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Journal of Bacteriology, November 2006, p. 7609-7616, Vol. 188, No. 21
0021-9193/06/$08.00+0     doi:10.1128/JB.01116-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Localization of the Transglutaminase Cross-Linking Sites in the Bacillus subtilis Spore Coat Protein GerQ{triangledown}

Alicia Monroe and Peter Setlow*

Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington, Connecticut 06030

Received 26 July 2006/ Accepted 16 August 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Bacillus subtilis spore coat protein GerQ is necessary for the proper localization of CwlJ, an enzyme important in the hydrolysis of the peptidoglycan cortex during spore germination. GerQ is cross-linked into high-molecular-mass complexes in the spore coat late in sporulation, and this cross-linking is largely due to a transglutaminase. This enzyme forms an {varepsilon}-({gamma}-glutamyl) lysine isopeptide bond between a lysine donor from one protein and a glutamine acceptor from another protein. In the current work, we have identified the residues in GerQ that are essential for transglutaminase-mediated cross-linking. We show that GerQ is a lysine donor and that any one of three lysine residues near the amino terminus of the protein (K2, K4, or K5) is necessary to form cross-links with binding partners in the spore coat. This leads to the conclusion that all Tgl-dependent GerQ cross-linking takes place via these three lysine residues. However, while the presence of any of these three lysine residues is essential for GerQ cross-linking, they are not essential for the function of GerQ in CwlJ localization.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bacillus subtilis is a gram-positive soil bacterium that, in response to starvation, undergoes the process of sporulation to form remarkably resistant spores (20). Early in sporulation, a division septum is placed asymmetrically, generating a larger compartment termed the mother cell and a smaller compartment termed the forespore. After a series of highly regulated events, the mother cell lyses, releasing the mature spore into the environment. Although metabolically dormant, the spore retains its ability to sense nutrients, and when they become available, the spore will move through the processes of germination and outgrowth until it has been converted back into a growing cell (21).

One event that occurs late in sporulation is the formation of a multilayered, proteinaceous structure surrounding the spore known as the coat. Electron microscopy has revealed that the coat of B. subtilis spores is composed of two major layers: a thicker and layered outer coat and an inner coat, which is composed of several fine lamellae (7, 9). In B. subtilis spores, the coat is comprised of more than 40 proteins that together play a role in both spore resistance and spore germination, as the coat allows small nutrient molecules to access germinant receptors that lie beneath the coat and excludes or inactivates a variety of potentially toxic molecules (6, 7, 22).

The coat proteins are synthesized in the mother cell during sporulation and are then layered onto the developing forespore in a series of steps that are coupled to developmental events that drive spore formation (6). Once the coat is assembled, the proteins work together to provide spore resistance. In most cases, the deletion of any single coat protein does not have a substantial effect on spore resistance or germination, suggesting that either many coat proteins have redundant functions or their function is not easily assessed in the laboratory. Exceptions to this include at least five coat morphogenic proteins, SpoIVA, SpoVID, SafA, CotH, and CotE. The loss of any of these proteins has significant and in some cases dramatic effects on coat assembly and final coat structure (6, 9).

Studying the coat and all of its protein components has posed a significant challenge, as at least 30% of the total coat protein is insoluble (5, 6). While a number of coat proteins can be extracted from wild-type spores via the combined action of reducing agents, denaturing agents, and detergents, a number of coat proteins are not solubilized by these treatments (9). This observation has led to speculation that there are covalent cross-links between a number of coat proteins (2). One candidate cross-link is that formed by the enzymatic activity of transglutaminase (Tgl), which catalyzes the formation of an {varepsilon}-({gamma}-glutamyl) lysine isopeptide bond between lysine donor residues and glutamine acceptor residues in proteins. Tgl activity has been detected in sporulating cells of B. subtilis, and a single tgl gene from this species has been identified (10, 12). Purified B. subtilis Tgl is able to cross-link {alpha}-casein and bovine serum albumin in vitro (23), and recent evidence suggests that Tgl and the cross-links that it forms are present in the spore coat (11, 12, 25).

There is also evidence that Tgl is involved in the cross-linking of a specific B. subtilis coat protein, GerQ. This latter protein is found largely in the insoluble fraction of the coat and is necessary for the proper localization of CwlJ, an enzyme important in the hydrolysis of the peptidoglycan cortex during spore germination (18, 19). GerQ is cross-linked into high-molecular-mass complexes in the spore coat late in sporulation, but in tgl strains, GerQ cross-linking is significantly reduced (19). GerQ contains a large number of lysine and glutamine residues in its amino-terminal region (18, 19); thus, it has many of the residues necessary for Tgl-catalyzed cross-linking. In this work, we identify the region of GerQ where cross-linking takes place. GerQ is shown to be a lysine donor, and its cross-linking takes place through any one of three lysines (K2, K4, or K5) present near the amino terminus of the protein. We further show that these lysine residues, while essential for cross-linking, are not essential for GerQ function in localizing CwlJ. These results support and strengthen our hypothesis that GerQ is a substrate for Tgl.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Strains and plasmids used in this study. The B. subtilis strains and plasmids used in this study are listed in Table 1. All strains are isogenic with strain PS832, a prototrophic derivative of strain 168. B. subtilis strains prepared in this work were generated by transformation with chromosomal or plasmid DNA as described previously (1). Escherichia coli strain DH5{alpha} was used for the production of plasmids (15). DNA sequencing was carried out on all plasmid DNA to confirm inserts and mutations.


