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Journal of Bacteriology, August 2003, p. 4883-4890, Vol. 185, No. 16
0021-9193/03/$08.00+0 DOI: 10.1128/JB.185.16.4883-4890.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Y and Its Target Promoters
Department of Microbiology, Cornell University, Ithaca, New York 14853-8101,1 Department of Biology, San Francisco State University, San Francisco, California 94132,2 Experimental Station E328/148B, DuPont Central Research and Development, Wilmington, Delaware 198823
Received 3 April 2003/ Accepted 20 May 2003
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ABSTRACT |
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Y is of unknown function. We demonstrate that the sigY operon is expressed from an autoregulatory promoter site, PY. We selected for transposon-induced mutations that upregulate PY transcription in an attempt to identify genes involved in Y regulation. The resulting insertions disrupted yxlC, the gene immediately downstream of sigY. However, the phenotype of the yxlC::Tn10 insertion was due to polarity on the downstream genes of the sigY operon; a nonpolar insertion in yxlC did not lead to derepression of PY. Further analyses revealed that both yxlD and yxlE encoded proteins important for the negative regulation of Y activity. A comparison of the transcriptomes of wild-type and yxlC::Tn10 mutant strains revealed elevated expression of several operons. However, only one additional gene, ybgB, was unambiguously identified as a direct target for Y. This was supported by analysis of direct targets for Y transcription with whole-genome runoff transcription followed by macroarray analysis. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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factors function as global regulators of a variety of stress responses often triggered by changes in the cell envelope (12). In some organisms, this particular family of regulators has expanded to include large numbers of paralogues. The Bacillus subtilis genome encodes seven ECF factors, Mycobacterium tuberculosis encodes 10, and, remarkably, Streptomyces coelicolor encodes at least 50. In most cases, the function of these factors is not yet known.
In B. subtilis, most studies to date have concentrated on three of the ECF factors, X, W, and M. The roles of these factors have been investigated by phenotypic analysis of mutant strains altered in activity (14, 15), identification of target operons (3, 4, 6, 17, 18, 30), and identification of signals that function to induce the various regulons (7, 28, 30, 32). The results indicate that X controls functions associated with modification of the cell envelope, while W and M control overlapping regulons that are induced by antibiotics that target the cell envelope (12). The W regulon is strongly induced by alkali shock (30), although this may be due to effects of high pH on cell wall synthesis.
Despite this progress, the roles of the other four ECF factors, Y, YlaC, V, and Z, are still mysteries. As one approach to defining the roles of these regulators, we generated mini-Tn10 transposon libraries to identify mutants with increased expression of Y, YlaC, V, or Z. In principle, selection for upregulation might identify proteins that interact directly with the operon control region (e.g., repressors) or genes that affect cell physiology in ways that trigger operon expression. In addition, since most of these operons are thought to be autoregulated by the encoded factor, insertions might identify negative regulators of factor activity (e.g., anti- factors).
Here we report the characterization of mutants that are derepressed for expression of sigY. We identified an insertion mutation, yxlC::Tn10, that activated expression of Y-dependent genes, including the autoregulated sigYyxlCDEFG operon. Using a combination of molecular genetic and genomic approaches, we identified genes within the sigY operon that regulate the activity of Y and characterized two Y target promoters.
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MATERIALS AND METHODS |
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Y activity.
