nucleotide sequence, and expression ofthe bacillus subtilisthe gram-positive bacterium bacillus...

Vol. 176, No. 21JOURNAL OF BACrERIOLOGY, Nov. 1994, p. 6518-65270021-9193/94/$04.00+0Copyright X) 1994, American Society for Microbiology

Cloning, Nucleotide Sequence, and Expression of theBacillus subtilis ion Gene

SABINE RIETHDORF,* UWE VOLKERt ULF GERTH, ANEIT WINKLER,4SUSANNE ENGELMANN, AND MICHAEL HECKER

Institut fur Mikrobiologie und Molekularbiologie, Emst-Moritz-Amdt-Universitat,17487 Greifswald, Germany

Received 6 June 1994/Accepted 22 August 1994

The kon gene of Escherichia coli encodes the ATP-dependent serine protease La and belongs to the family ofc32-dependent heat shock genes. In this paper, we report the cloning and characterization of the Ion gene fromthe gram-positive bacterium Bacillus subtilis. The nucleotide sequence of the Ion locus, which is localizedupstream of the heDL4XCDBL operon, was determined. The Ion gene codes for an 87-kDa protein consisting of774 amino acid residues. A comparison of the deduced amino acid sequence with previously described ion geneproducts from E. coli, Bacillus brevis, and Myxococcus xanthus revealed strong homologies among all knownbacterial Lon proteins. Like the E. coli ion gene, the B. subtilis Ion gene is induced by heat shock. Furthermore,the amount of Ion-specific mRNA is increased after salt, ethanol, and oxidative stress as well as after treatmentwith puromycin. The potential promoter region does not show similarities to promoters recognized by o+32 ofE. coli but contains sequences which resemble promoters recognized by the vegetative RNA polymerase EoA ofB. subtilis. A second gene designated orJX is suggested to be transcribed together with ion and encodes a proteinwith 195 amino acid residues and a calculated molecular weight of 22,000.

ATP-dependent proteases are involved in the regulation ofthe level of a number of proteins with short half-lives, such asSulA and RcsA of Escherichia coli (25, 26). In addition tobiologically active proteins, many damaged and abnormalproteins resulting from misfolding, premature termination, ordenaturation are subjected to ATP-dependent proteolytic deg-radation. During various stresses, increasing amounts of mis-folded and damaged proteins may accumulate. Hence, theATP-dependent proteases are also important during stress. InE. coli, two forms of energy-dependent proteolytic systems, theLon (La) (11, 14) and Clp (32, 36) proteases, have been fairlywell characterized. Lon (12, 22, 46) and ClpP (37) belong tothe family of heat shock proteins. The ATP-dependent serineprotease La is encoded by the ion gene of E. coli. Mutations inion result in a pleiotropic phenotype of E. coli cells, with anincreased sensitivity to UV light, mucoidy, filamentousgrowth, and defects in the lysogenicity of some bacterio-phages and in the degradation of regulatory or abnormalproteins (26).Although extracellular proteases have been a matter of

extensive investigation in various bacilli because of their indus-trial application, the knowledge of the structure and functionof ATP-dependent proteases in Bacillus species is still verylimited. Protein extracts of Bacillus subtilis cross-reacted withantibodies raised against the protease La of E. coli (4). Thegene coding for the Lon protease of Bacillus brevis was clonedand sequenced. However, in contrast to E. coli, the gene is notinduced by heat, and the insertion mutation does not cause anyobvious phenotype (34).We are interested in the response of B. subtilis to various

* Corresponding author. Phone: 03834/77271, ext. 210. Fax: 03834/883353.

t Present address: Department of Microbiology, The University ofTexas Health Science Center at San Antonio, San Antonio, TX78284-7758.

i Present address: Forschungsinstitut fuir Molekulare Pharmakolo-gie, 10315 Berlin, Germany.

stresses. According to their regulation, the heat shock proteinsof B. subtifis can be arranged into at least two groups (29).Gene products of the groESL and the dnaK operons representmembers of the first group. The heat induction of theseproteins requires the vegetative sigma factor orA of B. subtifis(9) and a conserved palindromic structure (CIRCE) justdownstream of the start point of transcription (50, 67, 70). Thealternative sigma factor orB of B. subtilis controls the heat shockinduction of the second group (6, 7, 8, 62). Preliminary dataindicate the existence of a third mode of induction of heatshock genes in B. subtilis. We suggest that neither aB nor thepalindromic structure is involved in the heat shock induction ofthese genes (39, 62).

In this report, the cloning and characterization of the heat-inducible ion gene of B. subtilis are described. The identificationand cloning of the B. subtilis ion gene are independentlyreported by Schmidt et al. in an accompanying paper (51). Thedata indicate that the Ion gene product, along with ClpP (62),and ClpC (39), belongs to the oB-independent heat shockproteins of B. subtilis, which are induced by heat and otherstress conditions. The ion operon contains a second openreading frame, orfX, which codes for a 22-kDa protein.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions. Thebacterial strains and plasmids used in this study are describedin Table 1. E. coli DH5aL and JM110 were routinely grown in acomplex medium and used as hosts for DNA manipulation. B.subtilis strains were cultivated under vigorous agitation at 37'Cin LB or in a synthetic medium described earlier (55). Cells ofB. subtilis were exposed to different stress conditions asdescribed by Volker et al. (62). Heat shock was achieved bytransferring exponentially growing cells from 37 to 48, 50, or52'C. The other stress conditions were provoked by exposingexponentially growing cells to either 4% (wt/vol) NaCl, 5%(vol/vol) ethanol, 0.02% (wt/wt) H202, or 20 Rg of puromycinper ml. The samples were taken during exponential growth

6518

on May 30, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

CHARACTERIZATION OF THE B. SUBTILIS LON GENE 6519

TABLE 1. Bacterial strains and plasmids

Strain or plasmid Genotype or descriptiona Reference(s) or source

E. coliDH5ot F' 480dlacZAM15 A(lacZYA-argF)U169 deoR recAl end4l hsdR17 (rK- mK+) supE44 27

thi-1 gyrA96JM110 leu F' (traD36 proAB+ lacIq lacZAM15) 68BL21(DE3) hsdSgal(XcIts857 indl Sam7 ninS lacUV5-T7 genel) 54

B. subtilisIS58 trpC2 lys-3 5ML6 trpC2 sigB::AHindIII-EcoRV::cat 33BGWLON1 trpC2 lys-3 lon::pJH101 This studyBD224 trpC2 recE4 17

