Sigma 54 Levels and Physiological Control of the Pseudomonas putida Pu Promoter

The cellular levels of the alternative sigma factor ${sigma}$ ⁵⁴ of Pseudomonasputida have been examined in a variety of growth stages andculture conditions with a single-chain Fv antibody tailoredfor detection of scarce proteins. The levels of ${sigma}$ ⁵⁴ were alsomonitored in P. putida strains with knockout mutations in ptsOor ptsN, known to be required for the C-source control of the ${sigma}$ ⁵⁴-dependent Pu promoter of the TOL plasmid. Our results showthat $~$ 80 ± 26 molecules of ${sigma}$ ⁵⁴ exist per cell. Unlike thatin relatives of Pseudomonas (e.g., Caulobacter), where fluctuationsof ${sigma}$ ⁵⁴ determine adaptation and differentiation when cells facestarvation, ${sigma}$ ⁵⁴ in P. putida remains unexpectedly constant atdifferent growth stages, in nitrogen starvation and C-sourcerepression conditions, and in the ptsO and ptsN mutant strainsanalyzed. The number of ${sigma}$ ⁵⁴ molecules per cell in P. putida isbarely above the predicted number of ${sigma}$ ⁵⁴-dependent promoters.These figures impose a framework on the mechanism by which Pu(and other ${sigma}$ ⁵⁴-dependent systems) may become amenable to physiologicalcontrol.

INTRODUCTION

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Introduction

Bacterial RNA polymerase (RNAP) holoenzymes are assembled bya common catalytic core enzyme that associates with a polypeptide( ${sigma}$ ) conferring promoter recognition specificity. The majorityof bacteria have alternative ${sigma}$ factors, most of which show homologywith the major ${sigma}$ factor of Escherichia coli ( ${sigma}$ ⁷⁰) (34). A differentclass is composed of a unique member ( ${sigma}$ ⁵⁴, encoded by rpoN) thatdiffers both in amino acid sequence and mechanism of transcriptionactivation (5). In essence, ${sigma}$ ⁵⁴-RNAP holoenzyme forms a stableclosed complex at the target promoter that is activated by aspecialized family of regulators (20, 25) in a nucleotide (nucleosidetriphosphate) hydrolysis-dependent manner (12, 26, 32).

A single copy of rpoN is found in the genome of many (but notall) bacterial species, including archetypical organisms suchas E. coli, Salmonella enterica serovar Typhimurium, Pseudomonasaeruginosa, and Bacillus subtilis. In Pseudomonas putida, rpoNexists as a single copy (24) and its expression is subject tonegative autoregulation (18). A variety of biological functionsare regulated by ${sigma}$ ⁵⁴, although it appears that under favorablegrowth conditions these functions are dispensable since rpoNmutants are viable in all species tested except Myxococcus xanthus(17). The roles of ${sigma}$ ⁵⁴ vary among various microbial species,a fact reflected in the expression profiles of the factor. Whilein E. coli the intracellular levels of ${sigma}$ ⁵⁴ are constant throughoutdifferent growth stages (15), in Caulobacter crescentus theintracellular levels of ${sigma}$ ⁵⁴ oscillate according to growth conditionsand cellular differentiation (4). Although P. putida does nothave a differentiation program, the number of niches in whichthis species thrives (water, soil, and plant roots) is so diverse(30) that bacteria must undergo major changes in their globalphysiological status during adaptation to the disparate habitats.In addition, many strains of P. putida have a versatile metabolismfor utilization of recalcitrant carbon sources (including aromaticcompounds such as xylene or phenol), and the genes for thismetabolism are often under the control of ${sigma}$ ⁵⁴-dependent promoters(e.g., the Pu promoter of TOL plasmid pWW0) (1). These promotersare subject to physiological regulation, becoming preferentiallyactive at the stationary phase of growth (a phenomenon referredto as exponential silencing) (9), and modulation depending uponavailable carbon sources (i.e., C-source repression) (8, 10,24, 27). Two genes, ptsO and ptsN, adjacent to rpoN in the P.putida chromosome, play a role in the C-source repression thatglucose and gluconate exert on the Pu promoter (11). Both ptsNand ptsO encode homologues of phosphoenolpyruvate:sugar phosphotransferasesystem family proteins IIA(Ntr) and NPr, respectively.

