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1 Graduate School of Information Science, Nara Institute of Science and Technology, Takayama 8916-5, Ikoma, Nara 630-0192, Japan
2 Faculty of Pharmaceutical Sciences, Setsunan University, Nagaotouge 45-1, Hirakata, Osaka 573-0101, Japan
Correspondence
Kazuo Kobayashi
kazuok{at}bs.naist.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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-independent terminator is located upstream of a stop codon for the extended kinA open reading frame in ATCC 6051. Northern blot analysis showed that transcription of kinA terminated at this terminator, and kinA mRNA is missing a stop codon in ATCC 6051. Moreover, deletion of tmRNA suppresses the sporulation defect in ATCC 6051. These observations indicate that in ATCC 6051 the absence of a stop codon in kinA mRNA affects sporulation, probably by leading to rapid degradation of KinA via the trans-translation process. In ATCC 6051, the kinA mutation affects sporulation but not other Spo0A-dependent phenomena such as biofilm formation, which can be activated by a low level of Spo0A
P. This is due to the fact that KinA activity is kept low during the exponential phase via transcriptional and post-translational regulation. Thus, the stop-codon-less kinA probably affects only sporulation. DNA sequencing of 30 B. subtilis strains revealed that another strain also produces stop-codon-less kinA mRNA. This observation suggests that the lack of a stop codon for kinA mRNA may give rise to a selective advantage under certain conditions.
P, phosphorylated Spo0ATwo supplementary tables are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, 1993). In response to nutrient depletion, a histidine kinase phosphorylates itself, and then this phosphate residue is sequentially transferred to Spo0F, Spo0B and finally Spo0A. Spo0A is a member of a response-regulator family of transcriptional regulators, and phosphorylated Spo0A (Spo0AP) activates transcription of early sporulation genes.
Spo0AP directly regulates about 120 genes required for various stationary-phase phenomena including sporulation, competence development, production of degradative enzymes, cannibalism and biofilm formation (Molle et al., 2003). Spo0A-regulated genes can be classified into two groups based on Spo0A dose dependency: low-threshold genes require a low level of Spo0AP for activation or repression of transcription, whereas high-threshold genes require a high level of Spo0AP for transcription (Fujita et al., 2005). A representative low-threshold gene is abrB, whose expression is repressed by a low level of Spo0AP. AbrB is a pleiotropic global repressor of many genes active in stationary phase (Hamon et al., 2004; Strauch, 1991). One target of AbrB is the sipW operon required for biofilm formation (Hamon et al., 2004). By contrast, a high level of Spo0AP is required for induction of sporulation genes such as the spoIIA, spoIIE and spoIIG operon genes (Fujita et al., 2005). Thus, the level of Spo0AP can determine cell fate.
In vivo and in vitro evidence has shown that five kinases, KinA, KinB, KinC, KinD and KinE, can phosphorylate Spo0F (Antoniewski et al., 1990; Jiang et al., 1999, 2000; Kobayashi et al., 1995; LeDeaux & Grossman, 1995; Perego et al., 1989; Trach & Hoch, 1993). At least under laboratory conditions, these kinases appear to have different roles in laboratory strain 168. KinA and KinB are major kinases for sporulation, since a kinA kinB double-mutant strain cannot make spores (Trach & Hoch, 1993). On the other hand, mutations in kinC, kinD or kinE reduce biofilm formation but not sporulation, whereas mutations in kinA or kinB do not affect biofilm formation (Hamon & Lazazzera, 2001; Jiang et al., 1999; Kobayashi et al., 1995; LeDeaux & Grossman, 1995; LeDeaux et al., 1995). The function of Spo0A in biofilm formation is to repress abrB transcription, since abrB inactivation can restore biofilm formation in a spo0A mutant strain (Hamon & Lazazzera, 2001). In addition, although the kinA kinB double mutation does not affect abrB transcription, in a kinA kinB kinC kinD quadruple-mutant strain, the level of abrB transcription is elevated (Jiang et al., 2000). Thus, KinC, KinD and perhaps KinE seem to produce a low level of Spo0AP for repression of abrB transcription.
