Expression, Abundance, and RNA Polymerase Binding Properties of the delta  Factor of Bacillus subtilis*

Francisco J. López de SaroDagger , Noriko Yoshikawa, and John D. Helmann§

From the Section of Microbiology, Cornell University, Ithaca, New York 14853-8101

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The delta  protein is a dispensable subunit of Bacillus subtilis RNA polymerase (RNAP) that has major effects on the biochemical properties of the purified enzyme. In the presence of delta , RNAP displays an increased specificity of transcription, a decreased affinity for nucleic acids, and an increased efficiency of RNA synthesis because of enhanced recycling. Despite these profound effects, a strain containing a deletion of the delta  gene (rpoE) is viable and shows no major alterations in gene expression. Quantitative immunoblotting experiments demonstrate that delta  is present in molar excess relative to RNAP in both vegetative cells and spores. Expression of rpoE initiates from a single, sigma A-dependent promoter and is maximal in transition phase. A rpoE mutant strain has an altered morphology and is delayed in the exit from stationary phase. For biochemical analyses we have created derivatives of delta  and sigma A that can be radiolabeled with protein kinase A. Using electrophoretic mobility shift assays, we demonstrate that delta  binds core RNAP with an apparent affinity of 2.5 × 106 M-1, but we are unable to demonstrate the formation of a ternary complex containing core enzyme, delta , and sigma A.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

RNA polymerase (RNAP)1 from bacteria consists of a multisubunit enzyme complex that is the main target for the regulation of gene expression. The catalytic core of RNAP is composed of the subunits beta , beta ', and alpha  and is sufficient to catalyze the polymerization of NTPs into RNA chains. In addition, a vast array of other proteins makes contacts with RNAP at one or more steps of the transcription cycle and modifies its activities in various ways (1). Such ancillary polypeptides modify RNAP during the promoter binding, initiation, elongation, and termination steps to effect changes in gene expression. The affinity of the various transcription factors for RNAP varies greatly during the transcription cycle; for example, sigma  factors have a high affinity for RNAP during the pre-initiation steps but very low affinity during elongation, whereas for NusA the opposite occurs (2).

In the Gram-positive bacterium Bacillus subtilis, purified preparations of RNAP are usually found associated with the 21.4-kDa delta  factor (3, 4). delta  was found to be essential, together with a phage-encoded sigma  factor, to reconstitute selective transcription of the phage SP01 genome in vitro (3). Further biochemical characterization of delta  activity has revealed complex effects on RNAP activity. For example, delta  reduces the extent of contact between RNAP and promoter DNA, as detected by DNA footprinting assays, and antagonizes the formation of the open complex (5-8). These results suggest that RNAP binds to the promoter as a Esigma delta complex. However, in solution sigma  and delta  bind to RNAP with negative cooperativity (9). In addition, delta  can either inhibit or stimulate in vitro transcription, depending on reaction conditions, and its stimulatory effects are clearly DNA template-dependent (7, 10-13).

delta has a very unusual tertiary structure with an amino-terminal half that is folded as a alpha /beta type of structure and a carboxyl-terminal half that is unstructured in solution, as observed by circular dichroism analysis (14). The carboxyl-terminal region is highly acidic, consisting mostly of aspartate (37%), glutamate (34%), and hydrophobic residues (25%) and is essential for displacing RNA bound to RNA polymerase (14). This RNA displacement activity may explain our previous observation that delta  enhances RNAP recycling in multiple cycle transcription reactions, thus stimulating overall transcription (13). These structural and functional studies suggest that delta  acts by mimicking single-stranded RNA and points to a role of delta  in the termination or recycling steps of transcription (14).

Despite the clear effects of delta  on in vitro transcription, initial attempts to demonstrate a role for delta  in vivo were unsuccessful. delta  mutant strains sporulate, lack auxotrophies, grow up to 49 °C, and plate phage SP01 normally (4). In this study, we describe the expression of the rpoE locus, the phenotype of an rpoE deletion mutant, and preliminary biochemical analysis of the interactions between delta  and RNAP.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- Restriction enzymes and T4 polynucleotide kinase were purchased from New England Biolabs, Inc.; avian myeloblastosis virus reverse transcriptase was from Promega Corp.; growth media were from Difco; and [alpha -32P]dATP and [35S]methionine were from NEN Life Science Products.

Growth Conditions-- Strains were grown with vigorous shaking at 37 °C in Luria broth (LB) (15) or in minimal medium (16), which is composed of 40 mM MOPS buffer, pH 7.4, 2% glucose (w/v), 2 mM potassium sulfate, pH 7.0, 10 µg/ml tryptophan, and 1× Bacillus salts (0.2 g of magnesium sulfate, 2 g of ammonium sulfate, 1 g of sodium citrate, and 1 g of potassium glutamate/liter). Antibiotic concentrations used were, for Escherichia coli, ampicillin at 100 µg/ml; for B. subtilis, erythromycin at 2 µg/ml; neomycin, 10 µg/ml; macrolides/lincomycin/streptogramin B with 1 µg/ml erythromycin and 25 µg/ml lincomycin.

