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Cloning and sequencing of the Bacillus megaterium spoIIA operon
Biochimie (1992) 74, 695-704 © Soci6t6 franfaise de biochimie et biologie mol6culaire / Elsevier, Paris
695
Cloning and sequencing of the Bacillus megaterium spollA operon YP Tao**, DSS Hudspeth, PS Vary* Department of Biological Sciences, Northern Illinois University,Dekaib, IL 60115, USA (Received 5 January 1992: accepted 20 February 1992)
Summary - - The spollA operon of Bacillus megaterium has been cloned and the nucleotide sequence determined. The spoliA sequence contains three open reading frames coding for putative proteins of 116 aa, 147 aa, and 253 aa; the first and the third genes are preceded by a ribosomal binding site. The genes are in the same order as those of B subtilis and B licheniformis. The deduced amino acid sequences of these three open reading frames show 78-92% homology with SpolIAA, SpolIAB and SpolIAC of B subtilis and B licheniformis. Northern hybridization revealed that B megaterium also has two spollA transcripts, 1.77 kb and 2.92 kb, auaining maximum expression at t~ and t3, respectively. In addition, homology to a possible penicillin binding protein gene upstream and the first part of a spoVA operon downstream has been identified on the 3.34-kb fragment. The spollA and the downstream spoVA promoter regions are highly conserved among these three species. Sequence analysis of the spoVA promoter revealed a region upstream to the -35 that is highly conserved across Bacillus species.
Bacillus megaterium / sporulation / spollA / spoVA / pbp
Introduction Cells of the Gram-positive genus Bacillus can differentiate into dormant spores upon nutrient deprivation [ 1, 2]. This process of spore formation in Bacillus provides a relative simple experimental system for use in the study of cellular development and differentiation. Sporulation is divided into six or seven distinct morphological stages and involves the coordinated expression of at least 50 spo loci [3, 4]. Many of the spo genes have now been cloned and sequenced, and the function of their gene products is beginning to be understood. Several mechanisms have been found to be involved in the complex regulation of these genes. For example, sequential appearance of new sigma factors that bind to core enzyme and confer different promoter specificity allows transcription of new sets of sporulation genes [5-7]. A second mechanism is the release of spo genes through a two-component system from the repressive effects of a repressor such as AbrB [8]. Increase of sigma factor concentration *Correspondence and reprints **Present address: Waksman Institute of Microbiology, Rutgers University, Piscataway, NJ 08854, USA. Abbreviations: bp, pase pair(s); X-gal, 5-bromo-4-chloro-3indolyl-[~-D-galacto pyranoside; IPTG, isopropyl-~-D-thiogalactopyranoside; rbs, ribosomal-binding site(s); PBP, penicillin-binding proteins; ORF, open reading frame; PG, peptidoglycan; aa, amino acid(s).
during sporulation is a third mechanism. For example, o H increases five-fold in stationary phase from vegetative phase levels because of increased translation or stability of the spoOH mRNA and decreased turnover of o H [9]. Auxiliary factors other than sigma factors can serve as activators or repressors of transcription such as the 'switch protein' SpoIIID [10], which is a transcriptional activator of the spolVCB promoter, but is also a repressor of cotA and corD genes. Other regulatory mechanisms include the activation of sigma factors or processing of pro-o factors into mature forms. Examples are oK and oE processing [11], as well as changes in oF activity, which is inhibited by SpoIIAB [12, 13], but then released by SpoIIAA, the antagonist of SpoIIAB [13]. It is obvious that there are controls at transcription: post-transcription, translation, and post-translation levels. In B subtilis, there exist at lea~t nine different sigma factors in phage-uninfected cells [6]. It was previously proposed that developmental gene expression during sporulation is controlled by cascade of sigma factors [5]. It is now well established that the program of sporulation gene expression is largely governed by the regulated appearance of new sigma factors in the following dependent order: oa --> oF --> aE --> Ot:; --> OK [7]. One of these, oF, is encoded by the spollAC (or sigF) gene. The spollAC gene is the promoter-distal gene of the tricistron operon in B subtilis called spollA, the promoter-proximal cistrons being spollAA and spollAB [14, 15]. Strains mutated in spollAC
696 form aberrantly septated sporangia that are either disporic or multiseptate cells, which suggests that o F is required for the transcription of genes involved in forespore engulfment [16]. Recently, Partridge et al [12] have shown by using an inducible promoter to activate spollA expression in vegetative cells, that oF itself is necessary and sufficient to direct spolllG (o~3) transcription. Their results also indicated that another function of o F (independent of its sigma factor function) is the processing of o E. The work of Schmidt et ai [13] has shown that S p o l I A A is an antagonist o f S p o I I A B and that SpoIIAB is, in turn, an antagonist of SpolIAC. They speculated that the S p o l I A A / S p o I I A B / S p o l I A C regulatory system could play a role in controlling the timing and/or the location (the forespore chamber of the sporangium) for oF-directed gene expression. Interestingly, Margolis et al [17] have shown that o F is only active in the forespore, yet is required for the processing o f o E in the mother cell. Thus, the spollA operon is o f great interest and of central importance because it helps to govern the transition from a single cell to a sporangium consisting of two cellular compartments with different developmental fates. Since B megaterium is very efficient at both sporulation and germination, it is well suited for the studies of sporulation and has been used extensively for biochemical analysis of both sporulation and germination (for review see [18]). It is also a more divergent species from B subtilis and B iicheniformis [19] so that a study of sporulation in this species provides a unique opportunity to also compare sporulation among Bacillus species. The information that emerges from the comparison should be very useful in elucidating conserved (and thus critical) regulation of sporulation and in understanding the evolution of sporulation. In the past few years, we have developed genetic techniques in this species and have isolated several mutants by transposon-mediated mutagenesis [20]. We have characterized several Tn917-1acZ fusion spo mutants [21 ] and showed by electron microscopy that one of these, spo-58, formed aberrant multiple spore septa and expressed ~-galactosidase activity at approximately to. We report here the cloning and sequencing of the spo-58 gene as well as the entire B megaterium spollA operon. The gene organization, expression, and the predicted amino acid sequences of the operon are then compared with those of B subtilis and B licheniformis. M a t e r i a l s a n d methods Strains and plasmids All B megaterium strains used in this study were derived from QM B1551 (ATCC 12872). PV361 (plasmidless, wild-type) was constructed by curing all seven resident plasmids (p-T)
[20]. PV447 (p-7 lac-3 iac-6) is a Lac- derivative of PV361 120]. PV510 (p-7 iac-3 lac-6 spo-58::Tn917-1acZ-cat) was constructed by selecting a Tn917-1acZ-cat fusion insertion spo mutant in PV447 and was used to isolate genomic DNA for cloning spollA mutant gene [21]. E coli strain TGI [supE hsdD5 thi (Alac-proAB)/F'(traD36 proA+B+ laciq lacZ AMI5)] (a gift from J Rapoport, Institute Pasteur) was used as host. Integratior~al plasmid pJHl02 was provided by JA Hoch (Scripps Clinic). Media LB (Luria-Bertani) medium [22] was used for plasmid isolation and growing E coli for transformation. M9 medium [23] with 25 ~tg/ml of thiamine was used for maintaining TGI with its F'. B megaterium strains were grown in supplemented nutrient broth (SNB) medium, described previously [24]. Antibiotic concentrations were 5 ttg/ml erythromycin, 250 I.tg/ml lincomycin (MLS), 50-100 I.tg/ml ampicillin, and 4 I.tg/ml chloramphenicoi. Nucleic acid isolation The miniscreen procedure of Kawamura et ai [25], a modification of the Birnboim and Doly procedure [26], was used to isolate small amounts of plasmid DNA. Larger-scale plasmid DNA preparation was done by the method of Birnboim and Doly [26]. The method of Bovre and Szybalski [27] was used to isolate B megaterium genomic DNA. RNA was prepared by the method of Wu et ai [40] with the following modifications. PV361 was grown in SNB broth at 30°C with shaking at 300 rpm. The time when growth deviated from exponential at ODt,60 was designated as to. PV361 cultures (60 ml) taken from to to t5 were harvested by centrifugation in centrifuge bottles containing a half volume (30 ml) of semi-frozen IX Spizizen salts [29] and RNA was isolated by the method of T Henkin (personal communication). Cells were disrupted by vortexing with glass beads in the presence of vanadyl ribonucleoside complexes [30] (BRL) to inhibit RNAase activity. Finally, RNA was resuspended in diethylpyrocarbonate-treated water (0.1% by volume), and RNasin (1301, Promega) was added to the final concentration of 2 U per ~tl. Nucleic acid hybridization The method of Grunstein and Hogness [31] was followed for colony hybridization using Nylon-I (BRL) or 85-mm nitrocellulose circles (Schleicher and Schuell). Prehybridization was done according to the protocol from Amersham. Hybridization was carried out in the same solution as above, with the addition of DNA probe labeled by random priming (BRL) using [tx-32p] dATP. Hybridization was carded out for at least 12 h at 65°C. Filters were then washed twice in 2 × SSC, 15 min each time, once in 2 x SSC with 0. ! % SDS for 30 min, and finally in 0.1 x SSC for 10 min. For autoradiography, the filters were wrapped in Saran wrap and exposed to Kodak XAR-film with intensifying screen (DuPont Lightning Plus) at -70°C for the desired time. Transfer hybridizations were done by the alkaline capillary blotting method of Reed and Mann [32]. The method of Ausubel et al [33] was used for Northern hybridizations with the following modifications. About 10 ~tg of RNA from each sample was loaded into a 1.2% agarose denaturing gel containing 37% formaldehyde, with an RNA ladder (0.24-9.5 kb, BRL) as a size marker. Transfer to Hyband-N+ nylon membrane (Amersham) instead of a nitrocellulose membrane was carded out in 20 x SSC overnight.
697 Prehybridization was at 65°C as described for colony hybridizations.
DNA manipulation Restriction enzymes were purchased from IBI, BRL or Boehringer Mannbeim and were used as recommended by the suppliers. Restriction DNA fragments were recovered from agarose gels by electroelution (IBI). The Random Primers DNA Labeling System (BRL) was used to radioactively label probes. The Stratagene Klenow Fill-in Kit was used to make blunt ends of insert or vector DNA which had 5'-overhangs. Dephosphorylation of vector DNA was carded out as described by Sambrook et al [23] using calf intestine alkaline phosphatase (Boehringer Mannheim). Ligation reactions were done using T4 DNA ligase (BRL) in 20 I.d volume at 12.5°C overnight with a 1:3 molar ratio of vector:insert DNA and 2 lal of T4 ligase.
The wild-type gene was then cloned by probing with the 1.64-kb AvaI-HindIII fragment from pYP4 (containing B megaterium D N A plus 10 bases from the distal end of Tn917). Southern hybridization and autoradiography of HindIII-digested PV361 genomic DNA identified a 3.34-kb band (data not shown). The fragments were recovered and ligated to pUC18 cut with HindIII and transformed into TG1. Among 475 white ampr colonies tested, two hybridized with the probe. Restriction analysis and Southern hybridization showed that one of them (termed pYPS, see fig 1 ) had the correct size insert (3.34 kb). The promoter region was subcloned by ligating a 0.7-kb HindIII-EcoRI fragment from pYP5 to HindIII-EcoRI digested pUC 19 to yield pYP9 (fig 1).