Recently, it has been recognized that the laboratory strain 168 is not representative of B. subtilis (Earl et al., 2007), as it has several features that distinguish it from undomesticated strains. For example, the 168 strain is deficient in swarming motility (Kearns & Losick, 2003), poly-
-glutamate synthesis (Stanley & Lazazzera, 2005), antibiotic production (Nakano et al., 1988; Tsuge et al., 1999) and biofilm formation (Branda et al., 2001). These differences are caused by genetic differences among strains in at least the sfp, swrA and degQ genes (Kearns et al., 2004; Nakano et al., 1992; Stanley & Lazazzera, 2005; Tsuge et al., 1999). One undomesticated strain, ATCC 6051 (NCIB 3610), can produce these antibiotics and form biofilms, and has been used by many laboratories to study multi-cellular behaviour. Unlike strain 168, strain ATCC 6051 forms aerial structures that serve as preferential sites for sporulation (Branda et al., 2001). However, sporulation is conditional in ATCC 6051. It sporulates well in MSgg minimal medium (Branda et al., 2001) but we have found that it does not make spores efficiently in rich sporulation media such as DSM and 2x SG. This makes ATCC 6051 different, as other undomesticated strains can form aerial structures with spores in DSM and 2x SG. Here, we demonstrate that a functionally relevant genetic difference exists between the kinA gene in strain 168 versus that in strain ATCC 6051.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. The B. subtilis ATCC 6051 and NBRC strains were obtained from the American Type Culture Collection (ATCC) and NITE Biological Resource Center (NBRC), respectively. All strains with a W prefix are derivatives of ATCC 6051. The B. subtilis (natto) strain BEST 195 was isolated from Miyagono natto (Itaya & Matsui, 1999). In all cases, B. subtilis strains were maintained on TBABM, 2x SG or LB (Difco) (Kobayashi, 2007). E. coli JM105 was used for construction and maintenance of plasmids. Antibiotics were used at the following concentrations: ampicillin, 30 µg ml–1; chloramphenicol, 5 µg ml–1; erythromycin/lincomycin, 0.5 µg ml–1 and 25 µg ml–1, respectively; kanamycin, 10 µg ml–1; spectinomycin, 100 µg ml–1.
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) and β-galactosidase activity of each transformant was examined on TBABM plates supplemented with 100 µg X-Gal ml–1. The spo mutation was mapped to the chromosomal region between ykuM and splA.
Construction of B. subtilis mutant strains.
Mutant strains were constructed using an overlap-extension PCR technique (Kobayashi, 2007). Antibiotic-resistance cassettes were amplified by PCR using the primers pUC-F and pUC-R from pCBB31(cat), pAE41(erm), pDG780(kan) and pDG1726(spc) (Guerout-Fleury et al., 1995; Kobayashi et al., 1995). As ATCC 6051 has low competence, the mutant alleles were first introduced into strain 168 and then transferred into ATCC 6051 by transformation with chromosomal DNA from the newly generated 168 mutant strains. Transformation of B. subtilis was carried out using a standard protocol (Dubnau & Davidoff-Abelson, 1971). Primers used for construction of mutant alleles are listed in Supplementary Table S1 (available with the online version of this paper).
The 3' region of kinA was amplified from 168 or ATCC 6051 chromosomal DNA by PCR using the primers kinA-F9 and kinA-R9. After digestion with EcoRI and HindIII, the resultant fragments were inserted into pCA191, a chloramphenicol-resistant (Cmr) derivative of pUC19, to generate pCAkinA168 and pCAkinA6051, respectively. These plasmids were then used to transform strain 168 and Cmr transformants were selected. The DNA sequence of kinA in the transformants was determined; kinA168 and kinA6051 strains were selected among these transformants
The promoter region of spoIID was amplified from ATCC 6051 chromosomal DNA by PCR using the primers spoIID-P-F and spoIID-P-R. After digestion with EcoRI and BamHI, the DNA fragment was inserted into pDLK2, which contains a lacZ reporter between the amyE-up and -down sequences. The resultant plasmid, pDLKspoIID, was used to transform strain 168 and a transformant harbouring amyE : : spoIIDp-lacZ was used in further analyses.
Northern blot analysis.
B. subtilis strains were grown in 2x SG at 37 °C and cells were harvested at the indicated time points. Total RNA was isolated via the method described by Igo & Losick (1986). Northern blotting was performed as described previously (Kobayashi, 2007). To prepare digoxigenin (DIG)-labelled RNA probes, DNA fragments were amplified by PCR using the primers described in Table S1.
Sporulation assay.
B. subtilis strains were grown in 2x SG medium at 37 °C and sporulation was assayed at 24 h after the end of the exponential phase (T24). The number of spores per ml culture was determined by identifying the number of heat-resistant c.f.u. (80 °C for 10 min) on LB plates. The number of viable cells was determined by counting the total number of c.f.u. at T24 with one exception: viable cells from strain ATCC 6051 and its derivatives were measured at T3 because the number of viable cells from this strain had decreased by T24.