B. subtilis strain HB0065 was transformed with pIC333 (27) to generate random mini-Tn10 libraries as described previously (28). Nine libraries were generated and plated onto LB containing spectinomycin, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal), and growth-inhibitory levels (6, 8, or 10 µg/ml) of chloramphenicol (Cm). Cmr mutants with elevated ß-galactosidase activity were isolated following 2 days of incubation at 37°C. Chromosomal DNA was extracted from each mutant and used to transform HB0065, with selection on LB plates with spectinomycin (Spc), neomycin, and X-Gal. Only mutants that had a high level of linkage between the mini-Tn10(spc) and elevated expression of ß-galactosidase expression were characterized further. Plasmids containing the mini-Tn10 element with a ColE1 origin and flanking B. subtilis chromosomal DNA were recovered by transformation into E. coli. DNA sequences upstream and downstream of the transposon were obtained with two primers (50 and 51) corresponding to the left and right ends of the mini-Tn10, respectively. We generated a sigY yxlC double mutant (HB0121) by transformation of chromosomal DNA from HB4245 (sigY::mls) into HB0119 (HB0120 cured of SPß; Table 1). The PY-cat-lacZ fusion was then introduced into this strain by transduction, and ß-galactosidase was measured. Construction of null mutants of yxlC, yxlCDEFG, yxlFG, yxlCDE, yxlDE, yxlD, and yxlE. Long-flanking homology PCR was used as described (29) to generate allelic replacement mutants for each gene or group of genes. In brief, approximately 1,000-bp genomic regions flanking the gene(s) to be deleted were amplified from CU1065 chromosomal DNA by PCR. The primers used are summarized in Table 2. Drug resistance cassettes were amplified by PCR from pDG646 (macrolide-lincomycin-streptogramin B, mls), pDG780 (kanamycin, kan), or pDG1513 (tetracycline, tet) (10).
For each mutant construction, equal amounts (approximately 200 to 300 ng) of purified upstream flanking fragment, downstream flanking fragment, and the corresponding drug resistance cassette were used in a joint PCR procedure as described (29), with either the Expand polymerase (Roche) or the HotStarTaq Master Mix kit (Qiagen). The resulting PCR products were purified and then directly transformed into B. subtilis wild-type strain CU1065, selecting for the corresponding antibiotic resistance. The generated mutant strains are listed in Table 1 and shown in Fig. 1.
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To compare different strains, three individual colonies were inoculated in LB medium with corresponding antibiotics and incubated at 37°C overnight. Then 50 µl of each overnight culture was used to inoculate 5 ml of warm LB medium. Samples were taken at an OD600 of 0.8, and ß-galactosidase activity was assayed. Averages and standard deviations were calculated for each strain.
Primer extension assays.
RNA was prepared from mid-logarithmic-phase cells (OD600 
0.5) with the Qiagen RNeasy mini kit; 100 µg of total RNA (from CU1065 or the sigY yxlC double mutant strain) or 10 µg of total RNA (from the yxlC::Tn10 mutant) and 2 pmol of end-labeled reverse primer were mixed for each primer extension experiment following the procedures described previously (4). For mapping the sigY transcriptional start site, the end-labeled reverse primer 340 was used. The PCR-amplified sigY promoter region (with primers 339 and 340) was sequenced with the same primer, and the reaction products were electrophoresed adjacent to the primer extension products. For ybgB, primer 777 was used, and primers 776 and 777 were used for amplification of the ybgB promoter region for the sequence ladder.
Microarray analysis. Total RNA was prepared from B. subtilis CU1065 and the yxlC::Tn10 mutant grown aerobically in LB medium. The cell cultures were grown to an OD600 of 0.4, and the cells were harvested immediately. The protocol for RNA isolation, cDNA synthesis, and slide hybridization was described previously (31). Each RNA preparation was used to make both indocarbocyanine- and indodicarbocyanine-labeled cDNA, and all hybridizations were done twice, once with each cDNA preparation, to control for differences in labeling between the two fluorophores. Since all PCR products were spotted twice on each slide, all signal intensities and calculated ratios are the averages of four values. Two microarray experiments (yxlC mutant versus wild type) were performed with RNA prepared from two independent cell cultures. Signal intensities were quantified with ArrayVision software (Molecular Dynamics) and assembled into Excel spreadsheets. Mean values and standard deviations were calculated with Excel. Genes with a standard deviation in expression values (fluorescence intensity) greater than the mean value were ignored. Complete datasets are available as supplementary material at http://www.micro.cornell.edu/faculty.JHelmann.html.
Overproduction and purification of Y protein.
