstI and XbaI sites flanking the hemA sequences, and ligated with the promoterless lacZ vector 3

pCF1010 that had been restricted with PstI and XbaI (Table 1). 4

Construction of plasmids with wild type or mutant prrA genes. The wild type prrA 5

gene was amplified from purified genomic DNA (1) using primers PrrA5'UP, 5’-6

GGCAAAGCCCTCGCTCGTC-3’, and PrrA3’DOWN, 5'-7

CTTGATCGCAGCCTCGAACCAG-3’, and ligated into the EcoRV site of pUI1087 (40) to 8

create plasmid pBRO86. This plasmid was then used in oligonucleotide-directed mutagenesis 9

with the primers PrrAD63KUP, 5'-GCCTATGCAGTGGTGAAGCTGCGGCTCGAGGAC-3', 10

and its complement, PrrAD63KDOWN, to create a mutant prrA gene coding for the D63K 11

amino acid-substituted protein. Using the same plasmid template, a truncated prrA' gene that 12

lacks sequences coding for the last 24 amino acids of PrrA was generated by PCR with the 13

primers PrrA5’UP and PrrATrunc, 5'-AAGCTTCAGACATTGCGGTCGCACATTTC -3'. 14

Subsequent oligonucleotide-directed mutagenesis of the prrA' gene with the primers 15

PrrAD63KUP and PrrAD63KDOWN further altered the sequences to encode a D63K amino acid 16

substitution and truncated protein. All of the prrA mutant alleles were excised from the vector 17

plasmids using the flanking HindIII and BamHI sites and ligated into HindIII-BamHI restricted 18

pRK415, creating plasmids pD63K, pPrrA' and pD63K-PrrA'. 19

Construction of R. sphaeroides mutant strains. For construction of the prrA null 20

mutant BRO107 and fnrLprrA double null mutant BRO108, in which the PrrA coding sequences 21

are deleted, the cre-lox site-specific recombinase vectors (25) were used. To generate the target 22

strain containing the loxP sites for the cre recombinase the suicide vector pBRO81 was 23

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constructed in which 491 bp upstream and 515 bp downstream of the PrrA coding sequences are 1

inserted into pCM184 such that they are correctly oriented 5' to 3' with respect to each other, 2

with the loxP-Knr-loxP DNA sequences positioned between them. This plasmid was mobilized 3

into wild type strain 2.4.1 and fnrL null mutant strain JZ1691 (Table 1). The plasmid pCM154 4

that carries the cre site-specific recombinase gene was then mobilized into suitable recombinant 5

candidates that were Knr but Tc

s. For several of these Tc

r exconjugants, which now scored as 6

Kns, the pCM154 plasmid was segregated away by culturing the cells in the absence of Tc. 7

Recombination events in both BRO107 and BRO108 were confirmed by PCR amplification of 8

their genomic DNA sequences using primers corresponding to sequences flanking the prrA 9

deletion and subsequently sequencing the PCR products. Additionally, the absence of PrrA was 10

confirmed by immunoblot analysis. 11

For construction of the wild type and prrA-D63K "knock-in" strains JZ4148 and JZ4141, 12

the wild type or mutant genes were isolated from pBRO86 and its mutagenized derivative 13

plasmid using PvuII then ligated to the suicide vector fragment of pSUP202 generated using 14

ScaI. The resulting recombinant pSUP-WT and pSUP-D63K plasmids were mobilized into the 15

prrA null mutant strain BRO107 and plasmid integrants were selected for using Tcr. PCR and 16

immunoblot analyses were used to confirm the presence and functionality of the wild type and 17

mutant prrA alleles. 18

PrrA purification and modification. The PrrA protein was purified from ER2566 with 19

plasmid pJC407 (4), an E. coli strain that expresses a PrrA intein/chitin-binding domain fusion 20

protein, using the IMPACT T7: One-Step Protein Purification System (New England Biolabs, 21

Waltham, MA). Binding of the fusion protein to chitin beads and release of the PrrA protein by 22

the intein cleavage reaction was carried out under conditions described previously (4). The 23

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purified protein was then concentrated, dialyzed against Storage Buffer (40 mM Tris-HCl, pH 1

7.9, 50 mM KCl, 5 mM MgCl2, and 1 mM DTT) and stored as described previously (4). 2

Treatment of PrrA with acetyl phosphate was as previously described (4); treatment with 3

the phosphate analogue BeF3- (37) was performed as described by Laguri et al. (21). BeF3

- was 4

generated in situ in reactions containing 30 µM PrrA, 2 mM BeCl2, 6 mM NaF, and 20 mM 5

MgCl2, in a total volume of 20 µl that were incubated for thirty minutes at 30°C. 6

Phosphoprotein enrichment. Affinity chromatography of phosphoproteins with the BD 7

Phosphoprotein or the TALON® PMAC Phosphoprotein Enrichment Kits (Clontech, Mountain 8

View, CA), was performed according to the manufacturer’s instructions except that an additional 9

final wash with 20 mM Tris-HCl, 0.5 M NaCl, pH 7.5 was included prior to elution to improve 10

specificity. Samples used were crude lysates of cells in Buffer A (a component of the 11

enrichment kits) and protease inhibitor cocktail (Sigma Chemical Co.) at the manufacturers’ 12

recommended concentrations. The crude cell lysates were prepared by passaging the cells 13

through an SLM-Aminco French pressure cell (Spectronic Instruments Inc., Rochester, NY) at 14

