JOURNAL OF BACTERIOLOGY, Feb. 1992, p. 1053-1054
Vol. 174, No. 3
0021-9193/92/031053-02$02.00/0 Copyright X 1992, American Society for Microbiology
Characterization of the Bacillus subtilis Sporulation Gene spoVK NANCY FAN, SIMON CUTTING, AND RICHARD LOSICK* Department of Cellular and Developmental Biology, The Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138 Received 18 September 1991/Accepted 18 November 1991
The sporulation gene spoVK of BacUlus subtilis was cloned by use of the insertional mutation spoVK::Tn917fkHU8. The spoVK gene was shown to be the site of an incorrectly mapped mutation called spoVJ517. Thus, a separate spoVJ gene as defined by the 517 mutation does not exist and is instead identical with spoVK.
Genes (spo) involved in the process of sporulation in Bacillus subtilis are classified according to the stage of sporulation at which their gene products are required in morphogenesis (9). Most of the early-acting sporulation genes have been well characterized, yet little is known about genes acting at the terminal stages of sporulation, particularly those whose products are required at stage V of spore coat formation. Currently 11 distinct stage-V sporulation loci are believed to exist. These are spoVA, -B, -C, -D, -E, -F, -G, -J, -K, -M, and -Q, of which only four, spoVA, -E, -G, and -J, have been cloned and sequenced (6, 8). In this work we report the cloning and characterization of spoVK, which was previously identified among a collection of insertional spo mutants generated by using the transposon Tn917. The spoVK::Tn917fQHU8 (10) insertional mutation was determined to represent a newly identified spo locus on the basis of its map position (1680) and its phenotypic effect, causing a block at stage V. We show that spoVK is also the site Qf a mutation (spoVJS17, herein renamed spoVK517) defining the spoVJ locus, which we show was incorrectly mapped by Hill (5) to 2500 on the chromosome. Thus, the spoVJ locus as defined by the 517 mutation does not exist and is instead identical with spoVK. To clone spoVK and the surrounding chromosomal DNA, we used a previously described method for the cloning in Escherichia coli of chromosomal DNA adjacent to the site of a Tn917 insertion (13). Briefly, strain KS8 (10), which contained the Tn9J7 insertion spoVK::Tn917fQHU8, was transformed with the linearized plasmid pTV20 and then selected for Cmr to replace the transposon with a Tn9O7 derivative bearing an E. coli plasmid replicon and the bla and cat genes. Using this spoVK-pTV20 chromosomal derivative, we were able to clone approximately 800 bp of chromosomal DNA flanking one side of the Tn917 insertion and extending to an upstream HindIII site (plasmid pNF1; Fig. 1). The cloned segment of DNA was then subcloned in the phage M13 vector mpl9 and subjected to dideoxy nucleotide sequence analysis. Nucleotide sequence analysis identified part of an open reading frame into which the transposon had inserted. To our surprise, our partial sequence of the open reading frame was identical with that recently reported by Foulger and Errington (4) for the B. subtilis spoVJ gene, which had previously been cloned and characterized by Errington et al. (3). The precise site of the Tn917Q1HU8 insertion was *
between the first and second base pairs of isoleucine codon 127 (5'-AQkTA-3') of the spoVJ open reading frame. Because spoVK and spoVJ appeared to be identical, we attempted to verify the map positions of the spoVK:: Tn917fQHU8 and the spoVJS17 mutations. Hill (5) claimed that spoVJS17 was located in the 250° region of the chromosome on the basis of cotransformation and cotransduction experiments with ilvB2 and cotransduction experiments with leuA. In an attempt to verify linkage to ilvB2, a phage PBS1 generalized transducing lysate was prepared from the original spoVJS17-bearing mutant (strain 517; spoVJS17 trpC2) of Hill (5), which was obtained from J. Errington (Oxford University), and used to transduce strain CU267 (trpC2 leuB16 ilvB2) to Ilv+ (2). Despite repeating the experiment several times, we were unable to observe cotransduction of spoVJS17 with the ilvB2 marker. Rather, spoVJS17 and spoVK::Tn917fQHU8 exhibited substantial cotransformation with glnA100; the levels of cotransformation (43 and 40%, respectively) agreed with that previously reported (10) for spoVK::Tn9J7Q1HU8, which is located at 1680 (Fig. 2). (Linkage to glnA was measured by preparing chromosomal DNA from strains 517 and KS8 [spoVK::Tn917fQHU8] and using these DNAs to transform competent cells of strain PY78 [glnA100] to prototrophy [2].) Thus, spoVJ, at least as defined by the 517 mutation, does not exist, but is instead identical to spoVK. The 517 mutation is hereafter designated spoVKS17. As further evidence that the mutation of Hill (5)