Bacterial Strains-- B. subtilis strains used were CU1065 (W168 trpC2 attSPbeta ), HB6002 (CU1065 rpoE::lacZ, generated using the pTKlac vector), HB6005 (CU1065 PrpoE::cat-lacZ at the SPbeta locus, generated using the pJPM122 vector (17)), HB6010 (CU1065 Delta rpoE::cm, generated by transformation of CU1065 with RP17 DNA (14)), HB6012 (HB6002 abrB::neo), and HB6013 (HB6002 sinR::kan). The rpoE mutant used in this study is a deletion/insertion containing a chloramphenicol resistance cassette replacing the segment of the rpoE gene between codons 6 and 142 (4). E. coli strains used were DH5alpha (supE44 Delta lacU169 phi 80 lacZDelta M15 hsdR17 recA1 endA1 gyrA96 thi-1 relA1) and, for protein overexpression, BL21/DE3 (BL21 with lambda DE3 (18)).

Generation of rpoE-lacZ Fusions-- Plasmids used for the generation of lacZ fusions to the rpoE promoter region were pTKlac (19) and pJPM122 (17). The cloned rpoE gene was obtained originally from plasmid pCBdelta 11 (4), a pBR322 derivative containing rpoE on a ~2-kilobase HindIII fragment. To generate a transcriptional fusion to lacZ at the rpoE locus, the rpoE-containing HindIII fragment from pCBdelta 11 was cloned into pBSKII+ (Stratagene) such that the rpoE gene was oriented toward the unique KpnI site. The resulting plasmid, pFL22, was digested with EcoRI and BglII, and the promoter-containing fragment was ligated to pTKlac and digested with EcoRI and BamHI to generate pFL23. Integration of pFL23 into the rpoE mutant strain RP3 (4) generates strain HB6001 (rpoE-lacZ), whereas integration into the wild type (CU1065) generates HB6002(rpoE-lacZ). For generating the lacZ transcriptional fusion at the SPbeta locus, a HindIII to SspI fragment of pFL20 (14) was cloned into pBSKII+ after digestion with HindIII and SmaI to generate pFL21. The promoter-containing fragment was excised by digestion with HindIII and BamHI and cloned into pJPM122 after digestion with the same enzymes to generate pFL60. pFL60 was first integrated into B. subtilis ZB307A (20), and a transducing lysate was used to transfer of the PrpoE-cat-lacZ operon fusion into wild type (CU1065) and rpoE mutant (HB6010) backgrounds to generate strains HB6005 and HB6006, respectively.

Overexpression of delta  in B. subtilis-- Overexpression of delta  in B. subtilis was achieved using the Pspac-containing plasmid, pDG148 (21). The rpoE gene was removed from plasmid pFL32 (14) as an EcoRI to EcoRV fragment and cloned into pBSKII+ after digestion with EcoRI and SmaI to generate pFL24. Then the rpoE gene was removed from pFL24 as an XbaI fragment and cloned into pDG148 after digestion with XbaI to generate pFL25.

Primer Extension Analysis-- RNA was purified using the RNeasy Total RNA kit purification system of Qiagen Inc. RNA from in vitro reactions was produced by mixing a polymerase chain reaction (PCR) fragment containing the rpoE promoter with 1 pmol of Bacillus RNA polymerase core enzyme supplemented with 10 pmol of purified sigma A, 1× transcription buffer (40 mM Tris acetate, pH 8.0, 50 mM ammonium acetate, 20 mM potassium acetate, 4 mM magnesium acetate, 0.1 mM dithiothreitol, and 0.04 mg/ml bovine serum albumin), and 0.1 mM NTPs, and incubating the mixture at 37 °C for 15 min. The "run-off" RNA was then purified using the Qiagen kit and precipitated with ethanol. A primer (5'-TCCTTTAGCTCTTCCTG-3'), centered at around 40 base pairs downstream of the translation start site, was labeled with [gamma -32P]ATP and polynucleotide kinase (New England Biolabs, Inc.) following standard procedures (15). The labeled primer was then hybridized to the RNA in hybridization buffer (60 mM NaCl, 50 mM Tris-Cl, pH 8.0, and 10 mM dithiothreitol) by heating to 90 °C for 1 min and then cooling to 25 °C over 2 h. Next, nucleic acids were precipitated with ethanol in the presence of 0.3 M sodium acetate, pH 5.2, redissolved in extension buffer (50 mM Tris-HCl, pH 8.3, 40 mM potassium chloride, 7 mM magnesium chloride, 1 mM dithiothreitol, and 0.1 mg/ml bovine serum albumin) supplemented with 10 mM dNTPs and 10 units of avian myeloblastosis virus reverse transcriptase, and incubated at 37 °C for 20 min. The resulting labeled DNA was then visualized by electrophoresis followed by autoradiography.

