Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
Correspondence
Jeff Errington
jeff.errington{at}path.oxford.ac.uk
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ABSTRACT |
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INTRODUCTION |
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The early mother-cell-specific -factor,
E, is encoded by the sigE (spoIIGB) gene and is expressed upon the initiation of sporulation from a promoter that is recognized by RNA polymerase containing the housekeeping
-factor
A, in conjunction with SpoOA, the key regulator for entry into sporulation (Kenney & Moran, 1987
; Satola et al., 1991
).
E-dependent gene expression is, however, not observed until 2 h after the initiation of sporulation as the protein is synthesized in an inactive form, which must undergo proteolytic cleavage to become active (LaBell et al., 1987
). The protease responsible for the cleavage is probably SpoIIGA (Jonas et al., 1988
; Stragier et al., 1988
), encoded by the gene spoIIGA upstream of, and co-transcribed with, sigE. Cleavage of pro-
E occurs only in the mother cell and requires the SpoIIR protein, which is expressed in the prespore from a
F-dependent promoter, thereby coupling the activation of
E to
F activity (Karow et al., 1995
; Londono-Vallejo & Stragier, 1995
). As the first
-factor to become active in the mother cell,
E is responsible for the expression of the genes encoding the late mother cell
-factor,
K, and SpoIIID, a transcription factor required for fine-tuning the regulation of many
E-dependent genes (reviewed by Errington, 1993
). Other
E-dependent genes are involved in the formation of the spore coat and cortex and some are necessary for proper germination (reviewed by Piggot & Losick, 2002
). At least one
E-dependent gene is involved in the regulation of transcription of sigG, encoding the late prespore-specific
-factor
G, because mutations in the spoIIG operon block transcription at the sigG promoter (Partridge & Errington, 1993
).
DNA microarrays are increasingly being used for transcriptional profiling in various organisms. This technique enables an overview of which genes are being expressed under particular conditions and can produce vast quantities of informative data. Recently, DNA microarrays have been used to identify genes of Bacillus subtilis dependent on B (Price et al., 2001
),
H (Britton et al., 2002
), SpoOA and
F (Fawcett et al., 2000
) and CodY (Molle et al., 2003
).
Here we used DNA microarrays to compare the transcriptional profile of two different E mutants to the profile found in wild-type cells. In addition to the identification of previously known
E-dependent genes, we were able to assign 124 additional genes, of which 88 are organized in operons, to the
E regulon. Furthermore, disruption of some of the previously unknown genes resulted in a defect in sporulation.
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METHODS |
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Media and growth conditions.
Nutrient agar (Oxoid) was used as a solid medium for growing B. subtilis. X-Gal (150 µg ml-1), chloramphenicol (5 µg ml-1), kanamycin (5 µg ml-1), erythromycin (1 µg ml-1) and lincomycin (25 µg ml-1) were added as required. E. coli strains were grown in 2x TY medium (Sambrook et al., 1989) or on nutrient agar plates (Oxoid) supplemented with ampicillin (100 µg ml-1). B. subtilis strains were induced to sporulate by the resuspension method as described previously (Partridge & Errington, 1993
; Sterlini & Mandelstam, 1969
).
-Galactosidase assay.
-Galactosidase was assayed using a method described by Errington & Mandelstam (1986)
; one unit of enzyme is the amount that releases 1 nmol 4-methylumbelliferone min-1.
Determination of sporulation frequency and microscopy.
The sporulation frequency was determined by counting phase-bright spores and total cells by phase-contrast microscopy in samples taken 7 h after induction of sporulation. Spore morphology was analysed by phase-contrast light microscopy and images were acquired and analysed with a Princeton Instruments Micromax 1300Y/HS CDD camera and METAMORPH version 3.6 software.
mRNA preparation.
Cells were harvested from 50 ml cultures 2 h after the initiation of sporulation, pelleted and frozen in liquid nitrogen. In parallel, a sample was taken and assayed for alkaline phosphatase activity as a means of checking E activity (Errington & Mandelstam, 1983
; Partridge & Errington, 1993
). The cell pellets were thawed at 37 °C in 1 ml TE containing 25 mg lysozyme ml-1 for 5 min; 1 ml 150 mM NaCl, 0·1 % SDS, 10 mM EDTA, 10 mg Pronase ml-1 was then added, mixed gently and the mixture incubated for a further 5 min. Nucleic acids were extracted with acid phenol at 60 °C and precipitated with 2·5 vols absolute ethanol for 2 h at -20 °C. The nucleic acid pellet was washed with 70 % ethanol, inverted and dried for 15 min on the bench, before being dissolved in 180 µl RNase-free H2O (Ambion). A 75 µl sample of material from this crude preparation was incubated with 3 µl DNase (RNase free; Promega) at 37 °C for 1530 min to remove excess genomic DNA. The RNA was then purified further using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions, the RNase-free DNase set was used at the appropriate step in the protocol. RNA was eluted in two aliquots of 30 µl RNase-free dH2O. The sample was checked for contaminating DNA by agarose gel electrophoresis.