700 lb/in2, and insoluble material was pelleted by centrifugation at 13,000 × g for 15 min at 4°C. 15

Immunoblot analysis. Nitrocellulose membranes (Micron Separations Inc., Westboro, 16

MA) were prepared by electrophoretic transfer of proteins resolved by PAGE using 12% gels 17

from Invitrogen (Carlsbad, CA). The membranes were then probed according to standard 18

procedures (15), using a 1:5000 dilution of primary PrrA or 1:10,000 dilution of primary HemA 19

rabbit antisera. In all cases, secondary antibody was alkaline phosphatase-conjugated goat anti-20

rabbit antiserum (Sigma Chemical Co.). Detection of immunocomplexes was carried out using 21

the ImmunO Alkaline Phosphatase Substrate BCIP/NBT (MP Biomedicals, LLC., Aurora, OH). 22

Measurements of relative band intensities were made using Kodak Image Analysis software. 23

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Electrophoretic Mobility Shift Assays (EMSAs). End-labeled DNA was generated by 1

PCR using primer pairs in which one of the primers is 5’ end-labeled with biotin. Suitable 2

concentrations of end-labeled DNA were determined by dot blot analysis as described previously 3

(30). The binding reactions contained approximately 150 nM biotin-labelled target DNA, 1.33 4

µM PrrA or BeF3--PrrA, 2.5% glycerol, 50 ng/µl poly (dA•dT), 40 mM TRIS pH 7.9, 50 mM 5

MgCl2, 2 mM EDTA, and varying amounts of unlabeled competitor DNA in a total volume of 20 6

µl, and were incubated 20 min at room temperature, followed by the addition of 5µl loading 7

buffer (Pierce Chemiluminescent EMSA Kit). The mobility shift assays were carried out 8

according to the manufacturer’s instructions with the exception of the poly (dA•dT) substitution 9

(Sigma Chemical Co.), for poly (dC•dG), which is recommended to reduce non-specific binding 10

of high G+C genomes. Sample electrophoresis, transfer, and detection of the biotin-labeled 11

DNA were performed as previously described (30). 12

ββββ-galactosidase activity assays. Assays of β-galactosidase activity levels in crude cell 13

lysates were performed as previously described (36). The lysates were prepared as described for 14

the phosphoprotein affinity chromatography with the exception that these cells were lysed in 15

phosphate buffer (0.1 M NaPO4, pH 7.7). 16

Protein concentrations. Protein concentrations were determined with Pierce BCA 17

Protein Assay reagents or the Bio-Rad Protein Assay Dye reagent concentrate (Hercules, CA), 18

using bovine serum albumin as a standard in all cases. 19

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RESULTS 1

In vivo analysis of the phosphorylation state of PrrA versus oxygen availability. 2

Binding affinity of PrrA towards either or both of its target sites within the hemA upstream 3

sequences (Fig. 1) may be affected by the phosphorylation state of the protein. To consider this, 4

we needed to first establish under what conditions phosphorylated PrrA is present in the cell. 5

We used a combination of phosphoprotein affinity chromatography and immunoblot analyses to 6

examine samples prepared from wild type 2.4.1 cells grown under different conditions, and 7

found that as the growth conditions became increasingly anaerobic the amounts of 8

immunodetectable PrrA eluted from the affinity columns increased (Fig. 2). Thus, in agreement 9

with measurements of the kinase activity of its cognate sensor partner protein PrrB, which is 10

inhibited under aerobic growth conditions (29), our results indicate that the levels of 11

phosphorylated PrrA in the cell increased as oxygen tensions decreased. 12

In vivo analysis of the phosphorylation state of PrrA in the presence of oxygen, in a 13

cytochrome cbb3 oxidase- mutant. The cytochrome cbb3 oxidase

- mutant strain JZ722 was 14

isolated, following the application of transposon mutagenesis, in a selection for mutants having 15

increased transcription from hemA upstream sequences under aerobic conditions (39). From 16

studies of this oxidase- mutant stemmed the hypothesis that the presence and activity of the 17

oxidase is linked to PrrBA activity (39). The most recent model of the relationship between 18

cytochrome cbb3 oxidase and PrrBA proposes that the aerobic inhibitory signal directed towards 19

the kinase activity of PrrB is generated from the oxidase (28). This inhibition of PrrB kinase 20

activity would then be absent in cells lacking the cbb3 oxidase. Consequently, there should be 21

more phosphorylated PrrA under aerobic conditions in mutant strain JZ722 than in wild type 22

strain 2.4.1. 23

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We used our phosphoaffinity/immunodetection technique to compare the levels of PrrA 1

eluted from the column following the application of lysates of wild type 2.4.1 and mutant JZ722 2

grown under highly aerobic conditions. We found that, while PrrA was detected in the eluate 3

from the JZ722 lysate, none was detected in the eluate from the 2.4.1 lysate (Fig. 3). Also, the 4

immunodetectable levels of HemA protein correlated with the amount of phosphorylated PrrA, 5

as the levels were higher in lysate prepared from mutant strain JZ722 than from wild type strain 6

2.4.1 (Fig. 3). 7

Thus, by either lowering oxygen tensions or by removing functional cbb3 oxidase, the 8

amount of phosphorylated PrrA apparently increased in the cell. Since PrrA is directly involved 9

in hemA transcription (30), these results also indicate that the higher level of hemA transcription 10

in transposon mutant strain JZ722, which was the basis for our mutant isolation process (39), is 11

attributable to the higher intracellular concentration of phosphorylated PrrA. 12