Deletion of the rpoE 5'-untranslated Region-- To delete the 5'-untranslated region preceding the rpoE gene, the promoter region was amplified using the PCR and pFL24 as a template with primers NY1 (5'-GGCCCGAAGCTTTAACGGAAAACATCTCTCAGTCGG-3') and NY2 (5'-CGGGAATTCCTTATACAAACCATACCTCTC-3'), and the resulting product was digested with HindIII and EcoRI (sites underlined). The 5'-end of the rpoE gene, together with the corresponding ribosome binding site, was amplified using primers NY3 (5'-GGCGAATTCTAGAAAGGGAGTGTCCGACCTTGGG-3') and #107 (5'-GCGGATCCTACTTGACTGTCGGCTGAG-3') and digested with EcoRI and BamHI. The two PCR products were cloned in a three-way ligation into pJPM122 digested with HindIII and BamHI to generate pNY50. Transformation of pNY50 (linearized with ScaI) into ZB307A generates HB6040. Transducing lysates, prepared from HB6040, were used to move the SPbeta -borne PrpoEDelta -cat-lacZ operon fusions into CU1065 (generating HB6041) and the rpoE mutant strain HB6010 (generating HB6042).

beta -galactosidase Assays-- To assay gene expression, rich medium (2xYT) or minimal medium was inoculated from an overnight culture by 1:100 dilution, samples were taken, and beta -galactosidase was assayed by the procedure of Miller (22).

Western Blots-- Immunoblots were carried out using rabbit anti-delta antibodies (14), goat horseradish peroxidase-coupled anti-rabbit secondary antibodies (Bio-Rad), and an enhanced chemiluminiscence detection system (NEN Life Science Products) following the instructions of the suppliers. Pure delta  was obtained as described (14), and quantitation of its concentration was carried out using the Bradford reagent (Bio-Rad). Extracts of B. subtilis CU1065 cells were prepared by sonication of cells after a treatment with lysozyme and centrifugation at 12,000 rpm for 10 min. Spores of CU1065 and HB6010 were prepared by extensive washing, and extracts were prepared as described (23).

Construction and Purification of PKA Derivatives-- Derivatives of delta  and sigma A were constructed by the addition of a protein kinase A recognition motif to the amino terminus of the protein (24). We used the PCR to place 11 additional codons at the 5'-end of the corresponding genes.

Oligonucleotides used for PCR amplification of the delta  gene and addition of the PKA sequence were a "PKA primer" (5'-AAAAGCTAGCCTGCGTCGTGCGTCCCTGGGTGATCAGGGTATCAAACAATATTCA-3') and a 3'-rpoE primer (5'-CGCGGATCCCGACTATGAAAGTCAAGATCG-3'). The PKA primer encodes a PKA recognition site (ASLRRASLGDE) between a 5'-NheI site (singly underlined) and a BclI site (doubly underlined) used in later constructions. The PCR product was cloned first into pET11c (Novagen) as an NheI to BamHI fragment to generate pFL70. To generate delta NPKA and delta CPKA, we cloned the rpoE gene from the pFL70 product as an XbaI to BamHI fragment into pBKSII+ (digested with XbaI and BamHI) to generate pFL80. For delta NPKA we digested pFL80 at two BglII sites internal to the rpoE gene, filled the recessed termini using the Klenow enzyme and all four dNTPs, and ligated the resulting blunt termini with T4 ligase, thus creating a new BspDI site and an in-frame stop codon in plasmid pFL82 as described previously for unmodified rpoE (14). To create delta CPKA, we digested pFL80 with BclI, partially digested with BglII, and ligated. A plasmid was identified with a deletion of the amino-terminal 109 amino acids of delta  and was designated pFL85. For protein overproduction, each modified rpoE gene was removed as an XbaI to BamHI fragment and cloned into pET11c to generate plasmids pFL72 (delta NPKA) and pFL75 (delta CPKA). The modified rpoE genes were verified by DNA sequencing.

For the construction of sigma APKA the following oligonucleotides were used as PCR primers in a reaction containing the cloned sigA gene as template: 5'-GGGCTCTAGACTGCCTCGTCGCTCCCTGGCTGATAAACAAACCCACGAC-3' and 5'-CGCGGATCCTTAATCGTCCAGGAAGCTACG-3'. The resulting PCR product was digested with XbaI and BamHI (sites underlined) and ligated into pET11c digested with NheI and BamHI to generate pFL90. The sequence of the modified sigA gene was verified by automated DNA sequencing.

Purification of delta PKA and sigma APKA was as described for the unmodified proteins (14, 25). Briefly, delta NPKA was purified from E. coli BL21/DE3 cells by passage of an extract of isopropyl-1-thio-beta -D-galactopyranoside-induced cells through an S-Sepharose chromatography column previously equilibrated with buffer A (50 mM HEPES, pH 8.2, 2 mM EDTA, and 5% glycerol in water). After washing and elution of delta NPKA with buffer A supplemented with 200 mM NaCl, the protein solution was precipitated with ammonium sulfate (60% saturation) and centrifuged. Further purification of delta NPKA was carried out by the passage of the protein through fast protein liquid chromatography Superdex 70 (size exclusion chromatography) and Mono-S (ion exchange chromatography) columns. PKA derivatives were labeled with [gamma -32P]ATP and protein kinase A (New England Biolabs) as described (24).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Structure of the rpoE Promoter Region-- The rpoE promoter region contains a predicted sigma A-type promoter element located ~90 base pairs upstream of the translational start site (Fig. 1A). Primer extension analysis (Fig. 1B) with RNA purified from vegetatively growing cells and from in vitro transcription assays using B. subtilis RNA polymerase containing sigma A shows that transcription starts with approximately equal frequency at either of two purine residues located 84 and 86 base pairs from the translation start site. The putative -10 region of the promoter conforms to a sigma A consensus promoter (26), but there is weak if any similarity in the -35 region.