Transcriptional profiling.
A commercially available microarray was used, consisting of 3925 B. subtilis ORFs spotted on a glass slide (Eurogentec), onto which two fluorescently labelled RNA populations were simultaneously hybridized. RNA (50 µg) was used as a template for first-strand cDNA synthesis using the CyScribe First-Strand cDNA Labelling Kit (Amersham Pharmacia). The volume of each RNA sample was reduced to 5 µl in a Heterovac vacuum desiccator. Then 0·5 pmol B. subtilis-specific RT primers (Eurogentec) and 1 µl RNasin RNase inhibitor (Promega) were added and the reaction was made up to a total volume of 11 µl using RNase free dH2O. This reaction mix was heated to 70 °C for 5 min, then allowed to cool gradually to 42 °C (over about 15 min) to anneal the primers to the RNA. After a further 5 min at 42 °C, 0·5 µl RNasin, 2 µl 0·1 M DTT, 1 µl dCTP nucleotide mix, 1 µl dCTP CYDye-labelled nucleotide and 1 µl Cyscript reverse transcriptase were added. Strain 2809 and strains 2810 and 2811 were labelled with Cy5 dCTP and Cy3 dCTP, respectively. The reaction was incubated at 42 °C for 2 h in the dark. mRNA was degraded by the addition of 2 µl 2·5 M NaOH to the sample and incubating at 37 °C for 15 min; the reaction was neutralized with 10 µl 2 M HEPES. Labelled cDNA was purified from degraded mRNA and unincorporated nucleotides using DyEx spin columns (Qiagen) according to the manufacturer's instructions.
The slide was prehybridized with 1 µl yeast tRNA (10 mg ml-1; Sigma) and 1 µl salmon sperm DNA (10 mg ml-1; Clonetech) in 20 µl 5x SSC, 0·2 % SDS (both RNase free; Ambion) at 42 °C for 2 h in a sealed cassette. The coverslip was removed by dropping the slide into 0·1x SSC and the slide was dried with compressed air. The two cDNA samples were reduced down to <5 µl volume in a vacuum desiccator, mixed together and the total volume was made to 20 µl with 5x SSC, 0·1 % SDS. This was pipetted onto the slide, a new coverslip was put on and the slide was incubated at 42 °C for approximately 20 h in a sealed cassette. The slide was washed twice in 0·1x SSC, 0·1 % SDS, followed by twice in 0·1x SSC, then dried with compressed air and scanned using a GenePix 4000B microarray scanner (Axon Instruments). The GenePix Pro software (Axon Instruments) was used to scan the slide, to overlay the grid of ORF names and to measure the signal intensities; the scanning voltage for each signal was adjusted to eliminate areas where the signal was saturated and to obtain an intensity ratio of as close to 1 : 1 (total red signal : total green signal) over the whole slide as possible. Whilst aligning the grid, any spots showing very low levels of fluorescence were discarded from further analysis. Finally, a list of genes and the corresponding ratios of the two fluorescent signals was generated. To calculate the ratio, for each fluor the background intensity immediately around the spot was subtracted from the intensity of the spot itself, and then the ratio of the two signals was calculated. The whole procedure was carried out twice with two independent RNA preparations and each ORF was spotted twice onto the slide; therefore, the final ratio for a given gene corresponds to the mean intensity of four spots.
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RESULTS |
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All genes with at least threefold higher expression in the wild-type than in the E mutant were assumed to be dependent on
E and are listed in Table 2
. We used a rather high threshold value of 3 to minimize false positives. Genes transcribed by
F, another sporulation-specific transcription factor, did not give any detectable signal or had values well below 3 (e.g. gpr, 1·4; spoIIQ, 0·3; dacF, 0·8; spoIIR, 0·1; sigG, 0·1; spoIVB, 0·2). A total of 155 genes fell into this category, of which 43 had been reported previously as being dependent on
E. The remaining 112 genes are organized in 88 operons and among these are 23 genes (Table 2
; in bold) that were tentatively assigned as being
E-dependent on the basis of a previous microarray analysis of
F-dependent genes (Fawcett et al., 2000
). Three genes, cotZ, spoVFB and spsK, were previously reported to be
K-dependent (Daniel & Errington, 1993
; Zhang et al., 1994
); detecting these in our arrays might be due to the fact that
E and
K have similar recognition sequences (Haldenwang, 1995
).
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Screening for genes required for efficient sporulation
Most of the E-dependent genes identified by the microarray analysis were genes with unknown function. Some of these are likely to be required for formation of the spore coat, the cortex, for proper germination, and at least one
E-dependent gene is postulated to be involved in the regulation of transcription of sigG, encoding the late prespore-specific
-factor
G. We therefore investigated the phenotypic effects of interrupting selected candidate genes. Candidates were prioritized on the basis of two criteria: the presence of predicted membrane-spanning domains in the protein, or recognition of the promoter by
E663. The products of several known sporulation genes dependent on
E (spoIIB, spoIID, spoIIM, spoIIP) are involved in prespore engulfment and are membrane proteins (Abanes-De Mello et al., 2002
; Frandsen & Stragier, 1995
; Perez et al., 2000
; Smith et al., 1993
). It seems likely that other
E-dependent genes involved in engulfment would have domains capable of inserting into the membrane. The class of genes dependent on
E but not affected by the
E663 mutation were screened because they were candidates for the effector responsible for control of sigG transcription (see above).
