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Journal of Bacteriology, April 2002, p. 1998-2004, Vol. 184, No. 7
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.7.1998-2004.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Analysis of the Bacillus subtilis spoIIIJ Gene and Its Paralogue Gene, yqjG

Takako Murakami, Koki Haga, Michio Takeuchi, and Tsutomu Sato^*

International Environmental and Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan

Received 29 August 2001/ Accepted 31 December 2001

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The Bacillus subtilis spoIIIJ gene, which has been proven to be vegetatively expressed, has also been implicated as a sporulation gene. Recent genome sequencing information in many organisms reveals that spoIIIJ and its paralogous gene, yqjG, are conserved from prokaryotes to humans. A homologue of SpoIIIJ/YqjG, the Escherichia coli YidC is involved in the insertion of membrane proteins into the lipid bilayer. On the basis of this similarity, it was proposed that the two homologues act as translocase for the membrane proteins. We studied the requirements for spoIIIJ and yqjG during vegetative growth and sporulation. In rich media, the growth of spoIIIJ and yqjG single mutants were the same as that of the wild type, whereas spoIIIJ yqjG double inactivation was lethal, indicating that together these B. subtilis translocase homologues play an important role in maintaining the viability of the cell. This result also suggests that SpoIIIJ and YqjG probably control significantly overlapping functions during vegetative growth. spoIIIJ mutations have already been established to block sporulation at stage III. In contrast, disruption of yqjG did not interfere with sporulation. We further show that high level expression of spoIIIJ during vegetative phase is dispensable for spore formation, but the sporulation-specific expression of spoIIIJ is necessary for efficient sporulation even at the basal level. Using green fluorescent protein reporter to monitor SpoIIIJ and YqjG localization, we found that the proteins localize at the cell membrane in vegetative cells and at the polar and engulfment septa in sporulating cells. This localization of SpoIIIJ at the sporulation-specific septa may be important for the role of spoIIIJ during sporulation.

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The formation of endospore, a dormant and resistant cell type, is an adaptive means by which Bacillus subtilis survives conditions of nutrient starvation. Endospore formation involves processes of temporal changes and cellular differentiation of two cells that start out with identical genomes (18). These processes eventually culminate in the formation of an environmentally resistant spore. B. subtilis, therefore, offers an excellent biological system for studying cellular differentiation. During sporulation, an ordered sequence of morphologic events takes place, starting with the formation of an asymmetrically positioned septum that divides the sporangium into two unequal compartments: the forespore and the mother cell (24). Each compartment contains a chromosome and engages in a sporulation-specific program governed by four different, sporulation-specific sigma factors, whose activities are tightly regulated both temporally and spatially. Just after septation, gene expression is controlled by ^F in the forespore and by ^E in the mother cell. Later in development, when the forespore has become engulfed by the mother cell, ^F and ^E are replaced by ^G and ^K, respectively (13, 24). Furthermore, differential gene expression between the two compartments is governed by the successive appearance of these factors, whose activities are coordinated in a criss-cross fashion: (i) the activation of ^F in the forespore leads to the appearance of ^E in the mother cell, (ii) the ^E in turn causes the activation of ^G in the forespore, and then (iii) ^G leads to the appearance of ^K in the mother cell (13, 24).

Under the control of these sigma factors are hundreds of sporulation-specific genes, including many whose functions are not yet known. One of these sporulation genes, spoIIIJ, has been sequenced, and the gene product was found to contain several putative transmembrane segments (4). Another significant characteristic of spoIIIJ is that its expression occurs during vegetative growth and at a low level during sporulation. Moreover, it has also been reported that mutations in spoIIIJ arrest sporulation at stage III and block the activity of the forespore-specific sigma factor, ^G (4), which can also maintain its own synthesis by recognizing upstream signals of its own reading frame in addition to the transcription from the ^F promoter. Full activation of ^G requires not only completion of engulfment but also the products of spoIIIJ, as well as the eight products of the ^E-controlled spoIIIA operon; the mutation of this operon also blocks the activity of ^G (10, 12, 24). Therefore, it has been assumed that the engulfment of forespore and function of these stage III genes may serve as a "checkpoint" for delaying ^G-directed gene expression until a critical time point in the sporulation process.