View larger version (46K):
[in this window]
[in a new window]
 
Fig. 1.   Identification of the promoter for rpoE. The sequence of the rpoE promoter region (A) is illustrated from -58 to +91 relative to the first transcription start site (+1). The two main transcription start sites are marked by arrows. The -10 region of the promoter and the putative ribosomal binding site (r.b.s.) are capitalized. The initial transcribed region can assume at least two possible secondary structures, and the corresponding inverted-repeat sequences are indicated (><). The extent of the deletion engineered in strains HB6041 and HB6042 is overlined and marked with a Delta . Note that the delta  gene begins with a TTG start codon. B shows primer extension analysis of the rpoE promoter using RNA template purified from cells (lane 1), RNA generated in vitro using B. subtilis core RNAP supplemented with purified sigma A (lane 2), or RNA generated in vitro with the addition of purified delta  to the transcription reaction (lane 3).

Expression of rpoE and Abundance of delta  in the Cell-- The level of expression of rpoE was monitored by beta -galactosidase assays of cells containing rpoE-lacZ transcriptional fusions. When measured at the rpoE locus (strain HB6002), expression reaches a maximum of 60-80 Miller units at the transition between logarithmic and stationary phase (Fig. 2). Other strains in which the fusion was placed at the SPbeta locus, either in wild type or in an rpoE mutant, produced essentially identical results (data not shown).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   Expression of the rpoE gene. beta -galactosidase activity was measured in a strain carrying a rpoE::lacZ fusion, HB6002 (black-square, right axis) as a function of growth phase as measured by optical density (, left axis). The x axis indicates time after inoculation (hours). A reproducible increase in expression is observed at the end of logarithmic growth.

Folding of the untranslated RNA molecule with the Mulfold program (27) shows that this region contains several inverted repeats predicted to form stable stem loops (estimated Delta G of -23.3 and -22.7 kcal/mol at 25 °C and 1 M NaCl) (Fig. 1A). To investigate the possible regulatory function of these sequences, a deletion mutation was engineered and placed in wild type (HB6041) or rpoE mutant (HB6042) cells. beta -galactosidase analysis of these two strains showed that rpoE expression is increased 2-fold in the absence of these sequences. However, as noted for the wild-type leader region, the presence or absence of delta  had no detectable effect on expression. In vitro transcription from the rpoE promoter showed that small RNA fragments about 50 nucleotides long are produced when these sequences are present, indicating that these stem loops might function as transcriptional terminators. As with the in vivo result, the presence or absence of delta  in the transcription reaction did not make a difference (data not shown). Thus, expression of rpoE is unaffected by delta , and we find no apparent autoregulation of this locus.

Quantitative immunoblots were performed to estimate the abundance of delta  protein. The antiserum used for these studies is highly specific and reacts with a single 21-kDa band present in wild-type cells but absent from rpoE mutant strains (14) (Fig. 3B, lane 1). When 10 µg of cell lysate is analyzed, the amount of delta  detected remains constant during growth at between 10 and 50 ng (Fig. 3A, lanes 1-3) as judged by comparison with the pure protein. We therefore estimate that delta  represents 0.3 ± 0.1% by weight of soluble cell protein. Because RNA polymerase (Mr = 337,000) represents 1% by weight of soluble cell protein (28), we conclude that delta  is an abundant protein (~104 molecules/cell) present in an approximately 5:1 molar excess relative to RNAP. Purified spores also contain similar levels of delta  (Fig. 3B, lane 2).


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 3.   Estimation of the abundance of delta  in vivo. Immunoblot analysis, using anti-delta antibodies, is presented for extracts from vegetative cells (A) and spores (B). A, lanes 1-3, 10 µg of soluble cell protein from B. subtilis CU1065 grown in LB medium supplemented with glucose and collected at mid-logarithmic phase, A600 0.3 (lane 1); transition state, A600 0.7 (lane 2); and early stationary phase, A600 0.9 (lane 3). Purified delta  was loaded in lane 4 (10 ng), lane 5 (50 ng), and lane 6 (100 ng) as standards. In B, 10 µg of soluble protein extracted from spores of the rpoE mutant HB6010 (lane 1) or the wild-type CU1065 (lane 2) were loaded.