Several lines of evidence indicate that the physiological controlof ${sigma}$ ⁵⁴-dependent promoters is partially mediated through changesin ${sigma}$ ⁵⁴ activity and/or protein levels. For instance, overexpressionof ${sigma}$ ⁵⁴ in P. putida allowed a partial relief of the exponentialsilencing of Pu (9). Also, modifications of the -12/-24 motifof Pu that improve its similarity to the consensus ${sigma}$ ⁵⁴ promotershave a positive effect on the transcription of this promoterin exponential phase (M. Carmona and V. Lorenzo, unpublisheddata). This suggests that recruitment of ${sigma}$ ⁵⁴-RNAP may be a limitingstep for Pu activation in vivo as it occurs in vitro (7). Further,activation of ${sigma}$ ⁵⁴-dependent promoters in E. coli was found todepend upon the function of the specific protease FtsH, thelack of which can be compensated for by overproduction of thesigma (6).

The observations above highlight the importance of accuratelyquantifying the number of ${sigma}$ ⁵⁴ molecules present in P. putidaat the different stages of growth and in culture media thatinfluence Pu promoter activity. Although such quantificationwas partially attempted in the past (9), the poor quality ofthe polyclonal antiserum employed flawed the conclusions andleft unanswered the question of the number of ${sigma}$ ⁵⁴ molecules percell and the connection of ${sigma}$ ⁵⁴ to Pu activity, in particularthe modulation of ${sigma}$ ⁵⁴ by C and N sources. By employing a dedicatedphage antibody (Phab) displaying a single-chain Fv (scFv) antibodyfragment with high affinity for ${sigma}$ ⁵⁴ from P. putida, we have determinedaccurately the number of ${sigma}$ ⁵⁴ molecules in P. putida cells atdifferent growth stages and in various culture conditions. Ourdata indicate that ${sigma}$ ⁵⁴ is one of the most invariable and leastabundant cell proteins, thereby restricting the mechanisms thatmay account for the physiological control of Pu.

MATERIALS AND METHODS

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Materials and Methods

Strains, antibodies, and general procedures. Standard methods were used to purify, analyze, manipulate, andamplify DNA (2). The E. coli strain XL-1 Blue (recA1 gyrA96relA1 endA1 hsdR17 supE44 thi1 lac [F' proAB lacI^q lacZ ${Delta}$ M15 Tn10]Tc^r; Stratagene) was used as a host for bacteriophages and phagemids.Phagemid pPC2 bears the sequence of the high-affinity anti- ${sigma}$ ⁵⁴scFv named C2 assembled in vector pCANTAB-5Ehis (13) (detailson scFv C2 are available upon request). scFv C2 specificallyrecognizes P. putida ${sigma}$ ⁵⁴ in enzyme-linked immunosorbent assaysand in Western blots. Depending on the conditions employed forproliferation, scFv C2 was produced as a distinct polypeptideor as a fusion with the pIII protein of the M13 phage. scFv-pIIIhybrids were displayed as multiple copies on M13 particles (namedPhab C2) by packaging the phagemids with Hyperphage (M13KO7 ${Delta}$ pIII,Km^r; Progen) (28). P. putida KT2442 and the P. putida ptsN::Km(10), ptsO::Km (11), and rpoN::Km (19) mutants were grown at30°C in the indicated media: Luria-Bertani broth (LB) (29),M9 plus CAA (M9 containing 0.2% [wt/vol] Casamino Acids; Difco)supplemented or not with 0.2% (wt/vol) glucose, high-nitrogenmedium (M9 plus CAA supplemented with 0.2% succinate), and low-nitrogenmedium (modified M9 medium containing 2 mM NH₄Cl and supplementedwith 0.2% succinate). The last two media were supplemented with0.05% (vol/vol) Triton X-100 to avoid cellular clumping.

