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FEMS Microbiology Letters 153 (1997) 63^69 Analysis of non-polar deletion mutations in the genes of the spo0K (opp) operon of Bacillus subtilis John R. LeDeaux 1, Jonathan M. Solomon 2, Alan D. Grossman* Department of Biology, Building 68-530, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Received 8 April 1997; revised 13 May 1997; accepted 13 May 1997 Abstract The spo0K (opp) operon of Bacillus subtilis encodes an oligopeptide permease that is required for uptake of oligopeptides, development of genetic competence, and initiation of sporulation. We made in-frame, non-polar deletion mutations in each of the first four genes of the five-gene spo0K operon and tested effects on oligopeptide transport, sporulation, and expression of competence genes. spo0KA, B, C, and D were required for sporulation, competence development, and oligopeptide transport. Disruption of spo0KE caused a less severe phenotype than did disruption of any of the other genes of the operon. Keywords : Deletion mutant; spo0K operon; Bacillus subtilis 1. Introduction The spo0K (opp) operon of Bacillus subtilis (Fig. 1) encodes an oligopeptide permease [1,2] that is required for the initiation of sporulation and the development of genetic competence (reviewed in [3,4]). Spo0K is a member of the large family of ATP-binding cassette (ABC) transporters that are involved in the import or export of a wide variety of compounds * Corresponding author. Tel.: +1 (617) 253-1515; Fax: +1 (617) 253-8699; E-mail: [email protected] 1 Present address: USDA-ARS, North Carolina State University, Department of Genetics, P.O. Box 7614, Raleigh, NC 27695-7614, USA. 2 Present address: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111, USA. in many di¡erent organisms [5]. The Spo0K oligopeptide permease in B. subtilis can import oligopeptides from 3^5 amino acids with apparently little, if any, speci¢city, and is required for cells to utilize oligopeptides as a source of amino acids (for example, see [6,7]). The function of each of the ¢ve gene products of the spo0K operon is inferred based on predicted protein sequence and analogy to many ABC transporters [5]. Spo0KA is the ligand-binding protein and is attached to the outside of the cell by a lipid anchor [1]; Spo0KB and Spo0KC are membrane proteins that form a complex through which the ligand is transported; Spo0KD and Spo0KE are the ATPases that are thought to provide energy for transport [1,2]. Previous work had shown that a variety of insertion mutations in each of the ¢rst four genes of the operon cause defects in oligopeptide transport, competence, and sporulation [1,2,6,8], and that the de- 0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 2 3 2 - 2 J.R. LeDeaux et al. / FEMS Microbiology Letters 153 (1997) 63^69 64 fects are similar to those caused by deletion of the entire operon. All mutations reported 2. Materials and methods (trans- poson insertion, integrative plasmid), except for a Strains are listed in Table 1 and plasmids in Table spo0KD null [1], are polar on downstream genes, 2 and Fig. 1. Molecular biological procedures were making it di¤cult to evaluate the necessity of each according to standard protocols [9]. Cell growth, of the genes individually. Null mutations in spo0KD sporulation, and cause viously described [2,7,10,11]. a phenotype similar to that caused by L-galactosidase assays were as pre- polar mutations in the upstream genes [1,2]. In contrast null mutations in spo0KE (the last gene of the operon, Fig. 1) cause a much less severe phenotype 3. Results and discussion [1,2]. To