In vitro analysis of the phosphorylation state of PrrA versus binding affinities for 13

sites I and II within the hemA upstream sequences. We used electrophoretic mobility shift 14

assays (EMSAs) to examine the affinities of PrrA and phosphorylated PrrA towards the two 15

binding sites within the hemA upstream sequences. Previously, phosphorylation of PrrA was 16

achieved by incubation of the purified protein with acetyl phosphate (30). However, it has been 17

reported that the efficiency of modification of PrrA with BeF3-, which emulates phosphorylation 18

of the D63 residue (22), is estimated to be 90%, compared to the approximately 20% efficiency 19

of acetyl phosphate treatments (22). Therefore, we expected BeF3- modification of PrrA would 20

increase the sensitivity of the EMSAs. The relative affinities of PrrA versus BeF3--PrrA towards 21

each binding site were evaluated by performing the mobility shift assays in the presence of 22

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varying amounts of unlabeled competitor DNA having sequences containing both binding sites I 1

and II (Fig. 4). 2

For binding site I, the unlabeled DNA competed more effectively with complexes 3

involving BeF3--PrrA than with unmodified PrrA, while the opposite was true for binding site II. 4

Since the same concentrations of protein and competitor DNA were used in the assays involving 5

equivalent amounts of labeled binding site I or II DNA, the results indicate that BeF3--PrrA 6

behaves differently from unmodified PrrA towards the two binding sites. They also argue that 7

unphosphorylated PrrA binds DNA, and that it binds with higher affinity for binding site I than 8

for binding site II, while BeF3--PrrA binds with higher affinity for site II than for site I. To 9

confirm that the method used to modify PrrA was not contributing to the outcome, we also 10

performed this analysis using acetyl phosphate-treated PrrA. While more protein was required, 11

since modification is less efficient, we again found that the modified protein has greater affinity 12

for binding site II than binding site I (results not shown). 13

While in vitro transcription assays involving both hemA (30) and cycA (the P2 promoter; 14

4) have demonstrated that unphosphorylated PrrA can bind DNA and activate transcription, other 15

studies were unable to detect DNA binding of unmodified PrrA to the cycA sequences in the 16

absence of RNA polymerase (22). Our comparisons between the two hemA binding sites 17

indicate that unphosphorylated PrrA has greater affinity for certain DNA sequences than others. 18

Consideration of the relative dissociation constants of BeF3--PrrA and untreated PrrA for binding 19

site I estimated from our EMSAs versus those reported for that of the cycA PrrA binding site (22) 20

reinforces this idea. The Kd of BeF3--PrrA for site I is approximately 3.30 µM, and since 21

unmodified PrrA binds with greater affinity than BeF3--PrrA to hemA binding site I, the Kd of 22

PrrA for site I is less than 3.30 µM. By contrast, the Kd of BeF3--PrrA for its target within the 23

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cycA upstream sequences is reported to be approximately 5 µM, while the Kd of unmodified PrrA 1

for that same sequence is approximately 1 mM (22). Thus, while the affinity of 2

unphosphorylated PrrA for one of its target sequences, hemA binding site I, is greater than 3

phosphorylated PrrA, affinity of unphosphorylated PrrA for another target sequence, the cycA 4

binding site, is 200-fold less than phosphorylated PrrA. 5

In vivo analysis of the role of phosphorylated and unphosphorylated PrrA in hemA 6

transcription regulation. Previously, we used hemA::lacZ transcription reporter plasmids to 7

examine the consequences of altering one or the other or both of the PrrA binding sites within 8

the hemA upstream sequences that are otherwise intact; i.e. they contain both P1 and P2 promoter 9

sequences. Those studies demonstrated that the two binding sites are of equal importance to 10

transcription under aerobic conditions, while anaerobically binding site II is of greater 11

importance (30). Interpreted within the context of our in vitro results, which indicate that the 12

amount of phosphorylated PrrA increases with decreasing oxygen tensions, the difference in 13

importance of the two binding sites correlates with the phosphorylation state of PrrA. Our in 14

vitro transcription assays also identified the upstream P1 promoter as the target for PrrA- and 15

phosphorylated PrrA-mediated activation (30). Therefore, the relative importance of PrrA 16

binding sites I and II versus oxygen availability should persist even in the absence of the 17

downstream P2 promoter. We examined this using an otherwise equivalent set of reporter 18

plasmids that were deleted of the P2 promoter sequences (Fig. 5). In the presence of oxygen, the 19

β-galactosidase activities were reduced to a similar extent when either binding site I or binding 20

site II is altered; activities were 0.3-fold and 0.2-fold those for the intact sequences, respectively. 21

By contrast, in the absence of oxygen, the impact of altering the two binding sites differed; 22

activities when binding site I was altered versus when binding site II was altered were reduced to 23

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0.8-fold versus 0.3-fold relative to the intact sequences. Therefore, regardless of the presence or 1

absence of the P2 promoter, the relative importance of the two binding sites depends on the 2

phosphorylation state of PrrA. 3

Since our phosphoafffinity/immunodetection results indicate that more phosphorylated 4