B. subtilis also possesses the SpoIIIJ paralogue, YqjG. The yqjG gene product is 37% identical to SpoIIIJ and has several transmembrane domains (25). Interestingly, SpoIIIJ/YqjG belongs to the Oxa1p homologues (25) that are present in the thylakoid membrane (16), in the inner mitochondrial membrane (5-8), and in the inner membrane of Escherichia coli (20, 23). One of the homologues, YidC protein, was recently identified as Sec-dependent or Sec-independent membrane protein translocase essential for maintaining viability in E. coli (20, 23). This information suggests that SpoIIIJ/YqjG is an essential gene for enhancing cell viability and membrane protein translocation. We obtained preliminary results supporting this assertion by studying the localization and associated effects of SpoIIIJ/YqjG inactivation.

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Bacterial strains, plasmids, and general methods. The bacterial strains and plasmids used in this study are listed in Table 1. Oligonucleotide primers are shown in Table 2. Transformation of B. subtilis was performed according to the method described by Dubnau and Davidoff-Abelson (2). The efficiency of sporulation was measured by growing B. subtilis cells in Difco sporulation (DS) medium (22) at 37°C for 24 h. The number of spores (CFU) per milliliter of culture was determined as the number of heat-resistant (80°C for 10 min) colonies on tryptose blood agar base. Plasmid constructions were made in E. coli JM105.

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TABLE 1. Bacterial strains and plasmids used in this study

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TABLE 2. Oligonucleotide primers used in this studya

Plasmid and strain constructions. Plasmids pMUTIIIIJ-S, pMUTyqjG-S, pMUTIIIJ2, and pMUTyqjG2 were constructed with primers IIIJ1S and IIIJ2S, QJG1S and QJG2, IIIJ3 and IIIJ-R, and QJG4 and QJG5, respectively, to amplify the internal fragments of spoIIIJ and yqjG with chromosomal DNA of B. subtilis 168 as a template. The PCR products and the plasmid pMUTinT3 which was used for the construction were completely digested with HindIII and BamHI and then ligated. The plasmid constructs were cloned in E. coli JM105 and selected on ampicillin-supplemented Luria-Bertani (LB) solid media. To construct pUCIIIJ::cat, a KpnI-BamHI fragment bearing the spoIIIJ gene was generated by PCR amplification with the oligonucleotide primers IIIJUP and IIIJDOWN and subcloned into pUC19 (28). A 1.0-kb SacI and HincII fragment of the cat gene of pCBB31 (21) was cloned into a XbaI site in the spoIIIJ gene of the obtained plasmid. Plasmids pJMIIIJ and pJMyqjG carrying the internal fragments of spoIIIJ and yqjG were constructed by amplifying an EcoRI-BamHI fragment with the oligonucleotide primers IIIJ2S and IIIJ-gfp-F and the oligonucleotide primers QJGE1 and QJG2, respectively, followed by cloning in pJM114 (17). Plasmid pCA191 was constructed by cloning a HpaII-BanIII DNA fragment of pC194 (3) containing the cat gene in pUC19. pIIIJ-green fluorescent protein (GFP) and pyqjG-GFP carrying the spoIIIJ-gfp and yqjG-gfp genes were constructed with the oligonucleotide primers IIIJ-gfp-F and IIIJ-gfp-R and the oligonucleotide primers QJGE1 and yqjG-gfp-R, respectively, to obtain EcoRI-BamHI-digestible PCR fragments. Next, BamHI-XbaI-digestible amplified fragments were generated, with chromosomal DNA of B. subtilis spoIIEpPE1 (11) as a template, with the primers gfp-sg-F2 and gfp-sg-R2. The inserts were then ligated with the EcoRI-XbaI site of pCA191. To obtain pMFIIIJ, the entire spoIIIJ coding fragment was amplified by PCR with the oligonucleotide primers IIIJ-F and IIIJ-R. We then attempted cloning this fragment in the ClaI-BamHI site of pMF20 but found that the ClaI site of the vector was inactivated by methylation. To surmount this problem, the HindIII site nearby was used after blunting of the cohesive ends. The ClaI site of the insert was also blunted. Thus, a ClaI (blunted)-BamHI PCR fragment was inserted in a HindIII (blunted)-BamHI site of the vector, resulting in the xylose-inducible plasmid pMFIIIJ.

