Vol. 172, No. 4JOURNAL OF BACTERIOLOGY, Apr. 1990, p. 1939-19470021-9193/90/041939-09$02.00/0Copyright © 1990, American Society for Microbiology

Complex Character of senS, a Novel Gene Regulating Expression ofExtracellular-Protein Genes of Bacillus subtilis

LIN-FA WANG AND ROY H. DOI*

Department ofBiochemistry and Biophysics, University of California, Davis, California 95616

Received 13 September 1989/Accepted 13 January 1990

The senS gene of Bacillus subtilis, which in high copy number stimulates the expression of severalextracellular-protein genes, has been cloned, genetically mapped, and sequenced. The gene codes for a highlycharged basic protein containing 65 amino acid residues. The gene is characterized by the presence of atranscription terminator (attenuator) located between the promoter and open reading frame, a strongribosome-binding site, and a strong transcription terminator at the 3' end of this monocistronic gene. Theamino acid sequence of SenS showed partial homology with the N-terminal core binding domain region ofbacterial RNA polymerase sigma factors and a helix-turn-helix motif found in DNA-binding proteins. The genecan be deleted without any effect on growth or sporulation.

The efficient expression of extracellular-protein genesinvolves a complex set of transcriptional regulatory factors.These include the products of genes such as sacU (18, 24),sacQ (1, 24, 50), sacV (26), prtR (29, 43, 52), hpr (19, 33), iep(42), sin (14), and senN (48, 49). Some of these factorscontrol the expression of genes by positive action, e.g., theproducts of sacU, sacQ, prtR, and senN; others have anegative effect, e.g., the products of hpr and sin. Uppromoter mutations or a high copy number of the positivefactor genes causes hyperexpression of extracellular proteingenes. On the other hand, deletion of a particular regulatoryfactor gene does not affect growth, sporulation, or thenormal level of expression of the extracellular-protein genes.This indicates that either the regulatory-factor gene is notessential or the absence of its function is compensated for bythe presence of the other regulatory factors.

Sequential deletion analyses have revealed the presence ofseveral sites upstream of the promoter of the extracellular-protein genes that appear to be the target sequences for theseregulatory factors (17). This upstream region may extend upto 250 base pairs (bp) (position -250) from the start point oftranscription (position + 1) and contain both positive andnegative regulatory target sites, and it is somewhat similar toupstream activating sites of eucaryotic promoters.We have recently identified a Bacillus natto transcrip-

tional regulatory-factor gene, senN, which in high copynumber stimulates the expression of a fairly wide spectrumof extracellular-protein genes (48, 49). In this paper wereport the isolation and characterization of a gene, senS(GenBank/EMBL accession no. M30611), from Bacillussubtilis that differs slightly in base sequence from senN,codes for a homologous but slightly different protein fromSenN, maps genetically in a unique position relative to theother regulatory factors, and has a complex gene structure.The deduced amino acid sequence of SenS showed a typicalhelix-turn-helix motif of DNA-binding proteins and hassignificant homology with several B. subtilis RNA polymer-ase sigma factors. The senS gene organization shows apromoter followed by a stem-and-loop region indicative of ap-independent transcription termination site, followed by theopen reading frame for SenS. This organization indicatesthat senS expression itself is highly regulated. Thus, the

* Corresponding author.

expression of extracellular-protein genes may be controlledby a cascade type of regulatory mechanism.

MATERIALS AND METHODS

Bacterial strains, plasmids, and media. Most of the B.subtilis strains used in this study were the same as thosedescribed previously (49). JM101 was used for production oflacZ fusion protein in Escherichia coli. Plasmid pMC1871was from Pharmacia. pRF373 was a kind gift from ReinholdBruchner (4). The 2 xSG medium (23) was used as thestandard sporulation medium for most of the expressionstudies.

Genetic mapping by using PBS1 transduction. To geneti-cally map the B. natto senN homologous locus in the B.subtilis chromosome, the EcoRI 1.8-kilobase (kb) fragmentcontaining the senN gene (48, 49) was subcloned into theintegration plasmid pCP115 (36) to create pCP115-N1.8. Thisplasmid was then used to transform B. subtilis DB2 (trpC2).Chloramphenicol-resistant (Cmr) transformants were se-lected on tryptose blood agar base plates containing 5 ,ug ofchloramphenicol per p.l. One of the transformants waspicked and named DB39. The integration of pCP115-N1.8 atthe homologous senS locus was further confirmed by South-ern blot analysis. A PBS1 lysate was then prepared from thisstrain and used to transduce the BGSC mapping-kit strains(7) to Cmr.

Molecular cloning of senS by gene conversion. With theprevious knowledge of the strong sequence homology be-tween senN and senS as revealed by Southern blot analyses(49) and the homologous integration described above, weused the gene conversion strategy to clone the B. subtilissenS gene. To do this, the N- and C-terminal parts of thesenN coding regions were deleted to form plasmids pWL71and pWL72 (Fig. 1). These plasmids, showing a SenS-phenotype, were then used to transform DB102 (21). Kan-amycin-resistant (Kmr) transformants with the Sen+ pheno-type restored by gene conversion were screened directly on2xSG plates containing kanamycin (5 ,ug/ml) and skim milk(1%).

In vitro synthesis of sen gene products. For in vitro analysisof the Sen proteins, the coding regions of senN (the EcoRV-BamHI fragment [49]; Fig. 2) and senS (the HindIII-BamHIfragment; Fig. 2) were subcloned into the Bluescribe plasmidfrom Stratagene so that the sen gene expression was under

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EcoRi ~~~~~~~~~PlasmidSenEcoRI EcoRV HincIl Sst BomHI Xbo1 EcoRl

pWL71 -pWL72 -

FIG. 1. Construction of plasmids pWL71 and pWL72 for cloningof senS by gene conversion. The upper part of the figure shows arestriction map of the B. natto senN region as a 1.8-kb EcoRIfragment originally cloned in pUBHR-N1.8 (49). The shaded regionrepresents the part which was essential for the Sen+ phenotype. Thelower part of the figure schematically illustrates the 5' and 3' enddeletions of the essential region in pWL71 and pWL72, respectively,which all showed a Sen phenotype. as shown at the right side of thefigure.

the control of the T7 promoter from the vector DNA as wellas its own promoter. These plasmids (pBP-senN and pBP-senS), after linearization by use of proper restriction en-zymes, were used as templates for in vitro transcription-amplification by using the T7 transcription kit from BethesdaResearch Laboratories, Inc. The amplified mixture, includ-ing the original plasmid template and the newly synthesizedmRNA, was then subjected to in vitro-coupled transcription-translation by using the Amersham expression kit in thepresence of [35S]Met. The labeled polypeptides thus pro-duced were separated onpolyacrylamide gel electrilowed by autoradiographispecified by the kit supplmodifications.