PlasmidspIB130 Cloning vector, Apr International Biotech-

nology Inc.pBluescriptIIKS/' Cloning vector, Apr StratagenepBluescriptIISK+I' Cloning vector, Apr StratagenepSPT19 Cloning vector, Apr Boehringer MannheimpJH101 Insertion vector, Apr, Tcr, Cmr 19pBL2637 pBL2 containing a 280-bp Sau3A fragment of chromosomal DNA from B. subtilis 64pIBI2637 pIBI30 containing the 280-bp Sau3A fragment of pBL2637 This studypBSLON pJH101 digested with EcoRV-AVal and ligated with the 290-bp XbaI-AVaI fragment This study

of pIBI2637pBSLON1 Obtained after plasmid rescue from EcoRI-digested chromosomal DNA of B. subtilis This study

BGWLON1pBSLON8 Obtained after plasmid rescue from NcoI-digested chromosomal DNA of This study

B. subtilis BGWLON1pBSLON9 pBluescriptIISK+ containing the 4.038-kb EcoRV-BglII fragment of pBSLON1 This studypSPTLON pSPT19 containing the 300-bp EcoRI-XbaI fragment of pIBI2637 This studypBSLON10 pBluescriptlIKS- containing the 770-bp PvuI-BamHI fragment of pBSLON1 This studypBSLON11 pBluescriptIlKS- containing the 467-bp NdeI fragment of pBSLON8 This studypWH703 Kmr, promoterless xylE and cat-86 18, 64pWHLON1 pWH703 containing the 800-bp EcoRI-HincII fragment of pBSLON10 This studypWHLON2 pWH703 containing the 497-bp EcoRI-HincII fragment of pBSLON11 This studya Abbreviations: Apr, Cmr, Kmr, and Tcr, resistance to ampicillin, chloramphenicol, kanamycin, and tetracylin, respectively.

prior to and after the shift at the times indicated in Fig. 4 and5. The time of the shift was set at zero.

Construction of an insertion mutation in the ion gene of B.subtilis. The 280-bp Sau3A fragment of pBL2637 (64) contain-ing an internal fragment located near the N-terminus-encodingpart of the ion gene of B. subtilis was ligated with theBamHI-digested vector pIBI30, resulting in plasmid pIBI2637.This plasmid was linearized with XbaI, the cohesive ends werefilled in with the Klenow fragment of DNA polymerase I, andthe plasmid was cut with AvaI. The 290-bp fragment wasinserted into the EcoRV-AvaI-digested integration vectorpJH101. The resulting plasmid, pBSLON, was used to trans-form competent cells of B. subtilis IS58. Chloramphenicol-resistant colonies were selected on agar plates containing 5 jugof chloramphenicol per ml. The integration of the plasmid wasverified by Southern blot analysis using a nonradioactive DNAlabeling and detection kit (Boehringer Mannheim).

Cloning of the ion gene and DNA sequence determination.Chromosomal DNA from B. subtilis BGWLON1, in which theIon gene is disrupted by the integration of plasmid pBSLON,was digested with EcoRI and NcoI, religated, and introducedinto E. coli DH5ot, generating plasmids pBSLON1 andpBSLON8 (Fig. 1). For sequencing, a 4.038-kb EcoRV-BglIIfragment from pBSLON1 was subcloned into the vector pBlue-scriptIISK+ digested with EcoRV and BamHI, yielding plas-mid pBSLON9. The sequence was determined by primerwalking using plasmids pBSLON1 and pBSLON8 or from a setof deletional plasmids of pBSLON9 generated by exonuclease

III/nuclease S1 digestion. Both strands were sequenced by thedideoxy-chain termination method of Sanger et al. (49), withplasmid DNA as the template.

Expression of orJX in E. coli. To express the orfX gene,plasmid pBSLON10 was transformed into E. coli BL21(DE3)(54), which carries the gene for the T7 RNA polymerase underthe control of an IPTG (isopropyl-,3-D-thiogalactopyranoside)-inducible lacUV5 promoter. For the construction of pBSLON10,the vector pBluescriptlIKS- was digested with EcoRV andligated with the 770-bp PvuI-BamHI fragment from pBSLON1previously treated with the Klenow fragment of DNA poly-merase I. The orientation of the cloned fragment was tested byEcoRI-SphI digestion. In pBSLON10, the DNA strand codingfor orJX can be transcribed by the T7 RNA polymerase.

Plasmid-harboring cells were cultivated in a synthetic me-dium (43) containing 100 jLg of ampicillin per ml. At an opticaldensity of 0.8 at 500 nm, IPTG was added to a final concen-tration of 1 mM, and 15 min later the culture was treated with200 jig of rifampin per ml. The bacteria were pulse-labeled for3 min with 10 tICi of L-[35S]methionine per ml both before and15 min after the addition of rifampin and lysed by boiling for3 min. Proteins of the cell extracts were fractionated on asodium dodecyl sulfate (SDS)-10 to 20% polyacrylamide gra-dient gel. Radioactively labeled proteins were detected byautoradiography (Fig. 3). To analyze the N-terminal proteinsequence, the bacteria were collected 2 h after the addition ofrifampin and lysed by boiling, and the proteins were trans-ferred from the gel onto a polyvinylidene difluoride membrane

VOL. 176, 1994


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

6520 RIETHDORF ET AL.

or

catIifPBSLON

amp

lon2637EcoR I

Ava ClapXbaI

xiion EcoRl

CaNRI NcoI

PBSLON8 L

chromosomol DNA

ton

integration of pBSLON

Bgll EcoRI

orfX orfY hemAXCDBLton

pBSLON 1

0 500 1000 bpsFIG. 1. Schematic representation of the chromosomal rearrangement after integration of plasmid pBSLON into the chromosomal Ion gene of

B. subtilis. Plasmids pBSLON1 and pBSLON8 were obtained after plasmid rescue using the restriction sites EcoRI and NcoI as described inMaterials and Methods.

by electroblotting. The protein of interest was sequenced on anApplied Biosystems A473a protein sequencer.