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  		TABLE 1. Strains and plasmids used in this study

 
For construction of plasmid pAM2, the promoter for gerQ was amplified by PCR (sequences of primers used in all PCRs are available upon request) from chromosomal DNA of strain PS832. The PCR product was cloned into plasmid pBC KS(–) (Stratagene, La Jolla, CA).

Plasmid pAM3, used for site-directed mutagenesis (see below), was constructed by PCR amplification of the open reading frame (ORF) of gerQ from chromosomal DNA of strain PS832 and cloning of the PCR product into plasmid pAM2 (behind the gerQ promoter). The promoter and ORF of gerQ were recovered as a BamHI-EcoRI fragment (BamHI site present in the 5' PCR primer for plasmid pAM2; EcoRI site present in the 3' PCR primer for plasmid pAM3) that was inserted between the same sites in plasmid pDG364 (4), creating plasmid pAM9. Plasmid pAM9 was used to transform B. subtilis strain KB29 to chloramphenicol resistance by a double-crossover event such that the amyE locus was replaced with the promoter and ORF of gerQ and a chloramphenicol resistance cassette to generate strain AM14. This same cloning strategy was used to produce plasmids pAM21, pAM22, pAM10, pAM11, pAM12, and pAM13, which contain the gerQ promoter followed by amino-terminal truncations of 5, 16, 23, 48, 60, and 86 residues, respectively, of GerQ in plasmid pDG364. These plasmids were then used to transform KB29 as described above to generate B. subtilis strains AM23, AM24, AM15, AM16, AM17, and AM18, respectively (Table 1).

Site-directed mutagenesis. The QuikChange site-directed mutagenesis kit (Stratagene) was used to make all mutants. Primers were designed based on the DNA sequence of plasmid pAM3. Mutagenesis was carried out using an Applied Biosystems GeneAmp PCR System 9700 under conditions previously described (3), except that 30 to 70 ng of template DNA was used instead of 10 ng. Following PCR, reaction mixtures were digested with DpnI and incubated for 3 h at 37°C to digest the methylated parent plasmids. Digestion reactions were used to transform E. coli DH5{alpha} competent cells, and successful mutagenesis was confirmed by DNA sequencing. Plasmid DNA was then subjected to the same cloning procedure outlined above to construct the final mutagenized strains of B. subtilis.

Growth of strains and spore preparation. E. coli and B. subtilis strains were grown in Luria-Bertani (LB) (15) medium at 37°C, supplemented with the appropriate antibiotics when necessary. Ampicillin and spectinomycin were used at a final concentration of 100 mg/liter; chloramphenicol was used at a final concentration of 5 mg/liter; tetracycline was used at a final concentration of 10 mg/liter; and erythromycin and lincomycin were used at final concentrations of 1 mg/liter and 25 mg/liter, respectively.

To prepare spores, B. subtilis strains were grown for 3 h in LB medium and then spread on 2x SG medium agar plates (16). Plates were incubated at 37°C for 5 days followed by 2 days at room temperature before harvesting. Spores were purified by sonication and repeated washing with distilled water as described previously (16). All spore preparations were free of vegetative and sporulating cells and germinated spores as seen by phase-contrast microscopy. GerQ cross-linking is a late event in sporulation (19). Previously, spores were grown in liquid medium to assess GerQ cross-linking (19); however, in the current work, spores were prepared on plates. It is more convenient to grow spores on plates, and because the spores remain on the plates for several days, it was likely that late events in sporulation would be complete at the time of harvest. Indeed, there was extensive GerQ cross-linking in our plate-grown spores (see Results).

Spore viability. B. subtilis spores in distilled water (optical density at 600 nm of 1.0) were heat activated (70°C, 30 min) and cooled on ice. LB agar plates with the appropriate antibiotics were spotted with 10-µl aliquots of serial dilutions of heat-shocked spores in sterile water. The colonies that formed were counted after 24 h of incubation at 30°C and/or 37°C. This assay measures spore viability, but since {Delta}gerQ strains grow normally, any defect seen in this assay would be due to a defect in spore germination (18).