The Phenotype of the rpoE Mutant-- The fact that the rpoE gene seems to be up-regulated in the transition state between the logarithmic and stationary phase (Fig. 2) might be indicative of a role of delta  in stationary phase phenomena. To investigate the consequences of an rpoE disruption on cell growth, we chose a mutation (RP17) in which most of the rpoE coding sequence is deleted (4). We find that rpoE mutants generate a striking colony morphology when incubated for extended times (3-4 days) on Luria agar at room temperature; colonies of HB6010 show a rough edge with a fractal geometry (Fig. 4B) in contrast to the isogenic parental strain, which has smooth edged colonies. In addition, after dilution of 24-h-old cells into fresh medium (1:100), the cells of HB6010 are markedly more elongated and tend to occur in aggregates, as seen under the light microscope (Fig. 4D), and this phenotype persists for several generations. HB6010 also shows a reproducible 30-min delay in entrance into the logarithmic phase (Fig. 5), as compared with CU1065, in either minimal medium or LB growth medium.


View larger version (93K):
[in this window]
[in a new window]
 
Fig. 4.   An rpoE mutant has an altered morphology. Growth pattern on agar plates of strains CU1065 (A) and HB6010 (B). Cells were diluted from an overnight culture, plated on Luria agar, and incubated at 37 °C for 12 h and at room temperature for a further 48 h. C and D, cells were grown overnight at 37 °C with vigorous shaking, diluted in fresh medium, and grown to an A600 of 0.2 (early logarithmic phase). In HB6010, a rpoE deletion mutant, cells were aggregated (clumped) and elongated with respect to wild type, features that gradually disappeared as the cultures reached an A600 of 0.5 and above.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5.   An rpoE mutant has a lengthened lag phase. Growth curves of strains CU1065 (black-diamond ) and HB6010 (black-square) when diluted 100:1 in fresh liquid LB medium after a 24 h incubation at 37 °C with vigorous shaking. Viable cell counts (colony forming units) demonstrated that viability is identical for both strains even after prolonged incubation. The average difference of time of entrance into logarithmic growth between CU1065 and HB6010 is 30 min.

In contrast with these results, most other attempts to define a phenotype for the rpoE mutant strain have not been successful. HB6010, a rpoE deletion mutant, sporulates with the same efficiency as CU1065, a wild-type strain, and the purified spores are equally sensitive to UV irradiation (data not shown). Growth rate in Luria broth, sporulation medium, or minimal medium is also indistiguishable between the two strains, as originally found by Lampe et al. (4). We did not detect any differences in motility or in resistance to H2O2 between CU1065 and HB6010 (data not shown). Double mutations of rpoE with other known regulators of transition state phenomena, such as abrB or sinR, did not result in phenotypes distinguishable from the phenotypes because of these mutations alone (data not shown). Finally, induction of delta  from the isopropyl-1-thio-beta -D-galactopyranoside inducible Pspac promoter (in pFL25) led to the overproduction of delta  protein as observed by immunoblot analysis but did not produce any appreciable changes in the growth rate or viability nor did it have any major effects on protein composition of the cell, as judged by a [35S]methionine pulse-chase experiment followed by SDS-polyacrylamide gel electrophoresis (data not shown).

Affinity of delta  for RNAP Core-- For use in biochemical studies, we placed a 9-amino acid recognition site for protein kinase A at the amino terminus of delta  to generate delta PKA. Overexpression and purification of delta PKA resulted in a protein that displaced nucleic acids as efficiently as wild-type delta  (14).

We used 4% native polyacrylamide gel electrophoresis to study the interaction of delta PKA with RNAP. As expected for a small protein with a -49 net charge, delta PKA migrated very fast, but its mobility was greatly reduced in the presence of increasing amounts of core RNAP (Fig. 6). Interestingly, two very closely spaced bands are observed suggesting that two distinct forms of RNAP-delta complex are present. Similar results are seen with E. coli RNAP (data not shown).


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 6.   Electrophoretic mobility shift assay of delta -PKA binding to RNAP. delta  protein (10 fmol) was labeled with 32P and mixed in a reaction with increasing concentrations of core RNAP from B. subtilis. Lane 1, no RNAP added. Lanes 2-6 contain 0.35, 0.7, 1.4, 2.8, and 5.8 pmol of RNAP, respectively, added in a 5-µl reaction. Buffer conditions are as described by Altmann et al. (38). All reactions were carried out at room temperature. Separation of free from complexed delta  followed in a 4% native polyacrylamide gel run in Tris-borate EDTA buffer (15).

Under these conditions 0.28 µM core enzyme binds less than 50% of the labeled delta , whereas 0.54 µM binds nearly all of the labeled delta . Because delta  is present at very low levels (2 nM) in these reactions, we can estimate the apparent dissociation constant as the concentration of RNAP required for half-maximal binding (assuming all RNAP can bind delta ). By interpolation, this is ~0.4 µM, corresponding to an association constant of 2.5 × 106 M-1. The presence of a single complex upon the addition of RNAP is consistent with a stoichiometry of 1:1 as previously observed (9, 14).