Pellicle formation.
Pellicle formation was examined in 2x SGG medium [2x SG plus 1 % (w/v) glycerol] as described previously (Kobayashi, 2007). Cells were observed via phase-contrast microscopy using a DMRE-HC microscope (Leica) combined with a digital CCD camera (1300Y, Roper Science).
Isolation of B. subtilis and related strains from soil.
To isolate wild Bacillus strains, soil samples were gathered from several places in the garden at Setsunan University. Approximately 2 g soil samples were dissolved in 2 ml of 10 mM Tris/HCl (pH 7.2) and then boiled at 95 °C for 5 min. From this, 0.1 ml of each sample was then spread onto LB plates and incubated at 37 °C. Forty-eight Bacillus strains were isolated based on sporulation ability and colony morphology. Because it is not known if these strains are B. subtilis, only strains that contained a kinA region that could be amplified by PCR using the primers kinA-F7 and kinA-R8 were used in the analysis. Notably, no specific DNA was amplified from Bacillus amyloliquefaciens or Bacillus licheniformis genomic DNA when tested by PCR with these primers (data not shown).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Moreover, transcription of the spoIIG operon was not induced in 6051 grown in 2x SG medium, suggesting that this strain has a defect in initiation of sporulation (Fig. 1a). To identify a genetic determinant responsible for the 6051 Spo– phenotype, we first sought to transfer the DNA region responsible for the Spo– phenotype from 6051 to 168, as 6051 is not suitable for genetic manipulation. To do this, 168 was transformed with chromosomal DNA isolated from 6051 amyE : : spoIID-lacZ (Kmr), and white and Spo– colonies were screened among Kmr transformants on TBABM plates containing X-Gal. One white, Spo– transformant was isolated and used for further analysis.
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). In a transformation assay, ykuM : : cat and splA : : cat alleles had >50 % linkage with the Spo– phenotype. Moreover, the cheV allele, which is located in the region between ykuM and splA, had >90 % linkage with the Spo– phenotype. As kinA, which encodes a histidine kinase for sporulation (Antoniewski et al., 1990; Perego et al., 1989), is located upstream of cheV, it seemed reasonable to propose that the kinA is responsible for the Spo– phenotype. To address this, the DNA region containing kinA was amplified by PCR and used for transformation. Spo+ transformants did appear after transformation of the Spo– hybrid strain with DNA containing kinA but did not appear after transformation with DNA lacking kinA (data not shown). Moreover, DNA sequencing of the entire kinA gene in strain ATCC 6051 revealed that a stop codon, TAA, present in strain 168 kinA is TAT (Lys) in strain 6051. The nucleotide difference results in a 201 bp extension of the kinA coding region in 6051, which extends the coding region into patA (Fig. 1b
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To ask if this difference is responsible for the Spo– phenotype, kinA gene in 168 (referred to as kinA168) was replaced by kinA of 6051 (referred to as kinA6051). It has been shown that initiation of sporulation is controlled by two kinases, KinA and KinB, in the laboratory strain 168. Mutations in kinA or kinB have little or no effect on sporulation but a combination of kinA and kinB mutations severely reduces sporulation (Trach & Hoch, 1993). As shown in Table 2, kinA6051 had a similar effect on sporulation with the kinA mutation in the 168 background; that is, kinA6051 reduced sporulation slightly on its own and dramatically when in combination with the kinB mutation. In the 6051 background, the kinB mutation had a striking negative effect on sporulation, decreasing to the level observed for the kinA kinB double-mutant strain (Table 2). Introduction of kinA168 restored the ability of 6051 to sporulate. Taken together, the results strongly suggest that kinA6051 is non-functional and is responsible for the Spo– phenotype of strain 6051.
The kinA6051 transcript lacks a stop codon
In strain 168, the kinA stop codon is followed by a -independent terminator sequence, which is conserved at the corresponding position of kinA6051 (Fig. 1b). Northern blot analysis revealed that the size of the kinA transcript is the same in 168 and 6051 and matches the predicted size of 1902 nt, which is based on the distance from the transcriptional start site to the terminator (Fig. 1c). These observations suggest that kinA transcription terminates at this terminator sequence upstream of a stop codon of the extended kinA in 6051. Thus, the kinA6051 transcript lacks a stop codon.