PrrA is present even under highly aerobic conditions in the cbb3- mutant strain JZ722 (Table 1), 5

we expected the pattern of β-galactosidase activities from the reporters having alterations within 6

the PrrA binding sites should follow suit; i.e., in the presence of oxygen, alterations to binding 7

site II should be more important in mutant strain JZ722 than in wild type strain 2.4.1. We 8

measured the level of β-galactosidase activities present in extracts of JZ722 with reporter 9

plasmids having alterations to site I or site II (30), and the results were as predicted; in the JZ722 10

background, β-galactosidase activities are 1.3-fold higher (89 ± 1 versus 68 ± 7 U mg-1

protein) 11

when binding site II is altered but 2.3-fold higher when binding site I is altered (369 ± 10 versus 12

158 ± 5 U mg-1

protein). Therefore, regardless of whether brought about by a reduction in 13

oxygen availability or by mutation, the more phosphorylated PrrA that is present in the cell, the 14

greater the relative importance of binding site II. 15

To directly examine the role of unphosphorylated PrrA in vivo, we needed to construct a 16

mutant strain having a prrA gene coding for PrrA mutant protein that is incapable of being 17

phosphorylated. In constructing this mutant, we considered the following. While Comolli et al. 18

(4) demonstrated that residue D63 of PrrA is the target for PrrB-mediated phosphorylation, a 19

D63A substitution leads to a loss of DNA binding activity as well a loss of phosphorylation. 20

However, Hemschemeier et al. (16) showed that a D to K substitution of the same residue in the 21

Rhodobacter capsulatus PrrA homolog RegA confers a loss of phosphorylation, but the mutant 22

protein is still capable of binding to DNA in vitro. We predicted that this would also be the case 23

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for D63K mutant PrrA protein, as RegA and PrrA share 91% similarity and 83% identity, and are 1

absolutely conserved within the helix-turn-helix motif of their DNA binding domains. 2

The availability of a prrA null mutant having no antibiotic resistance markers was also 3

required. Therefore, we used the cre-lox system (25) to construct mutant strain, BRO107, in 4

which the prrA coding sequences are completely excised and a single loxP site is left behind 5

(Table 1 and Materials and Methods). We then generated otherwise isogenic strains coding for 6

wild type- or D63K-PrrA, by introducing the suicide vector pSUP202 carrying either wild type 7

prrA and prrA-D63K mutant genes. In this way, the "knock-in" strains JZ4141 (wild type prrA) 8

and JZ4148 (prrA-D63K) were obtained. Application of our phosphoaffinity/immunodetection 9

technique to samples of cultures grown under anaerobic-dark conditions confirmed that the prrA 10

genes are present and functional; i.e., that PrrA or D63K-PrrA protein are produced and that 11

PrrA is phosphorylated while D63K-PrrA is not (Fig. 6). Note that this result also confirms the 12

specificity of the phosphoaffinity enrichment protocol for the phosphorylated form of PrrA. 13

Our set of hemA(P1)::lacZ transcription reporter plasmids having alterations to one or the 14

other of the two PrrA binding sites were then mobilized into JZ4141 and JZ4148, as well as the 15

parent prrA null mutant strain BRO107, and β-galactosidase activities were measured in extracts 16

of these exconjugants grown aerobically or anaerobically (Table 2). First, even in the prrA null 17

mutant strain BRO107, β-galactosidase activities were higher in extracts of cells grown in the 18

absence versus the presence of oxygen. The indirect activity of FnrL towards the P1 promoter 19

(11) could account for this anaerobic induction. Second, since activities were higher in cells 20

having D63K-PrrA than in cells lacking PrrA altogether, the unphosphorylated protein is capable 21

of binding DNA and activating transcription. Third, and consistent with all our other 22

transcription reporter plasmid data, under phosphorylating conditions (no oxygen), alterations to 23

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site II are more important for the wild type PrrA protein than for D63K-PrrA, which cannot be 1

phosphorylated. 2

Can unphosphorylated PrrA dimerize? The physical studies of Laguri et al. (22) 3

demonstrated that their PrrA is predominantly monomeric while their BeF3--PrrA is 4

predominantly dimeric, and so the presence of dimeric protein was also correlated with the 5

ability to detect BeF3--PrrA but not PrrA binding to the target site within the cycA upstream 6

sequences. However, our mobility shift assays indicate that unmodified PrrA has a higher 7

affinity for binding site I of hemA than BeF3--PrrA. These results suggest that either 8

unphosphorylated PrrA can dimerize and bind DNA or, far less likely, that monomeric PrrA is 9

binding to site I with affinities comparable to dimeric phosphorylated PrrA. Since the PrrA 10

protein used in our in vitro studies differs from that used by Laguri et al. (22) in that theirs 11

involved a PrrA protein having 21 amino acids added to its N-terminus including a polyhistidine 12

tag, while the purified protein used here has a Pro-Gly extension at its C-terminus (4), and since 13

the importance of the cytoplasmic environment with respect to PrrA or phosphorylated PrrA 14

dimer formation is not known, we undertook a genetic approach in R. sphaeroides as a means to 15

evaluate PrrA dimerization in the cell. 16

For this analysis, we constructed plasmids pD63K-PrrA coding for full-length PrrA 17

having a D63K substitution, pPrrA' containing a truncated prrA gene lacking sequences coding 18

for the DNA binding domain of PrrA (misses amino acid residues 160-184, including the entire 19

helix-turn-helix motif), and pD63K-PrrA' that carries a truncated prrA gene and codes for a 20