Protein analyses. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)was performed with standard protocols by using the Miniproteansystem (Bio-Rad). Whole-cell protein extracts from Pseudomonascells were prepared by harvesting the cells (10,000 x g, 5 min)from cultures grown in the indicated media and resuspendingthe cell pellet in 100 µl of H₂O. Next, 100 µl ofreducing 2x SDS sample buffer (120 mM Tris-HCl [pH 6.8], 2%[wt/vol] SDS, 10% [vol/vol] glycerol, 0.01% [wt/vol] bromophenolblue, 2% [vol/vol] 2-mercaptoethanol) was added to the samples,and the samples were boiled for 10 min, sonicated briefly ( $~$ 5s), and centrifuged (14,000 x g, 10 min) to eliminate the DNAviscosity and any insoluble material (e.g., peptidoglycans).Loading was normalized by the number of cells determined asCFU per milliliter after plating in LB-agar (1.5% [wt/vol])or by the total amount of protein (protein assay kit; Bio-Rad).Usually $~$ 1.25 x 10⁸ CFU or 10 µg of total protein was loadedper lane. Prestained standards (Kaleidoscope; Bio-Rad) wereused as markers of known molecular weight for the SDS-PAGE.After electrophoresis, the proteins were transferred to a polyvinylidenedifluoride membrane (Immobilon-P; Millipore) by using a semidrytransfer apparatus (Bio-Rad). After protein transfer, the membraneswere blocked for 2 h at room temperature (or for 16 h at 4°C)with MBT buffer (3% skimmed milk, 1% bovine serum albumin, and0.1% Tween 20 in phosphate-buffered saline [PBS]).

Immunodetection techniques. For detection of ${sigma}$ ⁵⁴ with the purified scFv C2, membranes withthe blotted proteins were incubated with 10 ml of MBT buffercontaining 500 ng of the antibody. Unbound scFvs were eliminatedby four washing steps of 5 min in 40 ml of PBS and 0.1% (vol/vol)Tween 20. Next, anti-E-tag monoclonal antibody (MAb)-peroxidase(POD) conjugate (1:5,000 in MBT buffer; Amersham Pharmacia Biotech)was added to detect the bound scFvs. After 1 h of incubation,the membranes were washed four times with PBS and 0.1% (vol/vol)Tween 20, the bound POD conjugates were developed by a chemiluminescencemixture of 1.25 mM luminol (Sigma) and 42 µM luciferin(Roche), and H₂O₂ was added at 0.0075% (vol/vol) in 100 mM Tris-HCl(pH 8.0). BM chemiluminescence blotting substrate (POD; Roche)was also used for developing the POD conjugates. After 1 minof incubation in the dark, the polyvinylidene difluoride membranewas exposed to an X-ray film (X-Omat; Kodak). For immunodetectionof ${sigma}$ ⁵⁴ with the M13 Hyperphage, the membranes were incubatedwith 30 ml of MBT buffer containing 5 x 10¹⁰ PFU of the phage.Unbound phages were eliminated by four washing steps of 5 minin 40 ml of PBS and 0.1% (vol/vol) Tween 20. Next, anti-M13MAb-POD conjugate (1:5,000 in MBT buffer; Amersham PharmaciaBiotech) was added to detect the bound M13 phages. After 1 hof incubation, the membranes were washed four times with PBSand 0.1% (vol/vol) Tween 20 and the bound POD conjugates weredeveloped with the BM chemiluminescence blotting substrate asdescribed above. In order to standardize the protein amountsloaded in each case, duplicate blots were subjected to incubationwith an anti-GroEL rabbit serum (1:5,000; kindly provided byJ. M. Valpuesta, Centro Nacional de Biotecnología) anddeveloped with anti-rabbit POD conjugate (1:5,000; Bio-Rad).