The sigY gene was PCR amplified from B. subtilis chromosomal DNA with oligonucleotides 141 and 142, designed to engineer an NcoI site upstream and a BamHI site downstream of the sigY gene. The PCR product was cloned into pET16x (Novagen) via the NcoI and BamHI sites to generate pKF85. The sequence of sigY in pKF85 was verified by DNA sequencing (Cornell DNA sequencing facility). Y was purified from E. coli strain BL21/DE3(pLysS) transformed with pKF85. Expression was induced by addition of 20 µM isopropylthiogalactopyranoside (IPTG) for 3 h, resulting in the formation of inclusion bodies. Y was purified from the inclusion bodies as follows: 2 ml of disruption buffer (50 mM Tris-HCl [pH 8.0], 2 mM EDTA, 0.1 mM dithiothreitol, 1 mM ß-mercaptoethanol, 233 mM NaCl, 10% glycerol) was added to a frozen pellet generated from 50 ml of induced culture. Then 0.4 ml of the resuspended cells was sonicated for 5-s pulses, 12 pulses total.
Inclusion bodies were collected by centrifugation at 13,000 rpm for 20 min at 4°C and resuspended twice in 10 ml of TEDG buffer(50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 0.5% [vol/vol] Triton X-100). The washed pellet was resuspended in 2 ml of TEDG with 0.4% Sarkosyl and gradually diluted to 20 ml with TEDG buffer to allow refolding of Y. The sample was then dialyzed against 200 ml of TEDG for 8 h at 4°C. A 1-ml Hi-trap heparin column was equilibrated with 3 ml of TEDG, and 1 ml of the dialyzed sample was loaded onto the column. The column was washed five times with 500 µl of TEDG. Y was eluted from the column with washes of increasing NaCl concentrations (50 to 500 mM NaCl) in TEDG buffer. Each eluate was tested for the presence of protein with the Bio-Rad protein detection assay, and peak fractions were collected. The renatured Y eluted with 0.5 M NaCl and was analyzed by polyacrylamide gel electrophoresis (PAGE) and confirmed to migrate at approximately 21.2 kDa, which is the predicted molecular mass for Y.
In vitro runoff transcription assay and microarray analysis.
The runoff transcription/macroarray analysis (ROMA) experiment was performed as described previously (6). Purified Y was added in 17-fold molar excess relative to the core RNA polymerase. With the Y autoregulated promoter as a template, we determined that the specificity of Y-dependent transcription was optimal between 100 and 150 mM KCl (data not shown). For the ROMA experiment, 100 mM KCl (final concentration) was used.
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RESULTS |
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Y is positively autoregulated.
Most ECF factors are positively autoregulated, often with an adjacent anti- factor gene that regulates activity (11, 12, 19). To determine whether sigY is autoregulated, we took advantage of the high level of expression from the sigY'-cat-lacZ reporter fusion in the yxlC::Tn10 mutant. When a sigY mutation was introduced into this genetic background, expression was reduced to the background level (Fig. 1, strain HB0122). Thus, the sigY'-cat-lacZ reporter fusion is also a reporter of Y-dependent transcriptional activity, PY-cat-lacZ.
The upstream region of sigY contains a candidate promoter similar to other promoters recognized by ECF factors (13). We mapped the transcription start site of sigY by primer extension, taking advantage of the high level of expression in the yxlC::Tn10 mutant. Transcription started from a C residue 9 bases downstream from the -10 region CGTC motif (Fig. 2A). Consistent with the ß-galactosidase result, this transcript was not detectable in the sigY yxlC double mutant even when 10 times more total RNA was used as the template. No other start sites were observed within the sigY regulatory region (250 bp upstream from the start codon). We conclude that Y positively autoregulates its own expression.
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Y autoregulatory promoter PY has consensus elements of TGAAC (-35) and CGTC (-10) with a 17-bp spacer (Fig. 2C). This is very similar to the consensus sequences recognized by other B. subtilis ECF factors, including W and X (Fig. 3). Although both X and W can recognize promoters with a CGTC motif in the -10 region (25), sigY has not been identified as part of either the X or W regulon (5, 6, 18), nor is sigY upregulated by the induction of other ECF factors (2; our unpublished results). These results suggest that other sequence features, in addition to those highlighted (Fig. 3), are important for promoter discrimination.
