Sep 13, 1989 - plasmid was then used to transform B. subtilis DB2 (trpC2). .... replace by a cait cartridge (1.4 kb in length) derived from. pC194, forming pWL30 ...
Vol. 172, No. 4
JOURNAL OF BACTERIOLOGY, Apr. 1990, p. 1939-1947
0021-9193/90/041939-09$02.00/0 Copyright © 1990, American Society for Microbiology
Complex Character of senS, a Novel Gene Regulating Expression of Extracellular-Protein Genes of Bacillus subtilis LIN-FA WANG AND ROY H. DOI* Department of Biochemistry 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 several extracellular-protein genes, has been cloned, genetically mapped, and sequenced. The gene codes for a highly charged basic protein containing 65 amino acid residues. The gene is characterized by the presence of a transcription terminator (attenuator) located between the promoter and open reading frame, a strong ribosome-binding site, and a strong transcription terminator at the 3' end of this monocistronic gene. The amino acid sequence of SenS showed partial homology with the N-terminal core binding domain region of bacterial RNA polymerase sigma factors and a helix-turn-helix motif found in DNA-binding proteins. The gene can be deleted without any effect on growth or sporulation.
The efficient expression of extracellular-protein genes involves 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 factors control the expression of genes by positive action, e.g., the products of sacU, sacQ, prtR, and senN; others have a negative effect, e.g., the products of hpr and sin. Up promoter mutations or a high copy number of the positive factor genes causes hyperexpression of extracellular protein genes. On the other hand, deletion of a particular regulatory factor gene does not affect growth, sporulation, or the normal level of expression of the extracellular-protein genes. This indicates that either the regulatory-factor gene is not essential or the absence of its function is compensated for by the presence of the other regulatory factors. Sequential deletion analyses have revealed the presence of several sites upstream of the promoter of the extracellularprotein genes that appear to be the target sequences for these regulatory factors (17). This upstream region may extend up to 250 base pairs (bp) (position -250) from the start point of transcription (position + 1) and contain both positive and negative regulatory target sites, and it is somewhat similar to upstream activating sites of eucaryotic promoters. We have recently identified a Bacillus natto transcriptional regulatory-factor gene, senN, which in high copy number stimulates the expression of a fairly wide spectrum of extracellular-protein genes (48, 49). In this paper we report the isolation and characterization of a gene, senS (GenBank/EMBL accession no. M30611), from Bacillus subtilis that differs slightly in base sequence from senN, codes for a homologous but slightly different protein from SenN, maps genetically in a unique position relative to the other regulatory factors, and has a complex gene structure. The deduced amino acid sequence of SenS showed a typical helix-turn-helix motif of DNA-binding proteins and has significant homology with several B. subtilis RNA polymerase sigma factors. The senS gene organization shows a promoter followed by a stem-and-loop region indicative of a p-independent transcription termination site, followed by the open reading frame for SenS. This organization indicates that senS expression itself is highly regulated. Thus, the *
expression of extracellular-protein genes may be controlled by 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 those described previously (49). JM101 was used for production of lacZ fusion protein in Escherichia coli. Plasmid pMC1871 was from Pharmacia. pRF373 was a kind gift from Reinhold Bruchner (4). The 2 x SG medium (23) was used as the standard sporulation medium for most of the expression
studies. Genetic mapping by using PBS1 transduction. To genetically map the B. natto senN homologous locus in the B. subtilis chromosome, the EcoRI 1.8-kilobase (kb) fragment containing the senN gene (48, 49) was subcloned into the integration plasmid pCP115 (36) to create pCP115-N1.8. This plasmid was then used to transform B. subtilis DB2 (trpC2). Chloramphenicol-resistant (Cmr) transformants were selected on tryptose blood agar base plates containing 5 ,ug of chloramphenicol per p.l. One of the transformants was picked and named DB39. The integration of pCP115-N1.8 at the homologous senS locus was further confirmed by Southern blot analysis. A PBS1 lysate was then prepared from this strain and used to transduce the BGSC mapping-kit strains (7) to Cmr. Molecular cloning of senS by gene conversion. With the previous knowledge of the strong sequence homology between senN and senS as revealed by Southern blot analyses (49) and the homologous integration described above, we used the gene conversion strategy to clone the B. subtilis senS gene. To do this, the N- and C-terminal parts of the senN coding regions were deleted to form plasmids pWL71 and pWL72 (Fig. 1). These plasmids, showing a SenSphenotype, were then used to transform DB102 (21). Kanamycin-resistant (Kmr) transformants with the Sen+ phenotype restored by gene conversion were screened directly on 2xSG plates containing kanamycin (5 ,ug/ml) and skim milk
(1%).
In vitro synthesis of sen gene products. For in vitro analysis of the Sen proteins, the coding regions of senN (the EcoRVBamHI fragment [49]; Fig. 2) and senS (the HindIII-BamHI fragment; Fig. 2) were subcloned into the Bluescribe plasmid from Stratagene so that the sen gene expression was under
Corresponding author. 1939
WANG AND DOI
1940
EcoRi EcoRV HincIl
EcoRI
Sst
J. BACTERIOL.
~~~~~~~~~PlasmidSen
EcoRl
Xbo1
BomHI
pWL71
-
pWL72 -
FIG. 1. Construction of plasmids pWL71 and pWL72 for cloning of senS by gene conversion. The upper part of the figure shows a restriction map of the B. natto senN region as a 1.8-kb EcoRI fragment originally cloned in pUBHR-N1.8 (49). The shaded region represents the part which was essential for the Sen+ phenotype. The lower part of the figure schematically illustrates the 5' and 3' end deletions of the essential region in pWL71 and pWL72, respectively, which all showed a Sen phenotype. as shown at the right side of the figure.