Effects of DNA and sigma  on the Interaction of delta  with RNAP-- We used labeled delta PKA and sigma PKA to investigate the interactions of delta  and sigma  with RNAP in the presence and absence of the strong trnS promoter (Fig. 7). As before, the addition of core RNAP (lane 2) leads to the appearance of a slower mobility species (Edelta ). To see whether we could detect formation of a Esigma delta complex, we added an excess of unlabeled sigma A. However, under these conditions, there was no "supershift" detected, suggesting that sigma A was unable to bind the Edelta complex (lane 4). However, delta  is displaced from the core by the presence of DNA (lanes 3 and 5). In a parallel series of experiments using radiolabeled sigma APKA, we observed the formation of a slow mobility complex corresponding to promoter-bound Esigma A (lanes 7 and 9) (data not shown), and this complex is stable to the addition of excess cold delta . We again failed to detect a delta -dependent supershift, suggesting that, under the conditions tested, binding favors complexes of Edelta or Esigma ·DNA but not Edelta ·DNA or Esigma delta ·DNA.


View larger version (42K):
[in this window]
[in a new window]
 
Fig. 7.   Electrophoretic mobility shift assay of delta PKA and sigma APKA interactions with RNAP. Under conditions similar to those described in Fig. 6, we used native 4% polyacrylamide gel electrophoresis to separate free from bound 32P-labeled sigma APKA and delta PKA in complexes with RNAP in the presence and absence of promoter DNA. On the left side of the panel, 10 fmol of labeled delta PKA was mixed with RNAP dialysis buffer (lane 1) or 0.1 pmol of B. subtilis core RNAP (lanes 2-5) in 5-µl reactions. In lanes 3-5, 1 pmol of a 165-base pair DNA fragment containing the trnS promoter of B. subtilis (8), sigma APKA (1 pmol, unlabeled), or both was added as indicated. Lanes 6 and 7 contain 0.1 pmol of 32P-labeled sigma A. RNAP (0.1 pmol), DNA (1 pmol), and unlabeled delta PKA (0.5 pmol) were added as indicated.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous analyses indicated that delta , a stoichiometric component of RNAP purified from B. subtilis, is dispensable for growth. Indeed, a rpoE mutant had no apparent phenotype (4). Nevertheless, delta  has large effects on the biochemical properties of RNAP (3, 5-8, 10-14, 29). In this paper, we confirm and extend these previous observations of the rpoE mutant phenotype, quantitate the levels of delta  in growing cells, and characterize the promoter region driving delta  expression. Finally, we demonstrate biochemically that delta  interacts with RNAP with an affinity comparable to other dissociable subunits.

Phenotype of the rpoE Mutant Strain-- Our analyses have revealed that an rpoE deletion mutant is unaltered in growth rate or viability under numerous conditions. However, we reproducibly detect an extended lag phase in the rpoE mutant strain, suggesting that delta  may play a role during growth phase transitions. This notion is also consistent with the observation that delta  is maximally expressed during the transition from growth to stationary phase. In addition, rpoE mutant cells retain an elongated morphology and tend to clump during early logarithmic phase growth. When grown on solid medium, the rpoE mutant has an enhanced tendency to produce rough edged colonies. The factors affecting colony morphology are complex (30, 31) and not well understood, so this phenotype is not very informative.

The relatively modest phenotype associated with an rpoE mutation is surprising for a subunit of RNAP. Another protein may be functionally redundant with delta , but attempts to identify such a protein by transposition mutagenesis were not successful (data not shown). Moreover, the availability of the complete genome sequence of B. subtilis does not reveal any paralogs (32). Nevertheless, delta  is conserved in several other Gram-positive organisms including Mycoplasma genitalium, Mycoplasma pneumoniae, Enterococcus faecalis, and Streptococcus pyrogenes. These homologs contain a statistically significant similarity to the amino-terminal domain of delta  and are typically associated with an acidic carboxyl-terminal domain. Previous work has revealed functionally equivalent proteins in other Bacilli (33), and a similarly sized protein was found to co-purify with RNAP from Staphylococcus aureus (34). Indeed, mutations in an S. aureus gene encoding a putative delta  homolog lead to a stationary phase survival defect (35). Thus, delta  seems to be a bona fide subunit of RNAP found in many different Gram-positive bacteria that may be important for long term survival or during growth phase transitions.

Interactions of delta  with RNAP-- delta is abundant in the cell during all stages of growth, at concentrations of approximately 104 copies/cell. This concentration would be sufficient to saturate the estimated amounts of RNAP in the cell. It also seems to be an abundant protein in spores, although the rpoE mutant sporulates and germinates with equal efficiencies as the wild type, and its spores are equally resistant to UV light. Induced expression of delta  from a plasmid did not alter patterns of gene expression in B. subtilis cells. Expression of rpoE is driven from a single sigma A-dependent promoter and is essentially constitutive under the conditions tested. We found no evidence for autoregulation.

