Copyright 0 1990 by the Genetics Society of America
Orientation of Genes in the Bacillus subtilisChromosome Daniel R. Zeigler and Donald H. Dean The Bacillus Genetic stock Center, Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210-1292 Manuscript received January10, 1990 Accepted for publication April 25, 1990 ABSTRACT The orientation of 96 genes on the Bacillus subtilis chromosome was deduced by the analysis of published data. Of these genes,9 1 were found to be oriented so that their promoterswere proximal to the chromosomal replication origin and their transcription termini to the replication terminus. Transcription of these genes would therefore be co-directional with replication. This chromosomal organization is consistent with the hypothesis advancedfor Escherichia coli that bacteria avoid headon collisions between RNA polymerase and DNA replication proteins by the appropriate orientation of their transcription units. 1
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HE genomes of the earliest living organisms were probably loose confederations of nearly autonomous genes (WOESEand Fox 1977). Very early in evolution, a process began that has resulted in the modern bacterial chromosome,acircular molecule composed of thousands of coordinately regulated and replicated genes. Current evidence strongly suggests that the chromosomal arrangement of modern bacteria is a product of natural selection. Despite the fact that major chromosomal rearrangements can and do occur in bacterial chromosomes, the overall order of genes is highly conserved among rather distantly related species (RILEYand ANILIONIS1978; ROTHand SCHMID1981).Whena gross rearrangementdoes occur, there can be a measurable decrease,sometimes drastic, in cell fitness as well (KONRAD1969; SCHMID and ROTH 1983; HILL and GRAY 1988).Not all rearrangements are detrimental, however, and one component of chromosomal stability may be a constraint, imposed by foldedchromosome structure,onthe ability of different segmentsof a circular molecule to come into physical contact with one another (SECALL and ROTH 1989). Thus an important objective of bacterial evolutionary genetics is to identify strategies of chromosomal organization common among the bacteria and to deduce theselective pressures that favor those strategies, One such organizational strategy was recently identified by BREWER (1 988). She was able to assign directions of transcription to over 500 Escherichia coli genes with respect tothe geneticmap of the organism. Almost three-fourths of these genesare oriented with their 5’ ends proximalto thereplication origin. Genes that are likely to be moderately to abundantly transcribed are even more biased in their orientation; over 90% of them have their 5’ ends origin-proximal. BREWER also noted thatmost E. coli plasmid and phage Genetics 125; 703-708 (August, 1990)
genomes display a similar bias in transcriptional orientation with respect to the origin. BREWERfurther proposed a possible selective advantage to this mode of chromosomal organization. Reasoning that collisions between RNA polymerase and DNA polymerase are unavoidable due to the their different rates of motion along DNA, she suggested that E. coli minimizes the effects of these collisions through its chromosomal organization. If a DNA polymerase complex were to collide with a slower moving RNA polymerase molecule from the rear, the rate of replication would only be slowed until the RNA polymerase completed transcriptionand left the helix. A head-on collision between polymerases could have more serious consequences, however. T h e replication complex might stall completely until transcription was finished. If an operonwere abundantly transcribed or unusually long so as to have multiple RNA polymerase molecules traversing it simultaneously, the replication fork might stall at the locus for a considerable length of time. Head-on collisions of polymerases would be avoided by just the organization BREWER observed, for genes with origin-proximal5’ endsare transcribed in the same direction as they are replicated. If E. coli has no means of efficiently resolving head-on collisions, then the selective pressure to avoid them might be considerable. We wished to examine the chromosomal organization of Bacillus subtilis, a Gram-positive organism quite unrelated to the enteric bacteria, to determine whether co-directionality of transcription and replication is a common strategy of chromosomal organization within the eubacterialkingdom. Over 500 genes have been located on the genetic map of B. subtilis (PICCOT1989; ZEICLER 1990). The DNA sequence has been reported for nearly 200 of these gencs. By comparing physical maps of sequenced genes with their genetic maps, we were able to deter-
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mine transcriptional orientation for 96 genes. Over 94% of these genes, 91 in all, were oriented so that their transcription and replication are codirectional. This observation strongly suggests that proper transcriptional orientation has been a critically important strategy during the evolution of the bacterial chromosome.