N-terminal sequence analexpressed in E. coli and B.was constructed for exprcanalysis of the N-terminalprotein. This was done bcontaining the promoter anresidues of senS (see Fig. eof pMC1871 to form an in-Iplasmid thus formed, pWLresulting PstI cassette of thmoved into shuttle plasmi(E. coli JM101 and B. Sliproduced SenS-LacZ fusi(activity. These in vivo-depurified by using the aminfinity chromatography tecthiogalactopyranoside-agaiously (5). The essentiallydetermined by SDS-PAGE7.5% SDS-PAGE; this prcblotting onto an Immobiloi

A.FE

B.IV H Ssti B

I

E V

FIG. 2. Comparison of re

fragment originally cloned frcfragment cloned by gene convparts are the regions essentialsolid black bar in panel B rep]B. subtilis. Restriction sites alB, BamnHI; and X, XbaI.

the method described previously (28). After the membranewas stained with Coomassie blue, the band corresponding toSenS-LacZ was cut out and directly subjected to an auto-mated sequence analysis by the Protein Structure Labora-tory at the University of California, Davis.

Disruption of the senS locus in the B. subtilis chromosome.The introduction of a senS deletion mutation in the B.subtilis chromosome was achieved by replacing the senSlocus with a cat cartridge through a homologous double-cross recombination event. For this purpose, the 2.4-kilo-base (kb) EcoRV-EcoRl fragment of pWL80 (see Fig. 2) wassubcloned into the HincII-EcoRI double cut pUC19 to formpWL26. The 0.4-kb HincII-BamHI fragment of pWL26containing the seniS gene (see Fig. 2) was then removed andreplace by a cait cartridge (1.4 kb in length) derived frompC194, forming pWL30. This plasmid, after linearizationwith SphI-EcoRI double digestion, was used to transformDB2 to Cmr. This mutant strain was named DB417.Other methods. Nick translation, Southern blot, dideoxy-

chain termination DNA sequencing (37), and other standardDNA techniques and enzyme assays were performed asdescribed in our previous communications (48, 49).

RESULTS

a 15% sodium dodecyl sulfate- Genetic mapping of the senS locus in B. subtilis. In ourophoresis (SDS-PAGE) gel. fol- previous report on the cloning and characterization of the B.ic analysis. Reaction conditions natto senN gene (49), we indicated that a B. siubtilis ho-liers were followed without any molog, named senS ("S" for subtilis), was detected by

Southern blot analysis. To further test the degree of se-lysis of SenS-LacZ fusion proteins quence homology between these two closely related speciessubtilis. A senS-lacZ gene fusion and to determine the genetic location of senS in relation toession studies as well as for the other known small regulatory genes with similar phenotypesamino acid sequence of the SenS (such as prtR, sacQ, etc.), an integration plasmid, pCP115-y subcloning the AlulT fragment, N1.8, was constructed by using the 1.8-kb EcoRI fragmentid the deduced first 26 amino acid from B. natto (49; also see Fig. 2). The recombinant plasmidi and 8), into the unique SinaI site was subsequently integrated into DB2 to form DB39 (trrpC2frame senS-lacZ gene fusion. The senS: :caut).,211, was digested with PstI. The The expected integration of pCP115-N1.8 at the senSie senS-lacZ fusion gene was then locus in DB2 was confirmed by Southern blot analysis (datad pRB373 to form pWL289. Both not shown). DB39 was then used to make a PBS1 donor,btilis DB403 harboring pWL289 lysate to be used in subsequent mapping experiments. Theon proteins with P-galactosidase recipients were the strains from the BGSC mapping kit-rived fusion proteins were then constructed by Dedonder et al. (7). The results are given inophenylthiogalactopyranoside af- Table 1 and Fig. 3. By three factor crosses, the c'at (senS)hnique on a 10-ml aminophenyl- marker was shown to be 40% linked to the tre-12 locus, androse column as described previ- 43% to glyB133, in the order of tre-senS-gly. A finer map-pure (more than 90% pure as ping, using B. subtilis 1A603 (spI3c2 trpC2 thiA78::Tn917)

) peak fractions were loaded to a (46) as recipient, indicated that senS was more than 97%)cedure was followed by electro- linked to the thiA78 locus. These data allowed us to locaten membrane (Millipore Corp.) by the senS locus at 700 on the B. slubtilis genetic map (34) and,

hence, to genetically define senS as a new gene differentfrom other known genes with similar phenotypes, includingsacQ (at 2850), sacU (3100), sac V (360), prtR (2000), hps (750),

X E and iep (3100).Cloning of the B. subtilis senS locus by gene conversion.