D63K-substituted polypeptide that also lacks the DNA binding domain. These plasmids were 21

mobilized into the prrA null mutant BRO107 and the wild type 2.4.1. 22

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Permissive conditions for growth of PrrA- mutants include aerobic and anaerobic-dark 1

conditions (with DMSO), whereas phototrophic conditions are nonpermissive (9). The prrA null 2

mutant strain BRO107 having any of the plasmids remained incapable of growing under 3

phototrophic conditions (results not shown), which indicates that PrrA in phosphorylated form is 4

essential for this growth mode. Therefore, if wild type polypeptides are dimerizing with the 5

products of the mutant alleles, the effective concentration of dimeric phosphorylated full-length 6

PrrA would be reduced and we expected this may manifest itself as a diminished capacity for 7

phototrophic growth. 8

We found that dark-aerobic and anaerobic (with DMSO) growth of wild type 2.4.1 cells 9

with the plasmids carrying the mutant prrA genes was indistinguishable from cells having the 10

plasmid vector alone. However, growth of cells having plasmids with any of the mutant prrA 11

genes is reduced under photosynthetic conditions relative to cells with the empty vector (Fig. 7), 12

which suggests that the defective proteins are able to interact with wild type PrrA. These results 13

are consistent with those previously reported for a multicopy analysis of a longer prrA' allele 14

coding for a protein truncated at amino acid residue 176 (9). 15

It could be argued that the dominant-negative behavior of the prrA-D63K gene is due to 16

interference of D63K-PrrA with phosphorylation of the wild type protein. We examined this 17

possibility using our phosphoaffinity/immunodetection protocol (Fig. 8). Notwithstanding the 18

presence of D63K protein, the wild type PrrA polypeptide could be phosphorylated, and digital 19

image analysis indicated there was a 1.0:1.0 ratio in the amount of phospho-PrrA detected in 20

both eluates. Since our in vivo and in vitro results indicate that unphosphorylated PrrA can bind 21

DNA, we propose the most parsimonious interpretation of the dominant-negative behavior of 22

prrA-D63K is that D63K-PrrA can dimerize with wild type PrrA. 23

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Dual regulation of hemA transcription by PrrA and FnrL. Previously, we have 1

shown that FnrL is required for anaerobic induction of the downstream P2 promoter and that 2

FnrL has an indirect role in transcription from the upstream P2 promoter as well (11). Our 3

results presented here and elsewhere (30) indicate that PrrA activity is directed towards the 4

upstream P1 promoter. To further examine the role of FnrL and PrrA in transcription emanating 5

from the P1 promoter, we determined the β-galactosidase activities in wild type and FnrL- cells 6

having our set of hemA(P1)::lacZ transcription reporter plasmids (Fig. 9). The results indicated 7

that, regardless of whether or not the PrrA binding sites were intact, P1 transcription was lower 8

in the absence of a functional fnrL gene. Therefore, both FnrL and PrrA are required for normal 9

regulated P1 transcription. 10

Oxygen control of hemA transcription – testing for other factors. To determine 11

whether or not PrrA and FnrL fully account for the regulated response of hemA transcription to 12

oxygen availability, we constructed the double mutant strain BRO108 that is incapable of 13

producing either PrrA or FnrL (Table 1). We then evaluated β-galactosidase activity using 14

reporter plasmids having both P1 and P2 promoters or having P1 alone. While we found that the 15

activity levels were somewhat higher in extracts of cells grown under highly aerobic and semi-16

aerobic conditions when both promoters were present (55 ± 2 and 65 ± 5 U mg-1

protein), 17

compared to the levels for P1 alone (1 and 3 U mg-1

protein), there is no appreciable response to 18

lowering oxygen tensions. Thus it would appear that removing PrrA and FnrL from the 19

cytoplasm essentially abolishes all oxygen control of hemA transcription. 20

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DISCUSSION 1

These studies were primarily directed towards understanding how PrrA acts differently 2

towards its two binding sites within the hemA upstream sequences. We have already shown by 3

in vitro transcription that unphosphorylated PrrA activates transcription, but activation is much 4

higher in the presence of phosphorylated PrrA (30). Therefore, we hypothesized that altering 5

phosphorylation could accomplish the difference in PrrA behavior towards the two binding sites. 6

To test this possibility, we first showed that the levels of phosphorylated PrrA protein in the cell 7

increase when oxygen tensions fall. We then examined how the phosphorylation state affects 8

PrrA affinity towards binding sites I and II. Our in vitro measurements indicate that 9

phosphorylated PrrA has greater binding affinity for site II than it does for site I, and the opposite 10

is true for unphosphorylated PrrA. The in vivo results suggest that the in vitro measurements 11

reflect what takes place in the cell, in that β-galactosidase activity assays indicate the levels of 12

transcription under aerobic conditions are equally affected by alterations to binding site I and II, 13

while anaerobic induction relies on intact binding site II. That D63K-PrrA mutant protein, which 14

cannot be phosphorylated, affects transcriptional events further confirms that unphosphorylated 15

PrrA can bind DNA. 16

Based on our findings with respect to binding sites I and II of hemA, we propose that the 17

PrrA binding sites of R. sphaeroides can be subdivided into two types; those for which 18

unphosphorylated PrrA has greater affinity, such as hemA binding site I, and those for which 19

phosphorylated PrrA has greater affinity, such as hemA binding site II. The existence of two 20

types of sites provides a plausible explanation as to why the ability to detect DNA binding by 21

unphosphorylated PrrA in vitro has not always been successful since our findings indicate that it 22

would depend on the DNA sequences that are used. For hemA, the presence of both kinds of 23