Spore decoating and spore protein extraction. Spores (6 mg [dry weight]) were decoated by incubation in 1 ml of decoating buffer (0.1 M NaCl, 0.1 M NaOH, 1% sodium dodecyl sulfate [SDS], 0.1 M dithiothreitol) for 30 min at 70°C. Spores were centrifuged, and the supernatant fluid was dialyzed overnight at 4°C against 0.33 M sodium acetate (pH 5.0) and then dialyzed against distilled water three times at 4°C for 4 h each time. The supernatant fluids were lyophilized, and the dry residue was suspended in 200 µl 2x SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer as described previously (19) (except that 0.002% bromophenol blue and 10% ß-mercaptoethanol were used). The spore pellets were washed 10 times with 1 ml distilled water and lyophilized.

Lyophilized intact (6 mg [dry weight]) or decoated (6 mg [dry weight] before decoating) spores were pulverized with 100 mg of glass beads in a dental amalgamator (Wig-L-Bug) for 20 pulses of 30 s each, with 30-s intervals between each pulse. Mechanically disrupted spores were suspended in 200 µl of 2x SDS-PAGE sample buffer and boiled for 5 min before SDS-PAGE and Western blot analysis were conducted (see below).

Western blot analysis. Protein extracts (from ~300 µg spores [5% of the total sample] unless stated otherwise) were run on SDS-PAGE gels (10% polyacrylamide), and proteins were transferred to an Immobilon-P membrane (Millipore). Immunological detection of GerQ on the membrane was performed as described previously (19).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cross-linking occurs within the first five amino-terminal residues of GerQ. GerQ has a large number of lysine and glutamine residues in its amino-terminal region (Fig. 1A) and thus has the required elements to form Tgl-catalyzed {varepsilon}-({gamma}-glutamyl) lysine isopeptide bonds with either itself or other spore coat proteins. We focused on the amino-terminal portion of the protein because the glutamine residues in GerQ are all localized to this region. Because there are many potential target residues in this region of GerQ that could be cross-linked by Tgl, it would have been too labor-intensive to individually mutate all of these residues. Instead, N-terminal truncation variants in which clusters of glutamine residues were deleted from the sequence were constructed. Initially, N-terminal truncations of 23 (strain AM15), 48 (strain AM16), 60 (strain AM17), and 86 (strain AM18) residues were made, and the variants were expressed from the amyE locus in a strain lacking wild-type gerQ. In addition, a strain that expressed the full-length GerQ protein from the amyE locus (AM14) was constructed via the same method to ensure that expression of GerQ from the amyE locus complemented a {Delta}gerQ strain.


Figure 1
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  		FIG. 1. (A) Amino acid sequence of GerQ. Glutamine residues are shown in boldface type. Positions of N-terminal truncations are indicated with arrows. The number above the arrow refers to the first residue after the truncation. Lysine residues 2, 4, and 5, which are important in GerQ cross-linking, are boxed and in boldface type. The more conserved C-terminal region of GerQ (as determined from B) is underlined. (B) Amino acid sequence alignment of GerQ from various species. Asterisks below the aligned sequences indicate identical residues in that column, double dots indicate well-conserved residues, and single dots indicate poorly conserved residues (as determined using the Clustal W website).

 
After mechanically disrupting the spores and subjecting total spore protein to Western blot analysis, the full-length gerQ replacement at the amyE locus (strain AM14) complemented a {Delta}gerQ strain, as normal GerQ cross-linking was seen (Fig. 2, lanes 1 and 2). We did not detect GerQ expression in strains with N-terminal truncations of 48, 60, or 86 residues (data not shown), because the protein may have been unstable or may not have been detected by our anti-GerQ antibody. However, deletion of the first 23 residues of GerQ (strain AM15) still allowed the generation of high levels of the GerQ monomer but with no detectable cross-linking (Fig. 2, lane 5). These results suggested that either a cluster of glutamines (Q7, Q9, Q10, Q12, Q18, Q21, and Q23), a cluster of lysines (K2, K4, and K5) (Fig. 1A), or both groups of these residues are responsible for the cross-linking in GerQ.


Figure 2
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  		FIG. 2. GerQ in total protein of spores of various strains. Total spore protein was extracted from identical amounts (6 mg [dry weight]) of mechanically disrupted spores by boiling in 2x sample buffer before running on SDS-PAGE gels. Proteins were transferred onto an Immobilon-P membrane, and GerQ was detected as described in Materials and Methods. The spores were from strains PS832 (wild type) (lane 1), AM14 ({Delta}gerQ::spc amyE::gerQ 1-181) (lane 2), AM23 ({Delta}gerQ::spc amyE::gerQ 6-181) (lane 3), AM24 ({Delta}gerQ::spc amyE::gerQ 17-181) (lane 4), AM15 ({Delta}gerQ::spc amyE::gerQ 24-181) (lane 5), and KB29 ({Delta}gerQ) (lane 6). Positions labeled a, b, and c show the positions of the GerQ monomer, an 18-kDa band, and a 15-kDa band, respectively. Note that lane 1 is from a separate gel. The numbered bars on the left side of the figure indicate the positions of molecular mass markers in kDa.