Quantification of ${sigma}$ ⁵⁴. The intensity of light emitted by the protein bands in the membranesdescribed above was quantified by employing the Quantity Onesoftware (Bio-Rad) and matched with a standard developed byusing purified ${sigma}$ ⁵⁴ protein (the kind gift of F. Bartels) runand processed under the same conditions. The absolute concentrationof purified P. putida ${sigma}$ ⁵⁴ was determined by amino acid analysisfor the standard curve. The protein sample was dried in a Speed-Vac(Beckman) and subsequently hydrolyzed in 6 N HCl-0.1% (vol/vol)phenol under vacuum conditions in a sealed glass tube for 24h at 110°C. The amino acid analysis of the dried hydrolyzedprotein sample was performed on a Beckman 6300 automatic analyzerto determine the amino acid composition as well as the proteinconcentration. Internal controls were performed with norleucine.This procedure allowed the accurate detection of 0.3 ng of ${sigma}$ ⁵⁴.

RESULTS AND DISCUSSION

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Results and Discussion

Rationale for quantification of ${sigma}$ ⁵⁴ of P. putida in vivo with a single-chain antibody. Antibody fragments assembled in M13 particles are particularlyuseful for the detection of proteins which are present in verysmall numbers in bacterial cells. This is because they can beeither produced as distinct molecules or attached to phage particles(33). When the latter are employed as antibody-like agents,the use of a secondary anti-M13 coat antibody affords an extraordinaryamplification from otherwise scarce target signals in the samples(22). Further, if the scFv is produced in an E. coli strainsubject to infection with a hyper helper phage (28), then theresulting phage pool is composed of particles displaying multiplescFv units. This multiplies the operative affinity and specificityof the antibody. On this basis, the antibody named scFv C2,targeted towards ${sigma}$ ⁵⁴ of P. putida, has been instrumental (bothas Phab C2 and as purified scFv) in monitoring and accuratelyquantifying the levels of the factor under various physiologicalcircumstances. This quantification was possible by simply matchingthe signals from cell extracts with a signal response standardset with purified ${sigma}$ ⁵⁴ protein.

Levels of ${sigma}$ ⁵⁴ in P. putida cells at different growth stages. The levels of ${sigma}$ ⁵⁴ in P. putida cells were investigated at differentpoints along the growth curve. To this end, P. putida cellswere grown at 30°C in rich liquid media (LB) and aliquotswere taken at different time points (Fig. 1). The protein extractsobtained from the cells were analyzed by SDS-10% PAGE and Westernblotting by using multivalent Phab C2 for detection (see Materialsand Methods). Loading of the gels was normalized so that 10µg of total protein was applied per lane. Detection ofGroEL polypeptide, whose levels per cell remain constant underthese conditions, was employed as an internal control for normalizationin the Western blots (Fig. 1, bottom inset). As shown in Fig.1, the levels of ${sigma}$ ⁵⁴ in P. putida cells were constant along thegrowth curve, at both exponential and stationary phases.

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FIG. 1. Intracellular levels of ${sigma}$ ⁵⁴ in P. putida at different points along the growth curve. P. putida cells were grown in LB, and samples of these cultures were taken at different time points (filled squares). Whole-cell protein extracts derived from these cells were analyzed by Western blotting ( $~$ 10 µg of protein was loaded per lane). These membranes were probed with the multivalent Phab C2 for detection of ${sigma}$ ⁵⁴ (top inset) or with a polyclonal antiserum against GroEL as an internal control (bottom inset). O.D. 600 nm, optical density at 600 nm.

Nitrogen starvation does not affect ${sigma}$ ⁵⁴ concentrations. In E. coli cells, the activity of RpoN is essential for growthunder nitrogen-limiting conditions. This is due to the requirementof the ${sigma}$ ⁵⁴ for the transcriptional activation of glnA, whichencodes the glutamine synthase, an enzyme responsible for theassimilation of ammonia at low concentrations (ca. 1 mM) (23).Similar to E. coli, P. putida rpoN mutant cells are unable togrow in media containing a low concentration of ammonia as thesole nitrogen source (19). Although low ammonia concentrationdoes not induce rpoN transcription (18), we speculated thatnitrogen starvation could otherwise affect the levels of the ${sigma}$ ⁵⁴ polypeptide in P. putida. Thus, protein extracts from P.putida cells grown in defined mineral media having low (2 mMNH₄Cl) or high (20 mM NH₄Cl) nitrogen content were analyzedby Western blotting by using the multivalent Phab C2 (see Materialsand Methods). As shown in Fig. 2, the intracellular contentof ${sigma}$ ⁵⁴ polypeptide remained invariable during P. putida growthin media with high or low ammonia concentration, even when thecultures reached the stationary phase. These data demonstratethat nitrogen starvation does not affect the constant intracellularlevel of ${sigma}$ ⁵⁴ protein in P. putida.