Promoter cloning and measurement of catechol 2,3-oxygen-ase activity. The promoter probe vector pWH703 (64) is aderivative of plasmid pPL703 (18) and contains the promoter-less catechol 2,3-oxygenase gene (xylE) from Pseudomonasputida and the promoterless cat-86 gene. For the constructionof pBSLON11, the vector pBluescriptlIKS- was digested withEcoRV and ligated with the 467-bp NdeI fragment frompBSLON8 which was previously treated with the Klenow frag-ment of DNA polymerase I. The direction of the cloned frag-ment in pBSLON11 was determined by sequencing. TheEcoRI-HincII fragments from pBSLON10 (800 bp) andpBSLON11 (497 bp) were inserted into the EcoRI-SmaI-digested vector pWH703, resulting in plasmids pWHLON1and pWHLON2. Transformation of protoplasts of B. subtilisBD224 was carried out as described by Chang and Cohen (10),and transformants were selected after regeneration of theprotoplasts on complex agar medium supplemented with 150jug of kanamycin per ml. Transformants containing a promoterupstream of the xylE gene developed a yellow color aftercolonies were sprayed with catechol. These cells were able togrow on plates with 10 jug of chloramphenicol per ml. Catechol2,3-oxygenase activity was measured as described by Volker etal. (64).

Analysis of transcription. Total RNA of the B. subtilisstrains was isolated from exponentially growing or stressedcells by the acid phenol method of Majumdar et al. (41), withsome modifications (62). Serial dilutions of total RNA weretransferred onto a positively charged nylon membrane by slotblotting and hybridized with digoxigenin-labeled RNA probesas instructed by the manufacturer (Boehringer Mannheim).The chemiluminograms were quantified with a personal den-sitometer from Molecular Dynamics. Induction ratios werecalculated by setting the value of the control to 1. Hybridiza-tion specific for Ion mRNA was conducted with a digoxigenin-

labeled RNA probe synthesized in vitro with T7 RNA poly-merase (noncoding strand) from linearized plasmid pSPTLON. This plasmid is derived from pSPT19 (BoehringerMannheim) and contains the 300-bp EcoRI-XbaI fragment ofpIBI2637. Hybridization specific for orX mRNA was carriedout by digoxigenin-labeling of the SphlI-linearized plasmid pBSLON10 by T3 polymerase. The RNAs synthesized in vitro fromthe coding strand were used as negative controls for thehybridization and did not yield any specific hybridization signal(data not shown).

Synthetic oligonucleotides complementary to the N-termi-nus-encoding region of the lon gene (PLON, 5'-GAAATATCCTGCTGAGTGGC-3') and orX (PORF, 5'-CCCGGCACATCCACA-3') were labeled with [y-32P]ATP and used asprimers for the primer extension analysis as described byWetzstein et al. (67).

General methods. Plasmid isolation, restriction enzymeanalysis, transformation of E. coli, exonuclease III digestionand filling in of the recessed 3' termini, and 5'-3' exonucleasedigestion by the Klenow fragment of DNA polymerase I wereperformed as described by Sambrook et al. (48). ChromosomalDNA from B. subtilis was isolated as described by Meade et al.(42). Transformation of natural competent B. subtilis cells wascarried out by the two-step protocol of Hoch (30).Computer analysis of sequence data. The sequence data

manipulations were performed with the Genetics ComputerGroup sequence analysis software package.

Nucleotide sequence accession number. The nucleotide se-quence data reported in this paper appear in the EMBL andGenBank nucleotide sequence databases under accessionnumber X76424.

RESULTS

Isolation of a strain with an insertion mutation in Ion.Experiments designed to clone promoter-containing DNA

NcoI

Le~

J. BACTERIOL.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


fragments of B. subtilis yielded a set of 15 fragments whichcaused heat induction of the reporter gene bglM, coding for the0-1,3-1,4-endoglucanase of Bacillus macerans (64). The nucle-otide sequence of the promoter-containing fragment of plas-mid pBL2637 exhibited 66.5% identity in a 282-bp overlap withthe N-terminus-encoding region of the B. brevis Ion gene (34).The 280-bp Sau3A fragment of pBL2637 which is internal tothe Ion-like coding sequence was used to construct the inte-grational vector pBSLON as described in Materials and Meth-ods. B. subtilis IS58 was transformed with pBSLON andselected for chloramphenicol resistance. Since pBSLON can-not replicate in B. subtilis, the chloramphenicol-resistant trans-formants should carry the plasmid integrated into the chromo-somal Ion gene by a Campbell-like recombination, creating adisruption of the ion gene. Neither mucoidy nor filamentationduring growth could be detected in the mutant strains. Fur-thermore, no significant differences in the growth parametersof the mutant strain B. subtilis BGWLON1 and the wild-typestrain B. subtilisIS58 were observed at a temperature of 37, 48,50, or 520C.

Nucleotide sequence of the B. subtilis Ion gene. The ion genewas cloned as described in Materials and Methods, the nucle-otide sequence was determined from plasmids pBSLON1,pBSLON8, and pBSLON9 in both orientations (Fig. 1). Figure2 displays the sequence of a 3,853-bp NdeI-HindIII fragment.The sequence downstream of the NsiI site (Fig. 2) is identicalto the sequence reported for the hemAXCDBL operon byHansson et al. (28) (Fig. 1). This finding is consistent with theresults of Schmidt et al. (51), who mapped the Ion gene of B.subtilis at position 245° of the genetic map (3). Computeranalysis of the sequence revealed the presence of two openreading frames (ORFs), which seemed to be transcribed in thesame direction as the hem operon. The first ORF starts with anATG codon at position 333 and might encode a proteinconsisting of 774 amino acids with a predicted molecular massof 86,600 Da and an isoelectric point of 6.13. The ATG codonat position 333 is preceded by a potential ribosome binding sitecomplementary to the 3' end of the 16S rRNA extending fromnucleotides 318 to 324 (Fig. 2). A comparison of orfl withdatabase entries established a high degree of identity withpreviously published sequences of bacterial Ion genes (seebelow). Therefore, orfl was designated Ion.The C-terminus-encoding part of an unknown ORF was

identified upstream of the ion gene. This ORF is followed by apotential factor-independent terminator structure (Go = -9.9kcal (1 kcal = 4.184 kJ)/mol) extending from nucleotides 151to 175.Another ORF with a length of 585 bp is located immediately

downstream of Ion (Fig. 2). This ORF was designated orfX.The translation initiation codon AUG of the oijX gene ispreceded by a Shine-Dalgarno sequence and overlaps the stopcodon of the ion gene by one nucleotide (Fig. 2). A potentialterminator (Go = -16.6 kcal/mol) was identified downstreamof orfX from bases 3232 to 3259 (Fig. 2). These data suggestthat ion and orX might comprise an operon.