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Y does not activate transcription of other ECF factors.
Next, we tested the effect of the yxlC::Tn10 insertion (leading to high in vivo Y activity) on the expression of autoregulatory promoters recognized by various ECF factors. We replaced the PY-cat-lacZ fusion in strain HB0120 with reporter fusions containing the known or putative autoregulatory regions for each of the other six ECF factors (Fig. 3 and data not shown). The results indicate that the yxlC::Tn10 insertion and consequent upregulation of Y activity do not lead to elevated expression of any of the other ECF factors. We conclude that, in general, Y does not regulate other ECF factors.
Effect of yxlC::Tn10 insertion is due to polarity.
In several well-characterized examples, the gene immediately downstream of an ECF factor gene encodes an anti- factor (12). Therefore, we hypothesized that yxlC might encode an anti- factor. However, when we engineered a yxlC::mls allelic replacement mutant (HB0917), we failed to observe an increase in Y activity (Fig. 1). Note that in this mutant the mls cassette was oriented to allow expression of downstream genes from the mls promoter. In light of this result, we hypothesized that the original Tn10 insertion was polar on downstream genes. sigY is the first of six codirectional genes, many with overlapping start and stop codons (Table 3), that likely constitute an operon.
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Y is negatively regulated by both YxlD and YxlE.
To determine which of the four downstream genes might have been affected by the yxlC::Tn10 insertion mutation, we constructed a series of allelic replacement mutants (Fig. 1). When the whole yxlCDEFG region was deleted, PY was derepressed (HB0918), indicating that Y is negatively regulated by one or more proteins encoded by the downstream genes. High-level expression from PY was also observed in the 
yxlCDE but not the yxlFG mutant. We conclude that YxlF and YxlG are not essential for PY regulation but might function as accessory factors, because expression was always lower in the yxlCDE than in the yxlCDEFG mutant.
To investigate the role of individual gene products of the yxlCDE region, three additional deletions (yxlDE, yxlD, and yxlE) were constructed (Fig. 1). The results indicate that both YxlD and YxlE are important for negative regulation of Y activity, with YxlD being the major negative regulator. Note that both YxlD and YxlE are small proteins predicted to associate with the cell membrane (Table 3). Thus, the signaling complex likely to regulate Y activity may be membrane localized.
Y regulon includes sigY operon and ybgB gene.
To identify other genes transcribed by Y, we used DNA microarray analysis to compare RNA populations from wild-type and yxlC::Tn10 mutant cells in two independent experiments. Overall, 99.5% of the expressed genes varied less than twofold in expression level, despite the nearly 100-fold effect of the yxlC insertion on expression of sigY itself. Only seven genes were significantly and reproducibly upregulated (>3.5-fold) in the yxlC mutant (Table 4). The most dramatic changes were the sigY (71-fold) and the yxlC (14-fold) genes. The interpretation of this finding is complicated by the fact that the mutant strain has a Tn10 insertion in the yxlC gene and the upregulation noted in the microarray study could, in principle, be due to countertranscription from the promoter of the spectinomycin resistance cassette in the Tn10 insertion (Fig. 1). Nevertheless, it is clear from the reporter fusion studies that the yxlC::Tn10 insertion leads to upregulation of PY. Therefore, we prefer a model in which the Tn10 insertion leads to upregulation of PY, which leads to elevated levels of sigY and the 5'-proximal part of yxlC.
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Y dependent (Fig. 2B). It is not yet known if the upregulation of ybgE is physiologically relevant, since it is separated from ybgB by a 212-bp intergenic region. This region has recently been shown to bind CodY, and ybgE is derepressed in a codY mutant (22). We suggest that the upregulation of ybgE is due to readthrough of the ybgB transcript into ybgE, which is otherwise repressed under our growth conditions.