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FIG. 2. Nitrogen starvation and ${sigma}$ ⁵⁴ levels in P. putida. Whole-cell protein extracts derived from P. putida cells grown in mineral media with high or low nitrogen content were obtained and analyzed by Western blotting as described in the legend to Fig. 1. Detection of ${sigma}$ ⁵⁴ and GroEL is shown in the top and bottom insets, respectively. O.D. 600 nm, optical density at 600 nm.

Effect of ptsN and ptsO mutations on levels of ${sigma}$ ⁵⁴ in P. putida. Mutation of ptsN or ptsO of P. putida, two genes adjacent toand downstream of rpoN, influences in opposite ways the C-sourcecontrol of the Pu promoter from pWW0. In a ptsN mutant, theactivity of Pu is not repressed by glucose, whereas a ptsO mutantstrain displays a phenotype of Pu repression even in the absenceof glucose (10, 11). Experimental evidence suggests the existenceof an equilibrium between the phosphorylated forms of PtsO,an NPr-like enzyme, and PtsN, a IIA(Ntr)-like enzyme. It isbelieved that glucose increases the share of phosphorylatedforms of PtsN, which is in turn responsible for the repressionof Pu by an undisclosed mechanism (10). In this context, weinvestigated whether P. putida ptsN and ptsO mutant strainshad altered intracellular levels of ${sigma}$ ⁵⁴ that could account fortheir phenotypes regarding the C-source repression of the Pupromoter. To this end, whole-cell protein extracts were preparedfrom P. putida KT2442 and the isogenic ptsN::Km and ptsO::Kmstrains, grown in M9 plus CAA medium supplemented or not withglucose (10 mM), and analyzed by immunoblotting with the multivalentPhab C2 as described above. The results from this experimentrevealed that neither the presence of glucose nor the ptsO orptsN mutation had an effect on the intracellular level of ${sigma}$ ⁵⁴polypeptide in P. putida (Fig. 3). Therefore, these data provethat C-source repression of Pu in P. putida is unrelated tochanges in the level of ${sigma}$ ⁵⁴.

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FIG. 3. Levels of ${sigma}$ ⁵⁴ in strains with mutations that affect the C-source regulation of Pu activity. Shown are Western blots to detect ${sigma}$ ⁵⁴ and GroEL (developed with Phab C2 and anti-GroEL serum, respectively) in whole-cell protein extracts ( $~$ 10 µg was loaded per lane) obtained from samples, harvested at the indicated time points, of cultures of wild-type (wt) P. putida and the isogenic ptsN and ptsO mutant strains grown in M9 plus CAA medium supplemented (top) or not (bottom) with 0.2% glucose.

Quantifying ${sigma}$ ⁵⁴ in P. putida cells. To accurately estimate the number of ${sigma}$ ⁵⁴ molecules per cell inP. putida, protein extracts derived from bacteria grown in differentmedia (e.g., LB, M9 with high or low nitrogen concentration,and M9 plus CAA with and without glucose) were analyzed by quantitativeWestern blotting. In all cases, P. putida cells were harvestedat stationary phase. In this assay, the signals obtained withpurified scFv C2 against a series of twofold dilutions of purified ${sigma}$ ⁵⁴ (from 5 to 0.3 ng) were employed to generate a standard curvewhich allowed the precise determination of the amount of ${sigma}$ ⁵⁴in the protein extracts normalized by numbers of CFU (Fig. 4).Quadruplicate experiments gave consistent results showing that $~$ 80 ± 26 molecules of ${sigma}$ ⁵⁴ exist per cell in P. putida,without significant variation with the different media analyzed.Interestingly, these numbers are within the range of, but lowerthan, those reported for E. coli ( $~$ 110 molecules/cell), whichalso remain roughly constant at exponential and stationary phases(15).