the control of the T7 promoter from the vector DNA as well as its own promoter. These plasmids (pBP-senN and pBPsenS), after linearization by use of proper restriction enzymes, were used as templates for in vitro transcriptionamplification by using the T7 transcription kit from Bethesda Research Laboratories, Inc. The amplified mixture, including the original plasmid template and the newly synthesized mRNA, was then subjected to in vitro-coupled transcriptiontranslation by using the Amersham expression kit in the presence of [35S]Met. The labeled polypeptides thus produced were separated on a 15% sodium dodecyl sulfatepolyacrylamide gel electri ophoresis (SDS-PAGE) gel. followed by autoradiographi ic analysis. Reaction conditions specified by the kit supplliers were followed without any
modifications. N-terminal sequence anallysis of SenS-LacZ fusion proteins expressed in E. coli and B. subtilis. A senS-lacZ gene fusion was constructed for exprcession studies as well as for the analysis of the N-terminal amino acid sequence of the SenS protein. This was done b y subcloning the AlulT fragment, containing the promoter anid the deduced first 26 amino acid residues of senS (see Fig. ei and 8), into the unique SinaI site of pMC1871 to form an in-Iframe senS-lacZ gene fusion. The plasmid thus formed, pWL,211, was digested with PstI. The resulting PstI cassette of thie senS-lacZ fusion gene was then moved into shuttle plasmi(d pRB373 to form pWL289. Both E. coli JM101 and B. Sli ,btilis DB403 harboring pWL289 produced SenS-LacZ fusi(on proteins with P-galactosidase activity. These in vivo-de-rived fusion proteins were then purified by using the amin ophenylthiogalactopyranoside affinity chromatography tec hnique on a 10-ml aminophenylthiogalactopyranoside-agai rose column as described previously (5). The essentially pure (more than 90% pure as determined by SDS-PAGE ) peak fractions were loaded to a 7.5% SDS-PAGE; this prc)cedure was followed by electroblotting onto an Immobiloin membrane (Millipore Corp.) by .
A.F E
B.II E
-
X
V H Ssti B
E
l
I
V
Hind Ill
H
B
X
E
Comparison of re striction maps for the 1.8-kb EcoRI fragment originally cloned frc im B. natto (A) and the 2.8-kb EcoRl fragment cloned by gene conversion from B. slbtilis (B). The shaded parts are the regions essential Ifor the Sen+ phenotype, whereas the solid black bar in panel B rep]resents the 1-kb insert only present in B. subtilis. Restriction sites alre: E, EcoRI; V, EcoRV; H, HindIlI; B, BamnHI; and X, XbaI. FIG. 2.
the method described previously (28). After the membrane was stained with Coomassie blue, the band corresponding to SenS-LacZ was cut out and directly subjected to an automated sequence analysis by the Protein Structure Laboratory 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 senS locus with a cat cartridge through a homologous doublecross recombination event. For this purpose, the 2.4-kilobase (kb) EcoRV-EcoRl fragment of pWL80 (see Fig. 2) was subcloned into the HincII-EcoRI double cut pUC19 to form pWL26. The 0.4-kb HincII-BamHI fragment of pWL26 containing the seniS gene (see Fig. 2) was then removed and replace by a cait cartridge (1.4 kb in length) derived from pC194, forming pWL30. This plasmid, after linearization with SphI-EcoRI double digestion, was used to transform DB2 to Cmr. This mutant strain was named DB417. Other methods. Nick translation, Southern blot, dideoxychain termination DNA sequencing (37), and other standard DNA techniques and enzyme assays were performed as described in our previous communications (48, 49).
RESULTS Genetic mapping of the senS locus in B. subtilis. In our previous report on the cloning and characterization of the B. natto senN gene (49), we indicated that a B. siubtilis homolog, named senS ("S" for subtilis), was detected by Southern blot analysis. To further test the degree of sequence homology between these two closely related species and to determine the genetic location of senS in relation to other known small regulatory genes with similar phenotypes (such as prtR, sacQ, etc.), an integration plasmid, pCP115N1.8, was constructed by using the 1.8-kb EcoRI fragment from B. natto (49; also see Fig. 2). The recombinant plasmid was subsequently integrated into DB2 to form DB39 (trrpC2 senS: :caut). The expected integration of pCP115-N1.8 at the senS locus in DB2 was confirmed by Southern blot analysis (data not shown). DB39 was then used to make a PBS1 donor lysate to be used in subsequent mapping experiments. The recipients were the strains from the BGSC mapping kit constructed by Dedonder et al. (7). The results are given in Table 1 and Fig. 3. By three factor crosses, the c'at (senS) marker was shown to be 40% linked to the tre-12 locus, and 43% to glyB133, in the order of tre-senS-gly. A finer mapping, using B. subtilis 1A603 (spI3c2 trpC2 thiA78::Tn917) (46) as recipient, indicated that senS was more than 97% linked to the thiA78 locus. These data allowed us to locate the senS locus at 700 on the B. slubtilis genetic map (34) and, hence, to genetically define senS as a new gene different from other known genes with similar phenotypes, including sacQ (at 2850), sac U (3100), sac V (360), prtR (2000), hps (750), and iep (3100). Cloning of the B. subtilis senS locus by gene conversion. Gene conversion or gene replacement is a technique based on the strong mismatch repair mechanism of B. sblbtilis (6, 20). Several cases have been reported in which gene conversion was used to construct specific deletion mutations (21, 51) and to clone particular DNA fragments containing inte[lar DNA fragme c ing te anetic tn 30, 41). We decided to use the esting genetic markers (21h gene conversion technique to clone the B. subtilis senS locus instead of using other conventional cloning techniques, based on the observation that the B. natto senN locus was highly homologous to senS of B. slbbtilis. The restoration of m
VOL. 172, 1990 TABLE 1. Three-factor transduction cross to map senS relative to tre and glyBa Recipient classes
Selection
Cmr
Order implied
Cmr
Gly
+ +
+
+
+ -
17 136 94 169
tre-sen-gly
+ + +
+ + -
+ +
4
tre-sen-gly
52
-
151
+ +
0 0
-
+ +
-
-
+ +
48 160
+ +
+ +
Gly+
No.
