Role of core promoter sequences in the mechanism of swarmer cell-specific silencing of gyrB transcription in Caulobacter crescentus
© England and Gober; licensee BioMed Central Ltd. 2005
Received: 01 March 2005
Accepted: 17 May 2005
Published: 17 May 2005
Each Caulobacter crescentus cell division yields two distinct cell types: a flagellated swarmer cell and a non-motile stalked cell. The swarmer cell is further distinguished from the stalked cell by an inability to reinitiate DNA replication, by the physical properties of its nucleoid, and its discrete program of gene expression. Specifically, with regard to the latter feature, many of the genes involved in DNA replication are not transcribed in swarmer cells.
We show that for one of these genes involved in DNA replication, gyrB, its pattern of temporal expression depends upon an 80 base pair promoter region with strong resemblance to the Caulobacter crescentus σ73 consensus promoter sequence; regulation does not appear to be affected by the general strength of the promoter activity, as mutations that increased its conformity with the consensus did not affect its cell-cycle expression pattern. Transcription from the gyrB promoter in vitro required only the presence of the σ73 RNA polymerase (from E. coli) and the requisite nucleoside triphosphates, although a distinct binding activity, present in crude whole-cell extracts, formed a complex gyrB promoter DNA. We also assayed the effect on gyrB expression in strains containing mutations in either smc or dps, two genes encoding proteins that condense DNA. However we found there was no change in the temporal pattern of gyrB transcription in strains containing deletions in either of these genes.
These experiments demonstrate that gyrB transcription does not require any auxiliary factors, suggesting that temporal regulation is not dependent upon an activator protein. Swarmer-specific silencing may not be attributable to the observed physical difference in the swarmer cell nucleoid, since mutations in either smc or dps, two genes encoding proteins that condense DNA, did not alter the temporal pattern of gyrB transcription in strains containing deletions in either of these genes. Rather a repressor that specifically recognizes sequences in the gyrB promoter region that are also probably essential for transcription, is likely to be responsible for controlling cell cycle expression.
The bacterium Caulobacter crescentus divides asymmetrically once during each cell cycle, yielding a sessile stalked cell and a motile swarmer cell [1, 2]. These two cell types differ not only in morphology but also in their ability to replicate their genomes and to divide. The stalked cell immediately reinitiates DNA replication and cell division. The swarmer cell, in contrast, is incompetent for these two processes until it sheds its single, polar flagellum and differentiates into a stalked cell. The stalked cell then passes through several predivisional stages, during which time the genome is replicated and segregated, the newly synthesized DNA is methylated, and a new flagellum is synthesized and constructed at the pole opposite the stalk [1–4]]. The asymmetry that distinguishes the two daughter cells is founded in the predivisional cell before cell separation occurs. Asymmetric cell division depends upon several general mechanisms, including intracellular protein and/or mRNA localization, regulated phosphorylation and proteolysis, and cell-cycle dependent transcription and translation.
The inability of the Caulobacter swarmer cell to replicate DNA is due in part to the activity of the response regulator CtrA, which binds to sites in the origin of replication (Cori) [5, 6]. CtrA is synthesized and phosphorylated when DNA replication is approximately half-way complete; the phosphorylated protein persists in the swarmer cell after cell division [7, 8]. Phosphorylated CtrA represses transcription of DNA adjacent to the Cori, that is necessary for replication initiation, and also prevents binding of the DnaA initiator protein [6, 9, 10]. When the swarmer cell differentiates into a stalked cell, CtrA is proteolyzed and DNA replication commences . In addition to its role in preventing DNA replication, CtrA is also a transcriptional activator of many genes that affect several different aspects of Caulobacter development. CtrA regulates transcription of its own gene, activates early genes in flagellar biogenesis, and represses ftsZ, the earliest known gene in cell division [5, 8, 11]. Interestingly, while microarray analysis has implicated CtrA in the control of approximately 25% of the cell-cycle regulated genes and it appears to directly regulate 55 operons, it does not regulate the transcription of several genes involved in DNA replication [12, 13]. Thus, control of DNA replication initiation also depends on CtrA-independent transcription regulation.