Transformations
Complementation analysis
E coli competent cells and transformation were as described by Sambrook et al [23]. The PEG-mediated protoplast transfor-
In order to prove that the correct gene had been cloned, the genes on the 3.34-kb fragment were introduced into the spo-58 mutant to see if it would complement the mutation. The 3.34-kb HindIII fragment was treated with PolI, Klenow fragment to generate blunt ends, and the fragment was cloned into pJM 102 cut with Sinai to yield pYP10. Vector p J M l 0 2 was constructed as an integrational plasmid in B subtilis (J Hoch, M Perego, personal communication). It has the ability to replicate autonomously in E coli, but not in Bacillus and contains a cat gene which can be selected in Bacillus. Plasmid p Y P I 0 (about 10 t.tg) was used to transform the spo-58 mutant (PVSI0). Four colonies were recovered on SNB containing 4 ~tg/ml Cm and were streaked and incubated on the same medium. After 24 h incubation, about 80% became free spores while no spores were detected in the mutant strain PV510 grown under the same conditions (but with MLS for maintaining Tn917). Therefore, the result verified genetically that the 3.34-kb fragment contains a region complementing the spo-58 mutant.
mation method of Von Tersch and Carlton [34] was used for megaterium except that cells were regenerated on non-selective RHAF plates overnight at 30°C, then replicated to SNB plus selective antibiotic plates. Transformants were seen after 24 h incubation at 30°C.
B
Doume-sirunued DNA sequencing The Sequenase version 2.0 (USB) protocol for dideoxysequencing was followed. After beat-denaturation, samples were loaded into sequencing gels containing 6% polyacrylamide or 5% 'Long Ranger' (AT Biochem, Malvern, PA) with 8.3 M urea. Electrophoresis was carried out in 1 x TBE buffer (0.1 M Tris base, 0.1 M boric acid, 2 mM Na2EDTA, pH 8.3). Primers were synthesized on an oligonucleotide synthesizer (ABI PCR-Mate). Results
Cloning of the B megaterium spollA genes We took advantage of the Tn917 insertion in spo-58 to clone the mutant gene by probing with a HindIlI-Aval fragment containing about 1 kb of the distal end of T n g 1 7 from plasmid p T V I [35]. Total chromosomal D N A from strain PV510 was cut with HindIII and hybridization with the probe gave a band of approximately 2.6 kb. HindIII-fragments in the 2.0-2.9 kb range were then recovered by electroelution, tested by dot hybridization, and ligated to HindIII-cut pUCI 8. E coli T G I competent cells were transformed and plated on LB amp X-gal plates. About 210 white ampr colonies were then tested by colony hybridization and two hybridized strongly with the original Tn917 probe. Restriction analysis and Southern hybridization showed that these two colonies had the correct insert (data not shown). One clone was saved as plasmid pYP4 (fig 1). This 2.6 kb insert consisted of about 1 kb from the Tn917-distal end and 1.6 kb from the spo-58 mutant downstream of the Tn917 insertion.
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Fig 1, Plasmids pYP4, pYP5 and pYP9. The solid triangle indicates the Tn917 insertion in spo-58. The bars indicate the two probes used in Northern hybridization (fig 4). Restriction sites are indicated as Cl (ClaD, HI (HgiAl), HIII (HindIII), RV (EcoRV), RI (EcoRI), BII (BgaIII), SI (Stul), PI (Pstl).
698 --
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GATATCGATGCTGCTTCTTGGTGGAAGATATTC~AACGTACATTCTCTATTTTCTCT~AAA~ATCTTAA~CAGCTAA%TGTGA~GA~ATAGAACTAGTTT 100 t
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-35 zeg£oa -10 zQg£on rbs TGTCGGTGAGAAGGATT~TATT~TGTCGGCAGCGAkAGACTGACTATA~GATTTCCGAC~A~TTTGTATAGT~AGTCTTTCGATTGAATT~GAATTCAA 200 AC~AG~TGTCTTGTTGGTTCGATTAACAGGAG~ATTAGAT~ATCATACGGCAG~G~GCTAAGA~GC~%ATCACCGAAGCGATAGAGAAGGAATCT~TC 300 AGcC•TTTGATTTTAAATTTACAGCATTTAACCTTTATG••CA•CTCTGGCTTGGGGGTTATTTT•GGGAGATACAAAC•GGTTCATAATAACGGCGGGG t
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AAATGGTTGTATGCGCTATTTCTCCTGCCATAGAACGTTTGTTTAATATGTCAGGCTTATTTAAAATTATTCGTTTTGAACCTAATGAAGCAAACGCG~T 500 TCAAAAATTGGGGGTGGCATAAT•AAAAATCAAATGAACCTCCAATTTTCCGCGCTCAGTCAAAATGAATCGTTTGCTCGCGTCACGGTAGCATCATTTA 600 TTACGCAGTTAGATCCAACAATGGATGAGTTAACAGAAATTAAAACAGTCGTATCTGAAGCAGTAACAAATGCTATTATTCACGGCTACGAAAGCAATTC 700 GGATGGCGTGGTATATATTTCTGTGACGCTTCATGAAGACGGTGTAGTCGAGCTGATGATTCGTGATGAAGGAATTGGTATCGATAACGTGGATGAAGCC 800 AA•CAGCCTTTATACACAACGAAACCAGATCTAGAACGTTCTGGAATGGGTTTCACCATAATGGAAAACTTTATGGACGAAATTCGAGTGGAGTCTACTG 900 rb. TATTAGAAGGTACGACCTTATATTTAAAAAAGCACTTAACAAATAGTAAAGCGCTATGTAATTAAGGAGACTTGCCAATGGATGTGGAGGTCAAAAAAAA 1000 TAAAAACGAACCGTATTTA~GGATCATGAAGTTAAAGACCTCATCAAGCGCAGTCAGCAAGGTGATCAAATTGCACGAGATACAATCGT~ABAAAAAT
II00
AT~G~CTCGTTT~TCGGTTGTTC~CGTTTTATTAACcGTGGTTAT~AGCCCGATGATTTATTTCAAATT~AT~TATA~G~CTATTAAAGTCCGTT~
1200
ATAAATTTGACTTATC~TAT~AT~TTAAATTTTCAA~ATAT~CTGTTCCAATGATTATTGGTGAAATTCAG~ATTTATTC~T~AT~AT~CAC~TAAA
1300
~QTTA~CC~TTCTCT~AAA~AAAT~A~CAATAAAATAC~TAAA~CAAAAQAC~A~CTTTCCAAATTACTT~QAC~c~TCCCTACA~TC~CTQA~QTTQ~A 1400 GAACATTTG~ATCTCACTCCTGAAGAA~TAGTGCTAGCTCAAGAAGCTAATCGT~CTCCTTC~TCTATTCACGA~ACCGTCTAT~AAAAT~AT~A~ATC
1500
CAATTACCC;TCTTGACCA~A TTGCTGAT;ATACGGAAG;GAAGTGGTT;GA TAAAATT;CCTTAAAAGIAGCAATTGAIGAACTTGAT:AGCGTGAAAA 1600 W
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TTGGTAAT~AA~GTCTGTTCAGAAACGT~AATTAGAAAATTTAAGAAAAT~CATACTACACGTACAGGGAGATAATGAATCGTAAGGTGTGATATGA~]~ 1900 fiGAAAACGTTCTTTATCTTA
1920
Fig 2. Nucleotide sequence of the coding and flanking regions of the B megaterium spoliA operon. The asterisks below nucleotides are positioned every 10 bases. The putative ribosomal-binding sites, start codons and promoter regions ate labeled and underlined; stop codons are overlined. The filled triangle indicates the position of the Tn917 insertion in the spollAC gene. The symbols <<< and >>> are used to show that these two regions complement each other to form the rho-independent terminator.
Sequencing of spoilA The sources of DNA for sequencing were the wildtype 3.34-kb HindIII fragment contained on plasmid
pYPS, the subcloned 0.7-kb EcoRI-HindIII promoter region on plasmid pYP9 and the mutant DNA on pYP4 (see fig l). The site of the transposon Tn917 insertion in spo-58 was determined by comparing the
699 sequence from pYP4 with that from pYP5. The sequencing strategy is also shown in figure 1 and was done using synthetic primers as well as commercially available universal primers for pUCI 8 and pUC 19. The entire spollA operon including its promoter as well as the downstream spoVA promoter region was sequenced in both directions and the sequence is shown in figure 2.
Analysis of gene products and regulatory signals A computer search of Genbank for homologous nucleotide sequences using the FASTA program of Pearson and Lipman [36] identified a striking homology over much of the sequence with the B subtilis spollA operon sequence (69% identity within the coding region). Preliminary sequencing of the flanking DNA has also shown extensive homology to the pbp gene of B subtilis upstream, and the first two genes of the spoVA operon (data not shown). The data suggest that the 3.34-kb fragment consists of the entire B megaterium spoliA operon, approximately half of a gene homologous to pbp at its 5' end, and DNA homologous to spoVAA and about half of spoVAB at its 3' end. Computer analysis of the spollA sequence indicated that the B megaterium spoliA is also tricistronic (spollAA, SpolIAB and spollAC). Three putative ribosomal-binding sites (rbs) have been found complementary to the 16S rRNA of B megaterium [37] and are gAGGAGGT (spollAA), AAGGAGac (spollAC) and AAGGtGtg (spoVAA) (the lower case letters denote differences from the 16S rRNA). The free energies of binding have been calculated according to the method of Tinoeo [381 a s - 1 8 . 8 , - 1 2 . 8 and -11.4 Kcal mol-t for spollAA, SpolIAB and spoVAA, respectively. No obvious rbs could be detected in front of the spollAB gene. The first two spollA genes overlap by one nucleotide and may.be translationally coupled. In contrast, there is a region of 12 nucleotides between the stop codon of SpollAB and the start codon of spollAC, and contains the rbs for spollAC. The spollAA gene starts with a GUG codon, while spollAB and spoilAC both start with an AUG codon. The spoVAA gene starts with an AUG codon, and has a putative ribosomal binding site preceding it. The spacing between pbp and spollAA, and between spollAC and spoVA of 94 and 159 nucleotides, respectively, differs markedly from B subtilis (see Distussion). A putative strong rho-independent terminator was found following the spoliAC stop codon. The free energy of the hairpin formation was calculated by the method of Zucker [39] as -24.4 Kcal mol-I. There was no obvious terminator found after the pbp stop codon, which may be explained by the observed pbp-spollA transcript detected by Northern hybridization (see below). A terminator of spollA transcription has also
been reported for both B subtilis and B licheniformis [40,411. The spollAA, spollAB and spollAC open reading frames code for predicted proteins with 116 aa (13 kDa), 147 aa (16 kDa) and 253 aa (29 kDa), respectively, and are compared in figure 3 to the corresponding proteins in B subtilis and B licheniformis of 117 aa, 146 aa, and 255 aa [15, 41]. Amino acid homology among the three organisms is very high. Conservation between B megaterium and B subtilis is 85, 89 and 92%, and between B megaterium and B licheniformis it is 79, 88 and 91% for SpolIAA, SpolIAB and SpolIAC, respectively.