Tre
_ -
Tre+
1941
senS REGULATION OF B. SUBTILIS GENES
+ +
tre-sen-gly
glyBJ33 metC3
the sen phenotype would be due to gene conversion on the multicopy plasmid. For this purpose, plasmids pWL71 and pWL72 were constructed as shown in Fig. 1. These plasmids were used to transform DB102 (6, 21) to Kmr, and positive transformants which restored the Sen activity by gene conversion were screened directly on skim milk plates containing kanamycin. In each case, about 1 to 2% of the total Kmr transformants showed the Sen+ phenotype, a frequency normally observed for gene conversion events (21, 22). Six positive clones from each transformation were picked for restriction map analyses. The results are shown in Fig. 2B. All twelve clones analyzed gave rise to the same restriction map, as shown. Upon EcoRI digestion, a 2.8-kb fragment was released, which was 1 kb longer than the 1.8-kb EcoRI fragment from B. natto containing the senN gene. However, this size matched the size observed previously in the Southern blot analysis (49). Since gene conversion works by a mismatch repair mechanism, the results suggested that the extra 1-kb sequence must be located between the two EcoRI sites. Further analyses with EcoRV, BamHI, Hincll, XbaI, HindIII, and SstI demonstrated that the restriction maps at
glyB
sen::cat
tre
p
-p
73% ~63%
73%
77%
98% _______________________
Stimulation (fold) with:
Enzyme
Alkaline protease (subtilisin) Neutral protease Amylase Alkaline phosphatase
senS
senN'
2.8
2.7
3.7 1.8 1.8
3.7 1.8 1.8
a Data from Wong et al. (49).
0
a Donor, DB39 (trpC2 senS::cat); recipient, BGSC5 (trpC2 tre-12).
TABLE 2. Comparison of stimulatory effects of multicopy senS and senN genes on the production of some extracellular enzymes
I
4~~
>99%
FIG. 3. Genetic map of the senS region of the B. subtilis chromosome, constructed from the three-factor crosses shown in Table 1. The arrow tails indicate the selected marker, and the genetic distances are shown as 100 minus the percentage cotransduction frequency. Reciprocal recombination values between senS::cat and either tre or glyB were unequal, with the genetic distance appearing smaller when senS::cat was the selected marker.
the two ends of the 2.8-kb "S" fragment (Fig. 2B) were the same as that for the 1.8-kb "N" fragment (Fig. 2A), except for the absence of the SstI site in the 2.8-kb fragment, which was later demonstrated to be caused by a single base pair change in this region (see later discussion). These results led us to the following conclusions: (i) we had cloned the B. subtilis senS locus, which is homologous but not identical to the B. natto senN region; (ii) all the data obtained were consistent with what would be expected for a gene conversion mechanism; (iii) the B. subtilis senS region contained an extra 1-kb sequence which was not present in that region of the B. natto genome. This 1-kb sequence (indicated by the solid box in Fig. 2B) was located between the EcoRV and Hincll sites and contained a HindIII site. To our knowledge, this was the first case in which cloning of a homologous chromosomal DNA fragment more than 1 kb (1,580 bp from pWL71 and 1,280 bp from pWL72) in size was successfully achieved by gene conversion transformation. Two clones (pWL77 and pWL80), one each from the transformants derived from pWL71 and pWL72, respectively, were chosen for further characterization. A deletion mapping similar to that for pUBHR-N1.8 (49) was conducted. The results showed that full Sen activity can be detected by using the 0.6-kb HindIII-BamHI fragment (data not shown), suggesting that the 1-kb insert located upstream of the B. subtilis senS region was not directly responsible for the Sen phenotype. The effects of the multicopy senS gene in plasmid pWL95 (a pUB18 derivative containing the 0.6-kb HindIII-BamHI fragment) on the production of extracellular proteases, amylase, and alkaline phosphatase were examined by using the same procedures as previously described for the senN gene (49). The results are shown in Table 2. These results indicated that is is difficult to distinguish senS from senN on a functional basis. Nucleotide sequence of senS and its flanking regions. To further characterize the senS coding region and to investigate the origin and properties of the upstream 1-kb insert, the EcoRV-BamHI fragments from pWL77 and pWL80 were subjected to DNA sequencing analysis. The nucleotide sequences obtained for these two clones were identical and are presented in Fig. 4. As predicted from the Southern blot data, the DNA sequence in the senS region is very homologous to that of the senN region (see Fig. 6), except for the 1-kb insert, which is actually 1,115 bp as determined by
sequencing.