 
To distinguish between the possibilities noted above, two additional N-terminal truncation variants of GerQ were constructed: a deletion of the first five residues (strain AM23) to examine the role of the lysines and a deletion of 16 residues (strain AM24) to examine the role of the glutamines and lysines. Western blot analysis of total spore protein showed that deletion of the first five residues alone was sufficient to eliminate GerQ cross-linking (Fig. 2, lanes 3 and 4). Note that the level of total GerQ protein in the truncation variants was reduced compared to that of wild-type spores. However, this reduction does not affect GerQ function (see below). These results suggested that lysine 2, 4, and/or 5 is responsible for all GerQ cross-linking in the spore coat.

Previously, analysis of GerQ in total spore extracts revealed the presence of an 18-kDa band thought to be a degradation product of GerQ (19). This band was also seen in strain AM14, in which full-length GerQ is expressed from the amyE locus (Fig. 2, lane 2). In addition, a band at approximately 15 kDa was seen in extracts of both wild-type and AM14 spores (Fig. 2, lanes 1 and 2). Interestingly, these bands were not present in the GerQ truncation variants (Fig. 2, lanes 3 to 5). We also saw faint bands at 25, 90, 100, and 150 kDa (Fig. 3, lanes 3 to 5); however, it appears likely that these bands are not due to a reaction with the antibody against GerQ (see below).


Figure 3
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  		FIG. 3. GerQ in extracts of spores of various strains. Identical amounts (6 mg [dry weight]) of spores from strains AM14 ({Delta}gerQ::spc amyE::gerQ 1-181) (lanes 1 and 2), AM23 ({Delta}gerQ::spc amyE::gerQ 6-181) (lanes 3 and 4), AM24 ({Delta}gerQ::spc amyE::gerQ 17-181) (lanes 5 and 6), AM15 ({Delta}gerQ::spc amyE::gerQ 24-181) (lanes 7 and 8), and KB29 ({Delta}gerQ) (lanes 9 and 10) were decoated by treatment at 70°C for 30 min in decoating buffer as described in Materials and Methods. Proteins removed by decoating (lanes 1, 3, 5, 7, and 9) and in the insoluble fraction of extracts (lanes 2, 4, 6, 8, and 10) from mechanically disrupted decoated spores were run on SDS-PAGE gels. Proteins were transferred onto an Immobilon-P membrane, and GerQ was detected as described in Materials and Methods. Positions labeled a, b, c, and d show the positions of a GerQ monomer, an 18/17-kDa band, a 16-kDa band, and a 15-kDa band, respectively. Note that lanes 7 to 10 are from a separate gel. The numbered bars on the left side of the figure indicate the positions of molecular mass markers in kDa.

 
GerQ truncation mutants are localized to the spore coat. While GerQ is present in the coats of wild-type spores as shown previously, most notably by the localization of a GerQ-green fluorescent protein fusion by fluorescence microscopy (18), it was possible that GerQ truncation variants were mislocalized. In wild-type spores or spores in which full-length GerQ is expressed from the amyE locus (strain AM14), decoating removes approximately 50% of the monomer and only a small fraction of the high-molecular-mass cross-linked GerQ (19). When spores containing the N-terminal truncations of GerQ (strains AM15, AM23, and AM24) were decoated, again, approximately 50% of the monomer was removed, as was seen for wild-type spores (Fig. 3, compare lanes 1 and 2 with lanes 3 to 8). Since a comparable level of GerQ was removed by the decoating of both wild-type spores and spores of the truncation variants, it is likely that all truncated GerQ variants were properly localized to the spore coat.

As mentioned above, a band at 18 kDa is present in total extracts from spores of the strain expressing full-length GerQ from the amyE locus and was also seen in material removed by decoating (Fig. 3, lanes 1 and 2). A corresponding band (16 kDa for strain AM24 and 15 kDa for strain AM15) was also removed by decoating of spores of GerQ truncation variants 17-181 (strain AM24) and 24-181 (strain AM15), although this band was less intense than that from wild-type spores (Fig. 3, lanes 5 and 7). A 17-kDa band was seen in the insoluble fraction of the extract from GerQ truncation variant 6-181 (strain AM23) but not in the material removed by decoating spores of this strain (Fig. 3, lanes 3 and 4). These lower-molecular-weight bands were less intense than those in extracts from wild-type spores, and their appearance in material removed by decoating or in total spore extracts was inconsistent. One possible explanation for this is that truncated GerQ is unstable at the high temperature and pH at which decoating takes place and that these bands represent degradation products of GerQ.