Transcriptional expression of spoilA It has been reported that B subtilis spollA has two transcripts expressed at different times [28, 42]. Savva and Mandelstam [42] reported that the smaller transcript (the spollA operon alone) becomes detectable at h, and that the larger transcript (a read-through product from the pbp gene) [28] is detectable at t3. To show that our gene is functional in vivo and to determine transcript size and time of expression, wild-type PV361 cells were grown in SNB broth and the RNA was isolated from to to ts, as described in Materials and methods. Northern hybridizations were carried out on duplicate filters using one probe within the pbp gene and another probe, a 0.63-kb EcoRI-Bglll fragment spanning parts of the spollAA gene and spoliAB gene (fig 1). As shown in figure 4 (right), two transcripts could be detected with the spoliA specific probe from stage to to ts. The smaller transcript was 1.77 kb and was detectable at to and most abundant at h, while the larger transcript was 2.92 kb, also detectable at to, but with the maximal amount between t2 and ta.5. In order to find out whether the larger transcript was from the 5' end of the cloned fragment, a 0.5-kb Hindlll-EcoRV fragment (from the pbp homologous gene at the 5' end of our 3.34-kb clone, see fig I) was found to hybridize with one band from to to t5 mRNA samples, and the band was exactly the same size as the larger band using the spoilA-specific probe mentioned above (fig 4, left). This indicates that two sizes of transcripts occur in B megaterium, as also reported in B subtilis.
Discussion The 3.34-kb HindIII fragment contains the entire B megaterium spollA operon. Preliminary sequencing also suggests that part of the B megaterium genes homologous to pbp and the spoVA region (spoVAA and part of spoVAB) are present. The observed overlap between spollAA and spollAB has also been reported in the corresponding regions of B subtilis and B licheniformis [41]. Similarly, the space between spollAB
700 Bm SpoIIAA Bs SpoIIAA B1SpoIIAA
VSLSIELEFK QDVLLVRLTG ELDHHTAEEL RSKITEAIEK ESISHLILNL MSLGIDMNVK ESVLCIRLTG ELDHHTAETL KQKVTQSLEK DDIRHIVLNL MSLGIDIHVK ~SVLCIRLTGELDHHTAET& ~KOVSGHLEQ TD~RHIVMNL
Bm SpoIIAA Bs SpoIIAA B1SpoIIAA
QHLTFMDSSG LGVILGRYKQ VHN~GGEMVV CAISPAIERL FNMSGLFKII EDLSFMDSSG LGVILGRYKQ IKQIGGEMVV CAISPAVKRL FDMSGLFKII IKOLGGEMIV C A I S P A V K R L F ~ A D ~
Bm SpoIIAA Bs SpoIIAA B1SpoIIAA
RFEPNEANAL QKLGVA** RFEQSEQQAL LTLGVAS* E L ~ Q S ~ Q R ~ ETLGVAS*
Bm SpoIIAB Bs SpoIIAB B1 SpoIIAB
MKNQMNLQFS ALSQNESFAR VTVASFITQL DPTMDELTEI KTVVSEAVTN MKNEMHLEFS ALSQNESFAR VTVASFIAQL DPTMDELTEI KTVVSEAVTN KTVVSEAVTN ~IOFT ZL~VTVAAFIAOL
Bm SpoIIAB Bs SpoIIAB B1 SpoIIAB
AIIHGYESNS D G W Y I S V T L HEDGVVELMI RDEGIGIDNV DEAKQPLYTT AIIHGYEENC EGKVYISVTL -EDHVVYMTI RDEGLGITDL EEARQPLFTT AIIHGYENSG OGNVYISVTL -EDHIVYLTI RDEGVGIPDL E~2~QELEX2
Bm SpoIIAB Bs SpoIIAB B1SpoIIAB
KPDLERSGMG FTIMENFMDE IRVESTVLEG TTLYLKKHLT NSKALCN* KPELERSGMG FTIMENFMDD VSIDSSPEMG TTIRLTKHLS KSKALCN* ~ F _ ~ ISIDSSPEM~ TTIHLTKHLS F ~ *
Bm SpoIIAC BS SpoIIAC B1SpoIIAC
M.DVEVKKNKN EPYLKDHEVK DLIKRSQQGD QIARDTIVQK NMRLVWSVVQ MDVEVKKNGK NAQLKDHEVK ELIKQSQNGD QQARDLLIEK NMRLVWSVVQ MDVEVKKENQ ~TOLKDHEVKELIKNSONGD ~ K ~ L L I E K ~
Bm SpoIIAC Bs SpoIIAC B1SpoIIAC
RFINRGYEPD DLFQIGCIGL LKSVDKFDLS YDVKFSTYAV PMIIGEIQRF RFLNRGYEPD DLFQIGCIGL LKSVDKFDLT YDVRFSTYAV PMIIGEIQRF ~ / ~ D . ~ ~ YDVRFSTYAV~II.~%~J.Q~
Bm SpoIIAC Bs SpoIIAC B1SpoIIAC
IRDDGTVKVS RSLKEMSNKI RKAKDELSKL LGRVPTVAEV AEHLDLTPEE IRDDGTVKVS RSLKELGNKI RRAKDELSKT LGRVPTVQEI ADHLEIEAED ~ RSLKELGNKI ~ S NGRIPTVOEIADYLEISSEE
Bm SpoIIAC Bs SpoIIAC B1SpoIIAC
VVLAQEANRA PSSIHETVYE NDGDPITLLD QIADHTEAKW FDKIALKEAI VVLAQEAVRA PSSIHETVYE NDGDPITLLD QIADNSEEKW FDKIALKEAI ~ V E S ~ ~ I C ~ OIADOSEEKW EDEIALKE~/
Bm SpoIIAC Bs SpoIIAC B1SpoIIAC
EELDEREKLI VYLRYYKDQT QSEVAARLGI SQVQVSRLEK KILKQMKLHM SDLEEREKLI VYLRYYKDQT QSEVAERLGI SQVQVSRLEK KI LKQIKVQM K ~ ~ K 2 O S E V A D R L G I Z.0_V_0_V. ~ L E~ KILKOIKNQ~
Bm SpoIIAC Bs SpoIIAC B1 SpoIIAC
NDT* DHTDG* ~HFES*
Fig 3. Alignment of SpolIAA, SpolIAB, and SpolIAC proteins of B megaterium (Bm), B subtilis (Bs), and B licheniformis (BI). Conserved amino acids are defined as the following groups [51 ]: (I, L, M, V), (H, K, R), (D, E, N, Q), (A, G), (F, Y, W), (S, T), (P) and (C). Positions at which three residues are identical or conserved are underlined.
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Kb
Fig 4. Northern hybridization of spollA transcripts. RNA was isolated as described in Materials and methods. Left: probed with pbp-specific DNA; right: probed with spoIIAA-spoIIAB-specific DNA (see fig l ).
and spollAC genes in each of these organisms is 12 nucleotides long, and the rbs preceding spoliAC suggests that B megaterium spollAC is also translationally independent. The spollA promoter regions of the three species, as shown in figure 5A, are very conserved. An 'OA box', TGNCGAA, where SpoOA binds to regulate gene expression [43], has been found preceding several B subtilis genes. A putative B megaterium OA box (TGACGAA) has been detected and matches that of B subtilis. Wu et ai [40] have reported that transcription of B subtilis spollA was dependent on spoOA. Recently, Hoch (personal communication) has shown that SpoOA acts as a positive regulator on B subtilis spollA exp,ession. It will be interesting to see if the B megaterium OA box has the same function. The nucleotide sequences between spollAC and spoVAA were compared among the three organisms. The putative open reading frame of B megaterium spoVA begins 159 base pairs (bp) downstream of the spollAC, instead of 166 bp for B licheniformis [41, 44], or 124 bp for B suhtilis [15, 44, 451. Moldover et al [44] compared the sequence of the spoVA upstream regions of B subtilis and B licheniformis, and discovered four regions that are perfectly conserved between these two species. These are an identical 20-bp region I, the -35 region, the -10 region and the region IV, respectively. They speculated that the unique 20-bp region may be a control region specific to spoVA, based on the fact that no such homology was detected between this region and the corresponding upstream regions of gerA and ssp genes, although all of these genes share extensive homology with the -35 and -10 regions of spoVA and are considered all transcribed by o<;. The putative B megaterium '20 bp region' preceding the -35 region of spoVA was detected (see fig 5B) as reported in the other two organisms; however, the sequences between B subtilis and B licheniformis are identical, while the B megaterium spoVA region matches 17 of the 20 nucleotides. The identification of this region in B megaterium, a much
more divergent species from B subtilis and B licheniformis [ 19], further suggests the important function of this region in spoVA regulation though it remains to be demonstrated. The putative -35 and -10 regions of B megaterium spoVA are detected and highly homologous to those of B subtilis and B licheniformis (fig 5B). Region IV, which is 8 bp in length and is identical between B subtilis and B licheniformis as reported by Moldover et al [44], may also be present in the B megaterium spoVA promoter region. It is the least conserved region of the four regions in the three species. Only five bp out of the eight nucleotides are conserved and the spacing between III and IV is eight nucleotides longer than in the other two species. Thus, the potential function of region IV in spoVA gene regulation is uncertain. Northern hybridization has confirmed the gene expression and temporal regulation at the transcriptional level and has determined the transcript size of spollA. The time of spollA expression from Northern hybridization corroborated well with the data of the ~-galactosidase activity assay, in which 13-galactosidase activity of the spo-58-1acZ fusion mutant was detected at to [21]. This is the first reported comparison of 13-galactosidase assays with transcriptional results in B megaterium. Savva and Mandelstam [42] reported that there were two spollA transcripts in B subtilis: 1.7 kb and 2.6 kb. The smaller transcript was first detected at t,, but peaked at t2, while the larger one was not detected until t~ and peaked at ts. Wu and Piggot [28] further showed that the larger transcript was a read-through product from the pbp gene upstream to spollA. However, they reported that this transcript appeared at t4 and was 2.9 kb instead of 2.6 kb. Our result agreed with most of the data from B subtilis since there were two spoilA transcripts in B megaterium and the sizes of the transcripts were similar [28]. However, in B megaterium both transcripts appeared earlier (both were present at to, possibly even earlier). The small transcript was detected about l h earlier than that of B subtilis, and the
702 2.92-kb transcript about 3-4 h earlier than the corresponding transcript of B subtilis. A simple explanation could be due to the sensitivity of the detecting methods. Another reason could be the different methods used to designate the onset of sporulation. Savva and Mendelstam [42] designated to by resuspension of the culture in sporulation medium while in
this study to was determined as the time when growth deviated from exponential phase. In a recent study of the role of a v in prespore-specific transcription, Partridge et al [12] reported that there must be a low level of expression of spoIIAC in vegetative cells. However, it is clear from the data presented here that the appearance and maximum expression of the larger
A
-35
OA b o x BmZIA BsZIA BIIIA
AACAGCTAAATGTGACGAAATAGAACTAGTTTTGTCGGTGAGAAGGATTCTATT TTTAAGTAATTATGCCGAATGACCACTAGTTTTGTCACGGTGAAGGAATTCATT TGGAGTGAATAATGCCGAACGGTCACTAGTTTTGTCACGGTGAAGGAATTTATA
BmIIA BsZZA BIZZA
-10 :bs CTGTCGGCAGCGAAAGACTGACTATAAGATTTCCG...AGGAGGTTTGTATAGTG CCGTCGAAATCGAAACACTCATTATCCGATCATAT.C~AATGAGC.ATG AAGTCTGAAGCGAAACACTCATTATCCGATTTAAACCAAGGAGGAATGAGG.AT$
X (20 n t box) BmV& BsVA B1VA
I":!: (-35)
TTGGTAATTAATGGTCTGTTCAGAAACG.TGAATTAGAAAATTTAAGAAAATC GTGGTAATTTATGGTCTTTTCGAGCGGA.TGAATGAGAACAAAATCGAACCA. GTGGTAATTTATGGTCTTTTGCGCCTTTCTGAATGATAAATGGGATGTACTT.
