At the onset of endospore formation in Bacillus subtilis, the DNA binding protein Spo0A activates transcription from two typesof promoters . The first type includes the spoIIG and spoIIE promoters, which are used by ^A-RNA polymerase, whereas the secondtype includes the spoIIA promoter, which is used by RNA polymerasecontaining the secondary sigma factor ^H . Previous genetic analyseshave identified specific amino acids in -helix E of Spo0A thatare important for activation of Spo0A-dependent, ^A-dependent promoters . However, these amino acids are not required for activation of the ^H-dependent spoIIA promoter . We now report the effectsof additional single-amino-acid substitutions and the effectsof deletions in -helix E . The effects of alanine substitutionsrevealed one new position [239] in Spo0A that appears to bespecifically required for activation of the ^A-dependent promoters.Based on the effects of a deletion mutation, we suggest that -helix E in Spo0A is not directly involved in interaction with ^H-RNA polymerase.

 
Initiation of endospore formation in Bacillus subtilis is controlled by the DNA binding protein Spo0A, which activates transcription from several promoters, including spoIIG [11], spoIIE [17],and spoIIA [2, 15], by binding to sites near the -35 regionof these promoters . The spoIIG and spoIIE promoters are used by ^A-RNA polymerase, whereas the spoIIA promoter is used byRNA polymerase containing the secondary sigma factor ^H . Previousstudies conducted by Buckner et al . [1] and Hatt and Youngman[4] identified a 14-amino-acid region in the C terminus of Spo0A[from residues 227 to 240] important for activation of Spo0A-dependent, ^A-dependent promoters . Specifically single-amino-acid substitutionsat positions G227, I229, S233, F236, and V240 result in reducedability to stimulate transcription of ^A-specific promoters whilehaving no effect on stimulation of ^H-dependent promoters [1,4] . In addition to these mutations that specifically impair ^A-dependent promoter activation, Buckner et al . [1] also report that a mutant form of Spo0A [S231F] suppresses the sporulationdefect of H359R and several other substitutions in ^A, againsuggesting that the region around residue 231 in Spo0A is importantfor ^A-dependent promoter activation . Interestingly, all mutationsin Spo0A affecting the ability of ^A-dependent RNA polymeraseto activate transcription cluster in -helix E, a flexible helixin the C terminus of the protein that is positioned away fromthe core structure of the protein [7, 18] [Fig . 1] . Taken together these results suggest that -helix E perhaps contacts ^A-RNA polymerasewhen bound to promoters to stimulate transcription.

 
It is not known whether all the amino acid residues in -helix E that are involved in stimulation of ^A-directed transcriptionhave been identified or whether any of the amino acids in -helix E play a direct role in activation of ^H-RNA polymerase . Therefore,we examined the effects of additional single-amino-acid substitutionsand the effects of deletions in -helix E on activation of ^A- and ^H-dependent promoters.

 
Bacterial strains and culture media. Routine microbiological manipulations and procedures were carriedout by standard techniques as described by Harwood and Cutting[3] . The concentrations of antibiotics used for selection onLuria broth or Difco sporulation media [DSM] were 5 µg/mlfor choramphenicol, 100 µg/ml for spectinomycin, and 100µg/ml for ampicillin . Cultures were in grown in Luriabroth, and sporulation was induced by nutrient exhaustion inDSM . Competent cells were prepared and transformed by the two-stepmethod as described by Harwood and Cutting [3].

The B . subtilis strains used [Table 1] are all derivatives ofJH642 and contain the trpC2 and phe-1 alleles . Plasmids derivedfrom pCB2 [1] were used for inserting various mutations at thewild-type spo0A locus.