outB and arol and is transcribed toward the origin (D. HENNER, personal communication). Cluster at 65": T h e operon p u r has been sequenced with a map order PurE-PurK-purB-purC-ORF-purOpurl-purF-purM-purN-purHu)-purD; transcription is from left to right (EBBOLE and ZALKIN 1987, 1989). Genetic mapping indicates the order oriC-purB-purFtre and oriC-purB-purH-tre (SAXILDand NYCAARD 1988), so the entire operon is transcribed away from RESULTS the origin. Cluster at 127": A single fragment from this region The orientation relative to the replication origin of has been cloned independently by two groups. One 96 genes could be determined by the examination of group subcloned and sequenced the spoVE gene from published literature.In all, 91 of thesegenes are the fragment (PIGGOT, CHAK and BUGAICHUK 1986; oriented so that,duringtheir transcription,RNA BUCAICHUK and PIGCOT1986). A second group subpolymerase would move in a direction away from the cloned and sequenced a portionof the fragment about replication origin. (Hereafter, these genes will be de10 kb from spoVE and obtained a geneorderof ORFscribed as being oriented "away" from the origin, and ftsA-ftsZ-ORF; their genetic mapping dataindicate the the minority class as beingoriented"toward"the order oriC-metC-spoVE-fts-pyrD (BEALL,LOWE and origin.) The results are presented graphically in FigLUTKENHAUS 1988). Comparison of sequences with ure 1. The genes fallin 13 clusters on the genetic the physical map of the sequence shows that each of map (PIGGOT1989; ZEIGLER 1990). In the narrative these genes and ORFs is oriented away fromthe that follows, the clusters are identified by map posiorigin. tion, by which they may also be located in Figure 1. Cluster at 195": ThyB and dfrA genes are co-tranCluster at 0":The nucleotide sequenceof the origin region reveals thegeneorder rpmH-oriC-"dnaA"scribed in that order (IWAKURA et al. 1988). Genetic "dnaN-ORF-recF-gyrB-gyrA-rrnO( 16s). Each gene, mapping indicates a map order of oriC-metB-ilvD-thyBincluding the open reading frame (ORF), is oriented dfrA-ilvA (MYODAet al. 1984), so transcription is away away from the origin,oriC (MORIYA,OGASAWARA and from the origin. YOSHIKAWA1985). The two rRNA-tRNA operons in Cluster at 200": A"super-operon" of aromatic the region are oriented oriC-rrnO-rrnA; each operon amino acid biosynthesis genes has been sequenced encodes five RNA molecules in the order 16s rRNA, (HENNER, BAND and SHIMOTSU 1984; HENNERet al. tRNA"', tRNAA'",23s rRNA, 5s rRNA (LOUCHNEY, 1986). The genes, in order of transcription, are LUNDand DAHLBERC1982;OCASAWARA,MORIYA trpE-trpD-trpC-trpF-trpB-trpA-hisH-tyrA-aroE. Genetic and YOSHIKAWA1983). mapping experiments have established the map order Cluster at 12": The major ribosomal protein cluster oriC-lys-aroF-aroB-trpE-trpA-hisH-tyrA-aroE(H0CHand of B. subtilis has been partially sequenced. The S10, NESTER 1973). Thus the entire cluster of genes is spc, and a operons are contiguous with a gene ortranscribed away from the origin. . . .der rplP-rpmC-rpsQ-rplN-rplX-rplE-rpsN-rpsHCluster at 204": Fine-point mapping of sporulation rpseE-rpmD-rplO-secY-infA-rpmJ-rpsM-rpsK-rpoA-rplQ-mutations in this region indicates a maporder of oriCO R F (HENKINet al. 1989; BOYLAN et al. 1988). The spoZlA-spoVA-lys (ERRINGTONand MANDELSTAM map order is oriC-cysA-rpsE-rpoA, so each of the genes 1983). Sequences disclose three genes in spoIIA and in the cluster is transcribed away from the origin (SUH, FORTand five in spoVA (FORTand ERRINGTON 1985; BOYLAN and PRICE 1986). PICGOT 1984). Comparison of genetic and physical Cluster at 29": The map order of genes in this maps shows that the operons are transcribedinderegion is oriC-amyE-outB-arol (FERRARI et al. 1985). lys and away from pendently of each other, toward These genes have been cloned on a contiguous fragthe origin. ment, which has been partially sequenced (YANG,GALCluster at 222": A fragment from this region conIZZI and HENNER 1983; ALBERTINI et al. 1987). Comtains three genes, P23-dnaE-rpoD (PRICE,GITT and parison of sequences with a physical map of the fragDo1 1983). Acomparison of the genetic map of these ment indicates a gene order of amyE-ORF-outB; an genes and their sequence shows that all three are additional ORF reported by ALBERTINI et al. (1987) transcribed away fromtheorigin (PRICEand DOI is probably spurious (D. HENNER, personal communicdd gene is also located in the region; it is 1985). The cation). The tmrB gene also maps in the region (NOpreceded by an ORF in the same orientation (SONG MURA et al. 1978; MORI et al. 1986). Comparison of and NEUHARD 1989). Ongoing sequencing studies its sequence (HARADA et al. 1988) with the restriction have shown that cdd and P 2 3 are separated by 6 kb map of the outB fragment indicates that it lies between
Orientation of B. subtilis Genes
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+0"
270"
90"
180"
Y
-* FIGURE 1 .-Location and transcriptional orientation of genes on the B. subtilis chromosome. Arrows pointin the direction of transcription, but are not lneilnt to indicate the number or relative length of the transcription units.
and aretranscribed in the same orientation (L. WANG, personal communication). Cluster at 246": Sequencing studies show five contiguous genes in this region, all transcribed from left to right as listed: rpmA-spoOB-obg-pheB-pheA (TRACH and HOCH 1989). Comparison of the genetic and physical maps of this region confirms that transcription must be directed away from the origin(FERRARI, TRACH and HOCH 1985; LAMONTand MANDELSTAM 1984).