XXX
XV
zbs
(-10) BmV& BsVA BIVA
CATACTACACGTACAGGGAGATAATGAATC.GTAAGGTGTGATATGA..ATG C A T A C T A C A T A ........ T A T A A C C A C C . . G A A A G A T G G T G A T C A A T G A T G C A T A C T A C A A C ........ T A T A A C C A T C A T A T . A G G A A G T G A C C C A . G A T G
Fig 5. Alignment of B megaterium (Bm), B subtilis (Bs), and B licheniformis (BI) promoter regions. A. Comparison with B subtilis [40] and B licheniformis [41] spollA promoters. B. Comparison with B subtilis and B licheniformis spoVA promoters (Moldover et al [44]). Lines indicate conserved regions between B subtilis and B licheniformis (solid) or all three species (dotted). Ribosomal binding sites and start and stop codons are underlined.
703
pbp-spoliA transcript in B megaterium was earlier than that observed in B subtilis by at least 3 h. In Bacillus, it has been shown that sporulation can be blocked by penicillin at two different stages, septum formation and also during spore cortex formation [46]. Penicillin binding proteins (PBPs) are enzymes involved in the metabolism of peptidoglycan. In B megaterium strain KM, Todd and Ellar [47, 48] reported that there were two sporulationspecific PBPs, PBP 3e and PBP 5a, both which were involved in cortex maturation. At present, it is unknown whether our PBP is either of these. If it is, it is unclear why the B megaterium pbp-spolIA transcript was present at to, and its product would not be needed until spore cortex maturation (stage IV). Illing and Errington [16] have recently shown that there is a small amount of peptidoglycan present in the spore septum. A pure speculation would be that our PBP from the pbp-spolIA transcript is involved in this kind of peptidoglycan synthesis. The presence of an 'OA box' in B megaterium spoIIA upstream region suggests that this species has an homologous spoOA gene. The promoter also suggests by its consensus sequences that B megaterium has a on gene and indeed, Fortnagle and coworkers have cloned the spoOH gene of B megaterium [49]. We have recently cloned and sequenced the spoVG gene from B megaterium, a gene transcribed in B subtilis by Ecja [52]. The promoter region of the putative spoVA operon is also conserved with a consensus sequence for ¢~, suggesting that this sigma factor is also present in B megaterium.
B megaterium, suggesting that it is, indeed, important. We have reported [50] tha~ several of the ssp gene loci have been conserved, even though the flanking genes have not. By comparing the limited number of sporulation genes from B megaterium with other bacilli, a pattern of extensive conservation of regulatory genes is suggested. The region that has been cloned containing the spollA operon is very conserved, although not identical, among three Bacillus species. It can also now be inferred, although not proven, that B megaterium has sigma factors oF, o'G, ¢~A,and ¢~s. Note added in the proof
A B megaterium gene homologous to spo0A has recently been cloned (P Youngmann, personal communication). Acknowledgments This research was supported by National Science Foundation ROW Career Advancement Award 8908929 (PSV), and NIH Fogarty International Fellowship 1 F06 TW01540-01, and by BRSG S07 RR071 awarded by the Biomedical Support Grant Program, Division of Research Resources, the National Institutes of Healths.
References l 2
Conclusion The B megaterium spollA operon has been cloned and its sequence is highly homologous to those of B subtiffs and B lichenijbrmis. We have shown that the spo58 mutant is in a gene homologous to the spollAC gene that codes for sigma factor oF in B subtilis. Comparison of the gene organization and gene expression of all the sporulation genes carried on the cloned fragment to the corresponding sporulation genes in B subtilis and B licheniformis revealed that these genes are very conserved in location, structure and fairly conserved in expression. B megaterium spollA operon contains three genes comparable in size (smaller by one to two amino acids) to the corresponding proteins in the other two species. There are also two spollA transcripts present in B megaterium, as reported in B subtilis, but with differences in timing of expression. The promoter regions of spollA and spoVA ope~ons are highly conserved among the three species. The 20 nucleotide conserved region of both B subtilis and B iicheniformis as identified by Moldover et al [44] as being unique to spoVA operon regulation, is also present, although less conserved, in
3 4
5 6
7 8 9
10
Freese E (1981) Initiation of bacterial sporulation. In: Sporulation and Germination (Levenson HS, Sonenshine AL, Tipper DJ, eds) Am Soc Microbioi, l - l 2 Soneshein L (1989) Metabolic regulation of sporulation and other stationary-phase phenomena. In: Regulation of Procaryotic Development: Structural and Functional Analysis of Bacterial Sporulation and Germination (Smith I, Slepecky RA, Setlow P, eds) Am Soc Microbiol, 109-130 Piggot PJ, Coote JG (1976) Genetic aspects of bacterial endospore formation. Bacteriol Rev 40, 908-962 Piggot PJ (I 989) Revised genetic map of Bacillus subtilis 168. In: Regulation of Procaryotic Development: Structural and Functional Analysis of Bacterial Sporulation and Germination (Smith I. Slepecky RA, Setlow P, eds) Am Soc Microbiol, I--41 Losick R, Pero J (1981) Cascades of sigma factors. Cell 25, 582-584 Moran CP Jr (1989) Sigma factors and the regulation of transcription. In: Regulation of Procaryotic Development: Sn'uctural and Functional Analysis of Bacterial Sporuiation and Germination (Smith I, Slepecky RA, Setlow P, eds), Am Soc Microbiol, 167-184 Stragier PRL (1990) Cascades of sigma factors revisited. Mol Microbioi 4, 1801-1806 Burbulys D, Trach KA, Hoch JA (1991) Initiation of sporulation in B subtilis is controlled by a multicomponent phosphorelay. Cell 64, 545-552 Healy J, Weir J, Smith I, Losick R (1991) Post-transcriptional control of a sporulation regulatory gene encoding transcription factor oH in Bacillus subtilis. Mol Microbiol 5,477-487 Kroos L, Kunkel B, Losick R (1989) Switch protein alters specificity of RNA polymerase containing a compartmentspecific sigma factor. Science 243, 526-529
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31
Stragier P, Kunkel B, Kroos L, Losick R (1989) Chromosomal rearrangement generating composite gene for a developmental transcription factor. Science 243, 507-512 Partridge SR, Foulger D, Errington J (1991) The role of o F in prespore-specific transcription in Bacillus subtilis. Mol Microbiol 5, 757-767 Schmidt R, Margolis P, Duncan L, Coppolecchia R, Moran CPJ, Losick R (1990) Control of developmental transcription factor o F by sporulation regulatory proteins SpolIAA and SpoIIAB in Bacillus subtilis. Proc Natl Acad Sci USA 87, 9221-9225 Errington J, Fort P, Mandelstam J (1985) Duplicated sporulation genes in bacteria. FEBS Lett 188, 184-188 Fort PJ, Piggot PJ (1984) Nucleotide sequence of sporulation locus spoliA in Bacillus subtilis. J Gen Microbiol 130, 2147-2153 tiling N, Enington J (1991) Genetic regulation of morphogenesis in Bacillus subtilis: Roles of o E and o r: in prespore engulfment. J Bacterioi ! 73, 3 ! 59-3169 Margolis P, Driks A, Losick R (1991) Establishment of cell type by compartmentalized acti~ ~tion of a transcription factor. Science 254, 562-565 Vary PS (1992) Development of genetic engineering in Bacillus megaterium. In: Biology of Bacilli (Doi R, ed) Butterworths, 253-311 Priest FG, Goodfeliow M, Todd C (1988) A numerical classification of the genus Bacillus. J Gen Microbiol 134, 1847-1882 Vary PS, Tao YP (1988) Development ofgenetic methods in Bacillus megaterium. In: Genetics and Biotechnology of Bacilli (Ganesan AT, Hoch JA, eds) Vol 2, Academic Press, 403-407 Tao YP, Vary PS (1991) Isolation and characterization of sporulation lacZ fusion mutants of Bacillas megaterium. J Gen Mic'robioi 137, 797-806 Levine M (1957) Mutations in the temperate phage P22 and lysogeny in Salmonella. Virol 3, 22-41 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular chining: A Laboratory Manual. Cold Spring Harbor LaboratoryPress, Cold "Spring Harbor, NY English ID. Vary PS (1986) Isolation of recombination detective and UV sensitive mutants of Bacillus megaterium. J Bacterio165, 155-160 Kawamura F, Wang LE Doi RH (1985) Catabolite-resistant sporulation (crsA) mutations in the Bacillus subtilis RNA polymerase ¢P~ gene (rpoD) can suppress and be suppressed by mutations in spoO genes. Proc Natl Acad Sci USA 82, 8124-8128 Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmids. Nucleic Acids Res 7, 1513-1523 Bovre K, Szybalski W (1971) Multistep DNA-RNA hybridization techniques. In." Methods in En:ymologv (Grossman L, Moldave K, eds) Vol XXI, Academic Press, 350-383 Wu JJ, Piggot PJ (1990) Regulation of late expression of the Bacilhas subtilis spoilA locus: evidence that it is cotranscribed with the gene for a putative penicillinbinding protein. In: Genetics and Biotechnology of Bacilli (Zukowski M, Ganesan Z, Hoch AT, eds) Vol 3 Academic Press, 321-327 Anagnostopoulos C, Spizizen J (1961) Requirements for transformation in Bacillus subtilis. J Bacteriol 8 !, 741-746 Kalman S, Duncan M, Thomas S, Price CW (1990) Similar organization of the sigB and spollA operons encoding alternate sigma factors of Bacillus subtilis RNA polymerase. J Bacteriol ! 72, 5575-5585 Grunstein M, Hogness DS (1975) Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci USA 72, 396 i-3965
32 Reed KC, Mann DA (1985) Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res 13, 7207--7221 33 Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1987) Current Protocols in Molecular Biology Vol 1, Wiley-Interscience, NY 34 Von "l'ersch MA, Carlton BC (1983) Megacinogenic plasmids of Bacillus megaterium. J Bacteriol 155, 872-877 35 Youngman PJ, Perkins JB, Losick R (1983) Genetic transposition and insertional mutagenesis in Bacillus subtilis with Streptococcus faecalis transposon Tn917. Proc Natl Acad Sci USA 80, 2305-2309 36 Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85, 2A.a.-~. 2448 37 Fliss ER, Setlow P (1984~ Complete nucleotide sequence and start sites for transcription and translation of the Bacillus megaterium protein C gene. J Bacteriol 158, 809-813 38 Tinoco I, Borer PN, Dengler B, Levine MD, Uhlenbeck OC, Crothers DM, Gmlla J (1973) Improved estimation of secondary structure in ribonucleic acids. Nature New Bio1246, 40--41 39 Zucker M (1989) On finding all suboptimal foldings of an RNA molecule. Science 244, 48-52 40 Wu JJ, Howard MG, Piggot PJ (1989) Regulation of transcription of the Bacillus subtilis spollA locus. J Bacterioi 171,692-698 41 Yudkin MD, Appleby L, Smith AJ (1989) Nucleotide sequence of the Bacillus iicheniformis homologue of the sporulation locus spollA of Bacillus subtilis. J Gen Microbiol 135, 767-775 42 Savva D, Mandelstam J (1986) Synthesis of spoliA mRNA and spoVA mRNA in Bacillus subtilis. J Gen Microbioi 132, 3005-301 ! 43 Strauch M, Webb V, Spiegelman G, Hoch JA (1990) The SpoOA protein of Bacillus subtilis is a repressor of the abrB gene. Proc Natl Acad Sci USA 87, 1801-1805 44 Moldover B, Piggot l-J, Yudkin MD (1991) Identification of the promoter and the transcriptional start site of the spoVA operon of Bacillus subtilis and BaciUus Iicheniformis. J Gen Microbiol 137, 527-53 I 45 Fort P, Errington J (1985) Nucleotide sequence and complementation analysis of a polycistronic sporulation operon, spoVA, in Bacillus subtilis. J Gen Microbiol 131, 1091-1105 46 Sowell MO, Buchanan CE (1983) Changes in penicillinbinding proteins during sporulation of Bacillus subtilis. J Bacterioi 153, 1331-1337 47 Todd JA, Ellar DJ (1982) Alteration in the penicillinbinding profile of Bacillus megaterium during sporulation. Nature (Lond) 300, 640-643 48 Todd JA (1983) Identification of penicillin-binding protein 5a of Bacillus megaterium KM as a DD-carboxypeptidase. Biochem J 214,653-655 49 Naumann G, Nertzke KD, Ribbe J, Honerlage W, Fortnagei P (1990) The Bacillus megaterium spoOH gene. Abstr. Gene Manipulations in Bacteria 53 50 Sussman MD, Vary PS, Hartman C, Setlow P (1988) Integration and mapping of Bacillus megaterium genes which code for small, acid-soluble spore proteins and their protease. J Bacteriol i 70,4942-4945 51 Helmann D, Chamberlin MJ (1988) Structure and function of bacterial sigma factors. Annu Rev Biochem 57, 839- 872 52 Hudspeth DSS, Vary PS (1992) spoVG sequence of Bacillus megaterium and Bacillus subtilis. Biochim Biophys Acta ! 130, 229-23 i