 
In order to create the spo0A deletion strain [EUAKB78], the 5'-flanking DNA of spo0A was PCR amplified with primers 0AUS5FOR [HindIII end] and 0ADS5REV [BclI end] and was cloned into HindIII-BclI-digestedpCB3 [1] to generate plasmid pAK53 . pAK53 was linearized withScaI and was transformed into JH642 as previously describedby Buckner et al . [1] . Chromosomal DNA was isolated from a spectinomycin[100 µg/ml]-resistant transformant by using the QiampDNA Mini Kit [Qiagen Inc., Valencia, Calif.] and was subjectedto PCR to determine if the gene replacement of spectinomycinfor spo0A occurred . The following PCR primers were used in combination,0AUUS5FOR and SpecREV or 0ADS2REVand SpecFOR, to confirm theallelic replacement of the spo0A with the spectinomycin gene.

The QuikChange site-directed mutagenesis kit [Stratagene, La Jolla, Calif.] was used to create mutations in Spo0A that resultedin single alanine substitutions . Briefly, pCB2 was used to createsingle alanine substitutions from amino acid positions 234 to239 of Spo0A . The combination of FOR and REV primers listedin Table 2 was used to make the single-amino-acid substitutions,and the resulting plasmids for each mutation were subjectedto sequencing by using the 0AUS4FOR and 0A3CREV primers to ensurethe presence of the desired mutation.

 
Overlapping PCR was used to create three different deletionswithin the coding sequence of Spo0A . In deletion 1, amino acidsfrom positions 225 to 244 were deleted, and in deletion 2, aminoacids from positions 233 to 241 were deleted, while in the thirdclass of deletion amino acids from positions 229 to 241 weredeleted . In the first round of PCR, the 5' end of the codingsequence [from the starting methionine to either amino acidposition S225, S233, or I229] was amplified with forward primer0AUSFOR in combinations with reverse primer 0ADS3REV, 0ADS4REV,or 0ADS5REV . This generated three PCR products that containedoverlapping complementary regions at their 3' end to primers0AUS2FOR, 0AUS3FOR, and 0AUS4FOR primers, respectively . In thesecond round, the 3' end of the Spo0A coding sequence [fromamino acid positions A244 or S241 to S267] was amplified withprimer 0AUS2FOR, 0AUS3FOR, or 0AUS4FOR in combination with 0ADS6REV.In the third step, the products of the first- and second-roundprimers were combined and reamplified with outer primers 0AUSFORand 0ADS6REV . This gave PCR products that now contained the entire spo0A sequence with deletions incorporated within them. The PCR products obtained from the third round were digested with BclI and HindIII and were cloned into BamHI-HindIII-digested pCB3, thus giving rise to three different integrational vectors [pAK21, pAK22, and pAK23] . To confirm that the deletions werein frame with the coding sequence, plasmids pAK21, pAK22, andpAK23 were sequenced with primer 0A3CREV.

Each mutant derivative of pCB2 was linearized with ScaI and was transformed into competent JH642 . Chromosomal DNA was prepared from spectinomycin-resistant colonies and was subjected to PCRwith primer sets 0AUUSFOR and SpecFOR and 0ADSREV2 and SpecREVto indicate that recombination occurred at the correct locationon the chromosome . The resulting PCR fragment was then sequencedwith 0AUS4FOR and 0A3CREV to confirm the presence of the desiredmutation.

In order to measure the effects that Spo0A mutations had on ^A- and ^H-RNAP holoenzyme-transcribed promoters, each of themutant strains was transduced with an SPß lysate containingeither an spoIIA-lacZ, spoIIG-lacZ, or abrB-lacZ reporter, aspreviously described by Henriques et al . [5] . All strains used are listed in Table 1.

Sporulation assay. Sporulation was induced by medium exhaustion in DSM as describedpreviously [12] . Sporulation efficiency was determined in 30-hcultures as the total number of heat-resistant [80°C for20 min] CFU compared with the total number of CFU before heattreatment . Data presented were from representative experiments.Similar results were obtained in at least three independentexperiments.

ß-Galactosidase activity. Cultures were grown in duplicate in DSM with the appropriateantibiotics to initiate sporulation . Two 300-µl aliquotsof each culture were collected, i.e., one to measure the opticaldensity and the other to assay for ß-galactosidase activity . Enzymatic activity is reported in Miller units [5].

