Department of Cell Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0101, Japan1
Faculty of Pharmaceutical Sciences, Setsunan University, 45-1, Nagaotoge, Hirakata, Osaka 573-0101, Japan2
Author for correspondence: Naotake Ogasawara. Tel: +81 743 72 5430. Fax: +81 743 72 5439. e-mail: nogasawa{at}bs.aist-nara.ac.jp
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ABSTRACT |
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Keywords: Gram-positive bacterium, sporulation, cell differentiation, green fluorescent protein
Abbreviations: GFP, green fluorescent protein
a Present address: Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-Ohkubo, Urawa, Saitama 338-8570, Japan.
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INTRODUCTION |
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With completion of the sequencing of the B. subtilis genome, we might be able to identify all the genes involved in the sporulation process described above. In a 108 kb segment of the B. subtilis genome that covers between rrnO and spo0H, 90 ORFs were found, 52 of which had unknown function (Ogasawara et al., 1994 ). To identify novel genes involved in sporulation, Northern analysis was done using probes encompassing each gene in this region. We searched for genes that were transcribed after the onset of sporulation and whose inactivation caused some effects on spore formation and germination. We characterized two genes, yaaH and yabG, which were specifically induced during sporulation and involved in germination of spores and assembly of the spore coat, respectively (Kodama et al., 1999
; Takamatsu et al., 2000
). We now report the function of the yabQ gene expressed during sporulation by E
E and which is involved in formation of the spore cortex.
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METHODS |
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Oligonucleotide primers 8158RTF (5'-CGGCCAAAAGCTTGTAAC-3') and 8158RTR (5'-GGAGGATCCATAATATGAATTCATCTATTCAG-3') were used to amplify a 208 bp segment of the yabP upstream region. The PCR product, restricted at HindIII and BamHI sites introduced by the primers, was inserted into the HindIII/BamHI-restricted pMUTINT3 (Vagner et al., 1998 ; Moriya et al., 1998
) to obtain plasmid pMU158RT. The resulting plasmid was transformed into B. subtilis 168 by a single cross-over recombination for erythromycin resistance (0·5 µg erythromycin ml-1), to yield strain ASK222.
An integration vector for green fluorescent protein (GFP) fusion was constructed as follows. A fragment encoding the C-terminal portion of YabQ and a fragment encoding the C-terminal portion of CotE were PCR-amplified using primers QGFPF (5'-GTCGTCGACATGCGGAGCGATCATC-3') and QGFPR (5'-GAAGAATTCTCTCTTCAAAAACCGTGTG-3'), and COTEGFPF (5'-GTCGTCGACCATTTCGCCGAATGG-3') and COTEGFPR (5'-GAAGAATTCTTCAGGATCTCCCAC-3'), respectively. The PCR products of 280 and 208 bp were digested by SalI and EcoRI and introduced between the SalI and EcoRI sites of pMm2 (Takamatsu et al., 2000 ) to generate plasmids pMmyabq and pMmcote, respectively. The resultant plasmids were transformed into B. subtilis 168 by a single cross-over for erythromycin resistance to yield strain ASK223 or ASK226 in which the yabQ or cotE genes were switched with the yabQ or cotEgfp genes, respectively. The yabQgfp fusion was transferred to the chromosome of B. subtilis P20 (Coot, 1972
) and B. subtilis RL48 (Zheng et al., 1988
) and the resulting strains were named ASK224 and ASK225, respectively. The gfpspoIVA (Price & Losick, 1999
) and cotEgfp fusions were transferred to the chromosome of ASK221 and the resulting strains were named ASK227 and ASK228, respectively. Transformation of B. subtilis was done as described by Kawamura et al. (1980)
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Sporulation medium and measurement of spore resistance.