A computer analysis of the sequence in both orientations revealed three open reading frames (ORFs) with significant size (more than 60 amino acids in length), as indicated in Fig. 5. However, only ORFi and ORF2 were preceded by putative translational initiation signals, i.e., a Shine-Dalgarno (SD) or ribosome-binding site (rbs) sequence followed
1942
J. BACTERIOL.
WANG AND DOI
I
EcoRV
AGTTCTTGGAAATTCTGATTTTCGATATCTGGCGAATTTACGTAGTCTCCCATCGTTTCTTTCGAAAGGGACGTTCTCAGCCCCTCAATCCAGCGGACA
()
TTTGTCTTTTTTCTCCAGGGGATGTCCAGTTTGTTAAGTATTCCTGGGCGATGATTGCGTCACGATAATAMAATGC CGTTTGGTCGGGAGCGAC C CGTC C
' n0n
GGCTGCnCCCGCCGAGTGCTTGCTGCCAGACACTGGCGTTTGATTCGGAGCGTGCTCTAAAAMGTGTTTTATTGTTGAGATCGCACGTTCTGATAATGGC SmaI TTTCAATGAAAGAGCCGGAGCG=CATT=TTGAGGCTGATTGCCTCCCGGGCTGTTAAAAAAGGTTACCGCTTCAATGAATGGCGTTGTTTTTACCA
-40O(
TTCCGCTTGACGGACTTCCTGCTTTCAATAAAGGCTTTAACAGT=TTTTAACTCTGTTFTTGGCCCGACAAATTGGCCGAGGGCTTCTATGCGGTTTAC
5 00
'300
AccI
TTCTTTAGGCCAAAACTCTATTGATGATGTAAGCCGGTCATCTGTATACGGGGCCCAGTTCTGCCACGTGTTATATACTTCCTCAAAATCATCCCATCCC 6600) CATGTAATAGAAAAAATCGACACTTGAGAGATGGGCACTGCTTTAAATGTCATGGAGGTGACTATGCCGAAATTGCCTCCTCCGCCTCCCTGAGACGCC C ,7 00 PvuII AAAATGTGGATGATTTGAACAGCTGACTGTAATCAGATCAGCGCCCTCTTTTTCGTCTGCTACGATCATCTCAAGCTGCACGAGGCTGTCGCAAGTAAGA 8800 CCGGCAGCCCTTGTTAAAAGTCCAATTCC C CCTCCGAGAGTTAAACCTGTGAGCC CTACATTAGCAATGGTGCCTGCGGGAAGC GTCAGGC CGTATTGC C
9 00
AGAGTGTCCGATAGACTTCTCCCAATTCAGCCCCCGCTTCAATATAGGCCAGCTTTTTATCCTGATTCACAGTTATTTTTTTCATCTCGCTTAAATCAAT 1000 AACAAGACCGTTATTTAAAAGGGAAAAGTTCTCATAGCTGTGTCTGCCGCCTCTAATACGGAAAGGCACACGGTTTTCACGCGCCCATTTCAGCGCATTG 1100 HindIII AGTGCATCCTGTTTGTTTGGCAAAACACAATGATGTCAGATCCTTCTAAGCTTAGGTTAATATTGGTTCTTGCTTCGTTATAGTCCGGATCATCCCGT 12900
I
GTCACGATACGTCCGGTCAATTTGTCTTTTCCACACTCCCACATCTCTTTCTCTCGTATTCTAGTTTCTCTAGCTTATGCGTCAGGGGAAAAGAGTGTA 1 30 0 HincII HincII TAAGGAAAAAGCGGGGATGCAATCTGATACAGTGTCAACACCCTCAAAAAATAGTTGACAGGTCGGTATTGTATGAATTAACATGGTCAGTACAAATTTT 1400 TCAAATTTATCGCGCTGATCGGAACACCGAAGGCTCTTATCGTTTAGATAAGGGCCTTTTTGTATGAAAAAGGGGGGATTATTGATGGGAGTCAAAAAA 1500
GAAAAGGGGAGAAAACGATTCAGGAAGCGAAAAACCTACGGGAATCAGATTTTGCCGCTTGAGCTGCTGATTGAAAAAAACAAACGAGAGATTATAAACA 1600
GCGCGGAACTCATGGAAGAAATTTATATGAAGATTGATGAGAAGCATACGCAATGTGTAACTAAATATAAAAAAACCCGCTGACTACAACGGGTTTTTGC 1700 BamHI
1,73 ATTTCTCCATTAAGAATCTTT AATCGGCAATCCAAGGCCTTCTGCCACGCGTTTTCCGTATTCAGGATCC FIG. 4. Nucleotide sequence of B. .scbtilis setnS and its flanking regions. The presented sequence starts at 25 bp upstream of the EsLoRV site and ends at the BamHI site as shown in Fig. 2B. The sequence was obtained by dideoxy sequencing of two separate clones, pWL77 and pWL80 (see text), with both strands sequenced (except for the 25 bp upstream of the EcoRV site). All the restriction sites used in the M13 subcloning were confirmed in other independent clones overlapping the corresponding sites. The sequence is presented from 5' to 3' in the same orientation as that for senS transcription. The start (ATG) and stop (TGA) codons of the SenS coding region are double underlined. The boundary of the 1-kb (actually 1,115 bp) insert is marked by two vertical arrows. Important restriction sites are shown above their corresponding recognition sequences.
by an in-frame initiation codon (usually ATG). But ORF3 may still be functional in vivo via a translational coupling mechanism, since its N terminus overlaps with the C terminus of ORF2. This, of course, needs to be experimentally tested. The C terminus of ORF3 is truncated at the 5' end of the sequence presented in Fig. 4. Several interesting features were observed by sequence comparison 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 SD sequence 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). There are two important mismatches in the coding regions. The ORF2 ORF 1
ORF3
Hnd III Hllac 11 EQ RV Sma I A1 I 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 solid line, with its arrowhead indicating the direction of translation. The putative transcription initiation sites (promoters) and translation initiation sites (proper combination of SD sequence and in-frame initiation codon) are represented by the small arrows and squares, respectively. The restriction map and other symbols are the same as those described in the legend to Fig. 2.