determine the relative contribution of each 3.1. Construction of non-polar deletion mutations spo0K cistron to the phenotypes associated with null alleles of the entire operon, we made in-frame, To make each non-polar deletion, we ¢rst made a non-polar deletion mutations in each of the ¢rst four plasmid that contained an in-frame deletion in the genes 1). gene of interest within sequences that overlapped Mutants were then tested for oligopeptide trans- either the 5P or the 3P end of the operon (see Fig. 1 port, and Table 2). Each deletion plasmid was integrated genes. in the operon sporulation, and (Tables 1 and expression of 2; Fig. competence into the spo0K locus in the chromosome by single Fig. 1. Map of spo0K region and plasmids used to make spo0K mutations. Restriction site abbreviations : A, ApaI ; Bg, BglII ; C, ClaI ; E, EcoRI ; Es, EspI ; N, NruI ; Nc, NcoI ; P, PvuII ; R, EcoRV ; Rs, RsrII ; S, SphI ; Sn, SnaBI ; Sp, SplI ; X, XcmI. The location of the likely promoter site of spo0K is indicated by an arrow and P. The location of the Tn917lac [2] insertion is indicated by a triangle and Tn. All clones are in the vector pJH101, except pJL28 (vector pDR63), pJL32 and pJL79 (vector pDR67). 65 J.R. LeDeaux et al. / FEMS Microbiology Letters 153 (1997) 63^69 crossover, selecting for plasmid-encoded chloramphenicol resistance. Integration of the three plasmids that extend past the 3P end of the operon, pJL34 for vspo0KC, pJL9 for vspo0KD, and pJL19 for vspo0KDE (Fig. 1), gave rise to both Spo3 and Spo transformants, indicating that these three deletions did indeed cause a Spo3 phenotype. For these three plasmids, integration upstream of the deletion endpoint would place the mutant gene under control of the operon promoter and separate the wild-type gene from the upstream promoter (Spo3 ). Integration downstream of the deletion endpoint would leave the wild-type operon intact (Spo ). For plasmids with inserts that extend past the 5P end of the operon, pJL18 for vspo0KA and pJL29 for vspo0KB (Fig. 1), all single crossover events should result in transformants that are phenotypically Spo , whether the integration occurred upstream or downstream of the deletion endpoints. However, Spo3 transformants could result from gene conversion events that replaced the wild-type copy of the gene with the mutant allele from the plasmid. For both vspo0KA (pJL18) and vspo0KB Table 1 B. subtilis strains used Strain Genotypea JH642 trpC2 pheA1 KI418 spo0KA : :Tn917lac JRL43 JRL131 JRL135 JRL179 JRL189 JRL221 Commentsb or reference vspo0KD43 vspo0KA131 vspo0KDE135 vspo0KA amyE : :(spo0KA vspo0KB189 vspo0KE221 : : neo [2] JH642 trf to JH642 trf to JH642 trf to cat) vspo0KD with pJL9 vspo0KA with pJL18 vspo0KDE with pJL19 JRL131 trf to CmR with pJL28 JH642 trf to vspo0KB JRL275 amyE : :(comG-lacZ neo) a.k.a. AG1046 [10] JRL293 amyE : :(srfA-lacZ [10,11] 61974 cat) JRL358 vspo0KC322 vspo0K357 : : neo vspo0K358 : : erm JRL476 amyE : :(srfA-lacZ JRL322 JRL357 JRL485 JRL486 JRL488 JRL489 JRL492 JRL493 JRL494 JRL584 JRL682 JRL685 JRL1082 JRL1102 JRL1103 JRL1104 JRL1105 JRL1114 JRL1115 JRL1117 JRL1118 with pJL29 JH642 trf to NeoR with pJL42 JH642 trf to vspo0KC with pJL34 [8] 61974 cat : :spc) vspo0KA amyE : :(comG-lacZ neo) vspo0KA amyE : :(srfA-lacZ spc) vspo0KD amyE : :(comG-lacZ neo) vspo0KD amyE : :(srfA-lacZ spc) vspo0KDE amyE : :(srfA-lacZ spc) vspo0K : : erm amyE : :(comG-lacZ neo) vspo0K : : erm amyE : :(srfA-lacZ spc) vspo0KE584 : : spc vspo0KE : : spc amyE : :(comG-lacZ neo) vspo0KE : : spc amyE : :(srfA-lacZ cat) vspo0KC amyE : :(srfA-lacZ cat) vspo0KB amyE : :(comG-lacZ neo) vspo0KC