695
Cloning and sequencing of the Bacillus megaterium spollA operon YP Tao**, DSS Hudspeth, PS Vary* Department of Biological Sciences, Northern Illinois University,Dekaib, IL 60115, USA (Received 5 January 1992: accepted 20 February 1992)
Summary - - The spollA operon of Bacillus megaterium has been cloned and the nucleotide sequence determined. The spoliA sequence contains three open reading frames coding for putative proteins of 116 aa, 147 aa, and 253 aa; the first and the third genes are preceded by a ribosomal binding site. The genes are in the same order as those of B subtilis and B licheniformis. The deduced amino acid sequences of these three open reading frames show 78-92% homology with SpolIAA, SpolIAB and SpolIAC of B subtilis and B licheniformis. Northern hybridization revealed that B megaterium also has two spollA transcripts, 1.77 kb and 2.92 kb, auaining maximum expression at t~ and t3, respectively. In addition, homology to a possible penicillin binding protein gene upstream and the first part of a spoVA operon downstream has been identified on the 3.34-kb fragment. The spollA and the downstream spoVA promoter regions are highly conserved among these three species. Sequence analysis of the spoVA promoter revealed a region upstream to the -35 that is highly conserved across Bacillus species.
Bacillus megaterium / sporulation / spollA / spoVA / pbp
Introduction Cells of the Gram-positive genus Bacillus can differentiate into dormant spores upon nutrient deprivation [ 1, 2]. This process of spore formation in Bacillus provides a relative simple experimental system for use in the study of cellular development and differentiation. Sporulation is divided into six or seven distinct morphological stages and involves the coordinated expression of at least 50 spo loci [3, 4]. Many of the spo genes have now been cloned and sequenced, and the function of their gene products is beginning to be understood. Several mechanisms have been found to be involved in the complex regulation of these genes. For example, sequential appearance of new sigma factors that bind to core enzyme and confer different promoter specificity allows transcription of new sets of sporulation genes [5-7]. A second mechanism is the release of spo genes through a two-component system from the repressive effects of a repressor such as AbrB [8]. Increase of sigma factor concentration *Correspondence and reprints **Present address: Waksman Institute of Microbiology, Rutgers University, Piscataway, NJ 08854, USA. Abbreviations: bp, pase pair(s); X-gal, 5-bromo-4-chloro-3indolyl-[~-D-galacto pyranoside; IPTG, isopropyl-~-D-thiogalactopyranoside; rbs, ribosomal-binding site(s); PBP, penicillin-binding proteins; ORF, open reading frame; PG, peptidoglycan; aa, amino acid(s).
during sporulation is a third mechanism. For example, o H increases five-fold in stationary phase from vegetative phase levels because of increased translation or stability of the spoOH mRNA and decreased turnover of o H [9]. Auxiliary factors other than sigma factors can serve as activators or repressors of transcription such as the 'switch protein' SpoIIID [10], which is a transcriptional activator of the spolVCB promoter, but is also a repressor of cotA and corD genes. Other regulatory mechanisms include the activation of sigma factors or processing of pro-o factors into mature forms. Examples are oK and oE processing [11], as well as changes in oF activity, which is inhibited by SpoIIAB [12, 13], but then released by SpoIIAA, the antagonist of SpoIIAB [13]. It is obvious that there are controls at transcription: post-transcription, translation, and post-translation levels. In B subtilis, there exist at lea~t nine different sigma factors in phage-uninfected cells [6]. It was previously proposed that developmental gene expression during sporulation is controlled by cascade of sigma factors [5]. It is now well established that the program of sporulation gene expression is largely governed by the regulated appearance of new sigma factors in the following dependent order: oa --> oF --> aE --> Ot:; --> OK [7]. One of these, oF, is encoded by the spollAC (or sigF) gene. The spollAC gene is the promoter-distal gene of the tricistron operon in B subtilis called spollA, the promoter-proximal cistrons being spollAA and spollAB [14, 15]. Strains mutated in spollAC
696 form aberrantly septated sporangia that are either disporic or multiseptate cells, which suggests that o F is required for the transcription of genes involved in forespore engulfment [16]. Recently, Partridge et al [12] have shown by using an inducible promoter to activate spollA expression in vegetative cells, that oF itself is necessary and sufficient to direct spolllG (o~3) transcription. Their results also indicated that another function of o F (independent of its sigma factor function) is the processing of o E. The work of Schmidt et ai [13] has shown that S p o l I A A is an antagonist o f S p o I I A B and that SpoIIAB is, in turn, an antagonist of SpolIAC. They speculated that the S p o l I A A / S p o I I A B / S p o l I A C regulatory system could play a role in controlling the timing and/or the location (the forespore chamber of the sporangium) for oF-directed gene expression. Interestingly, Margolis et al [17] have shown that o F is only active in the forespore, yet is required for the processing o f o E in the mother cell. Thus, the spollA operon is o f great interest and of central importance because it helps to govern the transition from a single cell to a sporangium consisting of two cellular compartments with different developmental fates. Since B megaterium is very efficient at both sporulation and germination, it is well suited for the studies of sporulation and has been used extensively for biochemical analysis of both sporulation and germination (for review see [18]). It is also a more divergent species from B subtilis and B iicheniformis [19] so that a study of sporulation in this species provides a unique opportunity to also compare sporulation among Bacillus species. The information that emerges from the comparison should be very useful in elucidating conserved (and thus critical) regulation of sporulation and in understanding the evolution of sporulation. In the past few years, we have developed genetic techniques in this species and have isolated several mutants by transposon-mediated mutagenesis [20]. We have characterized several Tn917-1acZ fusion spo mutants [21 ] and showed by electron microscopy that one of these, spo-58, formed aberrant multiple spore septa and expressed ~-galactosidase activity at approximately to. We report here the cloning and sequencing of the spo-58 gene as well as the entire B megaterium spollA operon. The gene organization, expression, and the predicted amino acid sequences of the operon are then compared with those of B subtilis and B licheniformis. M a t e r i a l s a n d methods Strains and plasmids All B megaterium strains used in this study were derived from QM B1551 (ATCC 12872). PV361 (plasmidless, wild-type) was constructed by curing all seven resident plasmids (p-T)
[20]. PV447 (p-7 lac-3 iac-6) is a Lac- derivative of PV361 120]. PV510 (p-7 iac-3 lac-6 spo-58::Tn917-1acZ-cat) was constructed by selecting a Tn917-1acZ-cat fusion insertion spo mutant in PV447 and was used to isolate genomic DNA for cloning spollA mutant gene [21]. E coli strain TGI [supE hsdD5 thi (Alac-proAB)/F'(traD36 proA+B+ laciq lacZ AMI5)] (a gift from J Rapoport, Institute Pasteur) was used as host. Integratior~al plasmid pJHl02 was provided by JA Hoch (Scripps Clinic). Media LB (Luria-Bertani) medium [22] was used for plasmid isolation and growing E coli for transformation. M9 medium [23] with 25 ~tg/ml of thiamine was used for maintaining TGI with its F'. B megaterium strains were grown in supplemented nutrient broth (SNB) medium, described previously [24]. Antibiotic concentrations were 5 ttg/ml erythromycin, 250 I.tg/ml lincomycin (MLS), 50-100 I.tg/ml ampicillin, and 4 I.tg/ml chloramphenicoi. Nucleic acid isolation The miniscreen procedure of Kawamura et ai [25], a modification of the Birnboim and Doly procedure [26], was used to isolate small amounts of plasmid DNA. Larger-scale plasmid DNA preparation was done by the method of Birnboim and Doly [26]. The method of Bovre and Szybalski [27] was used to isolate B megaterium genomic DNA. RNA was prepared by the method of Wu et ai [40] with the following modifications. PV361 was grown in SNB broth at 30°C with shaking at 300 rpm. The time when growth deviated from exponential at ODt,60 was designated as to. PV361 cultures (60 ml) taken from to to t5 were harvested by centrifugation in centrifuge bottles containing a half volume (30 ml) of semi-frozen IX Spizizen salts [29] and RNA was isolated by the method of T Henkin (personal communication). Cells were disrupted by vortexing with glass beads in the presence of vanadyl ribonucleoside complexes [30] (BRL) to inhibit RNAase activity. Finally, RNA was resuspended in diethylpyrocarbonate-treated water (0.1% by volume), and RNasin (1301, Promega) was added to the final concentration of 2 U per ~tl. Nucleic acid hybridization The method of Grunstein and Hogness [31] was followed for colony hybridization using Nylon-I (BRL) or 85-mm nitrocellulose circles (Schleicher and Schuell). Prehybridization was done according to the protocol from Amersham. Hybridization was carried out in the same solution as above, with the addition of DNA probe labeled by random priming (BRL) using [tx-32p] dATP. Hybridization was carded out for at least 12 h at 65°C. Filters were then washed twice in 2 × SSC, 15 min each time, once in 2 x SSC with 0. ! % SDS for 30 min, and finally in 0.1 x SSC for 10 min. For autoradiography, the filters were wrapped in Saran wrap and exposed to Kodak XAR-film with intensifying screen (DuPont Lightning Plus) at -70°C for the desired time. Transfer hybridizations were done by the alkaline capillary blotting method of Reed and Mann [32]. The method of Ausubel et al [33] was used for Northern hybridizations with the following modifications. About 10 ~tg of RNA from each sample was loaded into a 1.2% agarose denaturing gel containing 37% formaldehyde, with an RNA ladder (0.24-9.5 kb, BRL) as a size marker. Transfer to Hyband-N+ nylon membrane (Amersham) instead of a nitrocellulose membrane was carded out in 20 x SSC overnight.