Transcription of many DNA replication genes begins just before DNA replication initiates at the swarmer-to-stalked cell transition. These swarmer-cell-silenced genes include dnaA, dnaN (encoding the β subunit of DNA polymerase III), dnaX (γ and τ subunits of DNA polymerase III), dnaK (replication-initiation chaperone), and gyrB (B subunit of DNA gyrase) [14–18]]. The predicted promoter sequences for most of these genes align well with the published consensus sequence for σ73 . As the promoters of these DNA replication-associated genes appear to contain neither a CtrA binding site or any obvious elements that would suggest transcription activation in stalked cells, the mechanism responsible for their cell-cycle dependent regulation remains unknown.
In this study we analyze the role of the gyrB promoter in swarmer cell-specific silencing of gene expression. We map the site at which transcription of gyrB starts and create deletions to further delineate the promoter sequences required for temporal regulation of the gene to an 80 bp region. Using site-directed mutagenesis we further show that transcriptional silencing in swarmer cells does not appear to depend upon the overall strength of the promoter activity. In vitro experiments demonstrate that gyrB transcription does not require any auxiliary factors, suggesting that temporal regulation is not dependent upon an activator protein. However, gel-mobility shift assays demonstrate that an activity, present in C. crescentus crude whole-cell extracts and unrecognized by anti-RNAP antibody, is able to bind to the gyrB promoter. One possibility is that swarmer-specific silencing is linked to the observed difference in the physical characteristics between the swarmer cell nucleoids and stalked cell nucleoids [20–23]. Thus, a protein that affects the overall folding and structure of the nucleoid might also be regulating transcription. In this regard, we also assayed the effect on gyrB expression in strains containing mutations in either smc or dps, two genes encoding proteins that condense DNA. However we found there was no change in the temporal pattern of gyrB transcription in strains containing deletions in either of these genes.
Mapping and deletion analysis of the gyrB promoter
Based on the transcription start sites found by primer extension, alignment and comparison with several other known σ73 genes allowed identification of possible -10 and -35 promoter elements of gyrB (Fig. 1B) [14–16, 19, 24–26]. The consensus is derived from σ73 transcribed genes that are expressed at relatively constant levels throughout the cell cycle; expression of gyrB, along with other DNA-replication genes, varies during the cell cycle. In analyzing the gyrB promoter and surrounding DNA, we considered two broad possibilities for gyrB transcription regulation. gyrB expression might be controlled by the activity of a swarmer cell-specific factor that inhibits transcription or by a stalked cell-specific factor that activates transcription. An (A+T)-rich region, beginning approximately 60 bp upstream of the transcription start site, at first appeared to be a good candidate for a possible regulatory protein binding site. This region consists of two stretches of repeated thymidine residues and one stretch of repeated adenine residues, approximately phased with respect to each other on the DNA strand (data not shown). Such an arrangement could create a bend in the DNA, a secondary structure that is preferred by some DNA-binding proteins and that can affect transcription independent of any additional protein constituents [27, 28]. Another part of the gyrB gene that might possibly contain a cis-acting regulatory site is the extremely long (170 bp), untranslated leader sequence between the transcription and translation start sites. Alternatively, gyrB transcription might not depend on any remote DNA elements or sites for sequence-dependent DNA binding proteins. As has been reported, the putative gyrB promoter appears more similar to a subset of σ73 genes that mirror its temporal regulation than to those genes that are constitutively expressed throughout the cell cycle (Fig. 1B) [14, 17, 24, 26]. This would seem to suggest that temporal regulation is dictated by the promoter itself rather than by a more distantly located DNA element.
Mutagenesis of the gyrB promoter
Each mutated promoter was fused to a promoterless lacZ gene and expression levels were quantitatively measured by β-galactosidase assays (Fig. 4A). The mutations P6m2 and P6m3 resulted in transcription levels that were twice that of the wild-type level, while the P6mL2, -3, and -4 were transcribed at normal or (P6mL3) reduced amounts. The increased promoter activity of P6m2 and P6m3 and the decreased expression of P6mL3 support the proposed promoter sequence reported here (Fig. 1) as the P6m2 and P6m3 mutations increase the agreement with the σ73 consensus and while the P6mL3 mutation decreases the conformity. One possibility that could account for the increased promoter activity of the P6m2 and P6m3 mutant promoters was inappropriate expression in swarmer cells. Therefore, we wished to examine the temporal transcription patterns of these promoters in order to test this possibility (Fig. 4B). Surprisingly, transcription from the mutant promoters, though stronger, was still regulated in the wild-type manner during the cell cycle (Fig. 4B).