A band at 37 kDa was seen in the material removed by decoating and in the insoluble fraction of extracts from spores of the strain expressing full-length GerQ from the amyE locus (strain AM14) (Fig. 3, lanes 1 and 2). A corresponding band at 35 kDa (strain AM23), 33 kDa (strain AM24), and 31 kDa (strain AM15) was also seen in material removed by decoating spores of GerQ truncation variants 6-181, 17-181, and 24-181, respectively (Fig. 3, lanes 3, 5, and 7). The molecular weights of these bands correspond to the molecular weights of full-length dimeric GerQ or GerQ truncation variant dimers. To determine if these potential dimer bands were cross-linked by Tgl, we transformed strains AM14, AM23, AM24, and AM15 with chromosomal DNA from a {Delta}tgl strain and found that in all cases, this band was still present in total extracts from spores of the {Delta}tgl strains (data not shown). This result suggests that the proteins in this band are not cross-linked by Tgl. It will be important to examine these bands in greater detail in the future. Note that these potential dimer bands were also present in total extracts of spores of GerQ truncation variants (Fig. 2, lanes 3 to 5) but were seen only after overexposure of the X-ray film. In some cases, a band greater than 250 kDa can be seen in the material removed by decoating (Fig. 3, lane 3). It is possible that this band consists of very-high-molecular-mass complexes that contain cross-linked GerQ, and the amount of this material that enters the gel varies from experiment to experiment. Bands at 25, 90, 100, and 150 kDa were also seen in extracts of the truncation variants (Fig. 3, lanes 4, 6, 8, and 10). However, these bands are not GerQ species, as the bands were also present in extracts of {Delta}gerQ spores (Fig. 3, lane 10). The intensities of these nonspecific bands varied from experiment to experiment (compare Fig. 2, lanes 3 to 6, and Fig. 3, lanes 3 to 10) for reasons that are not clear. These nonspecific bands might indicate the existence of a gerQ homolog in the B. subtilis genome; however, a BLAST protein search did not reveal any such homolog.

GerQ cross-linking occurs via lysine 2, 4, or 5. Since truncation of five amino-terminal residues was sufficient to eliminate high-molecular-mass cross-linked GerQ species, single mutations of K2, K4, and K5 to both glutamine and alanine were made. However, Western blot analysis of total spore protein showed that GerQ cross-linking was not affected by the mutation of any one of these three lysine residues (Fig. 4, lanes 2 and 3; also data not shown). Similarly, no combination of double mutations of residues K2, K4, and K5 to glutamine or alanine disrupted GerQ cross-linking (Fig. 4, lanes 4 and 5; also data not shown). Note that both the 18-kDa and 15-kDa bands were present in all single- and double-mutant GerQ variants.


Figure 4
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  		FIG. 4. GerQ in total extracts of spores of various strains. Total spore protein was extracted from identical amounts (6 mg [dry weight]) of mechanically disrupted spores by boiling in 2x sample buffer before running on SDS-PAGE gels. Proteins were transferred onto an Immobilon-P membrane, and GerQ was detected as described in Materials and Methods. The spores were from strains AM14 ({Delta}gerQ::spc amyE::gerQ 1-181) (lane 1), AM47 ({Delta}gerQ::spc amyE::gerQ 1-181 K4Q) (lane 2), AM46 ({Delta}gerQ::spc amyE::gerQ 1-181 K2A) (lane 3), AM58 ({Delta}gerQ::spc amyE::gerQ 1-181 K2Q K5Q) (lane 4), AM67 ({Delta}gerQ::spc amyE::gerQ 1-181 K2A K5A) (lane 5), AM68 ({Delta}gerQ::spc amyE::gerQ 1-181 K2Q K4Q K5Q) (lane 6), and AM76 ({Delta}gerQ::spc amyE::gerQ 1-181 K2A K4A K5A) (lane 7). Note that lanes 1 and 2 were from one gel, lanes 3 and 4 were from a second gel, lane 5 was from a third gel, and lanes 6 and 7 were from a fourth gel. The numbered bars on the left side of the figure indicate the positions of molecular mass markers in kilodaltons.

 
Given the above-described results, we mutagenized K2, K4, and K5 together to either glutamine or alanine. In this case, either change was sufficient to eliminate GerQ cross-linking (Fig. 4, lanes 6 and 7). Note, however, that the levels of the triple-mutant proteins were lower than those of full-length GerQ as well as those of the N-terminal truncation variants (compare Fig. 4, lanes 6 and 7, to Fig. 2) for reasons that are not understood (see Discussion). Interestingly, in spores of both the N-terminal truncation variant strains AM15, AM23, and AM24 and the K2/K4/K5 triple mutants (strains AM68 and AM76), there was no GerQ band at 18 or 15 kDa (compare Fig. 2, lane 1, to Fig. 2, lanes 3 to 5, and Fig. 4, lanes 6 to 7).