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targets explains how PrrA in unphosphorylated form can assist in transcription under non-1

inducing conditions, and can increase transcription when phosphorylated. 2

The broader significance of these findings is that these same two modes of regulation of 3

hemA mediated by unphosphorylated and phosphorylated PrrA may apply to additional genes in 4

R. sphaeroides. Recently, an elegant description of the transcriptome and proteome of cells 5

lacking PrrA altogether has been reported (10). We hope that the present study will provide 6

impetus for further transcriptome and proteome investigations directed towards resolving the 7

gene sets subject to each of the two modus operandi of PrrA. 8

The ability to detect phosphorylated PrrA using the phosphoaffinity/immunodetection 9

technique also made it possible to evaluate the current model as to how the PrrBA two 10

component system senses and responds to changes in oxygen availability. The signal inhibiting 11

PrrB kinase activity towards PrrA is thought to emanate from the rate of electron flow through 12

cytochrome cbb3 oxidase; high flow rates generate high levels of the inhibitory signal, as would 13

occur under aerobic conditions (28). This model then predicts that if by mutation the oxidase is 14

absent from the cell altogether, phosphorylated PrrA should be present regardless of the presence 15

or absence of oxygen. We found that, while phosphorylated PrrA could not be detected in wild 16

type cells grown under highly aerobic conditions, it could be detected in cells lacking the 17

oxidase, which is consistent with the model. However, it has not escaped our notice that as the 18

levels of phospho-protein increase, either by lowering oxygen tensions or through the absence of 19

the oxidase, apparently so too do the total amounts of PrrA. To what degree this increase in PrrB 20

substrate availability contributes to the increase in phosphorylated PrrA present in the cell is not 21

yet known. Irrespective of how this comes about, the increase in phosphorylated PrrA levels 22

precisely explains why a mutant strain lacking functional cytochrome cbb3 oxidase would be 23

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identified in a selection for transposon mutants having higher levels of hemA transcription in the 1

presence of oxygen (39). Furthermore, consistent with our other measurements, the presence of 2

higher levels of phosphorylated PrrA in the oxidase- mutant was found to be of greater 3

significance for binding site II than for binding site I of hemA. 4

These studies also addressed the question as to whether or not unphosphorylated PrrA 5

protein can dimerize in the cell. Our demonstration that unphosphorylated protein can bind to 6

DNA with a dissociation constant close to that of the phosphorylated protein for site I of hemA 7

favors the possibility that the protein binds to DNA in dimeric form. Further, multicopy analysis 8

of a prrA gene coding for D63K-PrrA protein reveals that it behaves in a dominant-negative 9

fashion in wild type cells. Laguri et al. (22) demonstrated that the monomer-dimer equilibrium 10

is shifted towards the dimeric form for the phospho-protein, while the amount of dimeric 11

unphosphorylated PrrA was below the level that could be detected using their methods. We have 12

not examined the relative concentrations of dimer versus monomer. Importantly, however, since 13

we have shown that hemA P1 transcription in vivo is higher in the presence of prrA-D63K versus 14

in the absence of prrA, our findings demonstrate that the amount of dimeric unphosphorylated 15

PrrA present in the cell is physiologically relevant. 16

We now consider the role of FnrL in P1 transcription, the importance of which was made 17

starkly apparent by altering the PrrA binding sites such that the PrrA contribution is diminished 18

or abolished. In fact, the results probably underestimate the full scope of the contribution of 19

FnrL in anaerobic induction of P1, as we were limited to culturing under semi-aerobic 20

conditions, which are permissive for the growth of the FnrL- mutant bacteria. We regard the 21

contribution of FnrL to P1 transcription as indirect, and have therefore postulated the existence 22

of a factor that would directly modulate P1 transcription. Our results indicate that PrrA and FnrL 23

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are the only transcription factors involved in oxygen responsiveness of hemA and so suggest that 1

PrrA may be that factor. A central question is then how this might come about. It may be that 2

FnrL regulates prrA transcription, that it affects the degree of PrrA phosphorylation, or that FnrL 3

influences the PrrA or phosphorylated PrrA monomer-dimer equilibrium. It is also conceivable 4

that FnrL interacts with PrrA and thereby alters its ability to bind to DNA. Certain findings 5

already suggest that FnrL probably does not regulate the total amounts of PrrA in the cell; the 6

prrA upstream sequences do not contain an FNR consensus-like sequence and the transcriptome 7

profiles of the wild type versus FnrL- mutant bacteria grown under semi-aerobic conditions 8

indicate that the levels of prrA transcripts are the same (unpublished results). Further 9

experimentation will be required to confirm or eliminate other possibilities. 10

While these studies were limited to investigations of hemA regulation by both PrrA and 11

FnrL, it is known that the overlap between the PrrA and FnrL regulons encompasses many genes 12

in addition to hemA. Therefore, insights into how the combined actions of both of these DNA 13

binding proteins achieve the correct transcriptional response for hemA should contribute to our 14

understanding of the regulation of those other genes as well. 15 ACCEPTED


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ACKNOWLEDGEMENTS 1

We thank Y. Fedotova and B. Gliga, for their technical assistance, J. Eraso and S. Kaplan 2

for providing PRRA2, and T. Donohue and colleagues for generously providing the PrrA 3

intein/chitin-binding domain fusion protein expression system. We also wish to express our 4

gratitude for the outstanding technical support for the phosphoenrichment kits from Clontech. 5

This work was supported by MCB award no. 0320550 from the National Science Foundation. 6