ß-Galactosidase assay. The activity of ß-galactosidase was determined as previously described by the method of Miller (15) with o-nitrophenyl-ß-D-galactopyranoside (ONPG) as the substrate. Enzyme-specific activity is expressed in nanomoles of the substrate (ONPG) hydrolyzed per milligram per minute.

Fluorescence microscopy. Microscopy was performed as described by Webb et al. (26). For the acquisition of single pictures, all strains were grown at 37°C to t₆ (i.e., 6 h after the initiation of sporulation), and aliquots were observed with an Olympus BX50 microscope. Fluorescence microscopy images were visualized with a cooled charge-coupled device camera (Sensys) and a fluorescein isothiocyanate filter set (for GFP). Images were processed with Metamorph 4.1.5 software (Universal Image) and Adobe Photoshop 4.0.1J.

Protoplasting, protein fraction, and Western immunoblot analysis. Cultures were harvested from vegetative cultures grown in LB medium, at an optical density at 600 nm (OD₆₀₀) of 1.0. Cell fractionation from protoplasts was done as described by Wu and Errington (27). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by Western immunoblotting with anti-GFP antibody purchased from Molecular Probes, Inc.

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Sporulation of spoIIIJ paralogue yqjG mutant and expression pattern of yqjG. Initial studies focused on determining whether spoIIIJ and yqjG are sporulation genes by measuring the sporulation frequency of the spoIIIJ and yqjG mutants. The sporulation frequency was significantly decreased by the spoIIIJ mutation (Table 3). This observation is in agreement with the results reported by Errington et al. (4). However, the mutation of yqjG did not abolish the sporulation ability of the cell, indicating that the yqjG gene does not play any significant role in the process of sporulation. In addition, both mutations appeared to have no apparent effect on growth in DS medium or minimal medium even at the stationary phase (data not shown).

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TABLE 3. Sporulation frequencies of the spoIIIJ and yqjG mutants

We speculated that the inability of yqjG to complement the spoIIIJ mutation in spore formation might be due to its lower level of expression than that of spoIIIJ. To examine this possibility, we tested the sporulation frequency of the strain JG2 (Pspac-yqjG spoIIIJ::cat) under inducing and noninducing conditions with or without IPTG (isopropyl-ß-D-thiogalactopyranoside). The result showed that the overexpression of yqjG did not significantly restore sporulation efficiency in the spoIIIJ mutant (10² spores/ml in noninducing condition versus 10³ spores/ml in inducing condition). We further constructed a new strain in which the yqjG gene is under the control of xylose-inducible promoter and spoIIIJ is mutated. However, the result was similar to that obtained for the yqjG-inducing system in JG2. This result suggests that the yqjG gene might have a slightly overlapping activity with that of spoIIIJ but has a dissimilar function in spore formation.