Gene conversion or gene replacement is a technique basedl on the strong mismatch repair mechanism of B. sblbtilis (6,

Hind Ill H B X E 20). Several cases have been reported in which gene conver-sion was used to construct specific deletion mutations (21,striction maps for the 1.8-kb EcoRI 51) and to clone particular DNA fragments containing inte[-

im B. natto (A) and the 2.8-kb EcoRl tnanetic m lar DNA fragme c ing teersion from B. slbtilis (B). The shaded esting genetic markers(21h 30, 41). We decided to use theIfor the Sen+ phenotype, whereas the gene conversion technique to clone the B. subtilis senS locusresents the 1-kb insert only present in instead of using other conventional cloning techniques,re: E, EcoRI; V, EcoRV; H, HindIlI; based on the observation that the B. natto senN locus was

highly homologous to senS of B. slbbtilis. The restoration of

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TABLE 1. Three-factor transduction cross to map senS relativeto tre and glyBa

Recipient classesSelection No. Order implied

Tre Cmr Gly

Cmr + + + 17 tre-sen-gly+ + - 136_ + + 94- + - 169

Tre+ + + + 4 tre-sen-gly+ + - 52+ - + 0+ - - 151

Gly+ + + + 0 tre-sen-gly+ - + 0- + + 48- - + 160

a Donor, DB39 (trpC2 senS::cat); recipient, BGSC5 (trpC2 glyBJ33 metC3tre-12).

the sen phenotype would be due to gene conversion on themulticopy plasmid. For this purpose, plasmids pWL71 andpWL72 were constructed as shown in Fig. 1. These plasmidswere used to transform DB102 (6, 21) to Kmr, and positivetransformants which restored the Sen activity by gene con-version were screened directly on skim milk plates contain-ing kanamycin. In each case, about 1 to 2% of the total Kmrtransformants showed the Sen+ phenotype, a frequencynormally observed for gene conversion events (21, 22). Sixpositive clones from each transformation were picked forrestriction map analyses. The results are shown in Fig. 2B.All twelve clones analyzed gave rise to the same restrictionmap, as shown. Upon EcoRI digestion, a 2.8-kb fragmentwas released, which was 1 kb longer than the 1.8-kb EcoRIfragment from B. natto containing the senN gene. However,this size matched the size observed previously in the South-ern blot analysis (49). Since gene conversion works by amismatch repair mechanism, the results suggested that theextra 1-kb sequence must be located between the two EcoRIsites. Further analyses with EcoRV, BamHI, Hincll, XbaI,HindIII, and SstI demonstrated that the restriction maps at

tre sen::cat-p p

73%~63%

98%

73%

77%I

_______________________ 4~~>99%

glyB

FIG. 3. Genetic map of the senS region of the B. subtilis chro-mosome, constructed from the three-factor crosses shown in Table1. The arrow tails indicate the selected marker, and the geneticdistances are shown as 100 minus the percentage cotransductionfrequency. Reciprocal recombination values between senS::cat andeither tre or glyB were unequal, with the genetic distance appearingsmaller when senS::cat was the selected marker.

TABLE 2. Comparison of stimulatory effects of multicopy senSand senN genes on the production of some extracellular enzymes

Stimulation (fold) with:Enzyme

senS senN'

Alkaline protease 2.8 2.7(subtilisin)

Neutral protease 3.7 3.7Amylase 1.8 1.8Alkaline 1.8 1.8

phosphatase

a Data from Wong et al. (49).

the two ends of the 2.8-kb "S" fragment (Fig. 2B) were thesame as that for the 1.8-kb "N" fragment (Fig. 2A), exceptfor the absence of the SstI site in the 2.8-kb fragment, whichwas later demonstrated to be caused by a single base pairchange in this region (see later discussion). These results ledus to the following conclusions: (i) we had cloned the B.subtilis senS locus, which is homologous but not identical tothe B. natto senN region; (ii) all the data obtained wereconsistent with what would be expected for a gene conver-sion mechanism; (iii) the B. subtilis senS region contained anextra 1-kb sequence which was not present in that region ofthe B. natto genome. This 1-kb sequence (indicated by thesolid box in Fig. 2B) was located between the EcoRV andHincll sites and contained a HindIII site. To our knowledge,this was the first case in which cloning of a homologouschromosomal DNA fragment more than 1 kb (1,580 bp frompWL71 and 1,280 bp from pWL72) in size was successfullyachieved by gene conversion transformation.Two clones (pWL77 and pWL80), one each from the

transformants derived from pWL71 and pWL72, respec-tively, were chosen for further characterization. A deletionmapping similar to that for pUBHR-N1.8 (49) was con-ducted. The results showed that full Sen activity can bedetected by using the 0.6-kb HindIII-BamHI fragment (datanot shown), suggesting that the 1-kb insert located upstreamof the B. subtilis senS region was not directly responsible forthe Sen phenotype.The effects of the multicopy senS gene in plasmid pWL95

(a pUB18 derivative containing the 0.6-kb HindIII-BamHIfragment) on the production of extracellular proteases, amy-lase, and alkaline phosphatase were examined by using thesame procedures as previously described for the senN gene(49). The results are shown in Table 2. These results indi-cated that is is difficult to distinguish senS from senN on afunctional basis.

Nucleotide sequence of senS and its flanking regions. Tofurther characterize the senS coding region and to investi-gate the origin and properties of the upstream 1-kb insert, theEcoRV-BamHI fragments from pWL77 and pWL80 weresubjected to DNA sequencing analysis. The nucleotide se-quences obtained for these two clones were identical and arepresented in Fig. 4. As predicted from the Southern blotdata, the DNA sequence in the senS region is very homolo-gous to that of the senN region (see Fig. 6), except for the1-kb insert, which is actually 1,115 bp as determined bysequencing.A computer analysis of the sequence in both orientations

revealed three open reading frames (ORFs) with significantsize (more than 60 amino acids in length), as indicated in Fig.5. However, only ORFi and ORF2 were preceded byputative translational initiation signals, i.e., a Shine-Dal-garno (SD) or ribosome-binding site (rbs) sequence followed

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EcoRV IAGTTCTTGGAAATTCTGATTTTCGATATCTGGCGAATTTACGTAGTCTCCCATCGTTTCTTTCGAAAGGGACGTTCTCAGCCCCTCAATCCAGCGGACA ()

TTTGTCTTTTTTCTCCAGGGGATGTCCAGTTTGTTAAGTATTCCTGGGCGATGATTGCGTCACGATAATAMAATGC CGTTTGGTCGGGAGCGAC CCGTC C ' n0n

GGCTGCnCCCGCCGAGTGCTTGCTGCCAGACACTGGCGTTTGATTCGGAGCGTGCTCTAAAAMGTGTTTTATTGTTGAGATCGCACGTTCTGATAATGGC '300SmaI

TTTCAATGAAAGAGCCGGAGCG=CATT=TTGAGGCTGATTGCCTCCCGGGCTGTTAAAAAAGGTTACCGCTTCAATGAATGGCGTTGTTTTTACCA -40O(

TTCCGCTTGACGGACTTCCTGCTTTCAATAAAGGCTTTAACAGT=TTTTAACTCTGTTFTTGGCCCGACAAATTGGCCGAGGGCTTCTATGCGGTTTAC 500AccI

TTCTTTAGGCCAAAACTCTATTGATGATGTAAGCCGGTCATCTGTATACGGGGCCCAGTTCTGCCACGTGTTATATACTTCCTCAAAATCATCCCATCCC 6600)