Apart from ybgB, none of the other upregulated genes appeared to be direct targets for Y-directed transcription. The des gene encodes a cold-inducible membrane phospholipid desaturase, and its transcription is controlled by A (1). tyrZ is a monocistronic gene encoding a minor tyrosyl-tRNA synthetase. No obvious Y-dependent promoter was found upstream of tyrZ, and we failed to detect its transcription start site even in the yxlC mutant. The yvdF gene encodes a putative maltogenic amylase (98% identical to the BbmA sugar hydrolase from B. subtilis SUH4-2 [8]) and is the second gene of a large cluster. Most genes in this cluster seem to be involved in maltose or maltodextrin utilization. However, no obvious Y-dependent promoter was found upstream of either yvdF or the larger gene cluster, and we were unable to detect any transcription start site for yvdF in primer extension experiments. Since only the second gene in this region was induced in the yxlC mutant, this could be a false-positive, the result of cross-hybridization, or an indirect effect.
Analysis of Y regulon by ROMA.
With the microarray approach, we identified two operons (sigY-yxlCDEFG and ybgB) as direct targets for Y-directed transcription. As a complementary approach, we used reconstituted Y holoenzyme to identify in vitro targets in a whole-genome transcription study. As described previously (6), in the runoff transcription/macroarray analysis (ROMA) experiment, we generated 33P-labeled runoff transcripts with total genomic DNA and used these to probe a DNA macroarray (Sigma/GenoSys) containing 4,107 B. subtilis open reading frames.
We detected only three strong hybridization signals by ROMA: sigY, ybgB, and yabE (Fig. 4). In contrast, dozens of strong signals were detected when the W or X holoenzyme was used (5, 6). Hybridization to yabE is apparently due to an RNA generated by a factor contaminating the core preparation (E), since the signal appeared in both E and EY experiments. Additional weak signals generated in the EY experiment but lacking in the E alone experiment were also identified. These signals correspond to ybgE, the downstream gene of ybgB, and the genes downstream of sigY. These signals were reduced in intensity due to the use of restriction enzyme-digested DNA in the in vitro transcription reaction, which served to limit transcription to promoter-proximal genes. Significantly, the other three genes (yvdF, des, and tyrZ) that were induced in the yxlC::Tn10 mutant (Table 4) were not detected in ROMA, consistent with our conclusion that these are not likely to be direct targets for Y.
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Y directs transcription of a small regulon including only the sigY-yxlCDEFG and the ybgB genes (Table 3). Note that most of these gene products are small, hydrophobic proteins, suggestive of a role in transport or other membrane-associated functions. |
DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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factors activate a variety of stress responses that often involve changes in the cell envelope or transport or efflux across the cell membrane (reviewed in references 12, 21, and 24). In B. subtilis, significant progress has been made in defining the regulons controlled by X, W, and M, but the functions of the other four ECF factors are unknown (13). Here we demonstrate that Y controls a small regulon, including its own operon, and at least one other target gene, ybgB. However, the function of this regulon is not yet clear.
We have explored several strategies to decipher the regulatory roles of the multiple ECF factors in B. subtilis. Mutational analyses revealed that none of the seven factors is essential, and even multiply mutant strains often have only subtle phenotypes. Therefore, we focused our efforts on identifying target genes that are recognized by each factor with promoter consensus search, DNA microarray, and in vitro transcription-based strategies (3, 4, 6, 7, 15-18). In complementary experiments, we attempted to define the chemical and genetic factors that elicit factor activation (28). Together, these studies revealed that X controls several operons that modulate cell envelope properties, including the D-alanylation of teichoic acids (dltABCDE), phosphatidylethanolamine biosynthesis (pssA psd), and expression of autolysins (lytR) (4, 5, 18). The W regulon includes at least 30 operons, including several with roles in antibiotic resistance (3, 6). This regulon is induced by antibiotics acting on the cell wall or by alkali stress (7, 30). The regulon controlled by M has not been well defined, but it appears to overlap the X and W regulons and includes at least one gene that functions in antibiotic resistance (bcrC) (4, 23).