697 Prehybridization was at 65°C as described for colony hybridizations.
DNA manipulation Restriction enzymes were purchased from IBI, BRL or Boehringer Mannbeim and were used as recommended by the suppliers. Restriction DNA fragments were recovered from agarose gels by electroelution (IBI). The Random Primers DNA Labeling System (BRL) was used to radioactively label probes. The Stratagene Klenow Fill-in Kit was used to make blunt ends of insert or vector DNA which had 5'-overhangs. Dephosphorylation of vector DNA was carded out as described by Sambrook et al [23] using calf intestine alkaline phosphatase (Boehringer Mannheim). Ligation reactions were done using T4 DNA ligase (BRL) in 20 I.d volume at 12.5°C overnight with a 1:3 molar ratio of vector:insert DNA and 2 lal of T4 ligase.
The wild-type gene was then cloned by probing with the 1.64-kb AvaI-HindIII fragment from pYP4 (containing B megaterium D N A plus 10 bases from the distal end of Tn917). Southern hybridization and autoradiography of HindIII-digested PV361 genomic DNA identified a 3.34-kb band (data not shown). The fragments were recovered and ligated to pUC18 cut with HindIII and transformed into TG1. Among 475 white ampr colonies tested, two hybridized with the probe. Restriction analysis and Southern hybridization showed that one of them (termed pYPS, see fig 1 ) had the correct size insert (3.34 kb). The promoter region was subcloned by ligating a 0.7-kb HindIII-EcoRI fragment from pYP5 to HindIII-EcoRI digested pUC 19 to yield pYP9 (fig 1).
Transformations
Complementation analysis
E coli competent cells and transformation were as described by Sambrook et al [23]. The PEG-mediated protoplast transfor-
In order to prove that the correct gene had been cloned, the genes on the 3.34-kb fragment were introduced into the spo-58 mutant to see if it would complement the mutation. The 3.34-kb HindIII fragment was treated with PolI, Klenow fragment to generate blunt ends, and the fragment was cloned into pJM 102 cut with Sinai to yield pYP10. Vector p J M l 0 2 was constructed as an integrational plasmid in B subtilis (J Hoch, M Perego, personal communication). It has the ability to replicate autonomously in E coli, but not in Bacillus and contains a cat gene which can be selected in Bacillus. Plasmid p Y P I 0 (about 10 t.tg) was used to transform the spo-58 mutant (PVSI0). Four colonies were recovered on SNB containing 4 ~tg/ml Cm and were streaked and incubated on the same medium. After 24 h incubation, about 80% became free spores while no spores were detected in the mutant strain PV510 grown under the same conditions (but with MLS for maintaining Tn917). Therefore, the result verified genetically that the 3.34-kb fragment contains a region complementing the spo-58 mutant.
mation method of Von Tersch and Carlton [34] was used for megaterium except that cells were regenerated on non-selective RHAF plates overnight at 30°C, then replicated to SNB plus selective antibiotic plates. Transformants were seen after 24 h incubation at 30°C.
B
Doume-sirunued DNA sequencing The Sequenase version 2.0 (USB) protocol for dideoxysequencing was followed. After beat-denaturation, samples were loaded into sequencing gels containing 6% polyacrylamide or 5% 'Long Ranger' (AT Biochem, Malvern, PA) with 8.3 M urea. Electrophoresis was carried out in 1 x TBE buffer (0.1 M Tris base, 0.1 M boric acid, 2 mM Na2EDTA, pH 8.3). Primers were synthesized on an oligonucleotide synthesizer (ABI PCR-Mate). Results
Cloning of the B megaterium spollA genes We took advantage of the Tn917 insertion in spo-58 to clone the mutant gene by probing with a HindIlI-Aval fragment containing about 1 kb of the distal end of T n g 1 7 from plasmid p T V I [35]. Total chromosomal D N A from strain PV510 was cut with HindIII and hybridization with the probe gave a band of approximately 2.6 kb. HindIII-fragments in the 2.0-2.9 kb range were then recovered by electroelution, tested by dot hybridization, and ligated to HindIII-cut pUCI 8. E coli T G I competent cells were transformed and plated on LB amp X-gal plates. About 210 white ampr colonies were then tested by colony hybridization and two hybridized strongly with the original Tn917 probe. Restriction analysis and Southern hybridization showed that these two colonies had the correct insert (data not shown). One clone was saved as plasmid pYP4 (fig 1). This 2.6 kb insert consisted of about 1 kb from the Tn917-distal end and 1.6 kb from the spo-58 mutant downstream of the Tn917 insertion.
[
pYP9
I
F
,,,c
pYP5 Hill
I
RV RI
I I
I
pYP4-r
I I v,,
BII
HI
I
I
n
Iv,D] Sl
PI
Hill
I
I
I
I
1 ,500~
,
. i ~ ,iIP qlI
I !
Fig 1, Plasmids pYP4, pYP5 and pYP9. The solid triangle indicates the Tn917 insertion in spo-58. The bars indicate the two probes used in Northern hybridization (fig 4). Restriction sites are indicated as Cl (ClaD, HI (HgiAl), HIII (HindIII), RV (EcoRV), RI (EcoRI), BII (BgaIII), SI (Stul), PI (Pstl).
698 --
O&
box
GATATCGATGCTGCTTCTTGGTGGAAGATATTC~AACGTACATTCTCTATTTTCTCT~AAA~ATCTTAA~CAGCTAA%TGTGA~GA~ATAGAACTAGTTT 100 t
t
*
*
*
.
*
*
t
-35 zeg£oa -10 zQg£on rbs TGTCGGTGAGAAGGATT~TATT~TGTCGGCAGCGAkAGACTGACTATA~GATTTCCGAC~A~TTTGTATAGT~AGTCTTTCGATTGAATT~GAATTCAA 200 AC~AG~TGTCTTGTTGGTTCGATTAACAGGAG~ATTAGAT~ATCATACGGCAG~G~GCTAAGA~GC~%ATCACCGAAGCGATAGAGAAGGAATCT~TC 300 AGcC•TTTGATTTTAAATTTACAGCATTTAACCTTTATG••CA•CTCTGGCTTGGGGGTTATTTT•GGGAGATACAAAC•GGTTCATAATAACGGCGGGG t
t
*
.
.
.
.
~
400
t
AAATGGTTGTATGCGCTATTTCTCCTGCCATAGAACGTTTGTTTAATATGTCAGGCTTATTTAAAATTATTCGTTTTGAACCTAATGAAGCAAACGCG~T 500 TCAAAAATTGGGGGTGGCATAAT•AAAAATCAAATGAACCTCCAATTTTCCGCGCTCAGTCAAAATGAATCGTTTGCTCGCGTCACGGTAGCATCATTTA 600 TTACGCAGTTAGATCCAACAATGGATGAGTTAACAGAAATTAAAACAGTCGTATCTGAAGCAGTAACAAATGCTATTATTCACGGCTACGAAAGCAATTC 700 GGATGGCGTGGTATATATTTCTGTGACGCTTCATGAAGACGGTGTAGTCGAGCTGATGATTCGTGATGAAGGAATTGGTATCGATAACGTGGATGAAGCC 800 AA•CAGCCTTTATACACAACGAAACCAGATCTAGAACGTTCTGGAATGGGTTTCACCATAATGGAAAACTTTATGGACGAAATTCGAGTGGAGTCTACTG 900 rb. TATTAGAAGGTACGACCTTATATTTAAAAAAGCACTTAACAAATAGTAAAGCGCTATGTAATTAAGGAGACTTGCCAATGGATGTGGAGGTCAAAAAAAA 1000 TAAAAACGAACCGTATTTA~GGATCATGAAGTTAAAGACCTCATCAAGCGCAGTCAGCAAGGTGATCAAATTGCACGAGATACAATCGT~ABAAAAAT
II00
AT~G~CTCGTTT~TCGGTTGTTC~CGTTTTATTAACcGTGGTTAT~AGCCCGATGATTTATTTCAAATT~AT~TATA~G~CTATTAAAGTCCGTT~
1200
ATAAATTTGACTTATC~TAT~AT~TTAAATTTTCAA~ATAT~CTGTTCCAATGATTATTGGTGAAATTCAG~ATTTATTC~T~AT~AT~CAC~TAAA
1300
~QTTA~CC~TTCTCT~AAA~AAAT~A~CAATAAAATAC~TAAA~CAAAAQAC~A~CTTTCCAAATTACTT~QAC~c~TCCCTACA~TC~CTQA~QTTQ~A 1400 GAACATTTG~ATCTCACTCCTGAAGAA~TAGTGCTAGCTCAAGAAGCTAATCGT~CTCCTTC~TCTATTCACGA~ACCGTCTAT~AAAAT~AT~A~ATC
1500
CAATTACCC;TCTTGACCA~A TTGCTGAT;ATACGGAAG;GAAGTGGTT;GA TAAAATT;CCTTAAAAGIAGCAATTGAIGAACTTGAT:AGCGTGAAAA 1600 W
•
W
W
W
t
~
.
.
ATCTTAAAACAAATGAAGCTGCATATGAATGATACGTAATACT~AAGAAGCAATCCGTTTTGCTATTCAGAAGAATAGTGAAACGGATTGCTTTTTTTAT t
,
20 n~ box
.