The expression of spoIIIJ occurs during vegetative growth and at a low level during sporulation (4). In order to obtain comparative data on the expression of yqjG and spoIIIJ, we used pMUTinT3 to construct transcriptional fusion between lacZ and yqjG or spoIIIJ. The effect of the growth phase of cells on the expression of spoIIIJ and yqjG was measured with spoIIIJ-lacZ and yqjG-lacZ fusions (Fig. 1). In both fusion-integrated strains, ß-galactosidase activities were high during the exponential phase but began to decrease concomitantly with further transition from exponential to stationary phase in DS medium. However, the level of expression of yqjG-lacZ was four- to fivefold lower than that of spoIIIJ-lacZ. Besides, the expression patterns of these genes were not influenced by the mutation of each other (data not shown). These results suggest that both genes act predominantly during vegetative phase rather than at sporulation.

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FIG. 1. Expression patterns of spoIIIJ-lacZ and yqjG-lacZ. The specific activity of ß-galactosidase and the cell growth were monitored in SPOIIIJlacZ (A) or YQJGlacZ (B) carrying spoIIIJ-lacZ or yqjG-lacZ transcriptional fusions, respectively. Symbols: , OD600 values; •, ß-galactosidase activities.

Double inactivation of spoIIIJ and yqjG leads to the lethal phenotype. To test the physiological function of spoIIIJ and yqjG during vegetative phase, we first tried to introduce the spoIIIJ deletion allele into the yqjG mutant by transformation. However, no transformants were obtained, implying that the spoIIIJ yqjG double inactivation led to a lethal phenotype. To confirm this, conditional double mutants of spoIIIJ and yqjG (strains JG1 and JG2, respectively) in which spoIIIJ and yqjG were fused to an IPTG-inducible promoter were constructed by transforming spoIIIJ and yqjG with chromosomal DNA from strains YQJGK (yqjG::kan) and IIIJC (spoIIIJ::cat), respectively. Next, we investigated the growth of these strains in the presence or absence of IPTG (with neither spoIIIJ nor yqjG expressed). As we expected, the lethal phenotype was observed in the absence of the inducer; cells of both strains were able to grow in the presence of IPTG, but virtually no cells were observed in the absence of IPTG (Fig. 2). The few colonies that we observed in the absence of the inducer might be due to the spontaneous mutation in the spac promoter or the lacI gene in the integrated pMUTinT3 plasmid (data not shown). A similar result with IPTG-independent suppressor mutants in a pMUTin controllable promoter system has recently been reported by Pragai and Harwood (19). These results confirmed that spoIIIJ/yqjG is essential for growth and suggest that these genes are able to complement the function of each other during growth.

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FIG. 2. Growth defect of spoIIIJ yqjG double mutants. (A) Scheme showing the genetic construction of mutants. (B) Growth of spoIIIJ, yqjG, and spoIIIJ yqjG mutants on LB plates at 37°C for 20 h under conditions of repression (IPTG-) and induction (IPTG+).

spoIIIJ expression during sporulation is required for efficient spore formation. Since the spoIIIJ gene exhibits the strongest level of expression during vegetative growth, we sought to determine whether this timing in expression is important for its function in spore formation. To address this issue, we constructed strain IIIJxyl2, in which the expression of the spoIIIJ gene is controlled by a xylose-inducible promoter, and then tested the influence of spoIIIJ induction before and after the initiation of sporulation on the efficiency of spore formation. As shown in Table 4, depletion of inducer for spoIIIJ expression after the initiation of sporulation caused a significant decrease in the number of heat-resistant spores regardless of spoIIIJ induction during vegetative growth, whereas the induction of spoIIIJ only after sporulation initiation was sufficient for efficient spore formation. Constitutive expression of spoIIIJ did not have any negative effect on sporulation. There was a considerable difference in the degree of sporulation deficiencies between the spoIIIJ::cat strain and the noninduced P xyl-spoIIIJ strain (compare Tables 3 and 4). This might be the result of the basal level expression of spoIIIJ from the xyl promoter. It is unlikely that this is due to the differences in selection markers for the spoIIIJ mutation, since both markers resulted in similar sporulation defects (data not shown). This result indicates that, although the expression of spoIIIJ is reduced as the cell enters the stationary phase and subsequently sporulation, the basal level of expression during sporulation is required for efficient spore formation, probably via activation of ^G. The preexisting SpoIIIJ protein appeared to be insufficient to enhance efficient sporulation, presumably because the protein rapidly undergoes degradation.