Previous analyses had established that delta , by virtue of its polyanionic carboxyl-terminal region, competes with nucleic acids for binding to RNAP (14). However, it was also observed that a rpoE mutant strain containing a truncated gene produced a stable protein corresponding to the amino-terminal domain of delta  and that this domain still co-purified with RNAP. Thus, determinants for core binding are likely to be present in both the amino- and carboxyl-terminal regions (14). delta  co-purifies with RNAP through several chromatographic steps and during late stages of purification tends to partition with fractions of RNAP containing alternative sigma  factors and not sigma A (36). In an in vitro competition assay, the presence of delta  biases the binding of the alternative sigma  factor gp28 (phage SP82) to RNAP over sigma A (9). Thus, a role for delta  in sigma  switching can be envisaged. A role for delta  in the events leading to the recycling of RNAP after transcription termination has also been documented; delta  has the unique ability of displacing RNA from binary complexes with RNAP, and this effect requires the presence of the carboxyl-terminal half of delta , delta C, which mimics RNA (14). Despite the abundant evidence indicating effects of delta  at the initiation complex (5, 6, 8, 11-13, 29), we were unable, using an electrophoretic mobility shift assay, to document the formation of the presumed Esigma delta complex in either the presence or absence of promoter DNA. This is consistent, on the other hand, with previous studies documenting negative cooperativity between sigma  and delta  (9) and with the fact that delta  mimics RNA and RNA displaces sigma  from RNAP (37).

Concluding Remarks-- The physiological role of delta  remains a mystery, although biochemical evidence suggests that this protein likely participates in both the initiation and recycling phases of transcription. As proposed in early studies, it is not yet excluded that delta  may function in sigma  factor switching, but such a role is clearly not essential. B. subtilis is now known to harbor no fewer than 17 sigma  factors including several whose functions are only just beginning to be deciphered. It will be interesting to determine if rpoE mutations are epistatic with any of these newly described regulators.

    FOOTNOTES

* This work was supported by United States Public Health Service Grant GM-47446 from the National Institutes of Health (to J. D. H) and by fellowships from the Hughes Undergraduate Summer Research Program (to N. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Laboratory of DNA Replication, Rockefeller University, Box 228, 1230 York Ave., New York, NY 10021. E-mail: desarof{at}rockvax.rockefeller.edu.

§ To whom correspondence should be addressed. Tel.: 607-255-6570; Fax: 607-255-3904; E-mail: jdh9{at}cornell.edu.