Cluster at 251": Genetic mapping studies indicate that the three polycistronic operons in this region are ordered (oriC)-sdh-ilv-leu (MACKEYand ZAHLER 1982). The sequences of these operonsreveal that sdh and ilv consist of three genes each and thatleu consists of four (MACKEY et al. 1984; PHILLIPS et al. 1987; S. A. ZAHLER, personal communication). It is apparent from comparison of genetic and physical maps that all three operons are transcribed away from the origin. An additional gene, gerE, together with an upstream
D. R. Zeigler and D. H. Dean
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ORF, lie between sdh and ilv in the same orientation (HASNAIN et al. 1985). Further sequencing has shown that sdh is preceded by three genes, trx-uvrB-ask, which are transcribed away from the origin, and anotherORF, whichliesin the opposite orientation (PETRICEK, RUTBERG and HEDERSTEDT 1989; CHEN, ZHANGand PAULUS1989). Cluster at 285”: Both gerA, which consists of three genes, and the citG gene have been cloned and sequenced; they are adjacent to each other, but their transcription is divergent (FEAVERS, MILESand MOIR 1985; MILES and GUEST 1985; ZUBERI, MOIR and FEAVERS 1987). Genetic mapping studies indicate the order oriC-cysB-gerA-citG-thrB(SMITH,MOIR and SAMMONS 1978). Thus citG is transcribed away from, and gerA toward, the origin. Cluster at 295”:The sacB gene andits 5’ regulatory region, sacR, have been cloned and sequenced (GAY et al. 1983; STEINMETZ et al. 1985).Unpublished PBSl transduction mapping by M. STEINMETZ (cited in GAYet al. 1983) suggests a map order of oriC-hissacB-sacR-smo-cysB. Based on this report, we tentatively oriented the sacB gene toward the origin. DISCUSSION
Orientation of genes: We were able to orient the direction of transcription for 96 B. subtilis genes. Of these genes,91 areoriented so that RNA polymerase, when transcribing them, moves in the same direction as a replication fork would move during replication. The set of sequenced genes includes representatives of almost every major class of genes in this organism. They encode biosynthetic, degradative, and central fueling reaction enzymes; components of DNA replication and repair pathways; RNA and proteinconstituents of the protein synthesis system; and gene products requiredfor celldivision andtheformation, germination, and outgrowth of spores. Unidentified long,ORFs are not included in the tally, but it is noteworthy that of the nine ORFs associated with the sample genes, eight are also oriented so that transcription of them would proceed away from the origin of replication. These observations, taken together with very similar results compiled for E. coli (BREWER1988), strongly suggest that the codirectionality of transcription and replication is a general feature of bacterial chromosomes. A 16s rRNA sequence analysis suggests that the last common ancestor of B. subtilis and E. coli lived billions of years ago, before the earth developed its aerobic atmosphere (FOXet al. 1980). Indeed, the phylogenetic distance between these two organisms is greater than the distance separating the recognized eukaryotic kingdoms (WOESE1987). That a common strategy of genomeorganization exists between two such diverse organisms implies that there
is a powerful selective advantage to it. We do not yet know ifthis strategy is a resultof convergent evolution or if it is a trait of the common ancestor thathas been conserved in the two lines of descent. T h e analysis of genomes from organisms in other eubacterial “phyla” (the cyanobacteria, for example) should help resolve this issue. The selective advantage inherent to this chromosomalorganization is not known with certainty, butthe polymerase-collision hypothesis remains an attractive possibility. Sporulation genes: A novel class of genes-perhaps approaching 150 in number-is required for endospore formation in B. subtilis (ERRINGTON, FORTand MANDELSTAM1985). These sporulation genes provide a unique opportunity to test the polymerase-collision hypothesis of genome organization, for most of them are transcribed in the complete absence of DNA replication. Following the environmental signals that initiate the sporulation program, the chromosome replicates once (BINNIEand COOTE 1983;SARGENT 1980). At about 2 hr after initiation under standard conditions, an asymmetric septum forms, partitioning the daughter chromosomesinto two unequal compartments. The smaller cell (the forespore) develops into the mature spore, whereas the larger one (the mother cell) ultimately lyses. As the spore develops, the forespore and mother cell embark on different programs of gene expression (reviewed by LOSICKand KROOS 1989). Once the septum forms, there are no further rounds of replication; the mature spore contains a single, complete chromosome (CALLISTER and WAKE 1974, 1976;WAKE 1976).Thus RNA polymerase does not oppose DNA polymerase after septum formation, regardless of the orientation of the transcription units. T h e polymerase-collision hypothesis predicts that genes transcribed early in sporulation would tend to be oriented so that transcription proceeds away from thereplication origin, whereas genes transcribed at a later stage would be oriented randomly. The timing of transcription is known for six of the sporulation operons oriented in Figure 1. T h e spoOB gene is expressed at high levels during logarithmic growth in B. subtilis; following the initiation of sporet al. ulation, expression declines markedly (BOUVIER 1984). T h e spoVE gene begins to be expressed less than 1 hr after initiation (BUGAICHUK 1987). The spoIZA operon, which consists of three genes, is transcribed beginning 1-2 hr after initiation of sporulation, about thetime the asymmetric septum is formed (SAVVA andMANDELSTAM1986; WU, HOWARD and PIGGOT1989). Transcription of the $OVA operon, comprised of five genes, and the gerA operon, comprised of three genes, begins about 3 hr into sporulation and is confined to the forespore compartment (ERRINGTON and MANDELSTAM1986; A. MOIR, per-
Orientation of B. subtilis Genes sonal communication, cited by LOSICKand KROOS 1989). Transcription of the gerA gene begins about 1 hr later and is confined to the mother cell (CUTTING and MANDELSTAM 1986). It is evident, then, that spoOB is transcribed while the chromosome is actively replicating, whereas spoVA, gerA and gerE are transcribed in the absence of replication. The timing of the other two operons relative to replication is less certain; spoZZA probably belongs in the postreplication class of genes, but because of its position near the terminus of replication one cannot ruleout thepossibility that its transcription begins before the replication fork has passed through the locus. It is interesting that, of these six operons, only gerA is transcribed toward the origin, and gerA falls quite definitely in the postreplication class. These data are of course too limited to warrant much speculation, butthey are atleast consistent with the polymerase-collision hypothesis. Because sporulation genes are being intensively studied, more information about theirorientation relative tothe replication origin should be forthcomingsoon. We thank D. HENNER, M. STEINMETZ, L. WANG andS. ZAHLER for communicating unpublished data to us. T h e Bacillus Genetic Stock Center is supported by National Science Foundation grant DIR-8820971 and by donations from the industrial sponsors who are acknowledged in our current strain catalog.
