Department of Biotechnology, Faculty of Engineering, Fukuyama University, 985 Sanzo, Higashimura-cho, Fukuyama 729-0292, Japan1
Author for correspondence: Y. Fujita. Tel: +81 849 36 2111. Fax: +81 849 36 2459. e-mail: yfujita{at}bt.fubt.fukuyama-u.ac.jp
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ABSTRACT |
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Keywords: Bacillus subtilis, genome, transcription, gene inactivation, gene expression
Abbreviations: ß-Gal, ß-galactosidase; DSM, defined nutrient sporulation medium; MM, glucose minimal medium; SD, ShineDalgarno
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INTRODUCTION |
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The first step in the common strategy adopted in the B. subtilis genome function project is that each participating group is responsible for the inactivation of the unanalysed genes in a certain region of the genome. This inactivation is being carried out through integrational mutagenesis of a pMUTIN series of plasmids carrying lacZ as a reporter gene, as illustrated in Fig. 1 (Vagner et al., 1998
). Segments of the target genes are cloned into one of the pMUTIN plasmids, and the genomic copy is inactivated through a single- or double-crossover event. The promoter activities of the inactivated genes are monitored by measuring ß-galactosidase (ß-Gal) activity in cells during growth and sporulation, in both a defined nutrient sporulation medium (DSM) (Schaeffer et al., 1965
) and a glucose minimal medium (MM). These two media were chosen for the ß-Gal monitoring because the catabolic and anabolic pathways are predominantly utilized in the growth in the nutrient and minimal media, respectively. Furthermore, several groups are also carrying out Northern blotting analysis of the unanalysed genes in their assigned regions under the same growth conditions as those for the ß-Gal monitoring, thereby constructing a transcription map of the region. These analyses are expected to provide valuable knowledge about the transcription organization, expression profiles and functions of the target genes. For the second step of the project, each of the laboratories has screened, or will screen, integrants constructed through easily conductable high-throughput protocols, mainly using plate tests.
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METHODS |
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Small target genes comprising less than approximately 200 bp were inactivated through a double-crossover event, which resulted in the deletion of part of their coding regions. This deletional inactivation was carried out as follows. The 5' portion of a target gene, including the SD sequence and translation-initiation codon, plus the 3' portion containing the translation-termination codon were amplified separately by PCR using the chromosomal DNA as template. The sequences of the two primer sets, which were designed to generate flanking EcoRI and BglII, and HindIII and EcoRI sites, are available from the web site. Each PCR product was cleaved with either EcoRI and BglII or HindIII and EcoRI, and the resulting two fragments were ligated into the HindIII/BamHI sites of pMUTIN2. The ligated DNA was digested with BamHI and BglII to avoid self-ligation, and then used for the transformation of E. coli strain C600 as described above. The resulting plasmids were linearized through EcoRI digestion, and then used for the transformation of B. subtilis strain 168 trpC2 as described above.
Cell growth conditions and ß-Gal assay.
B. subtilis cells with integrations in the desired target genes were grown on TBABG (tryptose blood agar containing 10 mM glucose) plates containing 0·3 µg erythromycin ml-1 at 30 °C overnight. The cells were inoculated into 50 ml DSM (Schaeffer et al., 1965 ) or MM in 200 ml Erlenmeyer flasks at OD600 0·05, and incubated at 37 °C in a water bath shaker. The composition of the MM (pH 7·0) was as follows: 0·4% glucose, 0·2% glutamine, 50 µg tryptophan ml-1, 62 mM K2HPO4, 44 mM KH2PO4, 11·4 mM K2SO4, 3·4 mM sodium citrate, 0·8 mM MgSO4, 50 µM CaCl2, 24 µM FeCl3, 13 µM ZnCl2, 3 µM Na2MoO4, 2·5 µM CuCl2, 2·5 µM CoCl2 and 1·32 µM MnSO4.
During growth and sporulation in DSM or MM, 1 ml aliquots (OD600 0·15·0) were withdrawn at 30 min or 1 h intervals, and the cells were collected by centrifugation and stored at -20 °C. The cells were suspended in 500 µl Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4 and 1 mM DTT) containing 10 µg DNase I ml-1 (Sigma) and 100 µg lysozyme ml-1 (Sigma), and incubated at 37 °C for 20 min. After centrifugation (27000 g, 20 min), the supernatants were used for spectrophotometric ß-Gal assays, as previously described by Atkinson et al. (1990) . One unit of ß-Gal activity was taken as the amount of the enzyme that produced 1 nmol o-nitrophenol min-1 at 25 °C.
Northern blotting.
B. subtilis strain 168 trpC2 cells grown in DSM or MM (OD600xml=100400) were harvested. RNA was then purified as described previously (Fujita et al., 1998 ). Northern blotting was also performed essentially as described previously (Sambrook et al., 1989
; Yoshida et al., 1997
), using total RNA and the 32P-labelled PCR product for the 5' portion of the ORF of the analysed gene as a double-strand-specific DNA probe; the primer sequences are provided on the web site.