    ABBREVIATIONS

The abbreviations used are: RNAP, RNA polymerase; MOPS, 4-morpholinepropanesulfonic acid; PCR, polymerase chain reaction; PKA, protein kinase A.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Uptain, S. M., Kane, C. M., and Chamberlin, M. J. (1997) Annu. Rev. Biochem. 66, 117-172[CrossRef][Medline] [Order article via Infotrieve]
  2. Gill, S. C., Weitzel, S. E., and von Hippel, P. H. (1991) J. Mol. Biol. 220, 307-324[Medline] [Order article via Infotrieve]
  3. Pero, J., Nelson, J., and Fox, T. D. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 1589-1593[Abstract]
  4. Lampe, M., Binnie, C., Schmidt, R., and Losick, R. (1988) Gene (Amst.) 67, 13-20[CrossRef][Medline] [Order article via Infotrieve]
  5. Achberger, E. C., Hilton, M. D., and Whiteley, H. R. (1982) Nucleic Acids Res. 10, 2893-2910[Abstract]
  6. Chen, Y.-F., and Helmann, J. D. (1997) J. Mol. Biol. 267, 47-59[CrossRef][Medline] [Order article via Infotrieve]
  7. Juang, Y. L., and Helmann, J. D. (1994) J. Mol. Biol. 235, 1470-1488[CrossRef][Medline] [Order article via Infotrieve]
  8. Juang, Y.-L., and Helmann, J. D. (1995) Biochemistry 34, 8465-8473[Medline] [Order article via Infotrieve]
  9. Hyde, E. I., Hilton, M. D., and Whiteley, H. R. (1986) J. Biol. Chem. 261, 16565-16570[Abstract/Free Full Text]
  10. Spiegelman, G. B., Hiatt, W. R., and Whiteley, H. R. (1978) J. Biol. Chem. 253, 1756-1765[Abstract]
  11. Dickel, C. D., Burtis, K. C., and Doi, R. H. (1980) Biochem. Biophys. Res. Commun. 95, 1789-1795[Medline] [Order article via Infotrieve]
  12. Dobinson, K. F., and Spiegelman, G. B. (1987) Biochemistry 26, 8206-8213[Medline] [Order article via Infotrieve]
  13. Juang, Y. L., and Helmann, J. D. (1994) J. Mol. Biol. 239, 1-14[CrossRef][Medline] [Order article via Infotrieve]
  14. López de Saro, F., Woody, A.-Y. M., and Helmann, J. D. (1995) J. Mol. Biol. 252, 189-202[CrossRef][Medline] [Order article via Infotrieve]
  15. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1990) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  16. Chen, L., James, L. P., and Helmann, J. D. (1993) J. Bacteriol. 175, 5428-5437[Abstract]
  17. Slack, F. J., Mueller, J. P., and Sonenshein, A. L. (1993) J. Bacteriol. 175, 4605-4614[Abstract]
  18. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Methods Enzymol. 185, 60-89[Medline] [Order article via Infotrieve]
  19. Kenney, T. J., and Moran, C. P. (1991) J. Bacteriol. 173, 3282-3290[Medline] [Order article via Infotrieve]
  20. Zuber, P., and Losick, R. (1987) J. Bacteriol. 169, 2223-2230[Medline] [Order article via Infotrieve]
  21. Stragier, P., Bonamy, C., and Karmazyn-Campelli, C. (1988) Cell 52, 697-704[Medline] [Order article via Infotrieve]
  22. Miller, J. H. (1972) Experiments in Molecular Genetics, pp. 352-255, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  23. Nicholson, W. L., and Setlow, P. (1990) in Molecular Biological Methods for Bacillus (Harwood, C. R., and Cutting, S. M., eds), pp. 391-450, John Wiley & Sons, Inc., New York
  24. Kelman, Z., Naktinis, V., and O'Donnell, M. (1995) Methods Enzymol. 262, 430-442[Medline] [Order article via Infotrieve]
  25. Chang, B. Y., and Doi, R. H. (1990) J. Bacteriol. 172, 3257-3263[Medline] [Order article via Infotrieve]
  26. Helmann, J. D. (1995) Nucleic Acids Res. 23, 2351-2360[Abstract]
  27. Walter, A. E., Turner, D. H., Kim, J., Lyttle, M. H., Muller, P., Mathews, D. H., and Zucker, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9218-9222[Abstract/Free Full Text]
  28. Burgess, R. R., and Jendrisak, J. J. (1975) Biochemistry 14, 4634-4638[Medline] [Order article via Infotrieve]
  29. Achberger, E., and Whiteley, H. R. (1981) J. Biol. Chem. 256, 7424-7432[Abstract/Free Full Text]
  30. Mendelson, N. H., and Salhi, B. (1996) J. Bacteriol. 178, 1980-1989[Abstract]
  31. Rudner, R., Martsinkevich, O., Leung, W., and Jarvis, E. D. (1998) Mol. Microbiol. 27, 687-703[CrossRef][Medline] [Order article via Infotrieve]
  32. Kunst, F., Ogasawara, N., Moszer, I., Albertini, A. M., Alloni, G., Azevedo, V., Bertero, M. G., Bessieres, P., Bolotin, A., Borchert, S., Borriss, R., Boursier, L., Brans, A., Braun, M., Brignell, S. C., Bron, S., Brouillet, S., Bruschi, C. V., Caldwell, B., Capuano, V., Carter, N. M., Choi, S. K., Codani, J. J., Connerton, I. F., Cummings, N. J., Daniel, R. A., Denizot, F., Devine, K. M., Duesterhoeft, A., Ehrlich, S. D., Emmerson, P. T., Entian, K. D., Errington, J., Fabret, C., Ferrari, E., Foulger, D., Fritz, C., Fujita, M., Fujita, Y., Fuma, S., Galizzi, A., Galleron, N., Ghim, S. Y., Glaser, P., Goffeau, A., Golightly, E. J., Grandi, G., Guiseppi, G., Guy, B. J., Haga, K., Haiech, J., Harwood, C. R., Henaut, A., Hilbert, H., Holsappel, S., Hosono, S., Hullo, M. F., Itaya, M., Jones, L., Joris, B., Karamata, D., Kasahara, Y., Klaerr-Blanchard, M., Klein, C., Kobayashi, Y., Koetter, P., Koningstein, G., Krogh, S., Kumano, M., Kurita, K., Lapidus, A., Lardinois, S., Lauber, J., Lazarevic, V., Lee, S. M., Levine, A., Liu, H., Masuda, S., Mauel, C., Medigue, C., Medina, N., Mellado, R. P., Mizuno, M., Moestl, D., Nakai, S., Noback, M., Noone, D., O'Reilly, M., Ogawa, K., Ogiwara, A., Oudega, B., Park, S. H., Parro, V., Pohl, T. M., Portetelle, D., Porwollik, S., Prescott, A. M., Presecan, E., Pujic, P., Purnelle, B., Rapoport, G., Rey, M., Reynolds, S., Rieger, M., Rivolta, C., Rocha, E., Roche, B., Rose, M., Sadaie, Y., Sato, T., Scanlan, E., Schleich, S., Schroeter, R., Scoffone, F., Sekiguchi, J., Sekowska, A., Seror, S. J., Serror, P., Shin, B. S., Soldo, B., Sorokin, A., Tacconi, E., Takagi, T., Takahashi, H., Takemaru, K., Takeuchi, M., Tamakoshi, A., and Tanaka, T. (1997) Nature 390, 249-256[CrossRef][Medline] [Order article via Infotrieve]
  33. Achberger, E. C., Tahara, M., and Whiteley, H. R. (1982) J. Bacteriol. 150, 977-980[Medline] [Order article via Infotrieve]
  34. Deora, R., and Misra, T. K. (1995) Biochem. Biophys. Res. Commun. 208, 610-616[CrossRef][Medline] [Order article via Infotrieve]
  35. Watson, S. P., Antonio, M., and Foster, S. J. (1998) Microbiology 144, 3159-3169[Abstract]
  36. Wiggs, J. L., Gilman, M. Z., and Chamberlin, M. J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 2762-2766[Abstract]
  37. Hansen, U. M., and McClure, W. R. (1980) J. Biol. Chem. 255, 9564-9570[Abstract/Free Full Text]
  38. Altmann, C. R., Solow-Cordero, D. E., and Chamberlin, M. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3784-3788[Abstract]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.