Eighteen genes of unknown function (cotZ, patA, ybfJ, ydcC, ydhF, yhaL, yhbH, yjaV, yjcA, ykpC, yloB, yndA, yodP, yqfD, yqhV, yrzE, ysnE and ywcB) were disrupted in a strain containing a lacZ fusion to sigG and in the wild-type background. All the deletion strains were screened for activity of the sigG : : lacZ fusion on nutrient agar plates supplemented with X-Gal, and the blueness of the colonies was compared with that of the parent strain. None of the genes tested had an effect on sigG expression that could be detected on plates (data not shown).
Screening the mutants based on colony appearance on nutrient agar plates revealed that 10 of the mutants might be affected in sporulation efficiency. These mutants were further analysed by inducing sporulation in liquid medium and scoring quantitatively for spore frequency by counting phase-bright spores by phase-contrast microscopy (Table 3). For five of the deletion strains (ydcC, yhaL, yhbH, yjaV and yqfD) a significant reduction in sporulation frequency was observed. Surprisingly, the ydcC mutant strain, which was severely affected when grown on nutrient agar plates (<0·5 % spores compared to the wild-type), produced around 23 % spores when sporulation was induced by the resuspension method. The most severe sporulation phenotype was exhibited by the yqfD mutant, which did not produce detectable numbers of spores (as examined by phase-contrast microscopy). Microscopic examination of the sporulating cultures revealed the existence of phase-dark spores that had probably failed to complete development to the phase-bright state. Disruption of yqfD had the strongest phenotype, producing no detectable phase-bright spores. Although the yhbH mutant strain produced 20 % phase-bright spores, phase-dark spores could also be detected readily. Fig. 2
shows images of sporulating cultures of wild-type, yqfD and yhbH mutant strain taken 7 h after the initiation of sporulation.
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DISCUSSION |
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Our data suggest that at least 178 genes (including the ones which were not detected by our microarrays) belong to the E regulon and 130 of these are organized in operons. Here, we identified 101 genes that had not previously been described as being under the control of
E. Among the newly identified genes are several transporters (citH, glnM, glnP and yknV), which could provide additional nutrients to the prespore. SodF (superoxide dismutase) and YocM (similar to small heat-shock protein) might provide protective properties to the sporulating cell. Only two of the identified genes, ykvU (similar to spore cortex membrane protein) and yqfD (similar to stage IV sporulation protein of B. licheniformis), show similarity to known proteins involved in sporulation. The majority of the newly identified genes are genes with unknown function. Interestingly, around 50 % of these genes (65 out of 124) encode putative membrane proteins. This might reflect the possibility that
E controls the expression of many genes involved in engulfment, formation of the spore coat and cortex, and possibly nutrient-scavenging functions that help to adapt the cell to starvation.
Disruption of some of the newly identified genes led to the identification of five additional proteins required for efficient sporulation (YdcC, YhaL, YhbH, YjaV and YqfD). Little is known about the function of these genes: ydcC, yhbH and yhaL have no function predicted from their sequences. Deletion of yqfD had the most severe sporulation phenotype as the mutant produced only phase-dark spores that failed to become phase-bright, indicating a block at a late stage in sporulation. yqfD, which encodes a protein with one potential membrane domain, probably plays a role after completion of engulfment in coat or cortex formation. It has been shown before that mutants that block engulfment also block activation of G, but the yqfD mutant was able to activate
G normally (Fig. 3
). In contrast, the reduced
G activity in the ydcC mutant might indicate that the YdcC protein, which also encodes a protein with one potential membrane domain, is involved in engulfment (Fig. 3
). The medium-dependent effect on sporulation in this mutant (hardly any spores were produced on nutrient agar plates), suggests that YdcC protein becomes essential for spore formation only under certain environmental conditions. A similar phenomenon has been reported previously for mutation of the spoIIAB and spoIIQ genes (Foulger et al., 1995
; Sun et al., 2000
). The increased
K activity found in the yhbH mutant suggests that the yhbH gene, which encodes a soluble protein, might be involved directly or indirectly in repression of certain
K-dependent genes. Alternatively the protein might affect the activity of
K by destabilizing the protein or changing its activity.
The challenge for the future is now to characterize more of the newly identified genes from our microarray analysis to get a better understanding of how the mother cell contributes to the formation of the spore and how differential gene expression and morphogenesis are co-ordinated in the two compartments.
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ACKNOWLEDGEMENTS |
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Received 9 April 2003;
revised 16 July 2003;
accepted 17 July 2003.