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inversion on cell fitness in Escherichia coli K-12. Genetics 119: 771-778. HOCH,J. A., and E. W. NESTER,1973 Gene-enzyme relationships of aromatic acid biosynthesis in Bacillus subtilis. J. Bacteriol. 116: 59-66. IWAKURA,M., W. KAWATA,K. TSUDAand T . TANAKA, 1988 Nucleotide sequence of the thymidilate synthase B and dihydrofolate reductase genes contained in one Bacillus subtilis operon. Gene 64: 9-20. KONRAD, E. B., 1969 The genetics of chromosomal duplications. Ph.D. thesis, Harvard University, Cambridge, Mass. LAMONT,I. L., and J. MANDELSTAM, 1984 Identification of a new sporulation locus, spolllF, in Bacillus subtilis. J. Gen. Microbiol. 1 3 0 1253-1261. LOSICK,R., and L. KROOS,1989Dependence pathways forthe expression ofgenes involved in endospore formationin Bacillus group of forespore-specific prosubtilis: identificationofa moters. J. Bacteriol. 171: 2708-2718. LOUGHNEY, K., E. LUND and J.E. DAHLBERG, 1982 tRNA genes are found between the 16s and 23s rRNA genes in E. subtilis. Nucleic Acids Res. 10: 1607-1624. MACKEY, C. J., and S . A. ZAHLER, 1982 Insertion ofbacteriophage SPP into the cztF gene of Bacillus subtilis and specialized transduction of the ilvBC-leu genes. J. Bacteriol. 151: 1222-1229. C. J., R. J.WARBURG,H. 0. HALVORSON and S. A. MACKEY, ZAHLER, 1984 Genetic and physical analysis of the ilvBC-leu region in Bacillus subtilis. Gene 32: 49-56. MILES,J. S., and J. R.GUEST,1985 Complete nucleotide sequence of the fumarase gene (citC) of Bacillussubtilis 168. Nucleic Acids Res. 13: 131-140. MORI,M., A. TANIMOTO, K. YODA,S. HARADA,N. KOYAMA, K. HASHIGUCHI, M. OBINATA,M. YAMASAKIand G. TAMURA, 1986 Essential structure in theclonedtransforming DNA that induces gene amplification of the Bacillus subtilis amyEtmrB region. J. Bacteriol. 166: 787-794. MORIYA,S . , N. OGASAWARA and YOSHIHAWA, H. 1985 Structure and organization of rRNA operons at the region of the replication origin of the E. subtilis chromosome. Nucleic Acids Res. 11: 6301-6318. MYODA,T. T., S. U. LOWTHER, V. L. FUNANGE and F. E. YOUNG, 1984Cloningandmapping of thedihydrofolatereductase gene of Bacillus subtilis. Gene 29: 135-143. NOMURA, S., K. YAMANE, T . SASAKI,M. YAMASAKI, G. TAMURA and B. MARUO, 1978 Tunicarnycin-resistant mutantsand chromosomal locations of mutational sites in Bacillus subtilis. J. Bacteriol. 136: 818-821. OGASAWARA, N., S. MORIYAand H. YOSHIKAWA, 1983 Structure and function of the region of the replication origin of the E. subtilis chromosome. 111. Nucleotide sequence of some 10,000 base pairs in the origin region. Nucleic Acids Res. 13: 22512265. PETRICEK, M.,L. RUTBERG and L. HEDERSTEDT, 1989T h e structuralgenefor aspartokinase I1 in Bacillussubtilis is closely linked to the sdh operon. FEMS Microbiol. Lett. 61: 85-88. S. HASNAIN, L. RUTBERCand J. PHILLIPS,M. K., L. HEDERSTEDT, R. GUEST,1987 Nucleotide sequence encoding the flavoprotein and iron-sulfur protein subunits of the Bacillussubtilis PY79 succinate dehydrogenase complex. J. Bacteriol. 169: 864-873. PIGGOT, P. J., 1989 Revised genetic map of Bacillus subtilis 168, pp. 1-42 in Regulation of ProcaryoticDevelopment:Structural and Functional Analysis of Bacterial Sporulation and Germination edited by 1. SMITH,R. A. SLEPECKY and P. SETLOW. American Society for Microbiology, Washington, D.C. 1986 Isolation PIGGOT,P. J., K. F. CHAK andV. D. BUGAICHUK, and characterization of a clone of the spoVE locus of Bacillus subtilis. J. Gen. Microbiol. 132: 1875-1881.