GerQ N-terminal truncation and triple mutants are functional. One obvious question is whether all GerQ variants are functional, particularly variants that exhibit no cross-linking. GerQ is necessary for the proper localization and thus the function of CwlJ, one of two cortex lytic enzymes (the other is SleB), either of which is essential for the hydrolysis of cortex peptidoglycan during spore germination (18). Consequently, in the absence of GerQ, SleB is essential for spore germination, and gerQ sleB spores germinate extremely poorly with nutrients and thus exhibit extremely low apparent viability (18). To test whether our full-length N-terminal truncation and triple-mutant GerQ proteins were functional, we introduced an sleB mutation into our gerQ mutant strains, checked for protein expression, and then assessed germination with nutrients. Western blot analysis of total spore protein showed that all GerQ variants were present at the same levels in all sleB spores but again at a reduced level compared to those in wild-type spores (data not shown). However, the level of the GerQ monomer was more comparable to that of wild-type spores than in other experiments (Fig. 4, lanes 6 and 7; also data not shown). In addition, there was no detectable band at either 18 or 15 kDa in spores of any of the GerQ variants with sleB. Importantly, spores of the various GerQ variants with sleB behaved like wild-type spores with regard to their viability (Table 2). If the full-length replacement and mutant strains had not functioned normally in CwlJ localization, we would have expected them to behave like {Delta}gerQ {Delta}sleB spores in which spore viability is reduced by more than 3 orders of magnitude (Table 2). However, this was not the case, as the viability of the GerQ variant spores was reduced by at most sevenfold, and the viability of the K2A/K4A/K5A GerQ triple-mutant spores (strain AM80) was essentially the same as that of wild-type spores (Table 2). Although mutation of lysine residues 2, 4, and 5 to alanine did not significantly affect viability, mutation of these lysine residues to glutamine caused a fivefold reduction in viability. These results suggest that mutation of lysine residues 2, 4, and 5 to glutamine may cause a structural change in GerQ such that its function is slightly inhibited. It is also possible that the reduction in viability of spores with GerQ truncations occurs because the first five residues of GerQ function to a small extent in CwlJ localization.


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  		TABLE 2. Viabilities of spores of various strainsa

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A number of coat proteins have been suggested to be involved in Tgl-mediated cross-linking (8, 19, 24-26). However, to date, Tgl-dependent cross-linking has been shown only for GerQ, making it the only known Tgl substrate in B. subtilis (19, 25). Recent work has also provided additional evidence for Tgl-dependent cross-linking of GerQ, although the number of Tgl-dependent cross-linked species present in spore extracts in this recent study was greatly reduced compared to that in our wild-type spores (13, 19). The reason for this difference is unclear but may be due to the use of different B. subtilis strains, different sporulation conditions, and/or a different anti-GerQ antibody (13).

This study supports the suggestion that GerQ, a spore coat protein in B. subtilis, is covalently cross-linked by Tgl. We provide evidence that the residues involved are lysines 2, 4, and 5 and that any one of these lysines can be used to cross-link GerQ to its binding partners. Moreover, we show that although these residues are necessary for GerQ cross-linking, they are not essential for GerQ function. The results presented here show that GerQ is a donor substrate in the Tgl-mediated cross-linking reaction in the spore coat of B. subtilis. Since GerQ is the only Tgl substrate in which the residues involved in cross-linking have been identified, GerQ may be a model protein for future studies of Tgl-dependent cross-link formation in the spore coat.

Positioning of the residues involved in cross-linking at the amino terminus of GerQ makes sense mechanistically. Tgl catalyzes the formation of {varepsilon}-({gamma}-glutamyl) lysine isopeptide bonds by first binding to the glutamine-containing substrate to form a {gamma}-glutamylthiolester, known as the acyl enzyme intermediate, with a release of ammonia (14). The lysine-containing substrate then reacts with the acyl enzyme intermediate to form the covalent cross-link (14). Having a lysine residue involved in cross-linking at the typically flexible amino terminus of the protein would likely facilitate the interaction of this lysine with the acyl enzyme intermediate. In the case of GerQ, three potential residues are present at the amino terminus, increasing the probability that an interaction with Tgl will occur and that a cross-link will be formed. It is possible that all three lysine residues are necessary for substrate recognition, but only one is used, albeit arbitrarily, to form the cross-link. Structural studies in the future will be helpful in understanding the precise interaction between GerQ and the acyl enzyme intermediate.

Previous work has shown that cross-linking is not necessary for GerQ function in the localization of CwlJ (19). In this work, we show that GerQ truncation variants and triple mutants are present as monomers that do not form cross-links but do localize CwlJ. One interesting possibility is that only the GerQ monomer is necessary for GerQ function. The level of the monomer in the GerQ variant and triple-mutant strains was reduced compared to that of wild-type spores but was sufficient for function. This suggests that GerQ does not have to be present at high levels in the spore to properly localize CwlJ.