CATGTAATAGAAAAAATCGACACTTGAGAGATGGGCACTGCTTTAAATGTCATGGAGGTGACTATGCCGAAATTGCCTCCTCCGCCTCCCTGAGACGCC C ,7 00PvuII

AAAATGTGGATGATTTGAACAGCTGACTGTAATCAGATCAGCGCCCTCTTTTTCGTCTGCTACGATCATCTCAAGCTGCACGAGGCTGTCGCAAGTAAGA 8800

CCGGCAGCCCTTGTTAAAAGTCCAATTCC CCCTCCGAGAGTTAAACCTGTGAGCCCTACATTAGCAATGGTGCCTGCGGGAAGCGTCAGGCCGTATTGCC 900

AGAGTGTCCGATAGACTTCTCCCAATTCAGCCCCCGCTTCAATATAGGCCAGCTTTTTATCCTGATTCACAGTTATTTTTTTCATCTCGCTTAAATCAAT 1000

AACAAGACCGTTATTTAAAAGGGAAAAGTTCTCATAGCTGTGTCTGCCGCCTCTAATACGGAAAGGCACACGGTTTTCACGCGCCCATTTCAGCGCATTG 1100HindIII I

AGTGCATCCTGTTTGTTTGGCAAAACACAATGATGTCAGATCCTTCTAAGCTTAGGTTAATATTGGTTCTTGCTTCGTTATAGTCCGGATCATCCCGT 12900

GTCACGATACGTCCGGTCAATTTGTCTTTTCCACACTCCCACATCTCTTTCTCTCGTATTCTAGTTTCTCTAGCTTATGCGTCAGGGGAAAAGAGTGTA 1 30 0HincII HincII

TAAGGAAAAAGCGGGGATGCAATCTGATACAGTGTCAACACCCTCAAAAAATAGTTGACAGGTCGGTATTGTATGAATTAACATGGTCAGTACAAATTTT 1400

TCAAATTTATCGCGCTGATCGGAACACCGAAGGCTCTTATCGTTTAGATAAGGGCCTTTTTGTATGAAAAAGGGGGGATTATTGATGGGAGTCAAAAAA 1500

GAAAAGGGGAGAAAACGATTCAGGAAGCGAAAAACCTACGGGAATCAGATTTTGCCGCTTGAGCTGCTGATTGAAAAAAACAAACGAGAGATTATAAACA 1600

GCGCGGAACTCATGGAAGAAATTTATATGAAGATTGATGAGAAGCATACGCAATGTGTAACTAAATATAAAAAAACCCGCTGACTACAACGGGTTTTTGC 1700BamHI

ATTTCTCCATTAAGAATCTTT AATCGGCAATCCAAGGCCTTCTGCCACGCGTTTTCCGTATTCAGGATCC 1,73

FIG. 4. Nucleotide sequence of B. .scbtilis setnS and its flanking regions. The presented sequence starts at 25 bp upstream of the EsLoRVsite and ends at the BamHI site as shown in Fig. 2B. The sequence was obtained by dideoxy sequencing of two separate clones, pWL77 andpWL80 (see text), with both strands sequenced (except for the 25 bp upstream of the EcoRV site). All the restriction sites used in the M13subcloning were confirmed in other independent clones overlapping the corresponding sites. The sequence is presented from 5' to 3' in thesame orientation as that for senS transcription. The start (ATG) and stop (TGA) codons of the SenS coding region are double underlined. Theboundary of the 1-kb (actually 1,115 bp) insert is marked by two vertical arrows. Important restriction sites are shown above theircorresponding recognition sequences.

by an in-frame initiation codon (usually ATG). But ORF3may still be functional in vivo via a translational couplingmechanism, since its N terminus overlaps with the C termi-nus of ORF2. This, of course, needs to be experimentallytested. The C terminus of ORF3 is truncated at the 5' end ofthe sequence presented in Fig. 4.

Several interesting features were observed by sequencecomparison between senS and senN, as presented in Fig. 6.First of all, the senS and senN regions are very homologous,more than 95% sequence identical. There are, however,several significant differences. The G to A change in the SDsequence at nucleotide (nt) 336 made the senS rbs (AG =

-20 kcal/mol, determined by the method of Tinoco et al.[44]) weaker than that of senN (AG = -25 kcal/mol). Thereare two important mismatches in the coding regions. The

ORF2

ORF3ORF 1

EQ RV Sma I A1 I Hnd III Hllac 11 lamHI

FIG. 5. ORFs deduced from the sequence data presented in Fig.4. The extent of each ORF is represented by the length of the solidline, with its arrowhead indicating the direction of translation. Theputative transcription initiation sites (promoters) and translationinitiation sites (proper combination of SD sequence and in-frameinitiation codon) are represented by the small arrows and squares,respectively. The restriction map and other symbols are the same asthose described in the legend to Fig. 2.

first one is at nt 397, which changed a Phe residue of SenNto a Tyr in SenS. The second one is more dramatic; it is amismatch which, at nt 528, changed the stop codon (TAG) ofsenN to a Tyr codon (TAT) in senS, leading to the presenceof 5 additional amino acid residues at the C terminus ofSenS. Finally, the mismatches in the terminator regions alsomade significant differences between the two genes in termsof the strength of the stem-loop structure, i.e., the stem-loopof senS is weaker than that of senN. The single base changeat nt 467 is the cause of the loss of the SstI recognition site(GAGTCT) in the senS coding region (see Fig. 2 andprevious discussion). As a conclusion, we should mentionthat although the sequence comparison demonstrated signif-icant differences between the two genes, there seemed to beno functional differences when these genes were introducedinto B. siubtilis on high-copy plasmids. But this does notexclude the possibility that they may have physiologicaldifferences when each exists as a single copy in vivo.When ORF1 (Fig. 5) was searched for a putative transla-

tion initiation site(s), two such regions were found. Thestronger one with a better SD sequence is shown in Fig. 6.The other weaker SD sequence (AAGGG) is at nt 310 to 314,which was also followed by an in-frame ATG codon at nt 325to 327. This ATG codon is seven amino acid residuesupstream of the one shown in Fig. 6. At this stage, theassignment of the initiation site as shown in Fig. 6 was purelybased on theoretical studies and a comparison of the se-quence data. This was, however, later proved correct byN-terminal sequence analyses to be presented later.One of the most puzzling, and perhaps most important,

features revealed by the sequence analyses was the discov-ery of two inverted repeat (IR) sequences between the