In vivo mutagenesis with EMS. The strain to be mutagenized was plated on DSM agar containingthe appropriate antibiotics and was incubated for 36 h at 37°C.A sterile piece of filter paper with 3 drops of ethyl methanesulfonate[EMS] [1.7 mg/ml; Sigma] was placed at the center of the plate.The cultures were further incubated for 24 h at 37°C, andthe plates were exposed to chloroform vapor for 15 min to killall nonsporulating cells . The plate contents were incubatedfor 48 h at 37°C to allow any spores to germinate . Colonies were seen only with the EMS-mutagenized strain EUAKB38 carrying the deletion 1 derivate of Spo0A . Single colonies [15] were picked from DSM plates and were streaked out to Luria broth plates . Chromosomal DNA was prepared from each of these coloniesand was used to transform EUAKB11 [wild type-Spo0A; spoIIA-lacZ]. The transformants were selected for spectinomycin resistance[the marker for the spo0A] and were scored for blueness . These transformations revealed that all the suppressor mutations were linked to the spo0A gene.

Chromosomal DNA was prepared from each of 15 strains, and the spo0A gene was amplified with primers 0AUS4FOR and 0A3CREV.The resulting PCR product was sequenced with primers to identifythe position of the new mutation . All sequencing was done atthe Emory Core DNA facility [Emory University, Atlanta, Ga.].

 
Identification of a new position in -helix E of Spo0A that is required for ^A-dependent promoter activation. To determine whether additional amino acids in -helix E areinvolved in stimulation of ^A-dependent promoters and if anyof these amino acids play a role in stimulation of ^H-directed transcription, we isolated mutants that produced single alanine substitutions at each position from 234 to 239 in Spo0A . Toassay the effects of the single alanine substitutions in Spo0Aon expression of Spo0A-regulated promoters, we transduced thesemutants with specialized SPß phage lysates that carriedeither fusions of spoIIG-lacZ [an Spo0A-activated, ^A-dependent promoter], spoIIA-lacZ [an Spo0A-activated, ^H-dependent promoter],or abrB-lacZ [an Spo0A-repressed promoter] . We also isolatedisogenic transductants of a strain containing a spectinomycinmarker linked to the wild-type spo0A allele and of a straincarrying a deletion of the spo0A locus . All of the strains werecultured in DSM, and the accumulation of ß-galactosidase was monitored during endospore formation . Three of the single alanine substitutions [L235A, G237A, and Y238A] had little effecton the expression of spoIIG-lacZ [Table 3] . These mutants alsoformed heat-resistant spores at frequencies similar to that of the wild-type strain [Table 3] . However, substitution ofmutations F236A and T239A resulted in reduced expression of spoIIG-lacZ and spore formation [Table 3; Fig. 2] . The T239Asubstitution caused increased expression of spoIIA-lacZ andhad little to no effect on expression of abrB-lacZ [Table 3;Fig. 2] . These latter results indicate that T239A replaced Spo0A functions as least as well as wild-type Spo0A in activating spoIIA transcription and in repressing abrB transcription . Therefore, the T239A substitution probably does not grossly alter structure, stability, or phosphorylation properties of the protein . Evidently, the side chain of T239 is required specifically for activation of the ^A-dependent spoIIG promoter.

 
Substitution of alanine for the phenylalanine at position 236[F236A] of Spo0A also reduced the expression of spoIIG-lacZ[Table 3] . However, this substitution also reduced expression of spoIIA-lacZ [Table 3] . This substitution did not preventrepression of abrB-lacZ expression; therefore, this form ofSpo0A probably accumulates and binds DNA like wild-type Spo0A.Substitution of serine for phenylalanine at position 236 is also reported to have similar ^A- and ^H-dependent effects inan independent screen conducted by Hatt and Youngman [4] . However,since replacement of phenylalanine at position 236 with eitherserine or an alanine reduced transcription from both the ^A-dependent and ^H-dependent promoters, it is not clear whether this substitutiondefines a position in Spo0A that is directly involved in ^A or ^H promoter activation or whether the substitution has a subtle, indirect effect on accumulation or phosphorylation of the protein.In summary, the effects of the alanine substitutions revealedone new position [T239] that appears to be specifically requiredfor activation of a ^A-dependent promoter and another amino acid[F236] that affects both ^A- and ^H-dependent promoter activation.However, in the latter case we cannot eliminate the possibilityof an indirect involvement of F236 in promoter activation.