Several different strategies have also been explored to define the role of Y. Direct comparison of the transcriptomes of wild-type and sigY null mutant strains did not reveal significant differences (data not shown). One interpretation of this result is that Y may not be active under the conditions of the experiment, and therefore very few genes (if any) were affected by the absence of the factor. For both the X and W regulons, the rules defining promoter recognition are reasonably well defined (25), and searching the genome for sequences resembling known target sites produced lists of candidate promoters, many of which turned out to be dependent on the expected factor (6, 17, 18). Similar search strategies have not been as successful for other ECF factors. While it is clear that the two known promoters recognized by Y are similar in sequence (Fig. 2C), searches based on the apparent consensus identified sites already classified as dependent on X, W, or both but very few additional candidates (data not shown). It remains possible that the Y regulon may overlap that recognized by X, W, or another ECF factor. However, this suggestion is not supported by either the in vivo transcriptome analysis or the ROMA studies reported here.
The present work was initiated with the goal of defining the genetic factors that negatively regulate Y activity. In similar studies with the autoregulatory X- and W-dependent promoters, we identified transposon insertions in genes for antibiotic biosynthesis, sugar isomerases, and multidrug efflux systems (28). With only one exception, the insertions affected X or W, but not both. Of those tested to date, none of these insertions affected Y activity (data not shown). Moreover, Y is not strongly activated by a variety of physical or chemical factors that activate X, W, and M (e.g., antibiotics, high salt concentrations, and extreme pH). Weak activation of sigY expression is observed in cells grown on minimal medium compared to rich medium, and this expression is Y dependent. Thus, the Y regulon appears to respond to different stresses than those known to activate other regulons.
In the present study, we only recovered insertions in the sigY operon itself, which focused attention on the regulatory roles of these cotranscribed genes. Our results indicate that the major negative regulators of Y activity, YxlD and YxlE, are two small membrane proteins. It is not yet clear whether these two proteins together form a multisubunit anti- factor or whether they act independently. Since their genes are cotranscribed with a predicted component of an ABC transporter (Table 3), we speculate that Y may be regulated by the activity of a membrane transport complex.
The dramatic effect of the yxlC::Tn10 insertion mutation on the activity of the Y autoregulatory promoter encouraged us to pursue global transcriptional profiling to identify other operons upregulated by elevated Y activity. Of the resulting candidate operons, only ybgB was clearly a direct target for Y-dependent transcription, and the function of this gene is not clear. Asai et al. (2) also used transcriptome analysis to define the regulons controlled by ECF factors. Their results, based on overexpression of individual ECF factors, support the idea that Y positively autoregulates its own expression and that of the downstream genes (yxlCDEFG and yxlH). The apparent upregulation of yxlH may be due to readthrough from the convergent sigY operon. Although these authors reported 10 additional genes as being upregulated by induction of Y, only one (tyrZ) was also identified in our comparison of wild-type and yxlC::Tn10 mutant strains, and our results suggest that the induction of many of these reported target genes may be due to indirect effects. It should be noted, for example, that their studies were done by inducing each factor and harvesting cells after 2 h of growth at 37°C, during which time both the control and experimental cultures likely entered the stationary phase.
Transcriptional profiling is a very powerful approach for defining the effects of regulatory proteins on gene expression, but some target operons may be missed. This can occur due to low expression levels (e.g., due to the inactivity of a needed activator), poor hybridization to target probes, and background expression from other promoter sites. Moreover, it is difficult to separate direct from indirect effects. As an independent approach to estimating the size of the Y regulon, we turned to ROMA, whole-genome in vitro transcription to generate Y-dependent transcripts followed by macroarray analysis to identify the corresponding genes. The results confirmed the transcriptional profiling studies; in both cases, the direct targets of Y regulon appeared to include only two operons, sigY-yxlCDEFG and ybgB. Further studies will be needed to define the physiological roles of these genes and their products.
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ACKNOWLEDGMENTS |
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Y overproduction plasmid. This work was supported by NIH grant GM-47446 (to J.D.H.), NIH MBRS SCORE grant GM-052588 (to L.M.-M.), and NIH MBRS RISE grant GM5-59298 (to C.B.).
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FOOTNOTES |
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