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1115 bp

EcoRV GABs GATATCTGGCgAATTACGTAGTCTCCCATCGTTTcttTCGAAAGTCCGGATCATCCCGT 60Bn GATATCTGGCcAATTTACGTAGTCTCCCATCGTTTaacTCGAAAGTCCGGATCATCCCGT 60

Bs GTCACGATACGTCCGGTCAATTTTGTCTTTTCCACACtCCCACATCTCTTTCTCTCGTAT 120Bn GTCACGATACGTCCGGTCATTTTGTCTTTTCCACACaCCCACATCTCTTCTCTCGTAT 120

Bs TcTAGTTCTCTAGCTTATGCGT0AGGGGAaAAGAGTGTATAAGGAM AAGCGGGGATGc l80Bn TtTAGTTCTCTAGCTTATGCGTCAGGGGAtAAGAGTGTATAAGGAAAAAGCGGGGATGt 180

-35Bs AATCTGATACAGTGTCAACaCCCTcAAAAAATAGTTGACAGGTCGGTATrGTATGAATTA 240Bn AATCTGATACAGTGTCAACtCCCTtAAAAAATAGTTGACAGGTCGGTATTGTATGAATTA 240

-10Bs ACA.TGTCAGTACAAA TCAAATTTCGCGCTGATCGGAACACCGAAGGCTCTTAT 300Bn ACATGGTCAGTACAAATTTTTCAMTTTATCGCGCTGATCGGAACACCGAAGGCTCTTAT 300

SD MetGlyValLysLys 5Am: Irr.TTPAlATAArz>-rTlrSlirrr,A rrsAA A lfrr"Wf A,.^ A^ . .r. .

1 2- .. ,,

3 4U'

43-

26-

18-

Bn CGTTTAGATAAGGGCCaGGGATTATTGATG AGTCAAMAAZ MetGlyValLysLys

360360

5

GluLysGlyArgLysArgPheArgLysArgLysThrQ1GlyAsnGlnIleLeuProLeu 25Bs GAAAAGGGGAGAAAACGATTcAGGAAGCGAMAAACCTii GGGAATCAGATTGCCGCTT 420En GAAAAGGGGAGAAAACGATTtAGGAGCGAAAAACCZg;GGGAATCAGATTTTGCCGCTT 420

GluLysGlyArgLysArgPheArgLysArgLysThrOiGlyAsnGlnIleLeuProLeu 25

GluLeuLeuIleGluLysAsnLysArgGluIleIleAsnSerAlaGluLeuMetGluGlu 45Bs GAGCTGCTGATTGAAAAAAACAAACGAGAGATTATAACAGCGCGGAaCTCATGGAAGAA 480Bn GAGCTGCTGATTGAAAAACAAACGAGAGATTATAACAGCGCGGAgCTCATGGMGM 480

GluLeuLeuIleGLuLysAsnLysArgGluIleI leAsnSerAlaGluLeuMetGluGlu 45

I1eTyrMetLysIleAspGluLysHisThrGlnCysValThrLysT..LysLysTrArg 65

Bs ATTTATATGAAGATTGATGAGAAGCATACGCAATGTGTAACTAAAO'AAA CCCGC 540Bn ATTTATATGAAGATTGATGAGAAGCATACGCAATGTGT AACTAIAAAACCCGC 540

IleTyrMetLysIleAspGluLysHisThrGlnCysValThrLys"*. 60

BsBn

BsBn

TGAcTaCAaCGGGTCTTcCATTAAGaATCMTTAATCGGCAMTCCAGG 600TGAtTtCAgcGGTTrGArCTaCATTAAGcATC TTMTCGGCMTCCAGG 600

CCTTCTGCCACGCGTTTTCCGTATTCAGGATCC 632CCTTCTGCCACGCGTTTTCCGTATTCAGGATCC 632

BamHI

FIG. 6. Sequence comparison of senS and senN genes, theirhomologous flanking regions, and their gene products. The DNAsequences are shown from 5' to 3', with the first nucleotide in theEcoRV recognition site as number 1. To facilitate the comparison,the same numbering coordinates are used for both sequences bylooping out the 1,115 bp insert for B. subtilis, as shown at the top.Amino acid sequences are shown above (for senS) and below (forsenN) their corresponding DNA sequences and are numbered fromthe first Met. Differences in DNA sequences are shown by lower-case letters, whereas changes in the amino acid sequences aremarked by dotted shading. The putative promoter and rbs sites areindicated by dots between the two DNA sequences. IR sequencesare shown by solid lines with inverted arrow heads. Abbreviations:Bs, B. subtilis sequence; Bn, B. natto sequence; SD, Shine-Dal-garno sequence.

promoter and rbs of the sen genes. These are shown in Fig.6 as paired solid lines with inverted arrows. The first long IRsequence forms a stable stem-loop structure, which was

followed by a stretch (six) of T's. This is a characteristicfeature of procaryotic p-independent termination sequences(35). The second shorter IR sequence was not as stable asthe first one. But there are two important features for thissecond IR sequence that make it significant. First, it over-laps with the rbs of the sen gene, which may play a role intranslational control of sen gene expression. Second, the lefthalf of this stem overlaps with the right half of the stem of thefirst IR sequence, which makes this organization somewhatlike an attenuator structure for other systems (2, 53). Al-though no classic attenuation mechanism has yet beendetected in this region, the second IR sequence may have aregulatory effect on the first IR sequence as a p-independenttranscription terminator.