D. H. Dean PRICE,C. W., and R. H. DOI, 1985Geneticmapping of rpoD implicates themajor sigma factor of Bacillussubtilis RNA polymerase in sporulation initiation. Mol. Gen.Genet. 201: 88-95. PRICE,C. W., M.A. GITTand R. H.Dol,1983 Isolation and physical mapping of the gene encoding the majorsigma factor ofBacillus subtilis RNA polymerase. Proc. Natl. Acad.Sci. USA 8 0 4074-4078. RILEY,M., and A.ANILIONIS, 1978 Evolutionof the bacterial genome. Annu. Rev. Microbiol. 32: 519-560. M. B. SCHMID,1981ArrangementandreROTH, J.R.,and arrangement of the bacterial chromosome. Stadler Symp. 13: 53-70. SARGENT,M. G.,1980Chromosome replication in sporulating cells of Bacillus subtilis. J. Bacteriol. 142: 491-498. SAVVA, D., andJ. MANDELSTAM, 1986 Use of a lacZ gene fusion to determine the dependence pattern and the spore compartment expression of sporulation operon spoVA in spo mutants of Bacillus subtilis. J. Gen. Microbiol. 132: 3005-301 1. SAXILD, H. H., and P. NYGAARD, 1988Gene-enzyme relationships of the purine biosyntheticpathway in Bacillussubtilis. Mol. Gen. Genet. 211: 160-167. SCHMID,M. D., and J. R. ROTH, 1983 Selection andendpoint distribution ofbacterialinversion mutations. Genetics 105: 539-557. SEGALL,A. M., andJ. R. ROTH, 1989Recombination between homologies in direct and inverse orientation in the chromosome ofSalmonella: intervals which are nonpermissive for inversion formation. Genetics 122: 737-747. SMITH,D. A., A. MOIR and R. SAMMONS, 1978Progress in genetics of spore germination in Bacillus subtilis, pp. 158-163 in Spores VII, edited by G. CHAMBLISS and J. C. VARY.American Society for Microbiology, Washington, D.C. SONG,B.-H., and J. NEUHARD, 1989 Chromosomallocation, cloning, and nucleotide sequence of the Bacillus subtilis cdd gene encoding cytidine/deoxyctyidine deaminase. Mol. Gen. Genet. 216: 462-468. M., D. LECOQ,S. AYERMICH, G. GONZY-TREBOUL and STEINMETZ, Bacillus P. GAY, 1985 T h e DNA sequence for the secreted subtilis enzyme levansucrase and its genetic control sites. Mol. Gen. Genet. 200: 220-228. SUH, J.-W., S. A. BOYLANand C. W. PRICE, 1986 Gene for the alpha subunit of Bacillus subtilis RNA polymerase maps in the ribosomal protein gene cluster. J. Bacteriol. 171: 2553-2562. TRACH, K., and J.A. HOCH, 1989 The Bacillus subtilis spoOB stage 0 sporulation operon encodes an essential GTP-binding protein. J. Bacteriol. 171: 1362-1 37 1. WAKE,R. G., 1976 Segregation of Bacillussubtilis chromosomes radioactively labeled during the first round of replication after germination of spores. J. Bacteriol. 127: 433-439. WOESE,C. R., 1987 Bacterial evolution. Microbiol. Rev. 51: 221271. WOESE,C. R.,and G. E. Fox,1977Theconcept ofcellular evolution. J. Mol. Evol. 10: 1-6. Wu, J.-J., M. G. HOWARD and P.J. PIGGOT,1989 Regulation of transcription of the Bacillus subtilis spolIA locus. J. Bacteriol. 171: 692-698. YANG,M., A. GALIZZI and D. HENNER, 1983 Nucleotide sequence of the amylase gene from Bacillus subtilis. Nucleic Acids Res. 11: 237-249. ZEIGLER, D. R., 1990 Bacillussubtilis 168, in Genetic Maps 1990: ACompilation of LinkageandRestrictionMaps of Genetically Cold Spring Studied Organisms, Vol. 5, editedby S . J. O’BRIEN. Harbor Laboratory, Cold Spring Harbor, N.Y. (in press). ZUBERI, A. R., A. MOIRand I. M. FEAVERS, 1987 T h e nucleotide sequence and gene organization of the gerA spore germination operon of Bacillus subtilis 168. Gene 51: 1-1 1. Communicating editor: J. R. ROTH
Orientation of Genes in the Bacillus subtilisChromosome Daniel R. Zeigler and Donald H. Dean The Bacillus Genetic stock Center, Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210-1292 Manuscript received January10, 1990 Accepted for publication April 25, 1990 ABSTRACT The orientation of 96 genes on the Bacillus subtilis chromosome was deduced by the analysis of published data. Of these genes,9 1 were found to be oriented so that their promoterswere proximal to the chromosomal replication origin and their transcription termini to the replication terminus. Transcription of these genes would therefore be co-directional with replication. This chromosomal organization is consistent with the hypothesis advancedfor Escherichia coli that bacteria avoid headon collisions between RNA polymerase and DNA replication proteins by the appropriate orientation of their transcription units. 1
T
,
HE genomes of the earliest living organisms were probably loose confederations of nearly autonomous genes (WOESEand Fox 1977). Very early in evolution, a process began that has resulted in the modern bacterial chromosome,acircular molecule composed of thousands of coordinately regulated and replicated genes. Current evidence strongly suggests that the chromosomal arrangement of modern bacteria is a product of natural selection. Despite the fact that major chromosomal rearrangements can and do occur in bacterial chromosomes, the overall order of genes is highly conserved among rather distantly related species (RILEYand ANILIONIS1978; ROTHand SCHMID1981).Whena gross rearrangementdoes occur, there can be a measurable decrease,sometimes drastic, in cell fitness as well (KONRAD1969; SCHMID and ROTH 1983; HILL and GRAY 1988).Not all rearrangements are detrimental, however, and one component of chromosomal stability may be a constraint, imposed by foldedchromosome structure,onthe ability of different segmentsof a circular molecule to come into physical contact with one another (SECALL and ROTH 1989). Thus an important objective of bacterial evolutionary genetics is to identify strategies of chromosomal organization common among the bacteria and to deduce theselective pressures that favor those strategies, One such organizational strategy was recently identified by BREWER (1 988). She was able to assign directions of transcription to over 500 Escherichia coli genes with respect tothe geneticmap of the organism. Almost three-fourths of these genesare oriented with their 5’ ends proximalto thereplication origin. Genes that are likely to be moderately to abundantly transcribed are even more biased in their orientation; over 90% of them have their 5’ ends origin-proximal. BREWER also noted thatmost E. coli plasmid and phage Genetics 125; 703-708 (August, 1990)
