Biochimica et Biophysica Acta, 1130 (1992) 229-231 © 1992 Elsevier Science Publishers B.V, All rights reserved 0167-4781/92/$05.00

BBAEXP 91)336

229

Short Sequence-Paper

spoVG sequence of Bacillus megaterium and Bacillus subtilis Deborah S.S. Hudspeth and Patricia S. Vary Department of Biological Sciences, Northern Illinois Unicersit.v, DeKaib. IL (USA) (Received 27 January 1992)

Key words: Sporulation specific gene; ( Bacillus )

We have sequenced the stage V sporulation specific gene spoVG in both Bacillus megaterium and Bacillus subtilis. The open reading frames encode polypeptides of 96 and 97 residues, respectively, and have an 88.6¢~ amino acid identity. Both genes have putative rho-independent terminators. No significant amino acid or nuclcotide homology of either gcnc was fimnd when compared with sequences contained in either the Genbank or EMBL data bases.

Bacillus is a genus of Gram-positive aerobic bacteria that forms spores by a relatively simple process of cell differentiation called sporulation, defined by six morphological stages. The genetics of this process has been extensively studied in B. subtilis where spo genes are designated with a Roman numeral indicating the stage at which mutations cause a block (e.g., spoOA, spoilA). One of these, spoVG, is of special interest because in B. subtilis it is transcribed at the onset of sporulation, yet its mutants phenotype cannot be detected until five hours later, at stage V [1,2]. it was originally cloned because of its presence as one of the early sporulation-specific messenger RNAs [2]. Although several laboratories have studied spoVG, these studies have been limited to its promoter region [3], and its complete gene has never been sequenced. In B. subtilis, spoVG has been shown to be under the control of the abrB regulatory protein [3] and transcribed by a

sigma H-containing RNA polymerase [4,5]. Its transcription is limited by mutations in the early genes spoOA, spoOB, spoOE, spoOF and spoOH [3,5,6]. The spoVG gene has been mapped in B. subtilis at 6° between the cam and tms genzs, a region that also includes abrB, spoOJ and spollE [1,7]. The mutant phenotype, as described by Rosenbluh et al. [1] is oligosporogenous (it produces 5-10% spores, whereas

The nucleotide sequence data reported in this paper writ appear in

the EMBL, Genbank and DDBJ Nucleotide Sequence Dalabases under the accession numbers X63377 and X62378 for B. megatermm and B. subtilis, respectively. Correspondence: P.S. Vary, Department of Biological Sciences, Northern Illinois University, Dc~alb, IL 60115, USA.

wild type producco 90%) alld the colonies are unusually dark brown. The few spores that are produced frequently lyse. The spoVG promoter has been sequenced and used in iacZ fusions for studies of the effects of the sigma H and abrB gene products on its expression (e.g., Refs. 3, 6, 8). B. megaterimn sporulates more efficiently than most other species of Bacillus [9]. Only 8% of the B. megaterium genome cross-hybridizes with that of B. subtilis [10]. Comparative studies should help to elucidate which genes and regulatory pathways are conserved between species and, thus, help determine those that are essential for sporulation. During the last few years our laboratory has developed a genetic system for B. megaterium and is investigating the regulation of genes involved in sporulation Ill,12]. We have initiated studies of spoVG in B. megaterium by cloning a 72{) bp HindlIl fragment from B. megaterium homologous to a B. subtilis spoVG-containing 640 bp Hindlll fragment [13], and by sequencing both clones for comparison. Physical maps and sequencing strategies are shown in Fig. I. The portion of the B. subtilis ,~poVG clone not previously sequenced has been completed as has the homologous B. megaterium spoVG clone. Our B. subtilis sequence agrees with the previously reported sequence of the promoter region [3,14,15] and with the sequence of the 5' region of an overlapping clone containing the tins gene [16]. Comparison of the nucleotide sequences of both species revealed an 80% nucleotide identity within the open reading frame. The B. megaterium clone does not include the spoVG promoter region because of an A to T transition relative to B. subtilis. This change introduced an Hindlll site 33 bases preceding the translational start site in B. megaterium. In addition, the

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TG ACCTAA'I'I~GTA~TAT C ~ , V I T I ~ ~ T A T

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subtilis

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EcoRl site at position 401 (see Fig. 2A) of the B. subtilis sequence (the origin of the tins containing clone) is not found in B. megater£urn because of a C to T transversion. Analysis of the deduced amino acid sequence revealed an 89.5% identity between the 96 amino acid R megateriurn and the 97 amino acid R subtil£s proteins (Fig. 2B). Of the ten amino acid differences seven are conserved substitutions as determined from the interpretation by Covello and Gray [17] of the algorithm of Grantham [18], indicating that spoVG is a highly conserved sporulation specific gene product. The deduced gene product is significantly polar in nature (60% polar residues in B. megaterium and 61% in B. s.bt£1is). It should be noted that transition is initiated at a methionine codon in B. megateriurn and a reline code in E subtilis. Searches of both the Genbank and EMBL data libraries revealed no significant homology with any published DNA or protein sequences. Hy-

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Fig. I. Restriction endonuclease maps of B. megateri, m (pDHVG) and B. subtilL~(pLS5.1) spoVG containing H£,dlll fragments cloned into pUC18. The boxed area delineates the spoVG open reading frame. Arrows indicate the direction and extent of sequence determined from oligonucleotide primers. A ,= Huelll. B = Bell, E = EcoRI, E* ~ EcoRi*. H B Hi, dill, P ~, Pt.,ll and R - Rsal.