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RESULTS AND DISCUSSION |
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Table 1 shows the expression levels of the 125 inactivated genes during growth and sporulation in DSM and MM. This table also shows the maximal ß-Gal activities of the integrant cultures during growth and sporulation in liquid DSM and MM, together with their expression patterns. More detailed results of the monitoring of ß-Gal synthesis can be found on the web site (http://bacillus.genome.ad.jp).
Among the 125 genes whose expression was examined by monitoring of ß-Gal synthesis, 80 genes were expressed in both DSM and MM, 7 in DSM only, 20 in MM only and 18 in neither medium (Table 1). Seventeen genes (yxaI, asnH, yxnB, yxbABC, iolFGHIJ, yxeE, hutM, yxiE, yxxG, yxkC and msmX) were strongly expressed in cells during growth and sporulation in DSM: the cells produced more than 100 U ß-Gal (mg protein)-1. Of these genes, iolFGHJ, yxeE and hutM were not expressed strongly in cells grown in MM. yxaL, yxiC, yxxD, yxjG, pepT and ywaA were expressed strongly in MM only. Table 1
also shows the expression patterns of the genes during growth and sporulation.
The expression profiles of the inactivated genes might not fully reflect their expression in vivo, because expression of the genes examined might be autoregulated or an inducer of an operon comprising several genes might be synthesized through the enzyme reaction(s) catalysed by their gene product(s). Moreover, it was observed that different locations of integration during mutagenesis affected the expression of lacZ (Y. Fujita & N. Yanai, unpublished results). It appears that the stability of an artificial mRNA encoding ß-Gal, whose synthesis starts from the promoter of the target gene, is sometimes affected by the location of the crossover.
Construction of a transcription map of the gntZywaA region
We performed Northern analysis of 119 genes to construct a transcription map of the gntZywaA region (Fig. 2). Total RNA was extracted from cells of strain 168 trpC2 grown in either DSM or MM and used for Northern blotting. The hybridization results are provided on the web site (http://bacillus.genome.ad.jp).
Fig. 2 shows the transcripts that were detected on Northern blots for the genes between gntZ and ywaA, together with their sizes, the times at which the cells were harvested and the medium used. The transcripts are placed beneath the analysed genes on the assumption that they were synthesized from upstream of them. When the same size of transcript was detected with multiple DNA probes from successive genes, the transcripts are appropriately placed beneath them according to their size (Fig. 2
). Of the 119 genes analysed, the transcripts of 77 genes were detected in both DSM and MM, 19 were detected in DSM only and 4 were detected in MM only. The transcripts of 19 genes were not detected in either medium.
A comparison of Table 1 and Fig. 2
indicates that the results of Northern blotting did not often coincide with those of ß-Gal measurement. Among 115 genes, the expression of which was analysed by both Northern blotting and ß-Gal measurement, the expression of 11 was detected only on Northern blotting and that of 14 was detected only on ß-Gal measurement. Moreover, more than 10 U ß-Gal (mg protein)-1 was synthesized by the integrants in nearly 30% of the instances where no transcript of the target genes was detected in DSM or MM. One explanation for this is that there are differences in stability between the mRNAs of different target genes. Sometimes, rRNAs (2·9 and 1·5 kb) can hamper the detection of specific mRNA. For five genes (yxeA, yxjL, yxjM, yxkA and aldY), we could not detect transcription by either ß-gal measurement or Northern blotting (Table 1
and Fig. 2
).
Northern experiments revealed the operon structure of this region. The 14 newly found polycistronic operons were as follows: yxaAB, yxaGH, yxaJKL, yxbBAyxnBasnHyxaM, yxbCD, yxcED, yxdJK, yxeFGH, yxeKLMNOPQ, yxeRyxxB, yxiMdeaD, yxjCDEF, yxiJI and yxkFmsmX. Recently, we detected cydABCD operon expression in cells grown on either solid DSM and MM or in liquid NSMP medium (Fujita & Freese, 1981 ) supplemented with glucose, but not in liquid DSM containing glucose or in liquid MM (Fig. 2
) (Winstedt et al., 1998
). As shown in Fig. 2
, we also detected the following polycistronic transcripts: 11·5 kb for iolABCDEFGHIJ, 4·5 kb for deoRdranupCpdp, 8·0 kb for hutPHUIGM, 4·0 kb for bglPHyxiE, 8·0 kb for wapAyxxG, and 2·5 kb for katByxiS. In addition, we found a 4·3 kb transcript covering sigYyxlCDEFG. We can not explain at the moment why this transcript continued to be synthesized to the oppositely oriented yxlH gene without termination of its synthesis between yxlG and yxlH. Table 1
also shows that there are very similar expression patterns for the integrants of the genes corresponding to polycistronic operons, except for yxaGyxaH, yxeRyxxB and katByxiS (Table 1
).
In conclusion, we emphasize that this systematic study of gene expression and organization has provided valuable information for comparison with the results of future microarray-based analysis.
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ACKNOWLEDGEMENTS |
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Received 13 September 1999;
revised 18 October 1999;
accepted 18 November 1999.