Transcription of gyrB does not require an activator
An activity, distinct from RNA polymerase, binds the gyrB promoter in vitro
Interestingly, we observed different results when we compared samples containing fresh extracts (and extracts that had been frozen only once) with those containing extracts that had been previously thawed and then refrozen. The non-RNAP binding activity was labile under conditions in which the extract had been subjected to one round of freeze-thaw (Fig. 6B, lanes 2 and 3 vs. lanes 4 and 5). Furthermore, when the extract that had been refrozen and thawed was used in the binding assay (Fig. 6B, lanes 2,3), the disappearance of the specific, labile protein band was accompanied by the appearance a new specific super-shifted band upon the addition of anti-RNAP antibody.
The role of DNA condensing proteins in the regulation of gyrB transcription
Previous studies have shown that, in addition to being unable to initiate DNA replication, C. crescentus swarmer cells also differ from stalked cells in their chromosomal architecture [21–23]. These experiments revealed that isolated swarmer cell nucleoids sediment faster in a sucrose gradient than those of stalked cells. One hypothesis is that the swarmer cell nucleoid possesses a different complement of DNA 'condensing' proteins such as histone-like proteins. The gel mobility shift activity present in crude cell extracts may reflect the binding of a protein that alters the overall architecture of the swarmer cell nucleoid. Two candidates for such an activity may be either Dps or SMC. In E. coli, the Dps protein is induced both in stationary phase and during starvation; it is able to physically protect the nucleoid from oxidative and nuclease damage, as well as affect transcription under these conditions [29–32]. SMC (S tructural M aintenance of C hromosomes), is a member of the eukaryotic SMC family [reviewed in [33–36]], large, multidomain proteins that associate with each other in an antiparallel fashion. This association occurs through long, coiled-coil domains that flank a central hinge region, resulting in protein dimers that are shaped like old-fashioned hairpins. Dimers of SMC associate with non-SMC factors to form specific complexes in eukaryotic cells, with functions in DNA organization and movement, ranging from condensation to dosage compensation to repair [33, 37]. Experiments in Bacillus subtilis have demonstrated that chromosomal DNA was visibly decondensed in smc mutants .
The Caulobacter dps gene is approximately 50 to 55% identical and 60 to 65% similar to dps genes in other organisms, including Burkholderia pseudomallei, Bordetella pertussis and Pseudomonas syringae [data not shown]. Furthermore, a search of the NCBI CDD (conserved domain database) revealed significant alignment with a COG (cluster of orthologous genes) for Dps/ferritin-like DNA-binding proteins. The gene appears to be the first in a two-gene operon; the expected product of the second gene is a conserved hypothetical protein of unknown function. A lacZ transcription fusion of a DNA fragment spanning 600 bp upstream of the predicted dps translation start site was expressed at a low level (350–400 units of β-galactosidase activity) in late log-phase cultures [data not shown]. Although it has been shown to be involved in DNA protection and starvation and stationary-phase survival in other organisms, it seemed possible that Dps might be involved in developmental events specific to the swarmer cell: the results of genome-wide DNA microarray experiments showed that dps mRNA is maximally present in swarmer cells [13, 30]. As one way to investigate a possible role for Dps in Caulobacter development, we engineered a strain in which 90% of the dps coding sequence was replaced with a gene conferring resistance to gentamicin. The strain was indistinguishable from wild-type cells in logarithmic growth and morphology, and we detected no overt aberration in swarmer-cell motility or nucleoid appearance (data not shown). Additionally, we constructed a strain with a null mutation in smc by disrupting the chromosomal copy of the s m c gene by insertion of a gene conferring spectinomycin resistance [see Methods]. The deletion was confirmed by Southern blot and by immunoblot with antibodies against SMC [data not shown].