Tn917
V
H
H
B
spoVK pNF1 0.5 kb
FIG. 1. Endonuclease restriction map of the spoVK region of the chromosome. The cleavage sites for HindIlI (H) and BgII (B) are shown; the open box below the map shows the position and direction of transcription of the spoVK open reading frame. The site of insertion of the transposon Tn917 in strain KS8 (spoVK:: Tn917Q1HU8) is shown. Chromosomal DNA flanking the Tn917flHU8 insertion that was cloned in plasmid pNF1 is indicated. The arrow above the map shows the extent and direction of nucleotide sequence determined in this study. Sequencing was by the dideoxy-chain termination method (11) with a phage M13 mpl9 clone containing the segment of DNA cloned in plasmid pNF1.
Corresponding author. 1053
1054
NOTES
J. BACTERIOL. 0
SpoVJ
locus called spoVH, which was claimed to be located in close proximity to spoVJ. However, the mutation defining spoVH (spoVH516) has been found to be an allele of spoVA (8), which is far away from the spoVJ-spoVH region of the chromosome. The spoVH locus was subsequently removed from the genetic map (8). (spoVH was cloned by Cutting and Mandelstam [1] before it was realized that it is identical to spoVA.) Also, no evidence from other laboratories has been reported for the existence of stage-V mutations in the reported positions of spoVJ and spoVH. If the original strains 516 and 517 are equivalent to those used more recently by ourselves and others, there is no ready explanation for the discrepancies in the results obtained.
%
spowVl
180
FIG. 2. Chromosomal positions of relevant genetic markers. The position of the spoVK locus at 168 min was established by cotransformation linkage with the glnA100 auxotrophic marker by the work of Sandman et al. (10) and confirmed in this study. The original (and incorrect) positions of two stage V (spoVJ and spoVH:) loci identified and mapped in the work of Hill (5) are also shown.
is an allele of spoVK, transformation with a 4-kb segment of DNA extending from the spoVK::Tn917QlHU8 transposon insertion in spoVK to a downstream EcoRI site was able to correct the sporulation defect of spoVK517-bearing cells. Another discrepancy with the results of Hill (5) concerns the phenotype of the 517 mutant strain. Hill (5) reported an unusual phenotype for strain 517, namely, that sporulating cells acquired resistance to lysozyme but not to organic solvents or heat. However, in our hands, sporulating cells of 517 and of the congenic (with the spo+ strain PY79) strains NF65 (containing the spoVK517 mutation) and KS8 (containing the spoVK::Tn917QkHU8 mutation) were equivalently defective in acquiring resistance to organic solvent, heat, and lysozyme (Table 1). Hill (5) also reported the discovery of a second stage-V TABLE 1. Resistance properties of spoVK mutants Viable
Strain Strain
mLl)a
to:
(CFU
ml-') PY79 (spo+) 517 (spoVJS17 trpC2) NF65b (spoVJ517) KS8 (spoVK::Tn917 QHU8)
Resistance (surviving CFU
count
Chloroform
Heat
Lysozyme
2.3 x 108 1.9 x 108 1.4 x 108 2.7 x 108
Vol. 174, No. 3
0021-9193/92/031053-02$02.00/0 Copyright X 1992, American Society for Microbiology
Characterization of the Bacillus subtilis Sporulation Gene spoVK NANCY FAN, SIMON CUTTING, AND RICHARD LOSICK* Department of Cellular and Developmental Biology, The Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138 Received 18 September 1991/Accepted 18 November 1991
The sporulation gene spoVK of BacUlus subtilis was cloned by use of the insertional mutation spoVK::Tn917fkHU8. The spoVK gene was shown to be the site of an incorrectly mapped mutation called spoVJ517. Thus, a separate spoVJ gene as defined by the 517 mutation does not exist and is instead identical with spoVK.