An amino acid sequence alignment of GerQ proteins from various Bacillus species has shown that this protein's sequence is not well conserved, especially in its amino-terminal region (18), although a more recent sequence alignment of this region revealed that lysines 2, 4, and 5 are conserved in GerQ from B. licheniformis (Fig. 1B). It is not known whether GerQ from this species is cross-linked, but if that is the case, this protein may use the same region of GerQ as a lysine donor as in B. subtilis. In contrast, GerQs from B. anthracis and B. cereus contain three glutamine residues in their amino-terminal region (Q3, Q4, and Q5) (18). Again, it is not known if GerQ is cross-linked in either of these species, but if cross-linking does occur, it may be through any of three amino-terminal glutamine residues rather than lysine residues. However, mutation of lysine residues 2, 4, and 5 to glutamine in B. subtilis GerQ was not sufficient for cross-linking, suggesting that the location of Tgl-mediated cross-linking may vary in GerQ proteins from different Bacillus species. It is also interesting that the amino acid sequence of GerQ from Oceanobacillus iheyensis does not contain any amino-terminal lysine residues and contains only one glutamine residue (Fig. 1B), suggesting that either this protein is not cross-linked by Tgl or the location of Tgl-mediated cross-linking is different in this species.

Now that the region in GerQ in which cross-linking occurs has been identified, it will be important to identify the cross-link as well as GerQ binding partners. Previously, an 18-kDa GerQ-derived band in spore coat fractions that was suggested to be a degradation product of GerQ was identified (19). This 18-kDa band was also observed in spores with full-length GerQ expressed from amyE and the single and double point mutants in lysine residues 2, 4, and 5. However, this 18-kDa band was not present in total spore extracts in which GerQ was not cross-linked. These results suggest that the 18-kDa GerQ-derived band may not be a GerQ degradation product but rather may be an internal cross-link in the GerQ monomer, causing the cross-linked protein to migrate faster on SDS-PAGE gels than the un-cross-linked protein. In support of this hypothesis, GerQ has 26 glutamine residues in its amino-terminal region, all of which have the potential to form an internal cross-link with lysine 2, 4, or 5. As mentioned above, the amino terminus of a protein is often a flexible region; thus, it is reasonable that a cross-link could form between this region and a more rigid part of the protein. However, we also saw bands at 17, 16, or 15 kDa (Fig. 3) in either the protein removed by decoating or the insoluble fraction of extracts from spores of GerQ truncation variants. One possible explanation for this is that the GerQ monomer is unstable under the decoating conditions and that, in the case of the truncation variant extracts, the lower-molecular-weight band does represent a degradation product. This hypothesis is strengthened by the fact that the band is present at a reduced level in the GerQ truncation variants compared to wild-type spores.

Previous work has shown that although GerQ cross-linking is greatly reduced in tgl strains, some Tgl-independent cross-links are still present (19). In this work, both Tgl-dependent and -independent cross-linking is greatly reduced in GerQ truncation and point mutation variants, suggesting that some Tgl-independent cross-linking may also involve the five amino-terminal residues of GerQ. It will be important to identify the source of these Tgl-independent GerQ cross-links in the future to determine if this is the case. In addition, it will also be important to identify other GerQ binding partners in the spore coat. Since the B. subtilis genome sequence is available, it should be possible to identify these binding partners with a combination of genetic and biochemical techniques, and this work is currently in progress.


   ACKNOWLEDGMENTS
 
We are grateful to Tom Carroll for his assistance with site-directed mutagenesis and for scientific discussions.

This work was supported by a grant from the NIH to P.S. (GM19698).


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030. Phone: (860) 679-2607. Fax: (860) 679-3408. E-mail: setlow{at}nso2.uchc.edu. Back