In Caulobacter crescentus, many of the genes involved in DNA replication, including dnaN, dnaX, dnaA, dnaC, dnaK and gyrB, are maximally expressed at the time that this process occurs [14–18, 24, 25]. Thus, transcription of these genes does not occur in swarmer cells, or in the swarmer pole of predivisional cells, and begins only when the swarmer cells differentiate into stalked cells or when DNA replication commences anew in the stalked cells at the completion of cell division. The means whereby this regulatory pattern is achieved remains unknown. In this study, we used gyrB as a representative gene with which to investigate the mechanism responsible for swarmer-specific transcription silencing in C. crescentus. As revealed by primer extension and in vitro transcription/primer extension assays, the transcription start site of the gene is separated from the translation start site by a fairly long leader sequence. There is apparent variation in the precise beginning of the transcript, as observed here and as reported elsewhere . The lack of a precise transcription start site may be due to other factors that influence gyrB regulation. For example, transcription of the gyrB gene in Caulobacter is induced by relaxation of the DNA , and this induction is distinct from the increased expression that occurs during the swarmer-to-stalked cell transition. It is also possible that the transcription start site varies depending upon undefined regulatory cues such as growth rate and/or growth phase of the culture.
Having delineated the gyrB promoter to a minimal, 80 base pair region, we compared this promoter to other σ73 promoters and to the published consensus sequence, taking care to explore different alignments based on the several possible start sites. The alignment shown [see Fig. 1] is that best fitting the major start site obtained by both primer extension and in vitro transcription/primer extension (for an alternative, see ). Examination of the -10 and -35 regions of the promoter, based upon our alignment, revealed that the gyrB promoter greatly differs from the C. crescentus σ73 consensus sequence. This suggested an interesting possibility for a swarmer-specific silencing mechanism. We reasoned that greater deviation from the consensus sequence might make these promoters slightly inferior binding sites for the σ73 RNAP, and thus slightly weaker. Such promoters might be more susceptible to a situation in which RNAP binding is partially inhibited or blocked, a possible scenario occurring in swarmer cells. Site-directed mutagenesis allowed us to test this possibility by creating versions of the gyrB promoter that more closely matched the consensus sequence and therefore should, in theory at least, have been transcribed at higher levels. However, while in two cases transcription from the mutated promoters was greater than from the wild-type promoter, temporal regulation of transcription was unaffected. Thus, it seems that the strength of the promoter does not intrinsically determine cell cycle regulation of gyrB.
The experiments presented here show that the gyrB promoter can be transcribed by E. coli σ70 RNA polymerase holoenzyme in the absence of additional protein factors, suggesting the unlikelihood of temporal regulation depending solely upon an activator. However, it is possible that a stalked-cell specific transcriptional activator is required to overcome a swarmer-cell specific repression complex or DNA topological conformation. When a probe containing the gyrB promoter was used in gel-mobility shift assays, two binding activities, both of which are components of crude Caulobacter extracts, were apparent. One of these activities was loosely identified as RNA polymerase based on the ability of anti-RNAP antibody to recognize and alter the migration of this band through the gel. The other binding activity was not recognized by the antibody. This second species was vulnerable to freeze-thaw treatment, as it was only present in extract that had not been re-frozen. One might imagine a scenario in which a swarmer-specific DNA protein(s) binds to the gyrB promoter and interferes with the ability of the polymerase to access the DNA.
Previous experiments with another swarmer cell-repressed promoter, the dnaX promoter, revealed the RRF motif, that by sequence comparison is located between the -10 and -35 elements in all of the swarmer cell-silenced DNA replication gene promoters . Within the RRF of the dnaX promoter, a C residue at position -21 appears to be involved in the temporal regulation of the gene . A substitution of T at this position results in a more than 3-fold increase in transcription and, more importantly, in a loss of swarmer-specific silencing of dnaX. Additionally it was shown that the RRF motif is bound by an unknown species found in crude extracts . The same shift was seen when DNA fragments encompassing the -10 and -35 regions of several different DNA-replication genes, including gyrB and dnaX, were used as probes in gel-mobility shift assays. It is possible that the binding activity reported here for the gyrB promoter is the same as that observed previously with the RRF motif. If this observed binding activity were responsible for the swarmer cell-specific silencing of gyrB transcription, the one might expect that swarmer cell extracts would be enriched in binding activity. However, we were unable to detect this activity in extracts derived from either isolated swarmer or stalked cells, probably owing to limitations in obtaining a sufficient quantity of material. The relevance of these DNA binding activities and a experimental definition of their binding sequences in similarly regulated promoters will need to be explored.