amyE : :(comG-lacZ neo) vspo0KDE amyE : :(comG-lacZ neo) vspo0KB amyE : :(srfA-lacZ cat) vspo0KB amyE : :(Pspac-spo0KB cat) vspo0KB amyE : :(Pspac-spo0KBC cat) vspo0KC amyE : :(Pspac-spo0KB cat) vspo0KB amyE : :(Pspac-spo0KBC cat) [8] JRL293 converted to SpcR CmS with pJL62 JRL131 trf to NeoR JRL131 trf to SpcR JRL43 trf to NeoR JRL43 trf to SpcR JRL135 trf to SpcR JRL358 trf to NeoR JRL358 trf to SpcR JH642 trf to SpcR with pJL75 JRL275 trf to SpcR JRL293 trf to SpcR JRL322 trf to CmR JRL189 trf to NeoR JRL322 trf to NeoR JRL135 trf to NeoR JRL189 trf to CmR JRL489 trf to CmR with pJL79 JRL189 trf to CmR with pJL32 JRL322 trf to CmR with pJL79 JRL322 trf to CmR with pJL32 a All strains are derived from JH642 and have the trp and phe mutations. b trf indicates transformation selecting for the indicated antibiotic resistance. Cm, Neo, and Spc refer to resistance to chloramphenicol, neomycin, and spectinomycin, respectively. J.R. LeDeaux et al. / FEMS Microbiology Letters 153 (1997) 63^69 66 Table 2 Plasmids Plasmid a Comments, source, or reference pAG58 Ap Cmp JH101 Ap Tc Cm ; integrative vector pGEMcat Ap Cm ; integrative vector pUC18erm Ap MLS ; source of erm cassette pBEST501 Ap Neo ; source of neo cassette pUS19 pJL62 Ap Spc ; integrative vector, source of spc cassette R S S R Ap Spc ; used to convert Cm Spc strains to Cm Spc [8] pDR63 Ap Cm ; vector used to recombine fragments into the chromosome at amyE by double crossover, derived from pDH32 [18] by deleting most of lacZ (from BamHI to BssHII) and deleting several restriction sites (from DraIII to SphI) pDR67 Ap Cm ; vector used to place fragments under control of the IPTG-inducible promoter Pspac and recombine into the pDR9 Ap Cm ; clone of spo0KB-E in pJH101 [2] pDR18 Ap Cm ; clone of spo0K promoter region from PvuII site upstream of the promoter to EcoRI site in spo0KA, cloned chromosome at amyE by double crossover [19] into pJH101 [2] a Ap, Tc, Cm, Neo, Spc, MLS refer to resistance to ampicillin, tetracycline, chloramphenicol, neomycin, spectinomycin, and erythromycin + lincomycin, respectively. Primary references for each of the ¢rst six plasmids are cited in [8]. 3 (pJL29), some transformants were Spo , indicating (Fig. 1). The partial diploid was phenotypically Spo that spo0KA and spo0KB are both required for spor- as ulation. plates. A complementation test was also performed Gene conversion events probably occurred in the judged on the by colony vspo0KB and phenotype vspo0KC on sporulation mutations to deter- transformants containing the plasmids that extend mine if they were polar on downstream genes. Two past pJL19, plasmids were made, one that had only spo0KB pJL34). However, these would be phenotypically in- under control of the IPTG-inducible promoter Pspac distinguishable from recombinants that were the re- (pJL79) and one that had both spo0KB and spo0KC sult of a single crossover. under the control of Pspac (pJL32). Both of the con- the 3P end of the operon (pJL9, To make strains that were missing plasmid sequen- structs were put at the nonessential amyE locus in vspo0KB vspo0KC strains. Both convspo0KB strain in an IPTG-deindicating that the vspo0KB dele- ces and had a single mutant allele, we screened for a both the second recombination event that excised the plasmid structs rescued the and left behind the deletion mutation. One or more pendent manner, and recombinants from the transformation of each tion is not polar on the downstream genes. In the plasmid was isolated and grown under non-selective presence of IPTG, the Pspac-spo0KB construct did Spo vspo0KC, but the Pspac-spo0KBC conditions (i.e. in the absence of chloramphenicol), not rescue the and plated on non-selective sporulation plates. Spo construct did, indicating that the colonies were chosen and then tested for sensitivity was not polar on spo0KD and spo0KE, and that two 3 to chloramphenicol to indicate loss of the plasmid. Strains JRL135 JRL322 JRL43 v ( spo0KD), v (vspo0KC) ( spo0KDE), JRL131 JRL189 v v ( spo0KA), ( spo0KB), and were made in this way. The de- letions in spo0KA, spo0KB, spo0KD, and spo0KDE vspo0KC deletion copies of spo0KB could not substitute for a loss of spo0KC. The vspo0KD deletion is not completely polar on spo0KE because the vspo0KD allele causes vspo0KDE allele a less severe phenotype than the (see below). were veri¢ed by PCR analysis (data not shown). None of the deletions in the ¢rst four genes of the 3.2. Mutant phenotypes spo0K operon were polar on downstream genes. A complementation test was performed on the vspo0- We measured the e¡ects of the various spo0K mu- KA mutation by putting spo0KA (under control of tations on transport (resistance to the toxic tripep- the spo0K promoter) at the amyE locus using pJL28 tide bialophos), sporulation, and competence. E¡ects J.R. LeDeaux et al. / FEMS Microbiology Letters 153 (1997) 63^69 67 Fig. 2. Expression of srfA-lacZ and comG-lacZ in di¡erent spo0K mutants. Strains were grown in S7 minimal medium with 1% glucose and 0.1% glutamate and amino acids (40^50 Wg/ml) for auxotrophic requirements. Note the di¡erent scales on the y-axis. A: srfA-lacZ. Wild-type (JRL293), ¢lled circles; vspo0KE : : spc (JRL685), open squares; vspo0KABCD : : erm (JRL494), open triangles. B: srfA-lacZ. vspo0KD (JRL489), ¢lled diamonds; vspo0KA (JRL486), ¢lled triangles; vspo0KB (JRL1105), open diamonds; vspo0KC (JRL1082), open circles; vspo0KDE (JRL492), ¢lled squares; vspo0KABCD : : erm (JRL494), open triangles (same data as in A). C: comG-lacZ. wild-type (JRL275), ¢lled circles; vspo0KE : : spc, (JRL682), open squares; vspo0KABCD : : erm (JRL493), open triangles. D: comG-lacZ. vspo0KD (JRL488), ¢lled diamonds; vspo0KA (JRL485), ¢lled triangles; vspo0KB (JRL1102), open diamonds; vspo0KC (JRL1103), open circles; vspo0KDE (JRL1104), ¢lled squares; vspo0KABCD : : erm (JRL493), open triangles (same data as in C). on competence were analyzed by measuring expression of srfA-lacZ and comG-lacZ. comG encodes components required for DNA uptake and is expressed only in the sub-population of cells that actually develops competence ([3,4,12], and references therein). Expression of comG (and other late com genes) is an excellent indicator of competence development per se [13]. Expression of a small open reading frame (comS) in the srfA operon is required for expression of the late com genes (comG) and development of competence [14,15]. srfA is expressed in virtually all of the cells, not just the sub-population 68 J.R. LeDeaux et al. / FEMS Microbiology Letters 153 (1997) 63^69 destined to become competent [12]. Its expression depends, in part, on spo0K [10,11,16] and two peptide pheromones that accumulate in culture medium as cells grow to high density [7,10,11]. 3.2.1. spo0KA^D The vspo0KA, vspo0KB, and vspo0KC non-polar deletions and the vspo0KDE deletion caused phenotypes that were indistinguishable from those caused by a vspo0KA^D : : erm mutation. The sporulation frequency (Table 3), expression of srfA-lacZ (Fig. 2A,B) and comG-lacZ (Fig. 2C,D) were similar in all of the mutants. In addition, transport of oligopeptides was defective in the mutants as all were fully resistant to 50 Wg/ml of bialophos. vspo0KD is virtually indistinguishable from spo0K null mutants in terms of sporulation and bialophos resistance consistent with results previously reported [1]. However, the vspo0KD mutation had a slightly less severe e¡ect on expression of srfA-lacZ (Fig. 2B) and comG-lacZ (Fig. 2D) than the other spo0K deletion mutations. 