-35
,
.
zegion
>>>>>>>>>>>>>>>>>>>
-10
zegion
<<<<<<<<<<<<<<<<<<<
1800 *
zbs
m~VAA
TTGGTAAT~AA~GTCTGTTCAGAAACGT~AATTAGAAAATTTAAGAAAAT~CATACTACACGTACAGGGAGATAATGAATCGTAAGGTGTGATATGA~]~ 1900 fiGAAAACGTTCTTTATCTTA
1920
Fig 2. Nucleotide sequence of the coding and flanking regions of the B megaterium spoliA operon. The asterisks below nucleotides are positioned every 10 bases. The putative ribosomal-binding sites, start codons and promoter regions ate labeled and underlined; stop codons are overlined. The filled triangle indicates the position of the Tn917 insertion in the spollAC gene. The symbols <<< and >>> are used to show that these two regions complement each other to form the rho-independent terminator.
Sequencing of spoilA The sources of DNA for sequencing were the wildtype 3.34-kb HindIII fragment contained on plasmid
pYPS, the subcloned 0.7-kb EcoRI-HindIII promoter region on plasmid pYP9 and the mutant DNA on pYP4 (see fig l). The site of the transposon Tn917 insertion in spo-58 was determined by comparing the
699 sequence from pYP4 with that from pYP5. The sequencing strategy is also shown in figure 1 and was done using synthetic primers as well as commercially available universal primers for pUCI 8 and pUC 19. The entire spollA operon including its promoter as well as the downstream spoVA promoter region was sequenced in both directions and the sequence is shown in figure 2.
Analysis of gene products and regulatory signals A computer search of Genbank for homologous nucleotide sequences using the FASTA program of Pearson and Lipman [36] identified a striking homology over much of the sequence with the B subtilis spollA operon sequence (69% identity within the coding region). Preliminary sequencing of the flanking DNA has also shown extensive homology to the pbp gene of B subtilis upstream, and the first two genes of the spoVA operon (data not shown). The data suggest that the 3.34-kb fragment consists of the entire B megaterium spoliA operon, approximately half of a gene homologous to pbp at its 5' end, and DNA homologous to spoVAA and about half of spoVAB at its 3' end. Computer analysis of the spollA sequence indicated that the B megaterium spoliA is also tricistronic (spollAA, SpolIAB and spollAC). Three putative ribosomal-binding sites (rbs) have been found complementary to the 16S rRNA of B megaterium [37] and are gAGGAGGT (spollAA), AAGGAGac (spollAC) and AAGGtGtg (spoVAA) (the lower case letters denote differences from the 16S rRNA). The free energies of binding have been calculated according to the method of Tinoeo [381 a s - 1 8 . 8 , - 1 2 . 8 and -11.4 Kcal mol-t for spollAA, SpolIAB and spoVAA, respectively. No obvious rbs could be detected in front of the spollAB gene. The first two spollA genes overlap by one nucleotide and may.be translationally coupled. In contrast, there is a region of 12 nucleotides between the stop codon of SpollAB and the start codon of spollAC, and contains the rbs for spollAC. The spollAA gene starts with a GUG codon, while spollAB and spoilAC both start with an AUG codon. The spoVAA gene starts with an AUG codon, and has a putative ribosomal binding site preceding it. The spacing between pbp and spollAA, and between spollAC and spoVA of 94 and 159 nucleotides, respectively, differs markedly from B subtilis (see Distussion). A putative strong rho-independent terminator was found following the spoliAC stop codon. The free energy of the hairpin formation was calculated by the method of Zucker [39] as -24.4 Kcal mol-I. There was no obvious terminator found after the pbp stop codon, which may be explained by the observed pbp-spollA transcript detected by Northern hybridization (see below). A terminator of spollA transcription has also
been reported for both B subtilis and B licheniformis [40,411. The spollAA, spollAB and spollAC open reading frames code for predicted proteins with 116 aa (13 kDa), 147 aa (16 kDa) and 253 aa (29 kDa), respectively, and are compared in figure 3 to the corresponding proteins in B subtilis and B licheniformis of 117 aa, 146 aa, and 255 aa [15, 41]. Amino acid homology among the three organisms is very high. Conservation between B megaterium and B subtilis is 85, 89 and 92%, and between B megaterium and B licheniformis it is 79, 88 and 91% for SpolIAA, SpolIAB and SpolIAC, respectively.
Transcriptional expression of spoilA It has been reported that B subtilis spollA has two transcripts expressed at different times [28, 42]. Savva and Mandelstam [42] reported that the smaller transcript (the spollA operon alone) becomes detectable at h, and that the larger transcript (a read-through product from the pbp gene) [28] is detectable at t3. To show that our gene is functional in vivo and to determine transcript size and time of expression, wild-type PV361 cells were grown in SNB broth and the RNA was isolated from to to ts, as described in Materials and methods. Northern hybridizations were carried out on duplicate filters using one probe within the pbp gene and another probe, a 0.63-kb EcoRI-Bglll fragment spanning parts of the spollAA gene and spoliAB gene (fig 1). As shown in figure 4 (right), two transcripts could be detected with the spoliA specific probe from stage to to ts. The smaller transcript was 1.77 kb and was detectable at to and most abundant at h, while the larger transcript was 2.92 kb, also detectable at to, but with the maximal amount between t2 and ta.5. In order to find out whether the larger transcript was from the 5' end of the cloned fragment, a 0.5-kb Hindlll-EcoRV fragment (from the pbp homologous gene at the 5' end of our 3.34-kb clone, see fig I) was found to hybridize with one band from to to t5 mRNA samples, and the band was exactly the same size as the larger band using the spoilA-specific probe mentioned above (fig 4, left). This indicates that two sizes of transcripts occur in B megaterium, as also reported in B subtilis.
Discussion The 3.34-kb HindIII fragment contains the entire B megaterium spollA operon. Preliminary sequencing also suggests that part of the B megaterium genes homologous to pbp and the spoVA region (spoVAA and part of spoVAB) are present. The observed overlap between spollAA and spollAB has also been reported in the corresponding regions of B subtilis and B licheniformis [41]. Similarly, the space between spollAB
700 Bm SpoIIAA Bs SpoIIAA B1SpoIIAA
VSLSIELEFK QDVLLVRLTG ELDHHTAEEL RSKITEAIEK ESISHLILNL MSLGIDMNVK ESVLCIRLTG ELDHHTAETL KQKVTQSLEK DDIRHIVLNL MSLGIDIHVK ~SVLCIRLTGELDHHTAET& ~KOVSGHLEQ TD~RHIVMNL
Bm SpoIIAA Bs SpoIIAA B1SpoIIAA
QHLTFMDSSG LGVILGRYKQ VHN~GGEMVV CAISPAIERL FNMSGLFKII EDLSFMDSSG LGVILGRYKQ IKQIGGEMVV CAISPAVKRL FDMSGLFKII IKOLGGEMIV C A I S P A V K R L F ~ A D ~
Bm SpoIIAA Bs SpoIIAA B1SpoIIAA
RFEPNEANAL QKLGVA** RFEQSEQQAL LTLGVAS* E L ~ Q S ~ Q R ~ ETLGVAS*
Bm SpoIIAB Bs SpoIIAB B1 SpoIIAB
MKNQMNLQFS ALSQNESFAR VTVASFITQL DPTMDELTEI KTVVSEAVTN MKNEMHLEFS ALSQNESFAR VTVASFIAQL DPTMDELTEI KTVVSEAVTN KTVVSEAVTN ~IOFT ZL~VTVAAFIAOL
Bm SpoIIAB Bs SpoIIAB B1 SpoIIAB
AIIHGYESNS D G W Y I S V T L HEDGVVELMI RDEGIGIDNV DEAKQPLYTT AIIHGYEENC EGKVYISVTL -EDHVVYMTI RDEGLGITDL EEARQPLFTT AIIHGYENSG OGNVYISVTL -EDHIVYLTI RDEGVGIPDL E~2~QELEX2
Bm SpoIIAB Bs SpoIIAB B1SpoIIAB
KPDLERSGMG FTIMENFMDE IRVESTVLEG TTLYLKKHLT NSKALCN* KPELERSGMG FTIMENFMDD VSIDSSPEMG TTIRLTKHLS KSKALCN* ~ F _ ~ ISIDSSPEM~ TTIHLTKHLS F ~ *
Bm SpoIIAC BS SpoIIAC B1SpoIIAC
M.DVEVKKNKN EPYLKDHEVK DLIKRSQQGD QIARDTIVQK NMRLVWSVVQ MDVEVKKNGK NAQLKDHEVK ELIKQSQNGD QQARDLLIEK NMRLVWSVVQ MDVEVKKENQ ~TOLKDHEVKELIKNSONGD ~ K ~ L L I E K ~
Bm SpoIIAC Bs SpoIIAC B1SpoIIAC
RFINRGYEPD DLFQIGCIGL LKSVDKFDLS YDVKFSTYAV PMIIGEIQRF RFLNRGYEPD DLFQIGCIGL LKSVDKFDLT YDVRFSTYAV PMIIGEIQRF ~ / ~ D . ~ ~ YDVRFSTYAV~II.~%~J.Q~
Bm SpoIIAC Bs SpoIIAC B1SpoIIAC
IRDDGTVKVS RSLKEMSNKI RKAKDELSKL LGRVPTVAEV AEHLDLTPEE IRDDGTVKVS RSLKELGNKI RRAKDELSKT LGRVPTVQEI ADHLEIEAED ~ RSLKELGNKI ~ S NGRIPTVOEIADYLEISSEE
Bm SpoIIAC Bs SpoIIAC B1SpoIIAC
VVLAQEANRA PSSIHETVYE NDGDPITLLD QIADHTEAKW FDKIALKEAI VVLAQEAVRA PSSIHETVYE NDGDPITLLD QIADNSEEKW FDKIALKEAI ~ V E S ~ ~ I C ~ OIADOSEEKW EDEIALKE~/
Bm SpoIIAC Bs SpoIIAC B1SpoIIAC
EELDEREKLI VYLRYYKDQT QSEVAARLGI SQVQVSRLEK KILKQMKLHM SDLEEREKLI VYLRYYKDQT QSEVAERLGI SQVQVSRLEK KI LKQIKVQM K ~ ~ K 2 O S E V A D R L G I Z.0_V_0_V. ~ L E~ KILKOIKNQ~
Bm SpoIIAC Bs SpoIIAC B1 SpoIIAC
NDT* DHTDG* ~HFES*
Fig 3. Alignment of SpolIAA, SpolIAB, and SpolIAC proteins of B megaterium (Bm), B subtilis (Bs), and B licheniformis (BI). Conserved amino acids are defined as the following groups [51 ]: (I, L, M, V), (H, K, R), (D, E, N, Q), (A, G), (F, Y, W), (S, T), (P) and (C). Positions at which three residues are identical or conserved are underlined.