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FIG. 4. Quantification of ${sigma}$ ⁵⁴ in P. putida. The intensity of light emission after chemiluminescence developing of a Western blot containing five serial twofold dilutions of purified ${sigma}$ ⁵⁴ from P. putida (upper blot) was used to generate a standard curve for quantification of ${sigma}$ ⁵⁴ in P. putida cells. Total protein extracts were prepared from P. putida grown in LB (graph), as well as cells grown in M9 plus CAA supplemented (+G) or not (-G) with 0.2% glucose and media with high (+N) or low (-N) nitrogen content (lower blot). About $~$ 1.25 x 10⁸ CFU was applied per lane of the gel (lanes 6 to 9). (Left panel) A standard curve is shown employing the log of the number of molecules of purified ${sigma}$ ⁵⁴ applied versus the log of the light intensity of their corresponding protein bands. The scFv C2 and anti-E-tag MAb-POD conjugate were used for detection as described above. Four independent experiments were performed and gave consistent results (standard deviation bars are included in the graph).

${sigma}$ ⁵⁴ levels and physiological regulation of Pu. The results presented above demonstrate that the level of ${sigma}$ ⁵⁴in P. putida is altogether constant at $~$ 80 molecules/cell throughoutany growth conditions. The number of ${sigma}$ ⁵⁴ molecules per P. putidacell is approximately twice the maximum number of ${sigma}$ ⁵⁴-dependentpromoters predicted in the genome of P. putida ( $~$ 50 promoters;I. Cases et al., unpublished data). The low number of ${sigma}$ ⁵⁴ moleculesis in contrast to the abundance of housekeeping sigma factor ${sigma}$ ⁷⁰ ( $~$ 750 molecules/cell in E. coli) (14). Because of this, itis plausible that the available pool of the ${sigma}$ ⁵⁴-containing formof the RNAP cannot saturate all ${sigma}$ ⁵⁴ promoters. Any conditionthat favors such an occupation may thus result in an increasedoutput of the promoter under activation conditions. This framework,in which the artificial increase of ${sigma}$ ⁵⁴ levels relieves the physiologicalcontrol of Pu (9), may simply reflect a higher occupation ofthe promoter by ${sigma}$ ⁵⁴-RNAP. Under normal in vivo conditions, ${sigma}$ ⁵⁴-RNAPalone fails to act on the Pu promoter and binding of the RNAPoccurs only by virtue of the recruitment caused by the integrationhost factor (3, 7, 31). Sigma factor competition in stationaryphase has been claimed as the major determinant of the physiologicalcontrol of another related ${sigma}$ ⁵⁴-RNAP promoter of Pseudomonas calledPo (16, 21). Since P. putida has as many as 24 sigma factors,versus the 7 found in E. coli (24), the role of sigma competitionin controlling the promoter output in vivo may be more dramaticthan anticipated (16, 21). A clear prediction of these notionsis that promoters with high affinity for ${sigma}$ ⁵⁴-RNAP may not undergomuch physiological control whereas those with a lower affinitymay be amenable to additional regulatory checks to promote ${sigma}$ ⁵⁴-RNAPbinding (Carmona and Lorenzo, unpublished). In any case, thelow number of ${sigma}$ ⁵⁴ molecules may contribute decisively to theability of the cells to rapidly adapt their metabolisms to changesin environmental conditions.

ACKNOWLEDGMENTS

We especially thank Frank Bartels (GBF, Germany) for his kindgift of purified ${sigma}$ ⁵⁴ from P. putida.

This work was supported by EU contracts ICA4-CT-2002-10011,QLK3-CT-2002-01933, and QLK3-CT-2002-01923, by grant BIO2001-2274of the Spanish Comisión Interministerial de Ciencia yTecnología (CICYT), and by the Strategic Research GroupsProgram of the Comunidad Autónoma de Madrid.

FOOTNOTES

* Corresponding author. Mailing address: Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco, Madrid 28049, Spain. Phone: 34 91 585 45 36. Fax: 34 91 585 45 06. E-mail: vdlorenzo{at}cnb.uam.es.

REFERENCES

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