:-:-::-

BS ~ T I T G TI~TACATC~ T A ~ ATIT~I~AA ACA~TGGTt~ ~ ,o,o,,o ,,,, ,o,,~ ,,o,o,,,,, o,,,,,oo • • ,oo,o ,, o, o, ,o

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Fig. 2. Sequence comparison of B. megateri.m and B. s.btilis spoVG. (A) The nucleotide sequence of the non-coding strand of both B. megateri.m (BM) and B. s.btilis (BS). Dashes are used to align the nucleotide sequences based on amino acid homology. The start and stop codons, relevant restriction sites, and promoters are underlined and identified. Arrows indicate dyed symmetry of the rho independent terminators. (B) Comparison of the deduced amino acid sequence of spoVG from both species. Amino acids are indicated using the single letter code. Colons indicate identities, periods conserved residues. Dashes represent gaps introduced to facilitate alignment.

R G

CA

U A

U-A C-G G-C O-U G-U A-U A-U A RR G A U-R C-G 8. B e ~ t ~ / u a A-U U-R -14,0 Keal sol -I C-G U-G G-C A-U G-C A-U -UAAACAUAUGAUACAUUA-UUUCUUUUUGAUU-

G C

G-C U-G C-G G O. e u b t i l i a G-C U-G -17,4 Keel mol "1 C-G A-U G-C G-C R-U -UAAAAAAUARCCAAAAAGGCA-UUUUUARUUCU-

Fig. 3. Putative rho-independent terminators of spoVG. Free energies of the most stable configurations determined were calculated according to

Zuker [21 ].

231

dropathy plots and analysis using the Chou and Fassman algorithm [29] indicated that spoVG does not contain the typical helix-turn-helix motif of a DNAbinding protein nor the hydrophobic a-helix typical of a membrane-bound protein. Putative rho-independent termination and ribosomal binding sites have been identified for both species. The free energy of binding of the ribosomal binding sites to the 3' end of the 16S ribosomal RNA (identical in B. megaterium and B. subtilis [20]) was determined according to Zuker [21]. This analysis indicated that B. megaterium binds the ribosome more stably (G = - 13.2 vs. - 9 . 0 for B. subtilis). The most stable rho-independent terminators and their free energies were also determined in this way and are shown in Fig. 3. The free energy for the B. megaterium structure is G = - 1 4 . 0 and for B. subtilis G = - 17.4. Our data, therefore, show a highly conserved gene for a potentially unique protein affecting sporulation. This work was supported in part by a NSF Career Opportunity for Women Grant #DM89089 (PSV) and by Abbott Laboratories. We would like to thank Dr. Mohammad Ashraf for production of synthesized oligonucleotide primers for sequencing and Dr. R. Losick for the generous gift of the pLS5 clone containing the B. subtilis gene. References l Rosenbluh, A., Banner, C.D.B., Losick, R. and Fitz-James, P.C. (1981) J. Bacteriol. 148, 341-351.

2 Segall, J. and Losick, R. (1977) Cell 11,751-761. 3 Zuber, P. and Losick, R. (1987) J. Bacteriol. 169, 2223-2230. 4 Carter, H.L. Ill and Moran, C.P. Jr. (1986) Proc. Natl. Acad. Sci. USA 83, 9438-9442. 5 Johnson, W.C., Moran, C.P. Jr. and Losick, R. (1983) Nature 302, 800-804. 6 Moran, C.P. Jr., Lang, N., Banner, C.D.B., Haldenwang, W.G. and Losick, R. (1981) Cell 25, 783-791. 7 Haidenwang, W.G., Banner, C.D.B., Ollington, J.F., Losick, R., Hoch, J.A., O'Connor, M.B. and Sonenshein, A.L. (1980) J. Bacteriol. 142, 90-98. 8 Ferrari, E., Henner, D.J., Perego, M. and Hoch, J.A. (1988) J. Bacteriol. 170, 289-294. 9 Millet, J. and Aubert. J.P. (1969) Annai. Inst. Pasteur (Paris) I ! 7 461-473. 10 Sharp, R.J., Bown, K.J. and Atkinson, A. (1980)J. Gen. Microbiol. 117, 201-210. 11 Vary, P.S. and Tao, Y.-P. (1988~ in Genetics and biotechnology bacilli (Ganesan, A.T. and l-loch. J.A., eds.), Vol. 2, pp. 403-407. 12 Tao, Y.-P. and Vary, P.S. (19t~l) J. Gen. Microbiol. 137, 797-806. 13 Ollington, J,F., Haldenwang, W.G., Huynh, T.V. and Losick, R. (1981) J. Bacteriol. 147, 432-442. 14 Carter, H.L. !11, Wang, L.-F., Doi, R.H. and Moran, C.P. Jr. (1988) J. Bacteriol. 170, 1617-1621. 15 Moran. C.P.Jr., Lang. N., Banner, C.D.B., Haldenwang, W.G. and Losick, R. (1981) Cell, 25, 783-791. 16 Nilsson, D., Hove-Jensen, B. and Arnvig, K. (1989) Mol. Gen. Genet. 218, 565-571. 17 Covello, P.S. and Gray, M.W. (1990) Nucleic Acids Res. 18. 5 | 89-5196. 18 Grantham, R. (1974) Science 143, 862-864. 19 Chou, P.Y. and Fasman, G.D. (1974) Biochemistry 113, 211-222. 20 Setlow, P. (1974) J. Bacteriol. 117, 1171-1177. 21 Zuker, M. (1989) Science '2,44, 48-52.