3.2.2. spo0KE In contrast to mutations in the rest of the operon, null mutations cause only a slight defect in sporulation (Table 3) [1,2]. A spo0KE insertion mutation was previously reported to cause a Com3 phenotype, where the competence of the cells was measured at a single time point during growth [2]. A more extensive analysis of competence gene expression and transformation frequency at di¡erent time points in the growth spo0KE Table 3 Sporulation frequency of di¡erent spo0K curve indicated that spo0KE mutants have delayed and somewhat reduced expression of srfA-lacZ (Fig. 2A), comG-lacZ (Fig. 2B) and transformation (data not shown). As previously reported [1], spo0KE mutants are able to transport peptides, as indicated by sensitivity to bialophos. However, we found that the spo0KE mutants were resistant to very low concentrations of bialophos (0.02^0.1 Wg/ml) while wild-type cells were sensitive to these concentrations. These results indicate that oligopeptide transport is slightly impaired in the spo0KE mutants. 3.3. ATPases of ABC transporters ABC transporters generally require two ATPase domains or subunits [5]. Some importers have two di¡erent ATPases (Opp), while others have two subunits of a single ATPase (His, Mal). We suspect that in the spo0KE mutant another ATPase is substituting for the missing subunit; perhaps a second subunit of Spo0KD replaces Spo0KE. Alternatively, an ATPase from a di¡erent transport system might substitute for the Spo0KE ATPase. Preliminary experiments indicate that the ATPases from app, which encodes a cryptic oligopeptide permease similar to Spo0K [6], are not substituting for Spo0KE. There is precedence for one part of an ABC transporter to work with another ABC transporter. The ligand-binding protein of the dipeptide permease Dpp can work with the membrane proteins of Spo0K to transport dipeptides: a mutation in dpp which is not polar on the gene encoding the ligand- mutants in DS medium Strain Genotype Spores/mla Percent sporulation Frequency relative to wild-type JH642 JRL131 JRL189 JRL322 JRL43 JRL221 JRL135 KI418 JRL358 WT vspo0KA vspo0KB vspo0KC vspo0KD vspo0KE : : neo vspo0KDE spo0KA : :Tn917 vspo0K : : erm 2.4 2.5 2.3 2.5 1.5 2.9 7.3 5.9 3.7 51 1 0.9 0.8 0.6 11 0.25 0.24 1.3 1 2.0 1.8 1.6 1.1 2.1 5.0 4.8 2.5 U10 U10 U10 U10 U10 U10 U10 U10 U10 8 6 6 6 6 7 5 5 6 U1033 U103 U103 U103 U10 U1033 U103 U10 2 2 2 2 1 3 3 2 a Data shown are from a single representative experiment, and similar results were obtained in at least three independent experiments. The sporulation frequencies of the spo0K mutants (except spo0KE) were typically 0.5^3% of wild-type. The sporulation frequencies of the spo0KE mutants were typically 20^50% of wild-type. The number of viable cells/ml ranged from 2.1 108 to 4.7 108 . U U J.R. LeDeaux et al. / FEMS Microbiology Letters 153 (1997) 63^69 binding protein of Dpp will still allow transport of dipeptides into the cell, but this transport is dependent on spo0K (F. Slack and A.L. Sonenshein, personal communication). Another example more pertinent to these results is the ability of UgpC, the single ATP-binding protein of the sn-glycerol-3-phosphate ABC transporter Ugp, to substitute for MalK, the single ATP-binding protein of the maltose ABC transporter [17] and the ability of MalK to substitute for UgpC [17]. The potential for mixing