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in Escherichia coli. FEMS Microbiological Review 75:399-428. 2

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TABLE 1. Bacterial strains and plasmids

Strain or Plasmid Relevant Characteristics

Reference or

source

E. coli

DH5α F- (φ80dlacZ∆M15) recA1 endA1 hsdR17 supE44 thi-1

gyrA96 relA1 deoR ∆(lacZYA-argF)U169

17

DH5αphe DH5α with phe::Tn10dCmr 9

ER2566 F- fhuA2 [lon] ompT lacZ::T7 gene1 gal sulA11 ∆(mcrC-

mrr)114::IS10R[mcr-73::miniTn10(TetS]2 R[zgb-

210::Tn10(Tets)] endA1 [dcm]

New England

Biolabs

HB101 F- ∆(gpt-proA)62 leuB6 supE44 ara-14 galK2 lacY1 6

S17-1 Conjugal donor; C600::RP4 2-(Tc::Mu)(Km::Tn7) pro

res mod+ (Tp

r Sm

r)

33

R. sphaeroides

2.4.1 wild type W. Sistrom

BR107 ∆prrA::loxP This study

BR108 JZ1691 with ∆prrA::loxP This study

JZ1678 ∆fnrL::ΩKnr 38

JZ1691 ∆fnrL::Ω SprSt

r 38

JZ722 cco::Tn5TpMCS 39

PRRA2 ∆prrA(BstBI-PstI)::ΩSprSt

r 8

JZ4141 wild type prrA knock-in, Tcr This study

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JZ4148 D63K-prrA knock in, Tcr This study

Plasmids

pBRO61 pCF1010 with PstI-XbaI fragment containing upstream

hemA sequences deleted of P2 and with alterations

within PrrA binding sites I and II (Fig.1)

This study


hemA sequences deleted of P2 (Fig. 1)

This study

pBRO81 pCM184 with 500 bp of upstream and downstream

sequences flanking the PrrA coding sequences inserted

into each of the two multiple cloning sites

This study

pBRO86 pUI1087 with prrA amplified from R. sphaeroides wild

type 2.4.1 inserted at the EcoRV site

This study



within PrrA binding site I (Fig. 1)

This study



within PrrA binding site II (Fig. 1)

This study

pBSIISK+ ColEI, Ap

r Stratagene

pCF1010 RSF1010 derivative; used for creating lacZ

transcriptional fusions, Tcr Sp

r/St

r

24

pCM157 cre expression plasmid, Tcr 25

pCM184 loxP, Knr Tc

r Ap

r 25

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pD63K pRK415 with HindIII-BamHI fragment containing

D63K-prrA

This study

pD63K-PrrA' pRK415 with HindIII-BamHI fragment containing

D63K-prrA'

This study

pJC407 PrrA intein/chitin-binding domain expression plasmid;

Apr

4

pPrrA' pRK415 with HindIII-BamHI fragment containing prrA' This study

pRK415 Broad-host-range plasmid; Mob+ Tc

r 19

pRK2013 IncP1 ColE1 Tra+ of RK2; Kn

r 6, 12

pSUP202 pBR325 derivative; Mob+ Ap

r Cm

r Tc

r 33

pSUP-D63K pSUP202 with prrA-prrA suicide vector; Mob+, Tc

r This study

pSUP-WT pSUP202 with wild type prrA; Mob+, Tc

r This study

pUI1087 pBSIISK+ with modified polylinker; Ap

r 40

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TABLE 2. β-Galactosidase activities in extracts 1

of R. sphaeroides with hemA(P1)::lacZ reporter plasmids. 2

______________________________________________________________________________ 3

β-galactosidase activity levels (U mg-1

protein) 4

30% oxygen 0% oxygen 5

Strain (genotype) Site Ia Site II Site I Site II 6

JZ4141 (wild type prrA) 46 ± 8 28 ± 4 547 ± 57 106 ± 11 7

JZ4148 (prrA-D63K) 20 ± 3 21 ± 2 65 ± 7 61 ± 6 8

BRO107 (prrA null) 9 ± 1 13 ± 1 24 ± 3 38 ± 4 9

aSite I and Site II refer to alterations to the two PrrA binding sites present in the reporter 10

plasmids; these are pBRO100 with alterations in site I and pBRO109 with alterations to site II 11

(Table 1). For further information about the strains and plasmids used, as well as growth 12

conditions used, see Materials and Methods. Reported are the mean values and the standard 13

deviations for duplicate assays of a minimum of three independent growth experiments. Units 14

are the same as in Fig. 5. 15 ACCEPTED


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Figure Captions 1

2

Figure 1. Description of the relevant hemA wild type and mutant upstream sequences used in 3

this study. The +1 sites of transcription from hemA promoters P1 and P2 (11, 26) are labeled as 4

(P1) and (P2). The FNR consensus-like sequence is also labeled. DNA sequences protected 5

from DNase I cleavage by PrrA, binding sites I and II (30), are shaded, and the identical 9 bp 6

motif within each of these protected regions is shown in reverse highlight. The P2 sequences 7

that were deleted using the same oligonucleotides described previously (11) are contained within 8

parentheses. DNA oligonucleotides used to create mutations within PrrA binding sites I and II 9

were previously described (30). Sequences corresponding to the primers used to generate DNA 10

that was investigated by the EMSAs are indicated with arrows; labeled binding site I DNA was 11

generated using primers "UP" and biotin-labeled "I", labeled binding site II DNA was generated 12

using primers "II" and biotin-labeled "DOWN", and unlabeled competitor DNA was generated 13

using primers "UP" and "DOWN". For further details regarding the oligonucleotides and their 14

use, see Materials and Methods. 15

16

Figure 2. Immunoblots of protein samples probed with anti-PrrA antisera. Samples examined 17

are either crude lysates ("Lysate") or protein eluted from a phosphoprotein affinity column 18