Difco sporulation (DS) medium (Schaeffer et al., 1965 ) was used to produce B. subtilis spores. Cells were grown in DS medium at 37 °C for 18 h after the end of exponential growth, and the spore resistance was assayed as follows. The culture was heated at 80 °C for 30 min, and treated with lysozyme (final concentration, 0·25 mg lysozyme ml-1) at 37 °C for 10 min, or with 10% (v/v) chloroform at room temperature for 10 min, as described by Nicholson & Setlow (1990)
. A portion of the sample was diluted in distilled water, plated on LuriaBertani agar, and incubated overnight at 37 °C. The number of survivors was determined by counting colonies.
Northern and primer extension analysis.
Total RNA was extracted from B. subtilis cells as described by Igo & Losick (1986) . In Northern analysis, hybridization and detection were performed using digoxigenin-labelled RNA probes (Asai et al., 2000
). Primer extension analysis was carried out using digoxigenin-end-labelled primers (Takamatsu et al., 2000
). DNA ladders for size markers were obtained using the same digoxigenin-end-labelled primers and a DIG Taq DNA sequencing kit (Boehringer Mannheim).
Phase-contrast and fluorescence microscopy.
An aliquot of the culture of B. subtilis cells in DS medium was transferred onto a microscope slide coated with poly-L-lysine. Images of phase-contrast and fluorescence from GFP were observed under an Olympus fluorescence microscope (AX70) with a U-MNIBA mirror cube unit. The images were captured with a cooled charge-coupled device camera (PXL-1400; Photometrics) and analysed using image processing software, IPLAB SPECTRUM (Signal Analytic Corporation).
Electron microscopy.
Glutaraldehyde was added to a final concentration of 0·3% (w/v) to the culture of B. subtilis cells allowed to sporulate for 8, 10 and 18 h after the onset of sporulation. The cells were collected by centrifugation and prefixed with 3% (w/v) glutaraldehyde in 50 mM phosphate buffer (pH 6·5) for 2 h at room temperature, followed by washing five times with the same buffer. Cells were then collected by centrifugation and fixed with a final concentration of 1% (w/v) osmium tetroxide in 50 mM phosphate buffer (pH 6·5) for 2 h at room temperature. After washing five times with distilled water, they were prestained for 2 h with 0·5% (w/v) uranyl acetate and embedded in 2% (w/v) agar. The agar block was dehydrated with ethanol and acetone, followed by embedding in Spurrs resin (Spurr, 1969 ) prior to thin sectioning. Thin sections were obtained using a Diatome diamond knife on a Leica ULTRACUT UCT, stained with 3% (w/v) uranyl acetate and then with lead citrate (Reynolds, 1963
), and examined under a Hitachi H7100 electron microscope.
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RESULTS AND DISCUSSION |
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Effects of inactivation of the yabP, yabQ or yabR gene on spore formation
Next, we examined the effects of inactivation of each of the three y genes in the yabPyabR operon on sporulation. The insertion of exogenous plasmid DNA, pMUTINT3 (Vagner et al., 1998 ; Moriya et al., 1998
), into the yabR gene by a single cross-over integration resulted in about a 10-fold reduction of sporulation frequency (data not shown); however, this was not further studied. Because inactivation of the yabP or yabQ gene by inserting the plasmid DNA leads to a polar effect on downstream divIC and yabR expression, we introduced an in-frame deletion within the yabP or yabQ gene by the two-step allele-replacement method (Asai et al., 2000
). A promoter for divIC that is active from the vegetative phase (T3 in Fig. 1
) was found within the yabQ gene by primer extension analysis (data not shown). Therefore, we avoided deleting the divIC promoter to obtain the yabQ deletion mutation. A strain carrying a deletion from codons 3 to 87 of the yabP structural gene (100 aa), ASK220, could sporulate and germinate, similar to the parental strain (data not shown). In contrast, a yabQ deletion mutant, ASK221, in which codons 4140 of the structural gene (211 aa) were deleted, produced abnormal spores sensitive to lysozyme, chloroform and, especially, heat treatments (Table 2
). Although transcription of the yabQ gene was weak during the vegetative phase in the wild-type cell (T1 and T2 in Fig. 1
), growth of the yabQ mutant was comparable to that of the parental strain. Thus we postulated that the defective phenotype of the yabQ mutant in sporulation was due to the loss of the functional yabQ product at the phase of sporulation.