Analysis of the sequence revealed the presence of a thirdORF (designated orfY) between orJX and hemA but oriented inthe opposite direction, with the translation initiation codon atposition 3760 (Fig. 1 and 2). It has the capacity to code for aprotein with 164 amino acid residues. No homologies to otherknown genes could be found. The significance of this ORFremains to be elucidated.Amino acid sequences of the B. subtilis Ion and orJX gene

products. The alignment of the deduced amino acid sequencewith known bacterial Lon protein sequences revealed overallidentities with B. brevis Lon (34) of 69%, E. coli La (2, 20, 56)

of 55%, Myxococcus xanthus LonV (57) of 55%, and M. xanthusLonD (21, 58) of 49%. This high degree of identity encom-passes almost the full length of the four sequences except theN-terminal region. The potential ATP-binding sites derivedfrom the proposed consensus sequences (GPPGVKT andQ-MKKAG--NPVFLL [12]), and the regions around thesesequences are strongly conserved and were found at the samepositions within the four proteins (12, 21, 34, 57, 58). Theserine residue at the position 677 (Fig. 2) corresponds to thepreviously described active serine site at position 679 of E. coliprotease La and is also conserved in the five sequences (1, 20,69).The nucleotide sequence of B. subtifis orX exhibits 62%

identity in a 488-bp overlap with a partially sequenced putativeofX gene in B. brevis (34) and significant identity with an ORFdownstream of the poLA gene of E. coli encoding the proteinYihA, the function of which is unknown (35, 47). Comparisonof the derived amino acid sequences by multiple sequencealignment established 59% identity in a 119-amino-acid over-lap to B. brevis OrfX and 37.5% identity in a 184-amino-acidoverlap to E. coli YihA. Schmidt et al. (51) indicate for B.brevis an error at position 2744 in the sequence published byIto et al. (34). The sequence has not been redetermined, butthe insertion of one base pair at this position would generatean ORF with 64t% identity in a 162-amino-acid overlap withthe corresponding OrfX of B. subtilis. Furthermore, the cor-rected version of OrfX of B. brevis displays a strong homologywith the actual N terminus of the B. subtilis OrfX (see below).

Expression of the orJX gene in E. coli. orX might encode aprotein with a calculated molecular weight of 22,000. To testthis assumption, we expressed the orX gene of B. subtilis in E.coli. For this purpose, plasmid pBSLON10, containing the T7promoter in front of the orfX gene, was transformed into E. coliBL21(DE3). Expression of the T7 RNA polymerase wasinduced by the addition of IPTG in the presence of rifampin,which prevented the initiation of transcription by the RNApolymerase of E. coii. Figure 3 displays an autoradiogram of aseparation of protein extracts on an SDS-polyacrylamide gelafter labeling of the proteins with L-[35S]methionine as de-scribed in Materials and Methods. A major band with anestimated molecular weight of 22,000 to 23,000 was detected inextracts from a culture of BL21(DE3) containing plasmidpBSLON10 when the cells were treated with rifampin after theaddition of IPTG (Fig. 3, lane 4). The same protein wasproduced to a lesser extent when these cells were treated withIPTG only (Fig. 3, lane 3). No protein band was found inextracts of BL21(DE3) containing plasmid pBluescriptIIKS-either after the addition of IPTG alone (Fig. 3, lane 1) or afterthe addition of IPTG and rifampin together (Fig. 3, lane 2).The N-terminal sequence of the major protein -MKVTK-

SEIVIS- confirmed the expected translational start point de-duced from the nucleotide sequence (Fig. 2).

Regulation of transcription of Ion and oryX. The Ion gene ofE. coli is induced by heat shock and other stresses. Therefore,we analyzed the influence of heat stress on the transcription ofion in B. subtiiis. Slot blot filters with total RNA of B. subtilisIS58 isolated before and after the stress were hybridized withdigoxigenin-labeled RNA probes obtained by in vitro tran-scription of the linearized plasmid pSPTLON with T7 RNApolymerase. A four- to sixfold increase in the amount ofIon-specific mRNA could be measured within 3 to 6 min aftera shift from 37 to either 48,50, or 520C (Fig. 4 and unpublishedresults). A similar induction was found after salt stress in acomplex medium (Fig. 4) but surprisingly not in a syntheticmedium (data not shown). The ion mRNA level was alsoenhanced after ethanol or oxidative stress (Fig. 4) and after

VOL. 176, 1994


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

Nd-I5&AVATCAGCAGGCCATCTTA~CÂTAGCAGTGCGCCAGATTATTGCAGTGAaGACGTTCCAAGAAGTGCTTGATGAAAmTTTGTAATCCGCCAACG GAAaCAAAAaCC

Y B N Q Q A I L K Q I D G I S I I A V K T F Q B V L D 8 I L V N P P T B Q K P P

TTCATATCGAMaTCAaTAAAGAATCTCCTTAAAC ACT~~TaAGlmTaGAACTTCmTTAAAAaCaG~CH I B I N K B 8 V -35 -10 81

mU AGC~~~tGA~~pCIS~~aTCTAAAGTCACAGT~~~CACATGGCAGAAGAATTAAAACGCAGCATCC

-35 -10 82 SD M A S S L K R S I P1 _>

Su3A.NdoICGCTCIAmTATT CGGTTCAGGCTCTTGAACAGGCAATGATGC C=AtTATTTTL L P L R C L L V Y P T M V L H L D V G R D R S V QA L B Q AL M H D H M I F L

TAGCCACTCCAGCAGAATCGATAAmCAAATTATT -lTA TT GA CCACGCACGTCTGTGTGTGA T Q Q D 8 I D 8 P G 8 D B I P T V G T Y T K I K Q M L K L P N G T I R V L V

TGGAGGOCGCGTAATACTT_=AAGC^GATAGC8 G L K R A H I V K Y N 8 H E D Y T 8 V D I Q L I H B D D 8 K D T 8 D E A L M R

.Sau3A *coRVGGACTTTGCTAGACCACTT5 %ACATÂAAAAmrCTAAAAAAATCTWWC1 OTGCACClCT L L D H F D Q Y I K I 8 K K I S A 8 T Y A A V T D I B B P G R M A D I V A S H

ATCTG=CCC_______ATAATL P L K L K D K Q D I L B T A D V K D R L N K V I D F I N N B K 8 V L S I B K K

I G Q R V K R S8 8 R T Q K 8 Y Y L R B Q M K A I QK L G D K G K T G B VQ

AGACGCTGAOAAAATCGMAÂG C _C G G lCICCGTCAAGTTCTGC GGAAAGCTCGS.TCCT L T D K I B B A G M P D H V K B T A L K B L N R Y E K I P S O S a SS S V I R