The condensed nature of nucleoids isolated from swarmer cells, relative to stalked cells, when subjected to centrifugation through a sucrose gradient, suggests that the two cell types differ in the proteins that affect the organization and folding of their chromosomes . Growth-phase dependent variation in the relative composition of the DNA-binding protein cadre in E. coli has been documented . A similar developmentally-regulated phenomenon might be responsible for silencing gyrB and other genes involved in DNA replication in Caulobacter swarmer cells. Proteins such as HU or H-NS bind DNA nonspecifically and have been shown to affect transcription regulation . While neither protein binds to a specific sequence on the DNA, each has a preference for certain secondary DNA structures, including gaps and junctions (HU) and (A+T)-rich bends (H-NS). Of particular interest, H-NS represses the virF gene by binding to two sites, one overlapping the RNAP binding site in the promoter and another 200 bp upstream . However, repression only occurs at temperatures below 32°C because below this temperature the DNA is more intrinsically bent, which allows interaction between H-NS bound at both sites. HU, the most abundant DNA-binding protein in bacteria, also regulates transcription. Similar to integration host factor, HU is thought to exert its influence by creating a bend in the DNA and thus affecting the activity of other, more specific transcription regulators [reviewed in [40, 42, 43]]. Based on searches of the published genome, we have concluded that C. crescentus does not possess a gene that might encode H-NS, nor does it appear to have a homolog of the closely related gene, stpA . C. crescentus, does however, possess genes encoding proteins that are closely homologous to the two subunits of E. coli HU (HUα and HUβ). We suspect that HU is probably not involved in the regulation of the DNA replication genes since we have recently found that the abundance of both HU subunits does not change during the course of the cell cycle [unpublished observation].
In order to determine whether or not two other well-known DNA condensation proteins might affect swarmer-specific transcription, we constructed strains in which smc or dps were disrupted. There is strong evidence that the large bacterial SMC protein, very different from the small DNA-binding proteins discussed above, is involved in chromosome condensation and partitioning, similar to its eukaryotic homologs [33, 36]. A deletion of the gene encoding SMC in Bacillus subtilis results in a temperature-sensitive strain with visibly decondensed chromosomes and defects in chromosome segregation [44, 45]. Similar results have also been reported in Caulobacter crescentus . Importantly, however, temporal transcription of gyrB was unchanged in the Δsmc mutant, indicating that SMC is not necessary for swarmer-specific transcriptional silencing.
The final target of our studies was the Dps protein. in E. coli, Dps protects DNA during periods of starvation or in stationary phase [31, 32]. When bound to DNA, Dps forms a crystalline structure that is proposed to render the DNA physically resistant to oxidative damage as well as degradation by some nucleases . Dps is also important for induced DNA protection during non-stationary phase growth, as dps mutants are more sensitive to hydrogen peroxide . The nature of the Caulobacter swarmer cell (DNA replication- and cell division-incompetent) makes it conceivable that the intracellular environment may be likened to that of a cell in stationary phase or starved for nutrients. Thus, perhaps Dps plays a different role in Caulobacter. Supporting this hypothesis, DNA microarray experiments have revealed that the dps mRNA level is greatest in swarmer cells and then decreases dramatically at the swarmer-to-stalked cell transition . A deletion of dps, however, did not have a visible effect on the Caulobacter cell cycle. Also, similar to the Δsmc strains, the temporal expression pattern of the gyrB promoter was unaffected in the Δdps strain.