-Helix E of Spo0A is not required for ^H-dependent promoter activation. Experimental results [1, 4] previously identified amino acidsubstitutions in -helix E of Spo0A that specifically reducedactivation of ^A-dependent promoters; however, no substitutionshave been described in this region that specifically affect ^H-dependent promoter activation . Therefore, it is not knownwhether -helix E is involved directly in ^H-dependent promoteractivation . To test the role of -helix E, we used oligonucleotide-directedmutagenesis to create alleles encoding three different deletionswithin this helix [Fig. 3] . The deletion limits and the placementsof linker glycine residues were designed to minimize the effectof deletions on the remaining structure [Fig . 3] . We then tested the effects of the deletions on expression of the Spo0A-regulated promoter-lacZ fusions . All three deletions abolished transcription from the spoIIA and spoIIG promoters [data not shown] . However,the three deletion derivatives of Spo0A repressed expression of abrB-lacZ [data not shown], suggesting that these deletion derivatives of Spo0A retained their ability to bind DNA.

 
We expected that deletion of -helix E would prevent activationof ^A-dependent promoters, since several amino acids in thisregion have been shown to be essential for ^A-dependent promoteractivation . However, deletion of -helix E also impaired theability of Spo0A to activate ^H-directed transcription . In orderto explore further the possibility that -helix E may be essentialfor stimulating ^H-directed transcription, we sought to identifyintragenic suppressors of the deletions . A selection for sporulation-proficientderivatives of the -helix E deletion strains failed, probablybecause sporulation would require restoration of both ^A-directed and ^H-directed transcription . However, during this procedurewe discovered that a single valine [GTT]-to-alanine [GCT] substitutionat position 8 [V8A] in the deletion 1 derivative of Spo0A suppressedthe effect of the deletion on ^H-dependent spoIIA-lacZ expression[Fig . 4] . The V8A substitution did not suppress the effect ofthe deletion on spoIIG-lacZ [Fig . 4] expression, nor did it restore formation of heat-resistant spores [data not shown]. Therefore, -helix E in the V8A-substituted Spo0A is not requiredfor activation of the ^H-dependent spoIIA promoter, while -helix E is required for activation of the ^A-dependent spoIIG promoter.

 
Since -helix E is not required for stimulation of ^H-directed transcription, at least not by the V8A-substituted Spo0A, how does the deletion of -helix E affect ^H-directed transcriptionand how does the V8A substitution suppress the effect of thedeletion on ^H-directed transcription? Immunoblot analyses ofSpo0A accumulation during sporulation revealed that the deletionderivatives of Spo0A accumulated to levels that were at leasttwo- to fourfold lower than that of wild-type Spo0A [data notshown] . These lower levels of accumulation may have been causedby small decreases in stability of the deletion derivativesof Spo0A and may have been compounded by the requirement ofSpo0A for stimulating transcription of its own structural gene[14] . However, the small decrease in accumulation of the Spo0Adeletion derivatives probably played no role in the reductionof Spo0A-dependent promoter activation . Immunoblot analysesshowed that the V8A substitution did not substantially increasethe accumulation of Spo0A [data not shown] . Nevertheless, theV8A substitution restored activation of the spoIIA promoterby the deletion 1 derivative Spo0A . One possible explanationfor these results is that the V8A substitution increases thefraction of the Spo0A that is phosphorylated, which allows the protein to stimulate transcription more efficiently . Consistent with this hypothesis is the observation by Stephenson et al.[13] that N12 of Spo0A is critical for interaction with theSpo0E phosphatase . Two highly conserved aspartate residues,D10 and D11, that form part of the acid pocket at the phosphorylationsite are located between N12 and V8 [6] . Therefore, it is possible that the V8A substitution may reduce interaction with the Spo0E phosphatase, resulting in higher levels of Spo0A phosphorylation.The equivalent residue of Spo0A V8 is normally a hydrophobicresidue in response regulator receiver domains and lies in thefirst element of the secondary structure, a ß-strandthat contributes to the positioning of the ß1-1 loopcontaining the residue D10-N12, and so this is our preferred explanation.