first one is at nt 397, which changed a Phe residue of SenN to a Tyr in SenS. The second one is more dramatic; it is a mismatch which, at nt 528, changed the stop codon (TAG) of senN to a Tyr codon (TAT) in senS, leading to the presence of 5 additional amino acid residues at the C terminus of SenS. Finally, the mismatches in the terminator regions also made significant differences between the two genes in terms of the strength of the stem-loop structure, i.e., the stem-loop of senS is weaker than that of senN. The single base change at nt 467 is the cause of the loss of the SstI recognition site (GAGTCT) in the senS coding region (see Fig. 2 and previous discussion). As a conclusion, we should mention that although the sequence comparison demonstrated significant differences between the two genes, there seemed to be no functional differences when these genes were introduced into B. siubtilis on high-copy plasmids. But this does not exclude the possibility that they may have physiological differences when each exists as a single copy in vivo. When ORF1 (Fig. 5) was searched for a putative translation initiation site(s), two such regions were found. The stronger 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 325 to 327. This ATG codon is seven amino acid residues upstream of the one shown in Fig. 6. At this stage, the assignment of the initiation site as shown in Fig. 6 was purely based on theoretical studies and a comparison of the sequence data. This was, however, later proved correct by N-terminal sequence analyses to be presented later. One of the most puzzling, and perhaps most important, features revealed by the sequence analyses was the discovery of two inverted repeat (IR) sequences between the
VOL. 172, 1990
senS REGULATION OF B. SUBTILIS GENES 1115 bp
EcoRV
-
GA
GATATCTGGCgAATTACGTAGTCTCCCATCGTTTcttTCGAAAGTCCGGATCATCCCGT GATATCTGGCcAATTTACGTAGTCTCCCATCGTTTaacTCGAAAGTCCGGATCATCCCGT
60 60
Bs Bn
GTCACGATACGTCCGGTCAATTTTGTCTTTTCCACACtCCCACATCTCTTTCTCTCGTAT
120 120
Bs Bn
TcTAGTTCTCTAGCTTATGCGT0AGGGGAaAAGAGTGTATAAGGAM AAGCGGGGATGc l80
Bs Bn
GTCACGATACGTCCGGTCATTTTGTCTTTTCCACACaCCCACATCTCTTCTCTCGTAT
TtTAGTTCTCTAGCTTATGCGTCAGGGGAtAAGAGTGTATAAGGAAAAAGCGGGGATGt
AATCTGATACAGTGTCAACtCCCTtAAAAAATAGTTGACAGGTCGGTATTGTATGAATTA
Bs En
26-
A^ .
. .
GluLysGlyArgLysArgPheArgLysArgLysThrQ1GlyAsnGlnIleLeuProLeu
GAAAAGGGGAGAAAACGATTcAGGAAGCGAMAAACCTii GGGAATCAGATTGCCGCTT GAAAAGGGGAGAAAACGATTtAGGAGCGAAAAACCZg;GGGAATCAGATTTTGCCGCTT
GluLysGlyArgLysArgPheArgLysArgLysThrOiGlyAsnGlnIleLeuProLeu
25 420 420 25
GluLeuLeuIleGluLysAsnLysArgGluIleIleAsnSerAlaGluLeuMetGluGlu
45
I1eTyrMetLysIleAspGluLysHisThrGlnCysValThrLysT..LysLysTrArg Bs ATTTATATGAAGATTGATGAGAAGCATACGCAATGTGTAACTAAAO'AAA CCCGC Bn ATTTATATGAAGATTGATGAGAAGCATACGCAATGTGT AACTAIAAAACCCGC
IleTyrMetLysIleAspGluLysHisThrGlnCysValThrLys"*.
65
540 540 60
Bs TGAcTaCAaCGGGTCTTcCATTAAGaATCMTTAATCGGCAMTCCAGG 600 Bn TGAtTtCAgcGGTTrGArCTaCATTAAGcATC TTMTCGGCMTCCAGG 600
CCTTCTGCCACGCGTTTTCCGTATTCAGGATCC CCTTCTGCCACGCGTTTTCCGTATTCAGGATCC BamHI
632 632
FIG. 6. Sequence comparison of senS and senN genes, their homologous flanking regions, and their gene products. The DNA sequences are shown from 5' to 3', with the first nucleotide in the EcoRV recognition site as number 1. To facilitate the comparison, the same numbering coordinates are used for both sequences by looping out the 1,115 bp insert for B. subtilis, as shown at the top. Amino acid sequences are shown above (for senS) and below (for senN) their corresponding DNA sequences and are numbered from the first Met. Differences in DNA sequences are shown by lowercase letters, whereas changes in the amino acid sequences are marked by dotted shading. The putative promoter and rbs sites are indicated by dots between the two DNA sequences. IR sequences are shown by solid lines with inverted arrow heads. Abbreviations: Bs, B. subtilis sequence; Bn, B. natto sequence; SD, Shine-Dalgarno sequence.