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TABLE 4. Influence of timing of spoIIIJ induction on sporulation efficiency

Localization of SpoIIIJ and YqjG. The spoIIIJ and yqjG products were predicted as membrane proteins, since the amino acid sequences of both gene products contain six transmembrane segments as determined by the SOSUI prediction system (9). Moreover, many homologues of these gene products, such as the E. coli YidC or the Oxa1 family in the higher eukaryotes, are known to localize at the membrane (5, 6, 7, 8, 16, 20, 23). In order to determine whether SpoIIIJ and YqjG are spatially located in the cell, we made translational fusions of spoIIIJ and yqjG to the gfp gene and then observed the location of the SpoIIIJ- and YqjG-GFP fusion proteins in B. subtilis by fluorescence microscopy. The activity of SpoIIIJ and YqjG is not inhibited by fusion to GFP, since both fusion proteins were able to grow in the spoIIIJ yqjG double mutant cell (data not shown). In addition, sporulation was not affected in either of the GFP fusion strains (data not shown). When growing cells were directly observed in DS medium, SpoIIIJ-GFP localized throughout the periphery of the cell (Fig. 3A). This result suggests that SpoIIIJ-GFP specifically localizes at the periphery of the cells, most probably within the cell membrane. In contrast, in the sporulating cells the green fluorescence of SpoIIIJ-GFP fusion was observed at locations around both the mother cell and outside of the forespore, suggesting that SpoIIIJ also localizes in the mother cell and forespore membrane. On the other hand, most of the cells containing YqjG-GFP showed rather faint fluorescence compared to that of SpoIIIJ, but the YqjG-GFP pattern obtained was similar to those of cells containing SpoIIIJ-GFP. We predicted that this low intensity of the fluorescence of YqjG-GFP could be due to a low level expression of the yqjG gene. Furthermore, we confirmed the cellular distribution of the GFP fusion proteins by Western immunoblotting with antibody to GFP (Fig. 3B). The localization of GFP fusion proteins was analyzed by the cell fractionation method (27). In both strains, a single fluorescent band was predominantly detected in the insoluble fraction (membrane) and only slightly detected in the soluble fraction (cytoplasm), confirming the predicted membrane association. These results indicate that these proteins localize and act at the cell membrane in vegetative cells and at the engulfment septa in sporulating cells.

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FIG.3. Localization of SpoIIIJ-GFP and YqjG-GFP fusion proteins. (A) Typical phase-contrast (a, c, e, g, i, k, m, and o) and fluorescence (b, d, f, h, j, l, n, and p) micrographs are shown. Strains carrying SpoIIIJ-GFP (a to h, strain JGFP) and YqjG-GFP (i to p, strain QGFP) were observed in the vegetative stage (a, b, i, and j) and at t2 (c, d, k, and l), t3 (e, f, m, and n), and t4 (g, h, o, and p). (B) Western blots of cell extracts were analyzed with an antibody that recognizes GFP. Lanes 1, insoluble fraction; lanes 2, soluble fraction.

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SpoIIIJ and YqjG belong to the Oxa1 homologue of E. coli YidC, but these proteins are also known to share some similarity with the Oxa1 homologues of Saccharomyces cerevisiae, Homo sapiens, and Arabidopsis thaliana (25). Recently, two Oxa1 homologues, Sp1 and Sp2, were identified in Schizosaccharomyces pombe (1). Interestingly, while the genes encoding Sp1 and Sp2 are together essential for cell growth, their double inactivation has been reported to be lethal (1). Among these homologues, E. coli YidC is the most characterized protein and recently was identified as essential for growth and as a Sec-dependent and -independent translocase. YidC is present in the inner membrane of E. coli. Mutations in yidC inhibit the insertion of Sec-dependent membrane proteins and cause minor defects in the export of secretory proteins (20).