genomes display a similar bias in transcriptional orientation with respect to the origin. BREWERfurther proposed a possible selective advantage to this mode of chromosomal organization. Reasoning that collisions between RNA polymerase and DNA polymerase are unavoidable due to the their different rates of motion along DNA, she suggested that E. coli minimizes the effects of these collisions through its chromosomal organization. If a DNA polymerase complex were to collide with a slower moving RNA polymerase molecule from the rear, the rate of replication would only be slowed until the RNA polymerase completed transcriptionand left the helix. A head-on collision between polymerases could have more serious consequences, however. T h e replication complex might stall completely until transcription was finished. If an operonwere abundantly transcribed or unusually long so as to have multiple RNA polymerase molecules traversing it simultaneously, the replication fork might stall at the locus for a considerable length of time. Head-on collisions of polymerases would be avoided by just the organization BREWER observed, for genes with origin-proximal5’ endsare transcribed in the same direction as they are replicated. If E. coli has no means of efficiently resolving head-on collisions, then the selective pressure to avoid them might be considerable. We wished to examine the chromosomal organization of Bacillus subtilis, a Gram-positive organism quite unrelated to the enteric bacteria, to determine whether co-directionality of transcription and replication is a common strategy of chromosomal organization within the eubacterialkingdom. Over 500 genes have been located on the genetic map of B. subtilis (PICCOT1989; ZEICLER 1990). The DNA sequence has been reported for nearly 200 of these gencs. By comparing physical maps of sequenced genes with their genetic maps, we were able to deter-
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mine transcriptional orientation for 96 genes. Over 94% of these genes, 91 in all, were oriented so that their transcription and replication are codirectional. This observation strongly suggests that proper transcriptional orientation has been a critically important strategy during the evolution of the bacterial chromosome.
outB and arol and is transcribed toward the origin (D. HENNER, personal communication). Cluster at 65": T h e operon p u r has been sequenced with a map order PurE-PurK-purB-purC-ORF-purOpurl-purF-purM-purN-purHu)-purD; transcription is from left to right (EBBOLE and ZALKIN 1987, 1989). Genetic mapping indicates the order oriC-purB-purFtre and oriC-purB-purH-tre (SAXILDand NYCAARD 1988), so the entire operon is transcribed away from RESULTS the origin. Cluster at 127": A single fragment from this region The orientation relative to the replication origin of has been cloned independently by two groups. One 96 genes could be determined by the examination of group subcloned and sequenced the spoVE gene from published literature.In all, 91 of thesegenes are the fragment (PIGGOT, CHAK and BUGAICHUK 1986; oriented so that,duringtheir transcription,RNA BUCAICHUK and PIGCOT1986). A second group subpolymerase would move in a direction away from the cloned and sequenced a portionof the fragment about replication origin. (Hereafter, these genes will be de10 kb from spoVE and obtained a geneorderof ORFscribed as being oriented "away" from the origin, and ftsA-ftsZ-ORF; their genetic mapping dataindicate the the minority class as beingoriented"toward"the order oriC-metC-spoVE-fts-pyrD (BEALL,LOWE and origin.) The results are presented graphically in FigLUTKENHAUS 1988). Comparison of sequences with ure 1. The genes fallin 13 clusters on the genetic the physical map of the sequence shows that each of map (PIGGOT1989; ZEIGLER 1990). In the narrative these genes and ORFs is oriented away fromthe that follows, the clusters are identified by map posiorigin. tion, by which they may also be located in Figure 1. Cluster at 195": ThyB and dfrA genes are co-tranCluster at 0":The nucleotide sequenceof the origin region reveals thegeneorder rpmH-oriC-"dnaA"scribed in that order (IWAKURA et al. 1988). Genetic "dnaN-ORF-recF-gyrB-gyrA-rrnO( 16s). Each gene, mapping indicates a map order of oriC-metB-ilvD-thyBincluding the open reading frame (ORF), is oriented dfrA-ilvA (MYODAet al. 1984), so transcription is away away from the origin,oriC (MORIYA,OGASAWARA and from the origin. YOSHIKAWA1985). The two rRNA-tRNA operons in Cluster at 200": A"super-operon" of aromatic the region are oriented oriC-rrnO-rrnA; each operon amino acid biosynthesis genes has been sequenced encodes five RNA molecules in the order 16s rRNA, (HENNER, BAND and SHIMOTSU 1984; HENNERet al. tRNA"', tRNAA'",23s rRNA, 5s rRNA (LOUCHNEY, 1986). The genes, in order of transcription, are LUNDand DAHLBERC1982;OCASAWARA,MORIYA trpE-trpD-trpC-trpF-trpB-trpA-hisH-tyrA-aroE. Genetic and YOSHIKAWA1983). mapping experiments have established the map order Cluster at 12": The major ribosomal protein cluster oriC-lys-aroF-aroB-trpE-trpA-hisH-tyrA-aroE(H0CHand of B. subtilis has been partially sequenced. The S10, NESTER 1973). Thus the entire cluster of genes is spc, and a operons are contiguous with a gene ortranscribed away from the origin. . . .der rplP-rpmC-rpsQ-rplN-rplX-rplE-rpsN-rpsHCluster at 204": Fine-point mapping of sporulation