In vitro-expressed products of senS and senN. The se-

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6- w

3-~

FIG. 7. In vitro synthesis of sen gene products. Lanes 1 though4 contain translation products obtained from no DNA (lane 1)(negative control), vector pBluescribe plus pBP (lane 2), pBP-senN(lane 3), and pBP-senS (lane 4). DNA templates (1 ,ug each) werefirst linearized by PvuII digestion and then transcribed by using theBRL T7 transcription kit. The "amplified" template-mRNA mix-tures were subsequently expressed by using the Amersham in vitroprocaryotic transcription-translation kit in the presence of [35S]Met.One third of the reaction mixtures was separated directly on a 15%SDS-PAGE gel, and this procedure was followed by autoradiogra-phy after drying the gel on filter paper. The numbers shown on theleft are molecular weight standards generated by the BethesdaResearch Laboratories prestained markers (low range). The arrowsnear lanes 3 and 4 indicate the bands which are absent in lanes 1 and2 and, hence, are produced by the coding regions of senN and senS,respectively.

quence data presented above revealed significant differencesat the C termini of SenN and SenS, i.e., SenS is 5 aminoacids larger than SenN, which makes about a 10% differencein their molecular masses due to their small molecular sizes.To further confirm this, an in vitro expression system wasused to synthesize [35S]met-labeled sen gene products. Sincethe stem-loop structure found between the promoter and thesen coding region was likely to reduce the transcriptionefficiency of the corresponding genes by the Amersham invitro transcription-translation kit, which is basically an E.coli cell-free lysate, an additional in vitro transcriptionstep-an mRNA "signal amplification"-was introduced byusing the T7 transcription system. T7 polymerase has theadvantage of being insensitive to bacterial transcriptiontermination signals (25; F. W. Studier, A. H. Rosenberg, andJ. J. Dunn, Methods Enzymol., in press). The results arepresented in Fig. 7. The band patterns in lanes 3 and 4 led usto the conclusions that (i) the senS gene product is indeedlarger than that of senN, as predicted from the sequencingdata, and (ii) the overall sizes of both protein productsdetermined by SDS-PAGE match the molecular massesdeduced from the ORFs by using the ATG codons as shownin Fig. 6, which are 7.9 kilodaltons (kDa) for SenS and 7.2kDa for SenN. The results, however, also revealed a bandaround 3 to 4 kDa, which was not expected from thesequence data. The identity of this polypeptide is thereforeunknown. Upstream deletions down to the stem-loop struc-ture of the senS coding region indicated that this polypeptide

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was not associated with the upstream sequence. Deletionsaffecting the rbs, hence the translation, of senS howeverabolished the 3.5-kDa band (data not shown). Therefore, the3.5-kDa polypeptide might be an artifact resulting fromspecific degradation of the sen gene product.

N-terminal sequence of senS gene products expressed in B.subtilis and E. coli. For expression studies of the single-copysenS gene in the B. subtilis chromosome and for N-terminalsequence analyses, a senS-lacZ fusion gene was constructedas described in Materials and Methods. pWL289, a B.subtilis-E. coli shuttle vector containing the fusion genecassette, was introduced by transformation into JM101 andDB104 for expression in E. coli and B. subtilis, respectively.SenS-LacZ proteins produced in these strains were thenpurified by the aminophenylthiogalactopyranoside affinitychromatography method (5). Starting from 500-mi late-logphase LB cultures (for B. subtilis, the culture medium alsocontained 0.5% glucose), about 0.5 mg (from B. subtilis) to 2mg (from E. coli) of fusion proteins was obtained. Theseproteins were more than 90% pure when checked by SDS-PAGE (data not shown). About 24 ,ug each, corresponding to200 pmol, of the B. subtilis- and E. coli-derived fusionproteins was run on a standard 7.5% SDS-PAGE, followedby electroblotting onto the polyvinylidene difluoride mem-brane (28). The membrane portions containing the SenS-LacZ band, after staining with Coomassie blue R-250, werecut out for direct automated sequence analysis. The resultsobtained for the first six amino acid residues were Gly-Val-Lys-?-Glu-Lys for the B. subtilis-derived fusion proteinand Gly-Val (Phe)-Lys-Lys-Glu-Lys for the E. coli-derivedone. These sequences matched exactly to the deducedN-terminal sequence (residues 2 through 7) of SenS, with itsfirst Met residue probably being removed after translation(see Fig. 6).

Transcriptional regulatory features of senS gene. Similarexperiments on promoter and terminator mapping as thosereported previously for the senN gene (49) were also con-ducted for the senS gene. Strong promoter activity wasdetected when the Sau3A (nt 40)-Sau3A (nt 278) fragmentwas cloned in the BamHI site of the promoter probe plasmidpWP19 (see Fig. 9). However, when the shorter Sau3A (nt40)-HincII (nt 196) was tested in a similar way, no promoteractivity was observed, indicating that the putative promoterwas located between the Hincll (nt 196) and Sau3A (nt 278)sites. This coincides well with the region predicted tocontain the putative -35 and -10 regions (nt 215 to 245; Fig.6) of B. subtilis F43 (alA) promoters (8). By using a similarmapping strategy and our terminator probe plasmid pWT19(48), a strong termination activity was located on the AluI (nt415)-BamHI (nt 620) fragment, a region showing a stem-loopstructure (see Fig. 6) characteristic of procaryotic p-indepen-dent transcription terminators.

Figure 8 is a schematic diagram showing the results fortesting the putative terminator-attenuator structure in frontof the SenS coding region revealed by sequence analysis.The test was done in our promoter-probing plasmid pWP19,which contains the pUC19 polylinker in front of a promot-erless subtilisin gene (aprE) (47). As the data indicated, thetranscription activity, monitored by the specific activity ofthe reporter gene product (subtilisin), was reduced dramat-ically when the stem-loop structure was included in pWP-AA compared with pWP-AS, which contained only thepromoter region. This might explain the relatively lowstimulatory effect (two- to fourfold) of the multicopy sengene on the expression of extracellular-protein genes incomparison with other similar regulatory genes (48, 49).