promoter and rbs of the sen genes. These are shown in Fig. 6 as paired solid lines with inverted arrows. The first long IR sequence forms a stable stem-loop structure, which was followed by a stretch (six) of T's. This is a characteristic feature of procaryotic p-independent termination sequences (35). The second shorter IR sequence was not as stable as the first one. But there are two important features for this second IR sequence that make it significant. First, it overlaps with the rbs of the sen gene, which may play a role in translational control of sen gene expression. Second, the left half of this stem overlaps with the right half of the stem of the first IR sequence, which makes this organization somewhat like an attenuator structure for other systems (2, 53). Although no classic attenuation mechanism has yet been detected in this region, the second IR sequence may have a regulatory effect on the first IR sequence as a p-independent transcription terminator. In vitro-expressed products of senS and senN. The se-
K-- i
-
1814-_
MetGlyValLysLys .r
lfrr"Wf A,.^
Bs GAGCTGCTGATTGAAAAAAACAAACGAGAGATTATAACAGCGCGGAaCTCATGGAAGAA 480 Bn GAGCTGCTGATTGAAAAACAAACGAGAGATTATAACAGCGCGGAgCTCATGGMGM 480 GluLeuLeuIleGLuLysAsnLysArgGluIleI leAsnSerAlaGluLeuMetGluGlu 45
Bs Bn
U'
180 240 240
5 360 Bn CGTTTAGATAAGGGCCaGGGATTATTGATG AGTCAAMAA 360 Z MetGlyValLysLys 5 A
,,
43-
-10 Bs ACA.TGTCAGTACAAA TCAAATTTCGCGCTGATCGGAACACCGAAGGCTCTTAT 300 Bn ACATGGTCAGTACAAATTTTTCAMTTTATCGCGCTGATCGGAACACCGAAGGCTCTTAT 300 SD
..
_
-35
AATCTGATACAGTGTCAACaCCCTcAAAAAATAGTTGACAGGTCGGTATrGTATGAATTA
Am: Irr.TTPAlATA Arz>-rTlrSlirrr,A rrsAA
2 3 4
1
Bs Bn
1943
6-
w
3-~ FIG. 7. In vitro synthesis of sen gene products. Lanes 1 though 4 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) were first linearized by PvuII digestion and then transcribed by using the BRL T7 transcription kit. The "amplified" template-mRNA mixtures were subsequently expressed by using the Amersham in vitro procaryotic 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 autoradiography after drying the gel on filter paper. The numbers shown on the left are molecular weight standards generated by the Bethesda Research Laboratories prestained markers (low range). The arrows near lanes 3 and 4 indicate the bands which are absent in lanes 1 and 2 and, hence, are produced by the coding regions of senN and senS,
respectively.
quence data presented above revealed significant differences at the C termini of SenN and SenS, i.e., SenS is 5 amino acids larger than SenN, which makes about a 10% difference in their molecular masses due to their small molecular sizes. To further confirm this, an in vitro expression system was used to synthesize [35S]met-labeled sen gene products. Since the stem-loop structure found between the promoter and the sen coding region was likely to reduce the transcription efficiency of the corresponding genes by the Amersham in vitro transcription-translation kit, which is basically an E. coli cell-free lysate, an additional in vitro transcription step-an mRNA "signal amplification" -was introduced by using the T7 transcription system. T7 polymerase has the advantage of being insensitive to bacterial transcription termination signals (25; F. W. Studier, A. H. Rosenberg, and J. J. Dunn, Methods Enzymol., in press). The results are presented in Fig. 7. The band patterns in lanes 3 and 4 led us to the conclusions that (i) the senS gene product is indeed larger than that of senN, as predicted from the sequencing data, and (ii) the overall sizes of both protein products determined by SDS-PAGE match the molecular masses deduced from the ORFs by using the ATG codons as shown in Fig. 6, which are 7.9 kilodaltons (kDa) for SenS and 7.2 kDa for SenN. The results, however, also revealed a band around 3 to 4 kDa, which was not expected from the sequence data. The identity of this polypeptide is therefore unknown. Upstream deletions down to the stem-loop structure of the senS coding region indicated that this polypeptide
1944
WANG AND DOI
was not associated with the upstream sequence. Deletions affecting the rbs, hence the translation, of senS however abolished the 3.5-kDa band (data not shown). Therefore, the 3.5-kDa polypeptide might be an artifact resulting from specific 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-copy senS gene in the B. subtilis chromosome and for N-terminal sequence analyses, a senS-lacZ fusion gene was constructed as described in Materials and Methods. pWL289, a B. subtilis-E. coli shuttle vector containing the fusion gene cassette, was introduced by transformation into JM101 and DB104 for expression in E. coli and B. subtilis, respectively. SenS-LacZ proteins produced in these strains were then purified by the aminophenylthiogalactopyranoside affinity chromatography method (5). Starting from 500-mi late-log phase LB cultures (for B. subtilis, the culture medium also contained 0.5% glucose), about 0.5 mg (from B. subtilis) to 2 mg (from E. coli) of fusion proteins was obtained. These proteins were more than 90% pure when checked by SDSPAGE (data not shown). About 24 ,ug each, corresponding to 200 pmol, of the B. subtilis- and E. coli-derived fusion proteins was run on a standard 7.5% SDS-PAGE, followed by electroblotting onto the polyvinylidene difluoride membrane (28). The membrane portions containing the SenSLacZ band, after staining with Coomassie blue R-250, were cut out for direct automated sequence analysis. The results obtained for the first six amino acid residues were GlyVal-Lys-?-Glu-Lys for the B. subtilis-derived fusion protein and Gly-Val (Phe)-Lys-Lys-Glu-Lys for the E. coli-derived one. These sequences matched exactly to the deduced N-terminal sequence (residues 2 through 7) of SenS, with its first Met residue probably being removed after translation
(see Fig. 6). Transcriptional regulatory features of senS gene. Similar experiments on promoter and terminator mapping as those reported previously for the senN gene (49) were also conducted for the senS gene. Strong promoter activity was detected when the Sau3A (nt 40)-Sau3A (nt 278) fragment was cloned in the BamHI site of the promoter probe plasmid pWP19 (see Fig. 9). However, when the shorter Sau3A (nt 40)-HincII (nt 196) was tested in a similar way, no promoter activity was observed, indicating that the putative promoter was located between the Hincll (nt 196) and Sau3A (nt 278) sites. This coincides well with the region predicted to contain the putative -35 and -10 regions (nt 215 to 245; Fig. 6) of B. subtilis F43 (alA) promoters (8). By using a similar mapping strategy and our terminator probe plasmid pWT19 (48), a strong termination activity was located on the AluI (nt 415)-BamHI (nt 620) fragment, a region showing a stem-loop structure (see Fig. 6) characteristic of procaryotic p-independent transcription terminators. Figure 8 is a schematic diagram showing the results for testing the putative terminator-attenuator structure in front of 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 promoterless subtilisin gene (aprE) (47). As the data indicated, the transcription activity, monitored by the specific activity of the reporter gene product (subtilisin), was reduced dramatically when the stem-loop structure was included in pWPAA compared with pWP-AS, which contained only the promoter region. This might explain the relatively low stimulatory effect (two- to fourfold) of the multicopy sen gene on the expression of extracellular-protein genes in comparison with other similar regulatory genes (48, 49).