GCAACTATATCGCTGCTlrCTCTTCC w 1 AAG~AGAA CTCTTGGACGAAGAGCACCACGGGCTTGAAAAAGTAAAAGN Y I DS L V a L PI T D E T D D K L D L KL A G R L L D E AH H G L B K V K B

AA GaT~Ga~CZCCCAGAAGCT.ACaAAAATCCCTGaAAGGCCCGATTCTCISTGTTAGGACCTCCAGGTG;TCGGAAAAACGTCTTTAGCCAAATCAATTGCAAAAAR I LS Y L A V Q 1 L T K S L K G P I L C L A G P P G V G K TS L A K 8 I A K 8

GCTTGGGACGCAATCGTCAGTCTCWACGAGGAGTTCWGOGTGAATCAGAGATACGCGCACCGACCTATGTCGGAGCAATGCCTGGACGTATTATTCAAGGGATGAAAAL G R K F V R I L G G V R D B 8 B I R G H R R T Y V G A L P G R I I Q G M K K

AAGCGGGCAAGCTG AATCCGGTCTT TCATTGATTTAGGGAGCCCATCATCCGCTATGCTTGAAGTGCTTGATCCAGAGCAAAACAGCAGCTa C K L N P V F L L D AI D K M S LD F R G D P 8 8 a M L TV L D P E Q N S 8 F

TCAGTGATIAALT G LLLGAGAAACCTTTGFSYVRSAT BSLT D H Y I 8 8 T F D L S K V L F I a T A N N L A T I P G P L R D R M E I I N I A

CAGGCTACACAGAAATAGAAAAACTTGAAATTGTAAAGGATCACTTGCTTCCAGCAAATCAAAGAACACGGGCTGAAGAAAAaGCAATC-TTCAGCTGCGTGATCAGGCGATTCTTGATAG Y T 8 IA I L E I V K D H L L P K Q I LE H G L K K SN L Q L R D Q A I L D I

TTATTTTT C C G A T TG a C ~ S l G A TI R Y Y VR SA G V R L SR Q L A A I C R K A A K A I V A L8 R K R I T V 0 B

AGAAGAACCTTCAAGRTTrTSATCOGAAAaCGCATTTTCAGATATGGACAAGCTGAAACAGAGGATCAAGrrGGTCSACTGACAGGGCTTGCG;TATACAACCGTTGGCGAGATACGCTTTK N L D F I GP R I F R Y G Q A BF I DS V G V V S G L A Y A T V G G D S L B

CGATTGAAGTATCGCTTTCACCGGGAAAAGGGAAATTAATCCTGCAGAAACTCGGGATGTTATGAGAGAGTCTGCTCAGGCTGCArrCAGCTATGTGCGATCCAAAAaCAGAAGAACI 8 V SL 8 P G K G K L I L T G K L L D V 0R 18 A Q A A F S Y V R S K T P B L

TTGGCATTGAACCTGACTTTCATGAGAAGTTATTCTATACATASTTACCAGGTG MCCCAAAAGATGCTCCCTCAGCCGG;TATTACGATGACGCGTGTTCTG=TG I 8 P D F H B K Y D I H I H V P G A VP P A G I TL A T A L V P A L

.PvuITAACOGGACGGG BlTrCGCGTGAAGTCGGCATGACTGGGAATAGcrSCGCGCCGCTTWOCTSiTq~=TAAAGGAAAAAGCGCTTGGCGCACATAGAGCGOGATTAAS G R A V 8 R E V G M T GA I T L R G R V L P I G G L KP K A L G A H R A G L T

CGACCASSATTCCGCCTAAAGATATAAAAGA _TAAGATATCCGGAAAGCGTCAGGGAGGGG CTGACATT ATATTGCTCCCCTTAG CACG 7TGGAlCATGCCTTAGT I I A I P S V RD G L T F I L A -H L D E V L B H A L V

TAiGVAAAATGAAAITCACAAAGTCAGAAATCGTGATCAGTGCAGTAAAACCGAACAGTACCCTGAAG'CC TCCATGCCGGAAGATCGAACCTAGAAAG E K K I

SD M K V T K S8E I V I 8 A V K P B Q Y P B G G L P e I A L A G R 8 N V G Korffl --->

ATCGTCTTTTATCAATCTTAATCAATQCGCAAAAATCTTGCGAGAACGTCATCA AAGCCGGAACCAAACGCTTAATTCTACATTAT G8 8 F I N 8 L I N R K N L A R T S 8 K P G K T Q S L N F Y I I N D B L H F V D V

GCCGGGCTACGGTTGCAAAGTGTCAAAG;T 1C CTA TATCACGACACGCGAGGAATTAAAAGCTGTGIGCAGATCGTTGATTTGCGP G Y G F A K V 8 K 8 8 R B A It G R M I B T Y I T T R B B L K A V V Q I V D L R

8phI

H A P 8 N D D V Q M Y B F L K Y Y G I P V I V I A T K A D R I P K G K V D K H A

C r Wl M C CCWAAGACG~aCTGATCT~rTTTCTTCC~rUCCINGCCGCACAAAXAATGATAAACCOCTAK V V R Q S L N I D P B D B L I L F 8 8 B T K K G K D B A N G A I K K M I N R

BjHIGAgaCTACC 1 CGTTTTATCAATAATAACAATAAACTGATTCCQATAAAGACAAACGGCCAATACCTCCTTCAGAACCTTOA~ATT

>>>>


FIG. 2. Nucleotide sequence of the B. subtilis Ion locus and deduced amino acid sequences of its gene products. Putative Shine-Dalgarnosequences, the potential -35 and -10 regions of the ion promoters, and restriction enzyme sites used in this study are underlined. The two possiblestart points of transcription, S1 and S2, of the Ion operon are indicated. Factor-independent terminators are labeled by arrows under thenucleotides. Termination codons are indicated by asterisks. The underlined potential promoter region and the Shine-Dalgarno sequence of orfYare derived from the nucleotide sequence. The putative active serine site is printed boldface and underlined.