Stop-codon-less mRNA prevents release of ribosomes from the mRNA, which in turn causes ribosomes to stall on the mRNA. Bacteria have a specific mechanism, termed trans-translation, for rescue of stalled ribosomes on incomplete or damaged mRNAs (Karzai et al., 2000; Withey & Friedman, 2003). The ssrA gene codes for tmRNA, which possesses both a tRNA-like domain and an mRNA-like coding sequence that together enable tmRNA to function as both a tRNA and an mRNA. This system rescues ribosomes stalled on the mRNAs and directs addition of a proteolysis tag to the C-termini of aberrant protein products, thus targeting them for proteolysis by the Clp protease complex (Gottesman et al., 1998; Wiegert & Schumann, 2001). Thus, the stop-codon-less mRNA of kinA6051 may be a substrate for tmRNA, leading to rapid degradation of KinA6051 protein. To address this possibility, the effect of a mutation in ssrA on sporulation in strain 6051 was examined. As shown in Table 3, the ssrA mutation restored sporulation in wild-type and kinB mutant strains, whereas the ssrA mutation did not restore sporulation in the kinA and kinA kinB mutant strains. Thus, the ssrA mutation suppresses the sporulation defect of strain 6051 in a kinA6051-dependent manner. These results indicate that the absence of a stop codon in kinA6051 mRNA affects sporulation, probably leading to rapid degradation of KinA6051 via the trans-translation process.
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P (Fujita et al., 2005; Hamon & Lazazzera, 2001). As has been shown for the laboratory strain (Hamon & Lazazzera, 2001), in strain 6051 pellicle formation appears to be controlled by other kinases, KinC and KinD, as kinC and kinD mutant strains form extremely thin pellicles (Fig. 2a, W924 and W925). However, unlike the situation in the laboratory strain, a mutation in kinE did not affect pellicle formation in strain 6051 (Fig. 2a, W885). Introduction of kinA168 did not affect pellicle formation in the wild-type strain and could not restore pellicle formation of kinC and kinD mutant strains (Fig. 2a, W1077 and W1078). However, when these strains harboured kinA168 they formed spores on pellicles efficiently, even in a kinC or kinD mutant background (Fig. 2b)
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), the abrB mutation bypassed the requirement for KinC and KinD in pellicle formation (Fig. 2a, W1045). Transcriptional analysis of abrB revealed that KinC and KinD are responsible for production of a low level of Spo0AP, which can repress abrB transcription (Fig. 3a). By contrast, mutation of kinA and kinB did not affect transcription of abrB (Fig. 3a), and introduction of kinA168 also did not affect abrB transcription (Fig. 3c, W1046). These observations suggest that KinA is distinct from KinC and KinD. KinA appears to be specifically required for sporulation, whereas KinC and KinD are involved in repression of abrB transcription.
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P controls repression of abrB transcription and direct induction of sporulation genes, but the latter requires a higher level of Spo0AP than the former (Fujita et al., 2005). We wondered why KinA168 cannot substitute for KinC or KinD despite the fact that KinA168 can accumulate enough Spo0AP to induce sporulation, even in the kinC or kinD mutant background (Fig. 2b). To address this, transcription of these kinases was compared by Northern blot analysis. When B. subtilis cells were grown in 2x SG, kinC and kinD were expressed when cells were in the exponential phase, whereas transcription of kinA, kinB and kinE was induced after the exponential phase (Fig. 3b).
As the phosphatases KipI and Sda inhibit activity of KinA (Burkholder et al., 2001; Wang et al., 1997), we hypothesized that KinA168 activity is inhibited by these phosphatases during the early growth phase, and therefore KinA168 cannot substitute for KinC or KinD. To address this, kinA168 was introduced into a kipI sda kinD triple-mutant strain. In the absence of kipI and sda, KinA168 could restore pellicle formation of the kinD mutant strain (Fig. 2a, W1136). Thus, as expected, KinA activity is inhibited by these phosphatases. Moreover, mutations of kipI and sda also partly restored pellicle formation of the kinD mutant strain alone (Fig. 2a, W1163), suggesting that these phosphatases also inhibit activity of other kinases.
To confirm that these effects resulted from regulation of abrB, transcription of this gene was examined. As was the case for pellicle formation, introduction of kinA168 did not affect abrB transcription in the wild-type and kinD mutant strains (Fig. 3c, W1046 and W1078). However, it should be noted that, unlike the case for pellicle formation, the kinD mutation did not affect abrB transcription in agitated culture (Fig. 3c, W925). By contrast, KinA168 could strongly repress transcription of abrB in the absence of kinD, kipI and sda (Fig. 3c, W1136). However, KinA168 did not affect transcription of abrB in the presence of kinD, even when kipI and sda were also absent (Fig. 3c, W1130). Since KinA and KinD phosphorylate Spo0F, these kinases may compete for Spo0F. Thus, these observations suggest that KinA is inhibited not only by the KipI and Sda phosphatases but also by competition with KinD in interaction with Spo0F.