SpoIIIJ and YqjG have almost all of the indicators and a high possibility of acting as translocases, since these genes are essential for viability, are homologues of YidC, and localize within the cell membrane. It is also possible that SpoIIIJ and YqjG functionally overlap and may function as backup genes for each other, but during sporulation it is impossible to substitute YqjG for SpoIIIJ, suggesting that some sporulation-specific protein that plays a role in sporulation is necessary for the function of SpoIIIJ. Besides, the expression level of spoIIIJ is fivefold higher than that of yqjG, implying that, of the two, it is SpoIIIJ that chiefly functions as a translocase. In addition, each gene is located at regions quite opposite in the chromosome. spoIIIJ is located at position 4,213.80 kb near the replication origin (0/4,214.81 kb), whereas yqjG is positioned at 24,831.33. (14). Recent bacterial sequencing data reveal that many bacteria possess the YidC homologue (e.g., in Pseudomonas putida, Staphylococcus aureus, and Salmonella enterica serovar Typhimurium). Similarly, in most of these bacteria the homologue genes are located close to the replication origin. In this context, it appears that yqjG might have moved to the current position from the spoIIIJ region through the occurrence of chromosomal rearrangement during evolution.

The spoIIIJ gene product expressed during vegetative phase was shown to be insufficient to enhance efficient spore formation. This finding, coupled with the amino acid similarity with the YidC transporter from E. coli, implies that the large amount of SpoIIIJ protein expressed during vegetative phase might be involved in a function(s) other than sporulation, such as the insertion of membrane proteins into the lipid bilayer. It is also likely that, during spore formation, SpoIIIJ might be involved in the transduction of the ^G activation signal from mother cell to the forespore.

spoIIIJ has been characterized as a sporulation gene (4), and we have shown here that SpoIIIJ localizes within the forespore membrane during sporulation. If SpoIIIJ acts as a translocase for the membrane proteins, we wondered what target protein SpoIIIJ integrates into the forespore membrane. The phenotype of spoIIIJ mutation is known to exhibit inhibition of subsequent events after completion of engulfment. Especially, the spoIIIJ mutations interfere with the activation of forespore-specific sigma factor ^G (4). Against this background, the possible target candidate for SpoIIIJ is likely to be (i) a stage III sporulation gene product, (ii) a membrane protein, or (iii) a product whose gene mutation positively influences ^G activity. The genes that fulfill these requirements are genes of the ^E-directed spoIIIA operon, which consists of the eight gene operon-encoding proteins SpoIIIAA to SpoIIIAH, all of which have been predicted to be membrane associated. Mutations in each of the eight genes block sporulation at stage III, after completion of engulfment, and prevent ^G activity. Among the eight proteins, SpoIIIAE, for instance, contains nine transmembrane segments as predicted by SOSUI. Following from our prediction, spoIIIJ may be needed for the functional expression of products of the spoIIIA operon. Since there is little detail on the localization of other spoIIIA products, further analysis is necessary in order to reveal the target of SpoIIIJ during sporulation.

 
We thank Richard Losick for providing B. subtilis strains, Samuel Amiteye for critical reading of the manuscript, and Shigeo Hosoya for technical assistance with the microscopic analysis.

This work was supported by a grant-in-aid for scientific research on priority areas ("Genome Biology") from the Ministry of Education, Science, Sports, and Culture of Japan.

 
* Corresponding author. Mailing address: International Environmental and Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan. Phone: 81-423-67-5706. Fax: 81-423-67-5715. E-mail: subtilis{at}cc.tuat.ac.jp.

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Journal of Bacteriology, April 2002, p. 1998-2004, Vol. 184, No. 7
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.7.1998-2004.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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