J. BACTERIOL.
IIr 9 u~ ~ ~ ~ snS Klilil
S
A
fImHi
RS H A
Plasmid Activity 0% pWP19 pWP-AA
7%/6
pWP-AS
100%/.
FIG. 8. Detection of a termination activity between the promoter and the coding region of senS gene. The upper part is a restriction map with symbols representing the promoter (bent arrow), rbs (solid square), and stem-loop structures. The coding region is shown by the shaded area. Restriction enzymes are: S, Sau3A; A, AluI; R, Rsal; and H, HaeIII. The lower part of the figure indicates the restriction fragments used to construct plasmids pWP-AA and pWP-AS, with the arrowheads indicating that the same transcription direction as that of aprE in pWP19 (47) was maintained during subcloning. After introduction into DB104 (21), the expression time courses of DB104(pWP-AS) and DB104(pWP-AA) were studied, with DB104(pWP19) as a negative control. Similar constitutive expression curves were obtained for both clones (see Results section). The average specific activity produced from relative specific activity of 7% was obtained for DB104(pWP-AA).
Time course studies of aprE expression in plasmids pWPAA and pWP-AS indicated that although there were more than 10-fold differences in activity, both constructs showed a similar expression pattern. The promoter was turned on during growth and continued to be expressed during the sporulation 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 temporal regulation, although the target genes controlled by SenS appeared to be regulated in a temporal fashion (12, 30, 32, 45). Deletion inactivation of the senS gene in the B. subtilis chromosome. To determine whether the senS gene was essential for growth, a deletion of the gene was constructed in 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 into the homologous chromosomal locus was confirmed by Southern blot analysis. Figure 9 shows the results of blots with EcoRI- and HindIII-digested chromosomal DNA. Lanes 1 and 3 show the EcoRI digestion patterns of DB2 and DB39 chromosomal DNA, respectively. Lanes 2 and 4 represent similar results obtained with HindIII digestion (lane 2 for DB2 and lane 4 for DB39). As predicted, the 2.8-kb EcoRI band in the wild-type strain (lane 1) was converted to a band around 3.8 kb (lane 3). The 1-kb increase in size was a result of the replacement of the 0.4-kb senS coding region by the 1.4-kb cat cartridge (see Materials and Methods for details of plasmid constructions). Similarly, in the HindIlI digestion, the upper band, which contains the senS coding region, showed lower mobility for DB39 DNA (compare lanes 2 and 4). The relative low intensity of the upper HindlIl bands was due to the shorter region of complementarity to the probe used, which was the EcoRVBamHI fragment (see Fig. 2B). DB39 grew normally, formed spores on 2xSG medium, and secreted apparently normal amounts of extracellular proteases. Sequence homology of SenS to RNA polymerase sigma factors. The highly charged nature, the basic amino acid (especially Lys)-rich composition of SenS, and its regulatory
1945
senS REGULATION OF B. SUBTILIS GENES
VOL. 172, 1990
R F R K R K T Y G N Q I L P L E L L I.EXC
SenS 11-31
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FIG. 11. Sequence alignment of DNA domains from several B. subtilis-DNA interacting proteins. The boxed regions indicate sequences with identical or chemically related amino acid residues. One gap, corresponding to position 28 of the SenS protein, was introduced to maximize the homology alignment. The numbers after the name of each sequence represent the regions used for this alignment. The complete sequence of each protein was derived from the same sources as those in the legend to Fig. 10, except for Sin (14) and SpollIC (11).
up 4U
2.3-, 1.9 -
data banks, no closely related homologs of SenS were found in other organisms. But significant partial homology was found between SenS and several known bacterial RNA polymerase a factors. A sequence comparison of SenS with those factors of B. subtilis origin is shown in Fig. 10. Due to its small molecular size, the SenS homologous regions in most of the or factors cover only part of the protein, mainly the region around the core binding domain, as postulated by several groups (10, 16, 39, 40). The functional significance of this homology awaits more detailed analyses of the SenS protein by genetic and biochemical approaches, which are currently being undertaken. Further analysis of the secondary structure of SenS revealed a helix-turn-helix motif from residues 11 to 31, which is characteristic of DNA-binding domains deduced from many DNA-binding proteins (31). This particular region, as shown in Fig. 11, showed not only structural similarity but also sequence homology to the DNA-binding domains of several B. subtilis RNA polymerase c factors and known DNA-binding proteins. It is interesting to note that in c factors the "core binding" and "DNA binding" domains are physically separated from each other in their primary sequences, while they seem to be overlapping in SenS. The origin and function of the upstream insertion sequence
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 DNA of DB2 and DB417, respectively. Lanes 2 and 4 contained the same DNA as those for lanes 1 and 3, digested with HindIII. The molecular weight markers are the phage 429 DNA digested with HindIII (49). See the text for a discussion of the results.