Although kinA transcription is low during the exponential phase (Fig. 3b), KinA168 strongly represses transcription of abrB in the absence of kipI, sda and kinD. This observation suggests the possibility that the kinase activity of KinA is stronger than that of KinD, which may enable KinA to induce sporulation. This possibility is supported by a previous report that, in vitro, KinC and KinD are less active on Spo0F than KinA; however, it should be noted that truncated forms of KinC and KinD were used in the study (Jiang et al., 1999).
Each kinase involved in the phosphorelay has a distinct function under the conditions we tested. These differences are caused by regulation at at least three levels: transcriptional regulation, control of kinase activity by phosphatases, and competition among kinases. By these regulations, KinA seems to be specific for sporulation that requires a high level of Spo0AP. Thus, the stop-codon-less kinA affects only sporulation but not other Spo0AP-related functions in strain 6051.
The kinA gene in other B. subtilis strains
We were interested to learn if the stop-codon-less form of the kinA gene exists in other B. subtilis strains. To address this, we determined the DNA sequence of the kinA 3' region in 30 B. subtilis strains: two B. subtilis subsp. subtilis strains, one B. subtilis subsp. spizizenii strain, three B. subtilis subsp. subtilis strains isolated from natto, and 24 strains newly isolated from soil. The DNA sequence of the stop codon region of kinA in each strain is shown in Fig. 4(a). Interestingly, the kinA sequence around a stop codon varied among strains, which can be classified into three groups. In the first group, the kinA stop codon region is the same as that of strain 168. In the second group, there is a 1 bp deletion downstream of the stop codon. And in the third group, there are two 1 bp deletions, and conceptual translation shows that the KinA protein produced by strains in this group is four amino acids longer than KinA168. These deletions are in a poly-A stretch around the position that corresponds to a stop codon for kinA168. The A-to-T substitution in kinA6051 also occurred in the poly-A stretch and, furthermore, kinA of NBRC 101239 has an A-to-G substitution in the poly-A stretch that does not affect the amino acid sequence. These observations imply that the sequence of the poly-A stretch may be susceptible to spontaneous mutations.
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). Although we did not determine the entire sequence of the insertion element, the sequence is significantly similar to that of IS660 in Oceanobacillus iheyensis HTE 83 (data not shown). To our surprise, the terminus of the IS insertion was the -independent terminator sequence and thereby, the IS insertion introduced a new potential -independent terminator, preceding the potential stop codon (Fig. 4c). NBRC 16449 sporulated well in 2x SG medium (see Supplementary Table S2, available with the online version of this paper). As is the case for ATCC 6051, the kinB mutation reduced sporulation in the NBRC 16449 background. Moreover, the ssrA mutation restored sporulation ability of the kinB mutant strain in a kinA-dependent manner. These observations suggest that NBRC 16449 also produces a stop-codon-less kinA mRNA.
It is easy to imagine that sporulation, which blocks an increase in cell number, can be disadvantageous under some conditions, particularly as in natural environments, bacteria live in competition with other organisms. The kinA mutation reduces the frequency of initiation of sporulation but does not affect phenomena such as biofilm formation, which is important for survival in natural environments. Moreover, the kinA mutation does not abolish sporulation: the kinA mutant strain produces spores at a low frequency in rich sporulation medium and makes spores normally in minimal medium (LeDeaux et al., 1995). These observations suggest that the kinA mutation may give rise to a selective advantage under certain conditions. However, ATCC 6051 and NBRC 16449 have unique variations that produce stop-codon-less kinA mRNAs. In addition, the stop-codon-less kinA is functional in an ssrA mutant background. These observations imply that the stop-codon-less kinA may be functional under certain conditions. However, we could not find conditions that induce sporulation in a stop-codon-less kinA-dependent manner. Investigating a possible function for the stop-codon-less kinA6051 will be a subject of future study.
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ACKNOWLEDGEMENTS |
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Edited by: M. Paget
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REFERENCES |
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Received 18 July 2007;
revised 27 August 2007;
accepted 14 September 2007.
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HindIII digest; lane 1). (c) The nucleotide sequence of the 3' region of kinA in strain NBRC 16449. The insertion is shaded and the