In summary, these results show that a core 80 bp gyrB promoter sequence is sufficient to confer a cell cycle-regulated pattern of transcription. In vitro assays indicated that gyrB transcription does not require any factors in addition to RNAP. Thus, the transcription of gyrB is likely to be regulated by a repressor protein in swarmer cells, and not an activator protein. This repressor may be related to the DNA binding activity present in crude cell extracts that bound specifically to the gyrB promoter region. Deletions in the genes encoding the global DNA binding proteins, SMC and Dps had no effect on viability, growth rate or the temporal transcription pattern of gyrB. Thus, the results of this study do not provide definitive support for the idea that DNA-binding proteins that alter the global architecture of chromosomal DNA are involved in transcriptional repression in the C. crescentus swarmer cell. However, it is possible that the condensation of the swarmer-cell nucleoid is the result of the combined activity of two or more proteins and this may be important in influencing gyrB transcription.
Bacterial strains, growth conditions, and plasmid construction
Strains and Plasmids
Rp4-2, Tc::Mu, Km::Tn7
F_, lacIq, proA+B+ lacZ _M15
NA1000 syn-1000 (synchronizable strain)
JG3003 syn-1000, smc7 (spec resistance)
JG3402 syn-1000, dps2 (gent resistance)
pRK290 derivative containing a promoterless lacZ gene
sacB counter selection vectors; (kanr)
500 bp, Pst I/Stu I gyrB fragment in pBluescript-KS+
904 bp Pst I/Hin dIII gyrB promoter fragment
1.65 kb Pst I/Eco RI fragment in placZ/290, gyrB-lacZ transcriptional reporter fusion
1.3 kb Pst I/Hin dIII fragment in placZ/290
942 bp Pst I/Hin dIII gyrB-lacZ transcriptional reporter fusion
904 bp Pst I/Hin dIII gyrB-lacZ transcriptional reporter fusion
167 bp Pst I/(native)Stu I gyrB-lacZ transcriptional reporter fusion
132 bp Pst I/(native)Stu I gyrB-lacZ transcriptional reporter fusion
180 bp Pst I/Hin dIII gyrB-lacZ transcriptional reporter fusion
80 bp Pst I/Hin dIII gyrB-lacZ transcriptional reporter fusion
The dps deletion strain was generated by the sacB selection method . In order to accomplish this, the dps gene was first isolated by PCR as two separate DNA fragments that were subcloned into pBluescript KS+, reconstituting the gene with a newly-created internal MluI site. The coding region was then disrupted by introducing a gentamicin resistance cassette from pKnock GM  into the unique MluI site. This entire fragment, dps2Δg, was then subcloned into the sacB selection vector, pNPTS139 [M.R.K. Alley, unpublished]. The plasmid, pNPTS-dps2Δg, harbored in E. coli strain S17-1, was introduced into C. crescentus strain NA1000 by conjugation. Transconjugants were selected from PYE agar plates containing kanamycin and naladixic acid (20 μg/ml). A 2 ml culture inoculated with several transformants was grown for approximately 8 hours in PYE without selection. The culture was then diluted 1/10 and plated (0.2 ml/plate) on PYE agar containing 2.5% sucrose. One to three hundred sucrose resistant colonies were than spotted onto two different growth media: PYE sucrose plates containing gentamicin and PYE plates containing kanamycin. Sucrose-resistant, kanamycin-sensitive, gentamicin-resistant colonies where isolated for further study. The deletion of dps in strain pJG3402 was confirmed by PCR. The smc deletion strain was also isolated by this basic method except that the smc gene was disrupted with a spectinomycin resistance cassette. The smc deletion was confirmed by Southern blot (data not shown) and by immunoblot with anti-SMC antibodies (not shown). Following allelic exchange, the disrupted dps and smc genes were each transduced into a new wild-type C. crescentus strain .
Cell cycle experiments
The transcription of the lacZ reporter fusions was assayed in cultures synchronized essentially as described previously . Isolated swarmer cells were suspended in M2 glucose medium and allowed to continue growth. At various time points, 5 ml portions of growing culture were removed and the proteins were pulse-labeled for 5 minutes with 35S-Trans-label (ICN). Labeled β-galactosidase was immunoprecipitated as described previously .