IIr 9 u~~~~~~~~snSKlilil S A RS H fImHiA

Plasmid ActivitypWP19

pWP-AApWP-AS

0%

7%/6100%/.

FIG. 8. Detection of a termination activity between the promoterand the coding region of senS gene. The upper part is a restrictionmap with symbols representing the promoter (bent arrow), rbs (solidsquare), and stem-loop structures. The coding region is shown bythe shaded area. Restriction enzymes are: S, Sau3A; A, AluI; R,Rsal; and H, HaeIII. The lower part of the figure indicates therestriction fragments used to construct plasmids pWP-AA andpWP-AS, with the arrowheads indicating that the same transcriptiondirection as that of aprE in pWP19 (47) was maintained duringsubcloning. After introduction into DB104 (21), the expression timecourses of DB104(pWP-AS) and DB104(pWP-AA) were studied,with DB104(pWP19) as a negative control. Similar constitutiveexpression curves were obtained for both clones (see Resultssection). The average specific activity produced from relative spe-cific activity of 7% was obtained for DB104(pWP-AA).

Time course studies of aprE expression in plasmids pWP-AA and pWP-AS indicated that although there were morethan 10-fold differences in activity, both constructs showed asimilar expression pattern. The promoter was turned onduring growth and continued to be expressed during thesporulation phase (we monitored the expression up to T3,i.e.A 3 h after the onset of sporulation) (data not shown).These results suggested that senS gene expression may be

regulated by an antitermination mechanism but not by tem-poral regulation, although the target genes controlled bySenS appeared to be regulated in a temporal fashion (12, 30,32, 45).

Deletion inactivation of the senS gene in the B. subtilischromosome. To determine whether the senS gene wasessential for growth, a deletion of the gene was constructedin vitro and was transfered into the B. subtilis chromosome.The details have been described in Materials and Methods.After transfer, the double crossover of the mutated gene intothe homologous chromosomal locus was confirmed bySouthern blot analysis. Figure 9 shows the results of blotswith EcoRI- and HindIII-digested chromosomal DNA.Lanes 1 and 3 show the EcoRI digestion patterns ofDB2 andDB39 chromosomal DNA, respectively. Lanes 2 and 4represent similar results obtained with HindIII digestion(lane 2 for DB2 and lane 4 for DB39). As predicted, the2.8-kb EcoRI band in the wild-type strain (lane 1) wasconverted to a band around 3.8 kb (lane 3). The 1-kb increasein size was a result of the replacement of the 0.4-kb senScoding region by the 1.4-kb cat cartridge (see Materials andMethods for details of plasmid constructions). Similarly, inthe HindIlI digestion, the upper band, which contains thesenS coding region, showed lower mobility for DB39 DNA(compare lanes 2 and 4). The relative low intensity of theupper HindlIl bands was due to the shorter region ofcomplementarity to the probe used, which was the EcoRV-BamHI fragment (see Fig. 2B).DB39 grew normally, formed spores on 2xSG medium,

and secreted apparently normal amounts of extracellularproteases.

Sequence homology of SenS to RNA polymerase sigmafactors. The highly charged nature, the basic amino acid(especially Lys)-rich composition of SenS, and its regulatory

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senS REGULATION OF B. SUBTILIS GENES 1945

1 234SenS 11-31

Sin :7-36SpoIIIC 95-114SpoIIAC 21-40SigA 332-351SigB 224-243SigE 206-225SigH 171-191

R F R K R K T Y G N Q I L P L E L L I.EXC

L S E L A E K A G V A K S Y S S I E RQ R E I A K E L G I S R S Y s R I E K

E L I K Q S Q N G D Q Q A R D LL I E KL E E V G K V F G V T R E R R Q I.EjQ K E T G D I L G I S Q M H V S R Lip RQKDVADMM GISQSYIISR tAXKKVLVLYLD IRSYQEWSDELRLH Eit

4.3-up

4U

2.3-,

1.9 - e

1.7-

FIG. 9. Southern blot of DB2(trpC2) and DB417(trpC2 senS::cat)chromosomal DNA using the 1-kb EcoRV-BamHI fragment of B.subtilis as the probe. Lanes 1 and 3 contained EcoRI-digested DNAof DB2 and DB417, respectively. Lanes 2 and 4 contained the sameDNA as those for lanes 1 and 3, digested with HindIII. Themolecular weight markers are the phage 429 DNA digested withHindIII (49). See the text for a discussion of the results.

effect at the level of transcription (49) suggested to us thatSenS might be a DNA-binding protein. This notion wasstrengthened by the sequence analysis results shown below.When we analyzed known protein sequences in various

SenS 1-65

SpoIIAC 11-74SpoIIIG 17-80SpoIVCB 28-91SigA 112-175SigB 8-71SigE 36-99SigH 12-75

FIG. 11. Sequence alignment of DNA domains from several B.subtilis-DNA interacting proteins. The boxed regions indicate se-quences with identical or chemically related amino acid residues.One gap, corresponding to position 28 of the SenS protein, wasintroduced to maximize the homology alignment. The numbers afterthe name of each sequence represent the regions used for thisalignment. The complete sequence of each protein was derived fromthe same sources as those in the legend to Fig. 10, except for Sin (14)and SpollIC (11).

data banks, no closely related homologs of SenS were foundin other organisms. But significant partial homology wasfound between SenS and several known bacterial RNApolymerase a factors. A sequence comparison of SenS withthose factors of B. subtilis origin is shown in Fig. 10. Due toits small molecular size, the SenS homologous regions inmost of the or factors cover only part of the protein, mainlythe region around the core binding domain, as postulated byseveral groups (10, 16, 39, 40). The functional significance ofthis homology awaits more detailed analyses of the SenSprotein by genetic and biochemical approaches, which arecurrently being undertaken.

Further analysis of the secondary structure of SenS re-vealed a helix-turn-helix motif from residues 11 to 31, whichis characteristic of DNA-binding domains deduced frommany DNA-binding proteins (31). This particular region, asshown in Fig. 11, showed not only structural similarity butalso sequence homology to the DNA-binding domains ofseveral B. subtilis RNA polymerase c factors and knownDNA-binding proteins.