{triangledown} Published ahead of print on 25 August 2006. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Anagnostopoulos, C., and J. Spizizen. 1961. Requirements for transformation in Bacillus subtilis. J. Bacteriol. 81:741-746.[Free Full Text]
  2. Aronson, A. I., and P. Fitz-James. 1976. Structure and morphogenesis of the bacterial spore coat. Bacteriol. Rev. 40:360-402.[Free Full Text]
  3. Carroll, T. M., and P. Setlow. 2005. Site-directed mutagenesis and structural studies suggest that the germination protease, GPR, in spores of Bacillus species is an atypical aspartic acid protease. J. Bacteriol. 187:7119-7125.[Abstract/Free Full Text]
  4. Cutting, S. M., and P. B. Vander Horn. 1990. Genetic analysis, p. 27-74. In C. R. Harwood and S. M. Cutting (ed.), Molecular biological methods for Bacillus. John Wiley & Sons Ltd., Chichester, England.
  5. Driks, A. 1999. Bacillus subtilis spore coat. Microbiol. Mol. Biol. Rev. 63:1-20.[Abstract/Free Full Text]
  6. Driks, A. 2002. Maximum shields: the assembly and function of the bacterial spore coat. Trends Microbiol. 10:251-254.[CrossRef][Medline]
  7. Driks, A. 2002. Proteins of the spore core and coat, p. 527-535. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and its closest relatives: from genes to cells. ASM Press, Washington, D.C.
  8. Henriques, A. O., B. W. Beall, and C. P. Moran, Jr. 1997. CotM of Bacillus subtilis, a member of the alpha-crystallin family of stress proteins, is induced during development and participates in spore outer coat formation. J. Bacteriol. 179:1887-1897.[Abstract/Free Full Text]
  9. Henriques, A. O., and C. P. Moran, Jr. 2000. Structure and assembly of the bacterial endospore coat. Methods 20:95-110.[CrossRef][Medline]
  10. Kobayashi, K., K. Hashiguchi, K. Yokozeki, and S. Yamanaka. 1998. Molecular cloning of the transglutaminase gene from Bacillus subtilis and its expression in Escherichia coli. Biosci. Biotechnol. Biochem. 62:1109-1114.[CrossRef][Medline]
  11. Kobayashi, K., Y. Kumazawa, K. Miwa, and S. Yamanaka. 1996. {varepsilon}- ({gamma}-Glutamyl)lysine cross-links of spore coat proteins and transglutaminase activity in Bacillus subtilis. FEMS Microbiol. Lett. 144:157-160.[CrossRef]
  12. Kobayashi, K., S. I. Suzuki, Y. Izawa, K. Miwa, and S. Yamanaka. 1998. Transglutaminase in sporulating cells of Bacillus subtilis. J. Gen. Appl. Microbiol. 44:85-91.
  13. Kuwana, R., N. Okuda, H. Takamatsu, and K. Watabe. 2006. Modification of GerQ reveals a functional relationship between Tgl and YabG in the coat of Bacillus subtilis spores. J. Biochem. (Tokyo) 139:887-901.[Abstract/Free Full Text]
  14. Lorand, L., and R. M. Graham. 2003. Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat. Rev. Mol. Cell Biol. 4:140-156.[CrossRef][Medline]
  15. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  16. Nicholson, W. L., and P. Setlow. 1990. Sporulation, germination and outgrowth, p. 391-450. In C. R. Harwood and S. M. Cutting (ed.), Molecular biological methods for Bacillus. John Wiley & Sons Ltd., Chichester, England.
  17. Paidhungat, M., K. Ragkousi, and P. Setlow. 2001. Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca2+-dipicolinate. J. Bacteriol. 183:4886-4893.[Abstract/Free Full Text]
  18. Ragkousi, K., P. Eichenberger, C. van Ooij, and P. Setlow. 2003. Identification of a new gene essential for germination of Bacillus subtilis spores with Ca2+-dipicolinate. J. Bacteriol. 185:2315-2329.[Abstract/Free Full Text]
  19. Ragkousi, K., and P. Setlow. 2004. Transglutaminase-mediated cross-linking of GerQ in the coats of Bacillus subtilis spores. J. Bacteriol. 186:5567-5575.[Abstract/Free Full Text]
  20. Setlow, P. 1994 Mechanisms which contribute to the long-term survival of spores of Bacillus species. Soc. Appl. Bacteriol. Symp. Ser. 23:49S-60S.[Medline]
  21. Setlow, P. 2003. Spore germination. Curr. Opin. Microbiol. 6:550-556.[CrossRef][Medline]
  22. Setlow, P. 2006. Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. J. Appl. Microbiol. 101:514-525.[CrossRef][Medline]
  23. Suzuki, S., Y. Izawa, K. Kobayashi, Y. Eto, S. Yamanaka, K. Kubota, and K. Yokozeki. 2000. Purification and characterization of novel transglutaminase from Bacillus subtilis spores. Biosci. Biotechnol. Biochem. 64:2344-2351.[CrossRef][Medline]
  24. Zhang, J., P. C. Fitz-James, and A. I. Aronson. 1993. Cloning and characterization of a cluster of genes encoding polypeptides present in the insoluble fraction of the spore coat of Bacillus subtilis. J. Bacteriol. 175:3757-3766.[Abstract/Free Full Text]
  25. Zilhão, R., R. Isticato, L. O. Martins, L. Steil, U. Völker, E. Ricca, C. P. Moran, Jr., and A. O. Henriques. 2005. Assembly and function of a spore coat-associated transglutaminase of Bacillus subtilis. J. Bacteriol. 187:7753-7764.[Abstract/Free Full Text]
  26. Zilhão, R., M. Serrano, R. Isticato, E. Ricca, C. P. Moran, Jr., and A. O. Henriques. 2004. Interactions among CotB, CotG, and CotH during assembly of the Bacillus subtilis spore coat. J. Bacteriol. 186:1110-1119.[Abstract/Free Full Text]

Journal of Bacteriology, November 2006, p. 7609-7616, Vol. 188, No. 21
0021-9193/06/$08.00+0     doi:10.1128/JB.01116-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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