treatment with puromycin, leading to the production of abnor-mal proteins in the cells (data not shown). This induction ofthe ion gene was significant, because the level of xynA mRNAdid not change in response to any of the conditions analyzed inthis study (data not shown). xynA codes for the enzymexylanase of B. subtilis (40). In a sigB mutant of B. subtilis, theion gene was induced at the same rate, indicating that theinduction of the B. subtilis ion gene may be independent of aB.The presence of a putative factor-independent terminator

upstream of ion indicates that the potential promoter shouldbe located between this terminator and the translation initia-tion codon for Ion at position 333 (Fig. 2). Furthermore,catechol 2,3-oxygenase activity could be measured (about 4 X102 iiU/108 cells) in the chloramphenicol-resistant cells of B.subtilis BD224 containing plasmid pWHLON2, suggesting thatthe essential promoter elements of the ion gene are locatedbetween the two NdeI sites in front of the Ion gene (Fig. 2). Incontrast, no activity of this enzyme (


heat salt ethanol H202

Ion atL ||| 62O

time (min) time (min) Urns (mn) time (min)

time (min) time (min) time (min) time (min)FIG. 4. Schematic representation of the increase of the Ion and ofX mRNA levels caused by different stresses. Bacteria were exposed to

different stresses as described in Materials and Methods. Serial dilutions of total RNA prepared from B. subtilis IS58 before (0 min) and differenttimes (3, 6, 9, 12, 15, 20, and 30 min) after the exposure to stress were bound to a positively charged nylon membrane and hybridized with thedigoxigenin-labeled antisense RNA probes specific for the corresponding genes. The hybridization signals were quantified with a personaldensitometer as described in Materials and Methods. The induction ratios of the mRNAs are shown.

subtilis is induced by heat shock. The intensity of the hybrid-ization signals after heat shock and the rate of heat inductionwere stronger at the putative transcriptional start point S2 thanat S1, leading to the suggestion of a preferable usage of S2under conditions of heat stress.The first hints for the existence of a B. subtilis ion gene were

obtained during the cloning of heat-sensitive promoters (64).The fragment which triggered the heat induction of a fusionpromoter was internal to ion and did not contain the promotersidentified in this study. Whether sequences in the structuralgene participate in the heat shock regulation of the B. subtilision gene should be investigated.The induction by heat and ethanol stress of a B. subtilis

protein cross-reactive to antibodies raised against La of E. coliwas previously observed by Arnosti et al. (4). The transcrip-tional analysis revealed that B. subtilis Ion is induced not onlyby heat shock but also by ethanol, salt, or oxidative stress (Fig.4) as well as after exposure of B. subtilis cells to puromycin

A

(data not shown). However, the increase of lon-specific mRNAafter the imposition of stress is lower than that observed formost other general stress genes (39, 62). Such a rather lowinduction ratio is in agreement with results obtained for E. coli.Goff et al. (22) measured a two- to threefold-higher level oftranscription of the ion gene after heat shock. Chuang andcoworkers (13) also found only a two- to threefold increase ofIon-specific mRNA after heat shock. Similar induction ratioswere found for E. coli Ion during ethanol stress or afterpuromycin treatment (23). But even a two- to threefoldincrease in ion transcription will considerably enhance the rateof degradation of abnormal proteins (23), whereas a stillhigher intracellular level of the protease La can be stronglydeleterious (24). Because of the toxic effects of the overpro-duced La protease in E. coli, the artificial production of largeamounts of this protein requires an inducible expressionsystem (56).