rpseE-rpmD-rplO-secY-infA-rpmJ-rpsM-rpsK-rpoA-rplQ-mutations in this region indicates a maporder of oriCO R F (HENKINet al. 1989; BOYLAN et al. 1988). The spoZlA-spoVA-lys (ERRINGTONand MANDELSTAM map order is oriC-cysA-rpsE-rpoA, so each of the genes 1983). Sequences disclose three genes in spoIIA and in the cluster is transcribed away from the origin (SUH, FORTand five in spoVA (FORTand ERRINGTON 1985; BOYLAN and PRICE 1986). PICGOT 1984). Comparison of genetic and physical Cluster at 29": The map order of genes in this maps shows that the operons are transcribedinderegion is oriC-amyE-outB-arol (FERRARI et al. 1985). lys and away from pendently of each other, toward These genes have been cloned on a contiguous fragthe origin. ment, which has been partially sequenced (YANG,GALCluster at 222": A fragment from this region conIZZI and HENNER 1983; ALBERTINI et al. 1987). Comtains three genes, P23-dnaE-rpoD (PRICE,GITT and parison of sequences with a physical map of the fragDo1 1983). Acomparison of the genetic map of these ment indicates a gene order of amyE-ORF-outB; an genes and their sequence shows that all three are additional ORF reported by ALBERTINI et al. (1987) transcribed away fromtheorigin (PRICEand DOI is probably spurious (D. HENNER, personal communicdd gene is also located in the region; it is 1985). The cation). The tmrB gene also maps in the region (NOpreceded by an ORF in the same orientation (SONG MURA et al. 1978; MORI et al. 1986). Comparison of and NEUHARD 1989). Ongoing sequencing studies its sequence (HARADA et al. 1988) with the restriction have shown that cdd and P 2 3 are separated by 6 kb map of the outB fragment indicates that it lies between
Orientation of B. subtilis Genes
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+0"
270"
90"
180"
Y
-* FIGURE 1 .-Location and transcriptional orientation of genes on the B. subtilis chromosome. Arrows pointin the direction of transcription, but are not lneilnt to indicate the number or relative length of the transcription units.
and aretranscribed in the same orientation (L. WANG, personal communication). Cluster at 246": Sequencing studies show five contiguous genes in this region, all transcribed from left to right as listed: rpmA-spoOB-obg-pheB-pheA (TRACH and HOCH 1989). Comparison of the genetic and physical maps of this region confirms that transcription must be directed away from the origin(FERRARI, TRACH and HOCH 1985; LAMONTand MANDELSTAM 1984).
Cluster at 251": Genetic mapping studies indicate that the three polycistronic operons in this region are ordered (oriC)-sdh-ilv-leu (MACKEYand ZAHLER 1982). The sequences of these operonsreveal that sdh and ilv consist of three genes each and thatleu consists of four (MACKEY et al. 1984; PHILLIPS et al. 1987; S. A. ZAHLER, personal communication). It is apparent from comparison of genetic and physical maps that all three operons are transcribed away from the origin. An additional gene, gerE, together with an upstream
D. R. Zeigler and D. H. Dean
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ORF, lie between sdh and ilv in the same orientation (HASNAIN et al. 1985). Further sequencing has shown that sdh is preceded by three genes, trx-uvrB-ask, which are transcribed away from the origin, and anotherORF, whichliesin the opposite orientation (PETRICEK, RUTBERG and HEDERSTEDT 1989; CHEN, ZHANGand PAULUS1989). Cluster at 285”: Both gerA, which consists of three genes, and the citG gene have been cloned and sequenced; they are adjacent to each other, but their transcription is divergent (FEAVERS, MILESand MOIR 1985; MILES and GUEST 1985; ZUBERI, MOIR and FEAVERS 1987). Genetic mapping studies indicate the order oriC-cysB-gerA-citG-thrB(SMITH,MOIR and SAMMONS 1978). Thus citG is transcribed away from, and gerA toward, the origin. Cluster at 295”:The sacB gene andits 5’ regulatory region, sacR, have been cloned and sequenced (GAY et al. 1983; STEINMETZ et al. 1985).Unpublished PBSl transduction mapping by M. STEINMETZ (cited in GAYet al. 1983) suggests a map order of oriC-hissacB-sacR-smo-cysB. Based on this report, we tentatively oriented the sacB gene toward the origin. DISCUSSION
Orientation of genes: We were able to orient the direction of transcription for 96 B. subtilis genes. Of these genes,91 areoriented so that RNA polymerase, when transcribing them, moves in the same direction as a replication fork would move during replication. The set of sequenced genes includes representatives of almost every major class of genes in this organism. They encode biosynthetic, degradative, and central fueling reaction enzymes; components of DNA replication and repair pathways; RNA and proteinconstituents of the protein synthesis system; and gene products requiredfor celldivision andtheformation, germination, and outgrowth of spores. Unidentified long,ORFs are not included in the tally, but it is noteworthy that of the nine ORFs associated with the sample genes, eight are also oriented so that transcription of them would proceed away from the origin of replication. These observations, taken together with very similar results compiled for E. coli (BREWER1988), strongly suggest that the codirectionality of transcription and replication is a general feature of bacterial chromosomes. A 16s rRNA sequence analysis suggests that the last common ancestor of B. subtilis and E. coli lived billions of years ago, before the earth developed its aerobic atmosphere (FOXet al. 1980). Indeed, the phylogenetic distance between these two organisms is greater than the distance separating the recognized eukaryotic kingdoms (WOESE1987). That a common strategy of genomeorganization exists between two such diverse organisms implies that there