Genes (spo) involved in the process of sporulation in Bacillus subtilis are classified according to the stage of sporulation at which their gene products are required in morphogenesis (9). Most of the early-acting sporulation genes have been well characterized, yet little is known about genes acting at the terminal stages of sporulation, particularly those whose products are required at stage V of spore coat formation. Currently 11 distinct stage-V sporulation loci are believed to exist. These are spoVA, -B, -C, -D, -E, -F, -G, -J, -K, -M, and -Q, of which only four, spoVA, -E, -G, and -J, have been cloned and sequenced (6, 8). In this work we report the cloning and characterization of spoVK, which was previously identified among a collection of insertional spo mutants generated by using the transposon Tn917. The spoVK::Tn917fQHU8 (10) insertional mutation was determined to represent a newly identified spo locus on the basis of its map position (1680) and its phenotypic effect, causing a block at stage V. We show that spoVK is also the site Qf a mutation (spoVJS17, herein renamed spoVK517) defining the spoVJ locus, which we show was incorrectly mapped by Hill (5) to 2500 on the chromosome. Thus, the spoVJ locus as defined by the 517 mutation does not exist and is instead identical with spoVK. To clone spoVK and the surrounding chromosomal DNA, we used a previously described method for the cloning in Escherichia coli of chromosomal DNA adjacent to the site of a Tn917 insertion (13). Briefly, strain KS8 (10), which contained the Tn9J7 insertion spoVK::Tn917fQHU8, was transformed with the linearized plasmid pTV20 and then selected for Cmr to replace the transposon with a Tn9O7 derivative bearing an E. coli plasmid replicon and the bla and cat genes. Using this spoVK-pTV20 chromosomal derivative, we were able to clone approximately 800 bp of chromosomal DNA flanking one side of the Tn917 insertion and extending to an upstream HindIII site (plasmid pNF1; Fig. 1). The cloned segment of DNA was then subcloned in the phage M13 vector mpl9 and subjected to dideoxy nucleotide sequence analysis. Nucleotide sequence analysis identified part of an open reading frame into which the transposon had inserted. To our surprise, our partial sequence of the open reading frame was identical with that recently reported by Foulger and Errington (4) for the B. subtilis spoVJ gene, which had previously been cloned and characterized by Errington et al. (3). The precise site of the Tn917Q1HU8 insertion was *
between the first and second base pairs of isoleucine codon 127 (5'-AQkTA-3') of the spoVJ open reading frame. Because spoVK and spoVJ appeared to be identical, we attempted to verify the map positions of the spoVK:: Tn917fQHU8 and the spoVJS17 mutations. Hill (5) claimed that spoVJS17 was located in the 250° region of the chromosome on the basis of cotransformation and cotransduction experiments with ilvB2 and cotransduction experiments with leuA. In an attempt to verify linkage to ilvB2, a phage PBS1 generalized transducing lysate was prepared from the original spoVJS17-bearing mutant (strain 517; spoVJS17 trpC2) of Hill (5), which was obtained from J. Errington (Oxford University), and used to transduce strain CU267 (trpC2 leuB16 ilvB2) to Ilv+ (2). Despite repeating the experiment several times, we were unable to observe cotransduction of spoVJS17 with the ilvB2 marker. Rather, spoVJS17 and spoVK::Tn917fQHU8 exhibited substantial cotransformation with glnA100; the levels of cotransformation (43 and 40%, respectively) agreed with that previously reported (10) for spoVK::Tn9J7Q1HU8, which is located at 1680 (Fig. 2). (Linkage to glnA was measured by preparing chromosomal DNA from strains 517 and KS8 [spoVK::Tn917fQHU8] and using these DNAs to transform competent cells of strain PY78 [glnA100] to prototrophy [2].) Thus, spoVJ, at least as defined by the 517 mutation, does not exist, but is instead identical to spoVK. The 517 mutation is hereafter designated spoVKS17. As further evidence that the mutation of Hill (5)
Tn917
V
H
H
B
spoVK pNF1 0.5 kb
FIG. 1. Endonuclease restriction map of the spoVK region of the chromosome. The cleavage sites for HindIlI (H) and BgII (B) are shown; the open box below the map shows the position and direction of transcription of the spoVK open reading frame. The site of insertion of the transposon Tn917 in strain KS8 (spoVK:: Tn917Q1HU8) is shown. Chromosomal DNA flanking the Tn917flHU8 insertion that was cloned in plasmid pNF1 is indicated. The arrow above the map shows the extent and direction of nucleotide sequence determined in this study. Sequencing was by the dideoxy-chain termination method (11) with a phage M13 mpl9 clone containing the segment of DNA cloned in plasmid pNF1.
Corresponding author. 1053
1054
NOTES
J. BACTERIOL. 0
SpoVJ
locus called spoVH, which was claimed to be located in close proximity to spoVJ. However, the mutation defining spoVH (spoVH516) has been found to be an allele of spoVA (8), which is far away from the spoVJ-spoVH region of the chromosome. The spoVH locus was subsequently removed from the genetic map (8). (spoVH was cloned by Cutting and Mandelstam [1] before it was realized that it is identical to spoVA.) Also, no evidence from other laboratories has been reported for the existence of stage-V mutations in the reported positions of spoVJ and spoVH. If the original strains 516 and 517 are equivalent to those used more recently by ourselves and others, there is no ready explanation for the discrepancies in the results obtained.
%
spowVl
180
FIG. 2. Chromosomal positions of relevant genetic markers. The position of the spoVK locus at 168 min was established by cotransformation linkage with the glnA100 auxotrophic marker by the work of Sandman et al. (10) and confirmed in this study. The original (and incorrect) positions of two stage V (spoVJ and spoVH:) loci identified and mapped in the work of Hill (5) are also shown.
is an allele of spoVK, transformation with a 4-kb segment of DNA extending from the spoVK::Tn917QlHU8 transposon insertion in spoVK to a downstream EcoRI site was able to correct the sporulation defect of spoVK517-bearing cells. Another discrepancy with the results of Hill (5) concerns the phenotype of the 517 mutant strain. Hill (5) reported an unusual phenotype for strain 517, namely, that sporulating cells acquired resistance to lysozyme but not to organic solvents or heat. However, in our hands, sporulating cells of 517 and of the congenic (with the spo+ strain PY79) strains NF65 (containing the spoVK517 mutation) and KS8 (containing the spoVK::Tn917QkHU8 mutation) were equivalently defective in acquiring resistance to organic solvent, heat, and lysozyme (Table 1). Hill (5) also reported the discovery of a second stage-V TABLE 1. Resistance properties of spoVK mutants Viable
Strain Strain
mLl)a
to:
(CFU
ml-') PY79 (spo+) 517 (spoVJS17 trpC2) NF65b (spoVJ517) KS8 (spoVK::Tn917 QHU8)
Resistance (surviving CFU
count
Chloroform
Heat
Lysozyme
2.3 x 108 1.9 x 108 1.4 x 108 2.7 x 108