Since the potential promoters upstream of Ion display a high

BA C G T co Co 3 6 9

AcAGAFA

DNA~~~~~~~~~~~~~~~~~~sequencing

~~~~~~~~~~~~~~~~~~~A¢W§:V;7&___ g I ~~~~~~~~~~~~~~AS

7 t9, S _ , .~~~~~~~~~~~~~~~~~a, X,, r it Ad5! I 11:_T

FIG. 5. Mapping the 5' end of the Ion mRNA by primer extension analysis before (37°C) and after (50'C) heat shock. RNA was isolated fromB. subtilis IS58 before (co) and at different times (3, 6, and 9 min) after heat shock. Equal amounts of total RNA (10 ,ug) were used for the primerextension analysis. The potential start points of transcription, S1 (A) and S2 (B), are marked with asterisks. Lanes A, C, G. and T show the dideoxysequencing ladder obtained with the same primer as used for primer extension. The sequence displayed is complementary to that determined byDNA sequencing.

J. BAC-1ERIOL.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


degree of similarity to promoters of housekeeping genes, thequestion arose as to which mechanism controls the stressinduction of the lon gene. According to our current knowledge,the stress genes of B. subtilis belong to at least two differentregulons (62). The well-known chaperones encoded by thegroESL and dnaK operons of B. subtilis (50, 67) belong to thefirst group. The vegetative form of RNA polymerase Eu43seems to be involved in the induction of both operons by heatshock (50, 67). In E. coli, on the other hand, the chaperonegenes are recognized by Eu32 (45). Chaperone genes of mainlygram-positive bacteria possess a 9-bp inverted repeat immedi-ately upstream of the coding region which is required for anormal heat shock response by the dnaK and groESL operons(70). Such an inverted repeat is not present in the regionimmediately upstream of Ion in B. subtilis. Hence, anothermechanism must be responsible for the stress induction oflon.Recently, the alternative sigma factor cuB has been shown to bea heat shock protein (6) which is responsible for the inductionof several stress proteins in B. subtilis (6, 7, 62). The ion genedoes not require this alternative sigma factor for its heat shockor stress induction, since the level of ion mRNA and theinduction ratio do not differ between a mutant with a deletionin sigB and the wild-type strain.

Apparently the ion gene might be a member of yet anotherclass of stress genes of B. subtilis. Similar to ion, clpP and clpCare induced by various stresses independent of the alternativesigma factor aB (39, 62). No CIRCE (70) was found in theregulatory region of the clpC operon (38). Unfortunately, thesequence of the regulatory region of clpP has not beenelucidated yet. Therefore, the presence of the CIRCE cannotbe excluded. However, clpP and clpC exhibit an inductionpattern distinctively different from that of groESL or dnaK ofB. subtilis (39, 62, 63).An insertional mutation in the ion gene of B. subtilis did not

lead to any obvious phenotypical changes. The mutation didnot impair growth at 48 or 500C in comparison with the wildtype (data not shown). Similar to B. brevis (34), features whichare characteristic of E. coli Ion mutants such as mucoidy,filamentous growth, and increased sensitivity to UV light (25,26) have not been observed in the B. subtilis ion mutant. In E.coli, these effects are due to a diminished rate of the degrada-tion of regulatory proteins such as SulA (31, 44, 52) or RscA(53, 59). The same pleiotropic phenotypic properties werefound in Salmonella typhimurium lon mutants (16). M. xanthuscarries two lon-related genes, lonV (57) and lonD or bsg4 (21,58). Whereas lonV is essential for vegetative growth (57), noeffects of a lonD mutation on vegetative growth could beobserved. However, lonD mutants can neither aggregate norform spores (58). Schmidt et al. (51) provide evidence for aninfluence of a mutation in the B. subtilis ion gene on theaccumulation and activity of the alternative sigma factor 'G,which is produced predominantly in the forespore. In anaccompanying report (51), it is proposed that the Lon proteaseprevents inappropriate synthesis of aG and hence the tran-scription of or -dependent genes.

Sequencing revealed the presence of an additional openreading frame, orX, immediately downstream of the lon gene.Our results suggest that this gene is transcribed together withlon. An inverted repeat downstream of orJX could act as afactor-independent transcriptional terminator of the putativeoperon. Slot blot hybridizations revealed a similar increase oflon-and orJX-specific mRNAs under various stress conditions.Furthermore, we could not find an internal promoter upstreamof orX either by primer extension or RNase protection assayor by subcloning a putative promoter region into a promoterprobe vector. The ofX gene codes for a protein of 195 amino

acids with a calculated molecular weight of 22,000. A productof about 22 kDa was produced in E. coli by the expression oforX, and the translation initiation codon postulated from thesequence data was confirmed by N-terminal sequencing. Inter-estingly, OrfX of B. subtilis, the homologous putative proteinfrom B. brevis, and the E. coli YihA protein contain threeconserved G regions of a class of GTP-binding proteins (51).

Initial experiments to analyze the role of B. subtilis OrfX didnot yield any clear evidence for a function of this protein in theregulation of the putative Ion operon itself (unpublished data).

ACKNOWLEDGMENTS

We are very grateful to R. Losick and R. Schmidt for critical readingof the manuscript and helpful discussions. We thank R. Schmidt and K.Altendorf for N-terminal sequencing of the OrfX protein, R. Losickfor providing the strain ML6, and R. Gloger, V. Grapentin, and A.Harang for technical assistance.

This work was supported by grants from the Deutsche Forschungs-gemeinschaft and the Fonds der Chemischen Industrie to M.H.

REFERENCES1. Amerik, A. Y., V. K. Antonov, A. E. Gorbalenya, S. A. Kotova, T. V.

Rotanova, and E. V. Shimbarevich. 1991. Site-directed mutagen-esis of La protease: a catalytically active serine residue. FEBS Lett.287:211-214.

2. Amerik, A. Y., L. G. Christyakova, N. I. Ostroumova, A. I.Gurevich, and V. K. Antonov. 1988. Cloning, expression andstructure of the shortened, functionally active lon gene of Esche-richia coli. Bioorg. Khim. 14:408-411.

3. Anagnostopoulos, C., P. J. Piggot, and J. A. Hoch. 1993. Thegenetic map of Bacillus subtilis, p. 425-463. In A. L. Sonenshein,J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and othergram-positive bacteria: biochemistry, physiology, and moleculargenetics. American Society for Microbiology, Washington, D.C.

4. Arnosti, D. N., V. L. Singer, and M. J. Chamberlin. 1986.Characterization of heat shock in Bacillus subtilis. J. Bacteriol.168:1243-1249.

5. Belitsky, B. R., and R. S. Shakylov. 1980. Amount of guanosinepolyphosphate and the level of stable RNA synthesis in Bacillussubtilis cells upon inhibition of protein synthesis. Mol. Biol.14:1343-1353.

6. Benson, A. K., and W. G. Haldenwang. 1993. The CB-dependentpromoter of the Bacillus subtilis sigB operon is induced by heatshock. J. Bacteriol. 175:1929-1935.

7. Boylan, S. A., A. R. Redfield, M. S. Brody, and C. W. Price. 1993.Stress-induced activation of the rB transcription factor of Bacillussubtilis. J. Bacteriol. 175:7931-7937.

8. Boylan, S. A., A. R. Redfield, and C. W. Price. 1993. Transcriptionfactor-aB of Bacillus subtilis controls a large stationary-phaseregulon. J. Bacteriol. 175:3957-3963.