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Synthesis of the spore cortex was blocked considerably in the yabQ mutant
Normal spores become phase-bright under the phase-contrast microscope after spore coat synthesis followed by core dehydration (Piggot & Coote, 1976 ; Errington, 1993
). However, large amounts of spores produced in the yabQ mutant were phase-dark and did not become phase-bright, even after further incubation (Fig. 3
). In wild-type cells, cortex synthesis was clearly observed under the electron microscope 8 h after the initiation of sporulation (Fig. 4ad
). In contrast, examination of the yabQ cells during sporulation revealed that synthesis of the spore cortex was blocked in them, resulting in production of defective sporangia that had almost entirely lost the spore cortex. In addition, the inner spore coat layer of the mutant spores seemed partially detached from the outer coat layer, suggesting that the YabQ deletion also affected the coat protein assembly (Fig. 4eh
). The spore cortex provides a heat-resistance property to the dormant spore. On the other hand, coat layers yield resistance to toxic solvents and lytic enzymes (Zheng et al., 1988
; Naclerio et al., 1996
; Takamatsu et al., 1999
). Thus the characteristics of the morphology of the yabQ mutant spore were compatible with the sensitivity of the spore to lysozyme, chloroform and heat treatments.
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Effect of the yabQ deletion on the expression of genes involved in cortex synthesis
We also asked if the YabQ protein might regulate the expression of genes involved in spore cortex synthesis. Several EE-dependent genes, mostly designated spoV, have been reported to participate in cortex synthesis. A family of so-called penicillin-binding proteins (PBPs) catalyses development of the peptidoglycan layer of the cortex (DacB and SpoVD) (Daniel et al., 1994
; Popham et al., 1999
). SpoVE protein, which is highly homologous with cell elongation protein (RodA), is also required for synthesis of the cortex peptidoglycan (Daniel & Errington, 1993
; Henriques et al., 1998
). Mutation in the spoVR gene causes a defect in cortex synthesis, although the precise function was not obvious (Beall & Moran, 1994
). Northern analysis of the expression of the dacB, spoVD, spoVE and spoVR genes in the yabQ mutant revealed that they were normally expressed and were independent of YabQ function (Fig. 6
).
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Role of the YabQ protein in spore formation
Extensive Northern analysis revealed EE-dependent transcription from the yabPyabQdivICyabR operon during sporulation. Deletion of the first ORF, the yabP gene, had no apparent effect on either sporulation or germination. However, deletion of the second ORF, yabQ, made spores sensitive to lysozyme, chloroform and, especially, heat treatment. Microscopic analysis showed that yabQ mutant spores possessed no obvious spore cortex layer and had an impaired spore coat, suggesting that yabQ plays an important role in synthesis of the spore cortex and coat. The YabQ protein was predicted to have five transmembrane domains and a signal sequence at the N-terminus region. YabQ fluorescence signals were observed surrounding the forespore independently of CotE and SpoVIA functions, suggesting that YabQ is associated with the forespore membrane. Expression of the genes participating in cortex synthesis and the sporulation sigma factors
E,
G and
K occurred normally in the yabQ deletion mutant. These findings suggest that the YabQ protein plays a direct role in cortex formation. An interesting possibility might be that YabQ is a transporter for components required for synthesis of the spore cortex and the defect affects coat assembly.
During preparation of this manuscript, Fawcett et al. (2000) reported analysis of genes expressed during sporulation by transcriptional profile analysis with a macro-array. They began building null mutations of possible sporulation genes thus identified, and deletion of yabP or yabQ was found to block sporulation at a late stage.
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ACKNOWLEDGEMENTS |
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Received 17 August 2000;
revised 30 November 2000;
accepted 13 December 2000.