is a powerful selective advantage to it. We do not yet know ifthis strategy is a resultof convergent evolution or if it is a trait of the common ancestor thathas been conserved in the two lines of descent. T h e analysis of genomes from organisms in other eubacterial “phyla” (the cyanobacteria, for example) should help resolve this issue. The selective advantage inherent to this chromosomalorganization is not known with certainty, butthe polymerase-collision hypothesis remains an attractive possibility. Sporulation genes: A novel class of genes-perhaps approaching 150 in number-is required for endospore formation in B. subtilis (ERRINGTON, FORTand MANDELSTAM1985). These sporulation genes provide a unique opportunity to test the polymerase-collision hypothesis of genome organization, for most of them are transcribed in the complete absence of DNA replication. Following the environmental signals that initiate the sporulation program, the chromosome replicates once (BINNIEand COOTE 1983;SARGENT 1980). At about 2 hr after initiation under standard conditions, an asymmetric septum forms, partitioning the daughter chromosomesinto two unequal compartments. The smaller cell (the forespore) develops into the mature spore, whereas the larger one (the mother cell) ultimately lyses. As the spore develops, the forespore and mother cell embark on different programs of gene expression (reviewed by LOSICKand KROOS 1989). Once the septum forms, there are no further rounds of replication; the mature spore contains a single, complete chromosome (CALLISTER and WAKE 1974, 1976;WAKE 1976).Thus RNA polymerase does not oppose DNA polymerase after septum formation, regardless of the orientation of the transcription units. T h e polymerase-collision hypothesis predicts that genes transcribed early in sporulation would tend to be oriented so that transcription proceeds away from thereplication origin, whereas genes transcribed at a later stage would be oriented randomly. The timing of transcription is known for six of the sporulation operons oriented in Figure 1. T h e spoOB gene is expressed at high levels during logarithmic growth in B. subtilis; following the initiation of sporet al. ulation, expression declines markedly (BOUVIER 1984). T h e spoVE gene begins to be expressed less than 1 hr after initiation (BUGAICHUK 1987). The spoIZA operon, which consists of three genes, is transcribed beginning 1-2 hr after initiation of sporulation, about thetime the asymmetric septum is formed (SAVVA andMANDELSTAM1986; WU, HOWARD and PIGGOT1989). Transcription of the $OVA operon, comprised of five genes, and the gerA operon, comprised of three genes, begins about 3 hr into sporulation and is confined to the forespore compartment (ERRINGTON and MANDELSTAM1986; A. MOIR, per-
Orientation of B. subtilis Genes sonal communication, cited by LOSICKand KROOS 1989). Transcription of the gerA gene begins about 1 hr later and is confined to the mother cell (CUTTING and MANDELSTAM 1986). It is evident, then, that spoOB is transcribed while the chromosome is actively replicating, whereas spoVA, gerA and gerE are transcribed in the absence of replication. The timing of the other two operons relative to replication is less certain; spoZZA probably belongs in the postreplication class of genes, but because of its position near the terminus of replication one cannot ruleout thepossibility that its transcription begins before the replication fork has passed through the locus. It is interesting that, of these six operons, only gerA is transcribed toward the origin, and gerA falls quite definitely in the postreplication class. These data are of course too limited to warrant much speculation, butthey are atleast consistent with the polymerase-collision hypothesis. Because sporulation genes are being intensively studied, more information about theirorientation relative tothe replication origin should be forthcomingsoon. We thank D. HENNER, M. STEINMETZ, L. WANG andS. ZAHLER for communicating unpublished data to us. T h e Bacillus Genetic Stock Center is supported by National Science Foundation grant DIR-8820971 and by donations from the industrial sponsors who are acknowledged in our current strain catalog.
LITERATURE CITED ALBERTINI, A. M., T . CARAMARI, D. HENNER,E. FERRARI andA. GALIZZI,1987 Nucleotide sequence of the outB locus of Bacillus subtilis and regulation ofits expression. J. Bacteriol. 169: 1480-1484. BEALL,B., M. LOWE andJ.LUTKENHAUS, 1988 Cloningand characterization of Bacillus subtilis homologs of Escherichia coli cell division genesjsZ andjsA.J. Bacteriol. 170: 4855-4864. BINNIE,C., and J. G. COOTE, 1983 Density gradient analysis of DNA replicated duringBacillussubtilis sporulation. Bacteriol. J. 156: 466-470. BOUVIER,J.,P.STRAGIER, C. BONAMYand J. SZULMAJSTER, 1984 Nucleotide sequence of the spo0B gene of Bacillus subtilis andregulation of its expression.Proc. Natl.Acad. Sci. USA 81: 7012-7016. BOYLAN,S. A., J.-W. SUH, S. M. THOMAS and C. W. PRICE, 1988 Gene encoding the alpha core subunit of Bacillussubtilis RNA polymerase is cotranscribed with the genes for initiation factor 1 and ribosomal proteins B, S13, S1 1 , and L17. J. Bacteriol. 171: 2553-2562. BREWER,B. J., 1988 When polymerases collide: replication and the transcriptional organizationof the E. coli chromosome. Cell 53: 679-686. BUGAICHUK, U. D., 1987 Studies of transcriptional regulation of the Bacillus subtilis developmental gene spoVE. J. Gen. Microbiol. 133: 2349-2357. BUGAICHUK, U. D., and P. J. PIGGOT,1986 Nucleotide sequence of the Bacillus subtilis developmental gene spoVE. J. Gen. Microbiol. 132: 1883-1 890. CALLISTER, H., and R. G. WAKE, 1974 Completed chromosomes in thymine-requiring Bacillus subtilis spores. J. Bacteriol. 102: 579-582. CALLISTER, H., and R. G. WAKE,1976 Homogeneity in Bacillus subtilis spore DNA content. J. Mol. Biol. 102: 367-371.
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