("Eluate") prepared from cultures of R. sphaeroides wild-type strain 2.4.1 grown under dark and 19

highly aerobic (30%), semi-aerobic (2%), or anaerobic (0%) conditions. A 4.1 mg protein 20

sample of each lysate was applied to the affinity columns. Immunoblots were prepared from the 21

crude lysate (18.0 µg protein) and equal sample volumes of the peak fractions eluted from each 22

column. 23

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1

Fig. 3. Immunoblots of protein samples probed with anti-PrrA or anti-HemA antisera. Samples 2

examined are either crude lysates ("Lysate") or protein eluted from phosphoaffinity columns 3

("Eluate") prepared from cultures of R. sphaeroides wild type strain 2.4.1 or cbb3 oxidase- 4

mutant strain JZ722 grown under dark and highly aerobic (30% oxygen) conditions. Antisera 5

used to probe the immunoblots are indicated in the figure. A 2.1 mg protein sample of each 6

lysate was applied to affinity columns. The immunoblot probed with anti-HemA antisera was 7

prepared using samples of crude lysate (21 µg protein). The immunoblots probed with anti-PrrA 8

antisera were prepared from samples of the crude lysates (2.0 µg protein) and equal sample 9

volumes of the peak fractions from each column. Further details are included in the Materials 10

and Methods. 11

12

Figure 4. Competition electrophoretic mobility shift assay results involving PrrA, target biotin-13

labeled DNA containing binding site I or binding site II sequences, and competitor unlabeled 14

DNA containing both sites I and II. The DNA used in the assays was generated by PCR and the 15

primers identified in Figure 1, and either 1.33 µM PrrA or BeF3

--PrrA was used. The amounts of 16

competitor DNA used are as indicated. For further details see Materials and Methods. 17

18

Fig. 5. β-Galactosidase activities in extracts of R. sphaeroides wild type 2.4.1 with 19

hemA(P1)::lacZ reporter plasmids having intact or altered PrrA binding sites that had been grown 20

under highly aerobic (30% oxygen) or anaerobic (0% oxygen) conditions. Reporter plasmids 21

used were "none": pBRO75, "I": pBRO100, "II": pBRO109, and "I & II": pBRO61. Alterations 22

to the hemA sequences present on the plasmids are detailed in Fig. 1, and additional information 23

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about the plasmids is included in Table 1. Vertical bars represent the standard deviations from 1

the means. Values represent duplicate assays of a minimum of three independent growth 2

experiments. One unit of enzyme activity is defined as 1 µmole of o-nitrophenyl-β-D-3

galactopyranoside hydrolyzed per minute. For further details regarding growth conditions used, 4

see Materials and Methods. 5

6

Fig. 6. Immunoblots of protein samples probed with anti-PrrA antisera. Samples examined are 7

either crude lysates ("Lysate") or protein eluted from TALON® PMAC magnetic beads 8

("Eluate"). The crude lysates were prepared from cultures of R. sphaeroides mutant strains 9

JZ4141 or JZ4148 (Table 1) grown under anaerobic-dark with DMSO conditions, and the eluate 10

was obtained by processing samples of crude lysates having 5.6 mg total protein. The 11

immunoblots were prepared using samples of the crude lysates (24.0 µg protein) and equal 12

sample volumes of the eluates. For further details, see Materials and Methods. 13

14

Figure 7. Growth of R. sphaeroides wild type strain 2.4.1 with the vector or plasmids coding for 15

the PrrA mutant proteins indicated, following incubation under the conditions shown. 16

17

Fig. 8. Immunoblots of protein samples probed with anti-PrrA antisera. Samples examined are 18

either crude lysates ("Lysate") or protein eluted from TALON® PMAC magnetic beads 19

("Eluate"). The crude lysates were prepared from cultures of R. sphaeroides wild type 2.4.1 with 20

either the vector pRK415 or plasmid pD63K-PrrA from cultures grown under anaerobic-dark 21

with DMSO conditions, and the eluate was obtained by processing samples of crude lysates 22

having 3.8 mg total protein. The immunoblots were prepared using samples of the crude lysates 23

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(23.0 µg protein) and equal sample volumes of the eluates. Further details are included in the 1

Materials and Methods section. 2

3

Fig. 9. β-Galactosidase activities in extracts of R. sphaeroides wild type strain 2.4.1 or FnrL- 4

mutant strain JZ1678 with hemA(P1)::lacZ reporter plasmids having intact or altered PrrA 5

binding sites that had been grown under semi-aerobic (2% oxygen) conditions. The reporter 6

plasmids used are as in Fig. 5. Additional information about the strains and plasmids is included 7

in Table 1. Vertical bars represent the standard deviations from the means for duplicate assays 8

of a minimum of three independent growth experiments. Values are the same as in Fig. 5. For 9

further details regarding growth conditions used, see Materials and Methods.10

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Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

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Fig. 5

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Fig. 6

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Fig. 7

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Fig. 8

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Fig. 9

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Documents

Regulation of the Rhodobacter sphaeroides 2.4.1 hemA gene by