Other possible mechanisms by which the V8A substitution restores activation of ^H-directed transcription by the -helix E deletionderivative of Spo0A would include creation of an interactionbetween the N-terminal domain of Spo0A and RNA polymerase thatcompensates for an interaction with RNA polymerase that was lost upon deletion of -helix E or a model in which the V8A affectsinteraction between the N- and C-terminal domains of Spo0A.We cannot eliminate the former model, but it seems unlikelythat substitution of valine for alanine, which effectively removesside chain volume, would establish a new interaction betweenproteins and seems likelier that the alanine substitution wouldeliminate an interaction, such as between Spo0A and Spo0E . Wealso cannot completely eliminate the latter model . However,if the V8A substitution affects the interaction between the C- and N-terminal domains of Spo0A, the effect on the conformation of the C-terminal domain would likely be very small . This effect would not likely be great enough to compensate for the absence of -helix E if this helix plays a direct role in stimulating ^H-RNA polymerase . Therefore, we conclude that -helix E in Spo0Aprobably is not directly involved in interaction with ^H-RNA polymerase . If -helix E does not interact with ^H-RNA polymerase,then another region of Spo0A probably interacts with ^H-RNA polymerase.Presently the best candidate for a region of Spo0A that interactswith ^H-RNA polymerase is at the extreme C terminus, where aminoacid substitutions at positions 257, 258, and 260 have beenshown by Rowe-Magnus et al . [9] and Perego et al . [8] to reduceactivation of the ^H-dependent promoter spoIIA . However, as Rowe-Magnuset al . [9] discuss in their paper, they could not eliminatean indirect role for this region in activation of ^H-dependent promoters.

 
This work was supported by Public Health Service grant GM54395from the National Institute of General Medical Sciences.

 
* Corresponding author . Mailing address: Department of Microbiology & Immunology, Emory University School of Medicine, Atlanta, GA 30322 . Phone: [404] 727-5969 . Fax: [404] 727-3659 . E-mail: moran@microbio.emory.edu.