It is interesting to note that in c factors the "core binding"and "DNA binding" domains are physically separated fromeach other in their primary sequences, while they seem to beoverlapping in SenS.The origin and function of the upstream insertion sequence

Gj K K !E K G R jIR F R KLS K

N A Q L K 1D H E V :Ki E L I K Q SQ N.GT.QQ A R .1) LI.P Vt.LLK NSjIE M R:K" L F R Q I Q D EI I.D.S A R E' KtLV...P Q P L S S S E E L K Y L E L M A KG D .. H A R N MK i

V N L S A K E E I A Y A Q LKI E Et E S K R RT-L i

T K T K.CKI E V D j L I S D Y Q T KIQD EiQ A Q T' R

P P P L S K D E E Q V L L MK. L P N ,qIA A R A IL

K E Q F C :;L E D E Q V I Et.l V H V 1 S D A L I Y L T

.N K'EI1N S E L M:.` ElI Y M K I

L .,L V W S V V Q R F L,., R G Y E P :D

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LL S V I Q R F 'N. g R G E Y V I, P.L

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A I.! LI.N L .V.S I :A.. K R Y V G R G M L FrDL

V Y T N L VD M L A: K K Y S K G K S F H [.-13: LRNILI;L VVIY I AR K F .1T G I N I L

KY R N F R A K R S Y F L I G AD RE I

H T Q C V T K

FQFQISIHRQISVQ

IGVGIGEGVGIG

EG

CIGC IGTIGNMGMIGTIGMIG

FIG. 10. Sequence homology of SenS with B. subtilis RNA polymerase sigma factors. The boxed regions indicate sequences with identical

or chemically related amino acid residues. One gap, corresponding to residue 25 of SenS, was introduced to maximize the homologyalignment. The numbers after the names of each sequence represent the regions used for comparison from the published complete sequences.The sources for complete sequences are SpollAC (13), SpoIIIG (27), SpoIVCB (39), SigA (15), SigB (3), SigE (38), and SigH (9).

SenS

SpoIIACSpolIIGSpoIVCBSigASigBSigESigH

Y KK TR-.R

L L ti vL M IC Si IL I K.G IL M[A VL L G A IL I1K::A VL YLK SI

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is unknown. By using the SmaI-HindIll fragment (see Fig. 4and 5) as a probe (whose sequence is unique for the 1-kbinsertion in B. subtilis), Southern blot analysis was con-ducted on the chromosomal DNA of B. natto BGSC 27A1(49) digested with EcoRI or HindIII. No homologous regionwas detected by this analysis, while a positive control of thesame amount of B. subtilis DB2 chromosomal DNA gaverise to a very strong signal under the same hybridizationconditions (data not shown). This suggested that the 1-kbsequence present in B. subtilis either was never present in B.natto or was lost from the chromosome of B. natto duringevolution. So far, nothing can be concluded about the originof this insertion sequence. A close look at the two ends ofthe insertion sequence (see Fig. 6) indicated that this regionwas flanked by a 2-bp (AG) direct repeat. Whether this hasanything to do with a transposonlike element is not known.When the deduced amino acid sequences of ORF2 and

ORF3 were searched for homology in several protein databanks, no significant homology was found to any knownprotein sequence. Chromosomal inactivation mutations, cre-ated by using a strategy similar to that used for the senScoding region, were also introduced at the restriction sitesSmaI, AccI, and HindIll (see Fig. 4 and 5), and no detect-able phenotypic changes were observed. The function of this1,115-bp insert is, therefore, unclear.

DISCUSSION

The characterization of senS indicates that it is similar tosenN but differs from other reported regulatory genes in basesequence, size, and genetic locus. The derived amino acidsequence of senS indicates that it is somewhat similar insize, charge, and composition to SenN (49), SacQ (1, 50),and PrtR (29, 52) but differs considerably from Sin (14) andHpr (33).The provocative properties of senS are its monocistronic

structure, the transcription termination-attenuation sitepresent between the promoter and the ORF for SenS, thevery strong rbs, the presence of potentially a second rbspreceding the initiation codon, and the very strong transcrip-tion termination signal at the end of the cistron. The pres-ence of a transcription attenuation site and the in vivodemonstration of its transcription termination function sug-gest that the expression of senS is regulated by someantitermination mechanism that allows readthrough into theORF, although definitive proof for the mechanism awaitsmore specific alteration of the terminator region upstream ofthe ORF. Thus, the SenS may play a role in some cascademechanism that controls the expression of a number ofextracellular protein genes. In preliminary experiments withtranslational fusion genes of the senS promoter-attenuatorregion and lacZ, we have not been able to determine anynutritional or growth conditions that stimulate senS geneexpression.The interesting properties of SenS are its high Lys con-

tent, its partial homology to RNA polymerase cu factors, andthe presence of a helix-turn-helix motif. These propertiessuggest that SenS may be able to bind to DNA and may havesome direct transcriptional regulatory function. We arecurrently purifying SenS and will determine whether it is infact a specific DNA-binding protein.The identification of senS and other regulatory genes for

the expression of extracellular-protein genes indicates thatthe regulation of these genes is as complex as that ofeucaryotic genes, which also have several upstream activat-ing sites and trans-activating factors. The use of upstream

deletion mutants and DNA footprinting techniques with theproducts of these B. subtilis regulatory genes may revealtheir possible mode of action.

Preliminary studies have shown that there is a critical levelof SenS that can be tolerated by B. subtilis and E. coli. If theattenuation site is removed and high expression of SenSensues, it is lethal for both B. subtilis and E. coli (unpub-lished data). The level of SenS made in the presence of highcopy numbers of the gene is sufficient for stimulating extra-cellular-protein genes, but it appears that when the level ofSenS exceeds the number of specific SenS sites (e.g.,putative specific binding sties on the DNA), then SenSaffects other essential functions (e.g., by nonspecific bindingto DNA) and is lethal to the cell. Thus, it appears that thecell must control the amount of SenS very precisely. Currentstudies are investigating this hypothesis.

ACKNOWLEDGMENTS

This research was supported in part by Public Health Servicegrant GM19673 from the National Institute of General MedicalSciences and by contract no. 860184 from Wyeth Laboratories.

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