Site-directed mutagenesis and molecular biology procedures
The initial template used for mutagenesis was the 180 bp gyrBP4 promoter fragment. The fragment was cloned into M13-BM20 and site-directed mutagenesis was performed on single-stranded DNA isolated from E. coli TG-1, as previously described . The mutant gyrB promoters were confirmed by sequencing performed according to the dideoxy chain-termination method . The β-galactosidase was assayed as previously described [57, 58], and the reported values represented the mean activity from at least three independently grown cultures assayed in triplicate. The standard deviation in all cases was less than 5%. All other routine molecular biology manipulations were performed as previously described .
Primer extension and in vitro transcription/primer extension
Primer extension, both singly and following in vitro transcription, was performed as previously described . The oligonucleotides (PE1: 5'-GCGTCGCCACGCGAACGC-3'; P E 2 : 5 ' – T G A G T T C G T C A G C C A G A G C – 3 ' ; B S 2 : 5 ' -GAGCAATATTACAGGATTCG-3') used in these experiments and for sequencing (data not shown) were complimentary to sequences near the 5' end of the predicted mRNA sequence and radioactively end-labeled with [γ-32P] ATP and T4 polynucleotide kinase . In each primer extension assay, the same primer was also used for a concurrent dideoxy sequencing reaction . For primer extension, total RNA was isolated from strain NA1000 (O.D. at 600 nm of 1.0) grown in M2 glucose minimal medium. Pelleted cells were resuspended in 20 mM sodium acetate, 1 mM EDTA (pH 5.4). 10% SDS was then added to a final concentration of 4%, followed by addition of an equal volume of phenol equilibrated with 20 mM sodium acetate, 1 mM EDTA (pH 5.4). Extraction, consisting of vortexing and incubation at 65°C for 10 minutes, was performed twice. The RNA was then ethanol precipitated from the aqueous phase, rinsed twice with 70% ethanol, and dried. Approximately 50 μg of the isolated RNA was used for primer extension.
In vitro transcription was performed as described . The template was a cesium chloride gradient-purified preparation of pBluescript KS+ containing either the 900 bp gyrBP3 promoter fragment (see Fig. 3) or an approximately 500 bp Pst I/Stu I fragment (gyrB500) containing the promoter and regions further upstream (Table 1). It was transcribed by either E. coli RNA Polymerase σ70 holoenzyme (Epicenter Technologies, Madison, WI). The resultant mRNA was then reverse transcribed from the PE2 primer, as detailed above.
Gel mobility shift assays
The crude extracts used in these experiments were prepared from mid-log phase, 0.5-liter cultures of wild-type Caulobacter crescentus (NA1000). The cells were collected by centrifugation and then rinsed three to four times with HEMGK (20 mM HEPES, pH 7.6; 0.1 mM EDTA, pH 8.0; 12.5 mM MgCl2; 5% glycerol; 100 mM KCl) before being re-suspended in 4 ml of the same. PMSF in 95% ethanol was then added to 0.1% and the cells were lysed by sonication. The lysate was cleared by centrifugation at 34,500 × g in a Sorvall SS-34 rotor; the cleared lysate, containing approximately 1 to 3 mg/ml protein, was immediately frozen (-80°C) in 0.4 ml aliquots. Unless otherwise indicated, each aliquot was thawed for use only once. The DNA probe was prepared by digesting pKSgyrB500 with Hin dIII (a site derived from the polylinker) and Pst I, purifying the fragment from a 1% agarose gel and then labeling with [α-32P] dGTP by filling in the Hin dIII site with DNA polymerase I, Klenow fragment (Promega). The gel shift assays were performed essentially as described, with some minor modifications . Briefly, each reaction contained 10–20 × 103 cpm of the labeled DNA probe and was incubated in Incubation Buffer (20 mM Tris-Cl, pH 7.5; 80 mM NaCl; 1 mM EDTA, pH 8.0; 0.1 mg/ml salmon-sperm DNA, sonicated) for 10 minutes at room temperature, after which loading dye was added and the samples run on a 6% non-denaturing polyacrylamide gel at 4°C. Where antibody to RNA polymerase  was included in the reaction, 1 to 3 μl were added and all samples were incubated an additional 5 minutes at room temperature.
J.C.E. was supported by USPHS predoctoral fellowship GM-07185. Work in our laboratory is supported by Public Health Service Grant GM48417 from the National Institutes of Health to J.W.G.
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