701
to
to.5 tl
t2
t3.5
t4
t5
2.92Kb--~
t o to. 5
t1
t2
t3. 5
t4
t5
-~-2.92 Kb ~ - 1.77
Kb
Fig 4. Northern hybridization of spollA transcripts. RNA was isolated as described in Materials and methods. Left: probed with pbp-specific DNA; right: probed with spoIIAA-spoIIAB-specific DNA (see fig l ).
and spollAC genes in each of these organisms is 12 nucleotides long, and the rbs preceding spoliAC suggests that B megaterium spollAC is also translationally independent. The spollA promoter regions of the three species, as shown in figure 5A, are very conserved. An 'OA box', TGNCGAA, where SpoOA binds to regulate gene expression [43], has been found preceding several B subtilis genes. A putative B megaterium OA box (TGACGAA) has been detected and matches that of B subtilis. Wu et ai [40] have reported that transcription of B subtilis spollA was dependent on spoOA. Recently, Hoch (personal communication) has shown that SpoOA acts as a positive regulator on B subtilis spollA exp,ession. It will be interesting to see if the B megaterium OA box has the same function. The nucleotide sequences between spollAC and spoVAA were compared among the three organisms. The putative open reading frame of B megaterium spoVA begins 159 base pairs (bp) downstream of the spollAC, instead of 166 bp for B licheniformis [41, 44], or 124 bp for B suhtilis [15, 44, 451. Moldover et al [44] compared the sequence of the spoVA upstream regions of B subtilis and B licheniformis, and discovered four regions that are perfectly conserved between these two species. These are an identical 20-bp region I, the -35 region, the -10 region and the region IV, respectively. They speculated that the unique 20-bp region may be a control region specific to spoVA, based on the fact that no such homology was detected between this region and the corresponding upstream regions of gerA and ssp genes, although all of these genes share extensive homology with the -35 and -10 regions of spoVA and are considered all transcribed by o<;. The putative B megaterium '20 bp region' preceding the -35 region of spoVA was detected (see fig 5B) as reported in the other two organisms; however, the sequences between B subtilis and B licheniformis are identical, while the B megaterium spoVA region matches 17 of the 20 nucleotides. The identification of this region in B megaterium, a much
more divergent species from B subtilis and B licheniformis [ 19], further suggests the important function of this region in spoVA regulation though it remains to be demonstrated. The putative -35 and -10 regions of B megaterium spoVA are detected and highly homologous to those of B subtilis and B licheniformis (fig 5B). Region IV, which is 8 bp in length and is identical between B subtilis and B licheniformis as reported by Moldover et al [44], may also be present in the B megaterium spoVA promoter region. It is the least conserved region of the four regions in the three species. Only five bp out of the eight nucleotides are conserved and the spacing between III and IV is eight nucleotides longer than in the other two species. Thus, the potential function of region IV in spoVA gene regulation is uncertain. Northern hybridization has confirmed the gene expression and temporal regulation at the transcriptional level and has determined the transcript size of spollA. The time of spollA expression from Northern hybridization corroborated well with the data of the ~-galactosidase activity assay, in which 13-galactosidase activity of the spo-58-1acZ fusion mutant was detected at to [21]. This is the first reported comparison of 13-galactosidase assays with transcriptional results in B megaterium. Savva and Mandelstam [42] reported that there were two spollA transcripts in B subtilis: 1.7 kb and 2.6 kb. The smaller transcript was first detected at t,, but peaked at t2, while the larger one was not detected until t~ and peaked at ts. Wu and Piggot [28] further showed that the larger transcript was a read-through product from the pbp gene upstream to spollA. However, they reported that this transcript appeared at t4 and was 2.9 kb instead of 2.6 kb. Our result agreed with most of the data from B subtilis since there were two spoilA transcripts in B megaterium and the sizes of the transcripts were similar [28]. However, in B megaterium both transcripts appeared earlier (both were present at to, possibly even earlier). The small transcript was detected about l h earlier than that of B subtilis, and the
702 2.92-kb transcript about 3-4 h earlier than the corresponding transcript of B subtilis. A simple explanation could be due to the sensitivity of the detecting methods. Another reason could be the different methods used to designate the onset of sporulation. Savva and Mendelstam [42] designated to by resuspension of the culture in sporulation medium while in
this study to was determined as the time when growth deviated from exponential phase. In a recent study of the role of a v in prespore-specific transcription, Partridge et al [12] reported that there must be a low level of expression of spoIIAC in vegetative cells. However, it is clear from the data presented here that the appearance and maximum expression of the larger
A
-35
OA b o x BmZIA BsZIA BIIIA
AACAGCTAAATGTGACGAAATAGAACTAGTTTTGTCGGTGAGAAGGATTCTATT TTTAAGTAATTATGCCGAATGACCACTAGTTTTGTCACGGTGAAGGAATTCATT TGGAGTGAATAATGCCGAACGGTCACTAGTTTTGTCACGGTGAAGGAATTTATA
BmIIA BsZZA BIZZA
-10 :bs CTGTCGGCAGCGAAAGACTGACTATAAGATTTCCG...AGGAGGTTTGTATAGTG CCGTCGAAATCGAAACACTCATTATCCGATCATAT.C~AATGAGC.ATG AAGTCTGAAGCGAAACACTCATTATCCGATTTAAACCAAGGAGGAATGAGG.AT$
X (20 n t box) BmV& BsVA B1VA
I":!: (-35)
TTGGTAATTAATGGTCTGTTCAGAAACG.TGAATTAGAAAATTTAAGAAAATC GTGGTAATTTATGGTCTTTTCGAGCGGA.TGAATGAGAACAAAATCGAACCA. GTGGTAATTTATGGTCTTTTGCGCCTTTCTGAATGATAAATGGGATGTACTT.
XXX
XV
zbs
(-10) BmV& BsVA BIVA
CATACTACACGTACAGGGAGATAATGAATC.GTAAGGTGTGATATGA..ATG C A T A C T A C A T A ........ T A T A A C C A C C . . G A A A G A T G G T G A T C A A T G A T G C A T A C T A C A A C ........ T A T A A C C A T C A T A T . A G G A A G T G A C C C A . G A T G
Fig 5. Alignment of B megaterium (Bm), B subtilis (Bs), and B licheniformis (BI) promoter regions. A. Comparison with B subtilis [40] and B licheniformis [41] spollA promoters. B. Comparison with B subtilis and B licheniformis spoVA promoters (Moldover et al [44]). Lines indicate conserved regions between B subtilis and B licheniformis (solid) or all three species (dotted). Ribosomal binding sites and start and stop codons are underlined.
703
pbp-spoliA transcript in B megaterium was earlier than that observed in B subtilis by at least 3 h. In Bacillus, it has been shown that sporulation can be blocked by penicillin at two different stages, septum formation and also during spore cortex formation [46]. Penicillin binding proteins (PBPs) are enzymes involved in the metabolism of peptidoglycan. In B megaterium strain KM, Todd and Ellar [47, 48] reported that there were two sporulationspecific PBPs, PBP 3e and PBP 5a, both which were involved in cortex maturation. At present, it is unknown whether our PBP is either of these. If it is, it is unclear why the B megaterium pbp-spolIA transcript was present at to, and its product would not be needed until spore cortex maturation (stage IV). Illing and Errington [16] have recently shown that there is a small amount of peptidoglycan present in the spore septum. A pure speculation would be that our PBP from the pbp-spolIA transcript is involved in this kind of peptidoglycan synthesis. The presence of an 'OA box' in B megaterium spoIIA upstream region suggests that this species has an homologous spoOA gene. The promoter also suggests by its consensus sequences that B megaterium has a on gene and indeed, Fortnagle and coworkers have cloned the spoOH gene of B megaterium [49]. We have recently cloned and sequenced the spoVG gene from B megaterium, a gene transcribed in B subtilis by Ecja [52]. The promoter region of the putative spoVA operon is also conserved with a consensus sequence for ¢~, suggesting that this sigma factor is also present in B megaterium.
B megaterium, suggesting that it is, indeed, important. We have reported [50] tha~ several of the ssp gene loci have been conserved, even though the flanking genes have not. By comparing the limited number of sporulation genes from B megaterium with other bacilli, a pattern of extensive conservation of regulatory genes is suggested. The region that has been cloned containing the spollA operon is very conserved, although not identical, among three Bacillus species. It can also now be inferred, although not proven, that B megaterium has sigma factors oF, o'G, ¢~A,and ¢~s. Note added in the proof
A B megaterium gene homologous to spo0A has recently been cloned (P Youngmann, personal communication). Acknowledgments This research was supported by National Science Foundation ROW Career Advancement Award 8908929 (PSV), and NIH Fogarty International Fellowship 1 F06 TW01540-01, and by BRSG S07 RR071 awarded by the Biomedical Support Grant Program, Division of Research Resources, the National Institutes of Healths.
References l 2
Conclusion The B megaterium spollA operon has been cloned and its sequence is highly homologous to those of B subtiffs and B lichenijbrmis. We have shown that the spo58 mutant is in a gene homologous to the spollAC gene that codes for sigma factor oF in B subtilis. Comparison of the gene organization and gene expression of all the sporulation genes carried on the cloned fragment to the corresponding sporulation genes in B subtilis and B licheniformis revealed that these genes are very conserved in location, structure and fairly conserved in expression. B megaterium spollA operon contains three genes comparable in size (smaller by one to two amino acids) to the corresponding proteins in the other two species. There are also two spollA transcripts present in B megaterium, as reported in B subtilis, but with differences in timing of expression. The promoter regions of spollA and spoVA ope~ons are highly conserved among the three species. The 20 nucleotide conserved region of both B subtilis and B iicheniformis as identified by Moldover et al [44] as being unique to spoVA operon regulation, is also present, although less conserved, in
3 4
5 6
7 8 9
10
Freese E (1981) Initiation of bacterial sporulation. In: Sporulation and Germination (Levenson HS, Sonenshine AL, Tipper DJ, eds) Am Soc Microbioi, l - l 2 Soneshein L (1989) Metabolic regulation of sporulation and other stationary-phase phenomena. In: Regulation of Procaryotic Development: Structural and Functional Analysis of Bacterial Sporulation and Germination (Smith I, Slepecky RA, Setlow P, eds) Am Soc Microbiol, 109-130 Piggot PJ, Coote JG (1976) Genetic aspects of bacterial endospore formation. Bacteriol Rev 40, 908-962 Piggot PJ (I 989) Revised genetic map of Bacillus subtilis 168. In: Regulation of Procaryotic Development: Structural and Functional Analysis of Bacterial Sporulation and Germination (Smith I. Slepecky RA, Setlow P, eds) Am Soc Microbiol, I--41 Losick R, Pero J (1981) Cascades of sigma factors. Cell 25, 582-584 Moran CP Jr (1989) Sigma factors and the regulation of transcription. In: Regulation of Procaryotic Development: Sn'uctural and Functional Analysis of Bacterial Sporuiation and Germination (Smith I, Slepecky RA, Setlow P, eds), Am Soc Microbiol, 167-184 Stragier PRL (1990) Cascades of sigma factors revisited. Mol Microbioi 4, 1801-1806 Burbulys D, Trach KA, Hoch JA (1991) Initiation of sporulation in B subtilis is controlled by a multicomponent phosphorelay. Cell 64, 545-552 Healy J, Weir J, Smith I, Losick R (1991) Post-transcriptional control of a sporulation regulatory gene encoding transcription factor oH in Bacillus subtilis. Mol Microbiol 5,477-487 Kroos L, Kunkel B, Losick R (1989) Switch protein alters specificity of RNA polymerase containing a compartmentspecific sigma factor. Science 243, 526-529
704 !i 12 13
14 15 16 17 18 19 20
21 22 23 24 25
26 27
28
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