effect at the level of transcription (49) suggested to us that SenS might be a DNA-binding protein. This notion was strengthened by the sequence analysis results shown below. When we analyzed known protein sequences in various SenS 1-65
SpoIIAC 11-74 SpoIIIG 17-80 SpoIVCB 28-91 SigA 112-175 SigB 8-71 SigE 36-99 SigH 12-75
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FIG. 10. Sequence homology of SenS with B. subtilis RNA polymerase sigma factors. The boxed regions indicate sequences with identical chemically related amino acid residues. One gap, corresponding to residue 25 of SenS, was introduced to maximize the homology alignment. 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).
or
1946
J. BACTERIOL.
WANG AND DOI
is unknown. By using the SmaI-HindIll fragment (see Fig. 4 and 5) as a probe (whose sequence is unique for the 1-kb insertion in B. subtilis), Southern blot analysis was conducted on the chromosomal DNA of B. natto BGSC 27A1 (49) digested with EcoRI or HindIII. No homologous region was detected by this analysis, while a positive control of the same amount of B. subtilis DB2 chromosomal DNA gave rise to a very strong signal under the same hybridization conditions (data not shown). This suggested that the 1-kb sequence present in B. subtilis either was never present in B. natto or was lost from the chromosome of B. natto during evolution. So far, nothing can be concluded about the origin of this insertion sequence. A close look at the two ends of the insertion sequence (see Fig. 6) indicated that this region was flanked by a 2-bp (AG) direct repeat. Whether this has anything 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 data banks, no significant homology was found to any known protein sequence. Chromosomal inactivation mutations, created by using a strategy similar to that used for the senS coding region, were also introduced at the restriction sites SmaI, AccI, and HindIll (see Fig. 4 and 5), and no detectable phenotypic changes were observed. The function of this 1,115-bp insert is, therefore, unclear. DISCUSSION The characterization of senS indicates that it is similar to senN but differs from other reported regulatory genes in base sequence, size, and genetic locus. The derived amino acid sequence of senS indicates that it is somewhat similar in size, charge, and composition to SenN (49), SacQ (1, 50), and PrtR (29, 52) but differs considerably from Sin (14) and Hpr (33). The provocative properties of senS are its monocistronic structure, the transcription termination-attenuation site present between the promoter and the ORF for SenS, the very strong rbs, the presence of potentially a second rbs preceding the initiation codon, and the very strong transcription termination signal at the end of the cistron. The presence of a transcription attenuation site and the in vivo demonstration of its transcription termination function suggest that the expression of senS is regulated by some antitermination mechanism that allows readthrough into the ORF, although definitive proof for the mechanism awaits more specific alteration of the terminator region upstream of the ORF. Thus, the SenS may play a role in some cascade mechanism that controls the expression of a number of extracellular protein genes. In preliminary experiments with translational fusion genes of the senS promoter-attenuator region and lacZ, we have not been able to determine any nutritional or growth conditions that stimulate senS gene expression. The interesting properties of SenS are its high Lys content, its partial homology to RNA polymerase cu factors, and the presence of a helix-turn-helix motif. These properties suggest that SenS may be able to bind to DNA and may have some direct transcriptional regulatory function. We are currently purifying SenS and will determine whether it is in fact a specific DNA-binding protein. The identification of senS and other regulatory genes for the expression of extracellular-protein genes indicates that the regulation of these genes is as complex as that of eucaryotic genes, which also have several upstream activating sites and trans-activating factors. The use of upstream
deletion mutants and DNA footprinting techniques with the products of these B. subtilis regulatory genes may reveal their possible mode of action. Preliminary studies have shown that there is a critical level of SenS that can be tolerated by B. subtilis and E. coli. If the attenuation site is removed and high expression of SenS ensues, it is lethal for both B. subtilis and E. coli (unpublished data). The level of SenS made in the presence of high copy numbers of the gene is sufficient for stimulating extracellular-protein genes, but it appears that when the level of SenS exceeds the number of specific SenS sites (e.g., putative specific binding sties on the DNA), then SenS affects other essential functions (e.g., by nonspecific binding to DNA) and is lethal to the cell. Thus, it appears that the cell must control the amount of SenS very precisely. Current studies are investigating this hypothesis. ACKNOWLEDGMENTS This research was supported in part by Public Health Service grant GM19673 from the National Institute of General Medical Sciences and by contract no. 860184 from Wyeth Laboratories. LITERATURE CITED 1. Amory, A., R. Kunst, E. Aubert, A. Klier, and G. Rapaport. 1987. Characterization of the sacQ genes from Bacillus licheniformis and Bacillus subtilis. J. Bacteriol. 169:324-333. 2. Bauer, C., J. Carey, L. Kasper, D. Lynn, D. Waechter, and J. Gardner. 1983. Attenuation in bacterial operons, p. 65-89. In J. Beckwith, J. Davies, and J. Gallant (ed.), Gene function in prokaryotes. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 3. Binnie, C., M. Lampe, and R. Losick. 1986. Gene encoding the
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senS REGULATION OF B. SUBTILIS GENES
1947
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