1. Buckner, C . M., G . Schyns, and C . P . Moran, Jr. 1998 . A region in the Bacillus subtilis transcription factor S po0A that is important for spoIIG promoter activation . J . Bacteriol . 180:3578-3583 .
2. Burbulys, D., K . A . Trach, and J . A . Hoch. 1991 . Initiation of sporulation in Bacillus subtilis is controlled by a multicomponent phosphorelay . Cell 64:545-552.
3. Harwood, C . R., and S . M . Cutting. 1990 . Molecular biology methods for bacillus . John Wiley & Sons, West Sussex, England.
4. Hatt, J . K., and P . Youngman. 1998 . Spo0A mutants of Bacillus subtilis with sigma factor-specific defects in transcription activation . J . Bacteriol . 180:3584-3591 .
5. Henriques, A . O., B . W . Beall, K . Roland, and C . P . Moran, Jr. 1995 . Characterization of cotJ, a ^E-controlled operon affecting the polypeptide composition of the coat of Bacillus subtilis spores . J . Bacteriol . 177:3394-3406.
6. Lewis, R . J., J . A . Brannigan, K . Muchova, I . Barak, and A . J . Wilkinson. 1999 . Phosphorylated aspartate in the structure of a response regulator protein . J . Mol . Biol . 294:9-15.
7. Lewis, R . J., S . Krzywda, J . A . Brannigan, J . P . Turkenburg, K . Muchova, E . J . Dodson, I . Barak, and A . J . Wilkinson. 2000 . The trans-activation domain of the sporulation response regulator Spo0A revealed by X-ray crystallography . Mol . Microbiol . 38:198-212.
8. Perego, M., J . J . Wu, G . B . Spiegelman, and J . A . Hoch. 1991 . Mutational dissociation of the positive and negative regulatory properties of the Spo0A sporulation transcription factor of Bacillus subtilis. Gene 100:207-212.
9. Rowe-Magnus, D . A., M . J . Richer, and G . B . Spiegelman. 2000 . Identification of a second region of the Spo0A response regulator of Bacillus subtilis required for transcription activation . J . Bacteriol . 182:4352-4355 .
10. Satola, S., P . A . Kirchman, and C . P . Moran, Jr. 1991 . Spo0A binds to a promoter used by sigma A RNA polymerase during sporulation in Bacillus subtilis. Proc . Natl . Acad . Sci . USA 88:4533-4537.
11. Satola, S . W., J . M . Baldus, and C . P . Moran, Jr. 1992 . Binding of Spo0A stimulates spoIIG promoter activity in Bacillus subtilis. J . Bacteriol . 174:1448-1453.
12. Schaeffer, P., J . Millet, and J . P . Aubert. 1965 . Catabolic repression of bacterial sporulation . Proc . Natl . Acad . Sci . USA 54:704-711.
13. Stephenson, S . J., and M . Perego. 2002 . Interaction surface of the Spo0A response regulator with the Spo0E phosphatase . Mol . Microbiol . 44:1455-1467.
14. Strauch, M . A., K . A . Trach, J . Day, and J . A . Hoch. 1992 . Spo0A activates and represses its own synthesis by binding at its dual promoters . Biochimie 74:619-626.
15. Trach, K., D . Burbulys, M . Strauch, J . J . Wu, N . Dhillon, R . Jonas, C . Hanstein, P . Kallio, M . Perego, T . Bird, et al. 1991 . Control of the initiation of sporulation in Bacillus subtilis by a phosphorelay . Res . Microbiol . 142:815-823.
16. Wu, J . J., M . G . Howard, and P . J . Piggot. 1989 . Regulation of transcription of the Bacillus subtilis spoIIA locus . J . Bacteriol . 171:692-698.
17. York, K., T . J . Kenney, S . Satola, C . P . Moran, Jr., H . Poth, and P . Youngman. 1992 . Spo0A controls the ^A-dependent activation of Bacillus subtilis sporulation-specific transcription unit spoIIE. J . Bacteriol . 174:2648-2658.
18. Zhao, H., T . Msadek, J . Zapf, Madhusudan, J . A . Hoch, and K . I . Varughese. 2002 . DNA complexed structure of the key transcription factor initiating development in sporulating bacteria . Structure [Camb.] 10:1041-1050.
19. Zuber, P., and R . Losick. 1987 . Role of AbrB in Spo0A- and Spo0B-dependent utilization of a sporulation promoter in Bacillus subtilis. J . Bacteriol . 169:2223-2230.

Free Online Full-text Article

What Is Environmental Microbiology?, What Is Amino Acid?, What Is Molecular Biology?, What Is Growth Medium?, What Is Salmonella?, s, Microbes, i, Bacteriology, o, Microbe, a, Microorganism, i, Microorganisms, a, Botulin, e, Multidrug resistant, i, Cryptococci, r, Escherichia coli, o, Clostridia, a, Escherichia coli, e, Bacteriophages, o, Saccharomyces yeast, e, Streptococcal, c, Microbial, s, Halophilic bacterium, s, Salmonella, o, Acinetobacter, o, Cryptococci, n, Escherichia coli, c, Cellulomanas, r, Micrococci, i, Yeasts, a, Pasteurella, e, Bacteroides, n, Pseudomonas aeruginosa




 

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

Last modified: May 25, 2005