subunits encoded by di¡erent transport operons increases the possible variation in transport complexes. Transporters that were previously thought to be homogeneous might actually exist in di¡erent forms, each with a potentially di¡erent function. Acknowledgments Bialophos was a generous gift from Meiji Seika Kaisha, Ltd., Pharmaceutical Research Center, Yokohama, Japan. We are grateful to F. Slack and A.L. Sonenshein for communicating unpublished results, and to members of our lab for useful discussions. J.M.S. was supported in part by a Howard Hughes predoctoral fellowship. A.D.G. was a Lucille P. Markey Scholar in Biomedical Sciences and this work was supported in part by a grant from the Lucille P. Markey Charitable Trust, and Public Health Services Grants GM41934 and GM50895 to A.D.G. from the NIH. References [1] Perego, M., Higgins, C.F., Pearce, S.R., Gallagher, M.P. and Hoch, J.A. (1991) The oligopeptide transport system of Bacillus subtilis plays a role in the initiation of sporulation. Mol. Microbiol. 5, 173^185. [2] Rudner, D.Z., LeDeaux, J.R., Ireton, K. and Grossman, A.D. (1991) The spo0K locus of Bacillus subtilis is homologous to the oligopeptide permease locus and is required for sporulation and competence. J. 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(1995) Isolation and characterization of kinC, a gene that encodes a sensor kinase homologous to the sporulation sensor kinases KinA and KinB in Bacillus subtilis. J. Bacteriol. 177, 166^175. [9] Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J. and Struhl, K. (1990) Current Protocols in Molecular Biology. John Wiley and Sons, New York. [10] Magnuson, R., Solomon, J. and Grossman, A.D. (1994) Biochemical and genetic characterization of a competence pheromone from B. subtilis. Cell 77, 207^216. [11] Solomon, J.M., Magnuson, R., Srivastava, A. and Grossman, A.D. (1995) Convergent sensing pathways mediate response to two extracellular competence factors in Bacillus subtilis. Genes Dev. 9, 547^558. [12] Hahn, J., Kong, L. and Dubnau, D. (1994) The regulation of competence transcription factor synthesis constitutes a critical control point in the regulation of competence in Bacillus subtilis. J. Bacteriol. 176, 5753^5761. [13] Hahn, J., Luttinger, A. and Dubnau, D. (1996) Regulatory inputs for the synthesis of ComK, the competence transcription factor of Bacillus subtilis. Mol. Microbiol. 21, 763^775. [14] D'Souza, C., Nakano, M.M. and Zuber, P. (1994) Identi¢cation of comS, a gene of the srfA operon that regulates the establishment of genetic competence in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 91, 9397^9401. [15] Hamoen, L.W., Eshuis, H., Jongbloed, J., Venema, G. and van Sinderen, D. (1995) A small gene, designated comS, located within the coding region of the fourth amino acid-activation domain of srfA, is required for competence development in Bacillus subtilis. Mol. Microbiol. 15, 55^63. [16] Hahn, J. and Dubnau, D. (1991) Growth stage signal transduction and the requirements for srfA induction in development of competence. J. Bacteriol. 173, 7275^7282. [17] Hekstra, D. and Tommassen, J. (1993) Functional exchangeability of the ABC proteins of the periplasmic binding proteindependent transport systems Ugp and Mal of Escherichia coli. J. Bacteriol. 175, 6546^6552. [18] Shimotsu, H. and Henner, D.J. (1986) Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis. Gene 43, 85^94. [19] Ireton, K., Rudner, D.Z., Siranosian, K.J. and Grossman, A.D. (1993) Integration of multiple developmental signals in Bacillus subtilis through the Spo0A transcription factor. Genes Dev. 7, 283^294.