9. Chang, B.-Y., K.-Y. Chen, and C. H. Liao. 1994. The response of aBacillus subtilis temperature-sensitive sigA mutant to heat stress. J.Bacteriol. 176:3102-3110.

10. Chang, S., and S. N. Cohen. 1979. High frequency transformationof Bacillus subtilis protoplasts by plasmid DNA. Mol. Gen. Genet.168:111-115.

11. Charette, M., G. W. Henderson, and A. Markovitz. 1981. ATPhydrolysis-dependent activity of the lon (capR) protein of E. coliK12. Proc. Natl. Acad. Sci. USA 78:4728-4732.

12. Chin, D. T., S. A. Gof, T. Webster, T. Smith, and A. L. Goldberg.1988. Sequence of the lon gene in Escherichia coli. A heat-shockgene which encodes the ATP-dependent protease La. J. Biol.Chem. 263:11718-11728.

13. Chuang, S.-E., D. L. Daniels, and F. R. Blattner. 1993. Globalregulation of gene expression in Escherichia coli. J. Bacteriol.175:2026-2036.

14. Chung, C. H., and A. L. Goldberg. 1981. The product of the Ion(capR) gene in Escherichia coli is the ATP-dependent protease,protease La. Proc. Natl. Acad. Sci. USA 78:4931-4935.

15. Desautels, M., and A. L. Goldberg. 1982. Liver mitochondriacontain an ATP-dependent, vanadate-sensitive pathway for the

VOL. 176, 1994


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


degradation of proteins. Proc. Natl. Acad. Sci. USA 79:1869-1873.16. Downs, D., L. Waxman, A. L Goldberg, and J. Roth. 1986.

Isolation and characterization of lon mutants in Salmonella typhi-murium. J. Bacteriol. 165:193-197.

17. Dubnau, D., and C. Cirigliano. 1974. Genetic characterization ofrecombination-deficient mutants of Bacillus subtilis. J. Bacteriol.117:488-493.

18. Duvall, D. J., D. M. Williams, P. S. Lovett, C. Rudolph, N.Vasantha, and M. Guyer. 1983. Chloramphenicol-inducible geneexpression in Bacillus subtilis. Gene 24:171-177.

19. Ferrari, F. A., A. Nguyen, D. Lang, and J. A. Hoch. 1983.Construction and properties of an integrable plasmid for Bacillussubtilis. J. Bacteriol. 154:1513-1515.

20. Fischer, H., and R. Glockshuber. 1993. ATP hydrolysis is notstoichiometrically linked with proteolysis in the ATP-dependentprotease La from Escherichia colt. J. Biol. Chem. 268:22502-22507.

21. Gill, R. E., M. Karlok, and D. Benton. 1993. Myxococcus xanthusencodes an ATP-dependent protease which is required for devel-opmental gene transcription and intercellular signaling. J. Bacte-riol. 175:45384544.

22. Goff,. S. A., L. P. Casson, and A. L Goldberg. 1984. Heat shockregulatory gene htpR influences rates of protein degradation andexpression of the lon gene in Escherichia coli. Proc. Natl. Acad. Sci.USA 81:6647-6651.

23. Goff, S. A., and A. L. Goldberg. 1985. Production of abnormalproteins in E. coli stimulates transcription of Ion and other heatshock genes. Cell 41:587-595.

24. Goff, S. A., and A. L Goldberg. 1987. An increased content ofprotease La, the Ion gene product, increases protein degradationand blocks growth in Escherichia coli. J. Biol. Chem. 262:4508-4515.

25. Goldberg, A. L. 1992. The mechanism and functions of ATP-dependent proteases in bacterial and animal cells. Eur. J. Bio-chem. 203:9-23.

26. Gottesman, S., and M. R Maurizi. 1992. Regulation by proteoly-sis: energy-dependent proteases and their targets. Microbiol. Rev.56:592-621.

27. Hanahan, D. 1983. Studies on transformation of Escherichia coliwith plasmids. J. Mol. Biol. 166:557-580.

28. Hansson, M., L. Rutberg, L. Schr6der, and L Hederstedt. 1991.The Bacillus subtilis hemAXCDBL gene cluster, which encodesenzymes of the biosynthetic pathway from glutamate to uropor-phyrinogen III. J. Bacteriol. 173:2590-2599.

29. Hecker, M., and U. Volker. 1990. General stress proteins inBacillus subtilis. FEMS Microbiol. Ecol. 74:197-213.

30. Hoch, J. A. 1991. Genetic analysis in Bacillus subtilis. MethodsEnzymol. 204:305-320.

31. Huisman, O., R D'Ari, and S. Gottesman. 1984. Cell-divisioncontrol in Escherichia coli: specific induction of the SOS functionSfiA protein is sufficient to block septation. Proc. Natd. Acad. Sci.USA 81:4490-4494.

32. Hwang, B. J., W. J. Park, C. H. Chung, and A. L Goldberg. 1987.Escherichia coli contains a soluble ATP-dependent protease (Ti)distinct from protease La. Proc. Natl. Acad. Sci. USA 84:5550-5554.

33. Igo, M., M. Lampe, C. Ray, W. Schafer, C. P. Moran, and RLosick. 1987. Genetic studies of a secondary RNA polymerasesigma factor in Bacillus subtilis. J. Bacteriol. 169:3464-3469.

34. Ito, K., S. Udaka, and H. Yamagata. 1992. Cloning, characteriza-tion, and inactivation of the Bacillus brevis lon gene. J. Bacteriol.174:2281-2287.

35. Joyce, C. M., and N. D. Grindley. 1982. Identification of two genesimmediately downstream from thepoLA gene of Escherichia coli. J.Bacteriol. 152:1211-1219.

36. Katayama, Y., S. Gottesman, J. Pumphrey, S. Rudikoff, W. P.Clark, and M. R. Maurizi. 1988. The two-component ATP-dependent Clp protease of Escherichia coli: purification, cloning,and mutational analysis of the ATP-binding component. J. Biol.Chem. 263:15226-15236.

37. Kroh, H. E., and L. D. Simon. 1990. The ClpP component of Clpprotease is the u32-dependent heat shock protein F21.5. J. Bacte-riol. 172:6026-6034.

38. KrUger, E., and T. Msadek. Unpublished results.

39. KrUger, E., U. Volker, and M. Hecker. 1994. Stress induction ofclpC in Bacillus subtilis and its involvement in stress tolerance. J.Bacteriol. 176:3360-3367.

40. Lindner, J. C., J. Stuilke, and M. Hecker. 1994. Regulation ofxylanolytic enzymes in Bacillus subtifis. Microbiology 140:753-757.

41. Majunidar, D., Y. J. Avissar, and J. H. Wyche. i991. Simultaneousand rapid isolation of bacterial and eukaryotic DNA and RNA-anew approach for isolating DNA. BioTechniques 11:94-101.

42. Meade, H. M., S. R Long, G. B. Ruvkunm, S. E. Brown, and S. E.Ausubel. 1982. Physical and genetic characterization of symbioticand auxotrophic mutants of Rhizobium meliloti induced by TnSmutagenesis. J. Bacteriol. 149:114-122.

43. Mitchell, J. J., and J. M. Lucas-Lenard. 1980. The effect ofalcohols on guanosine 5'-diphosphate-3'-diphosphate metabolismin stringent and relaxed Escherichia coli. J. Biol. Chem. 255:6307-6313.

44. Mizusawa, S., and S. Gottesman. 1983. Protein degradation inEscherichia coil: the Ion gene controls the stability of the SulAprotein. Proc. Natd. Acad. Sci. USA 80:358-362.

45. Neidhardt, F. C., and R A. VanBogelen. 1987. Heat shockresponse, p. 1334-1345. In F. C. Neidhardt, J. L. Ingraham, K. B.Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.),Escherichia coli and Salmonella typhimurium: cellular and molec-ular biology. American Society for Microbiology, Washington, D.C.

46. Phillips, T. A., R A. Van Bogelen, and F. Neidhardt. 1984. Ion geneproduct of Escherichia coli is a heat-shock protein. J. Bacteriol.159.283-287.

47. Polayes, D. A., P. W. Rice, M. M. Garner, and J. E. Dahlberg. 1988.Cyclic AMP-cyclic AMP receptor protein as a repressor of tran-scription of the spf gene of Escherichia coli. J. Bacteriol. 170:3110-3114.

48. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecularcloning: a laboratory manual, 2nd ed. Cold Spring Harbor Labo-ratory, Cold Spring Harbor, N.Y.

49. Sanger, F. S., S. Nicklen, and A. R Coulson. 1977. DNA sequenc-ing with chain-terminating inhibitors. Proc. Natd. Acad. Sci. USA74:5463-5467.

50. Schmidt, A., M. Schiesswohl, U. Volker, M. Hecker, and W.Schumann. 1992. Cloning, sequencing, mapping, and transcrip-tional analysis of the groESL operon from Bacillus subtilis. J.Bacteriol. 174:3993-3999.

51. Schmidt, R., A. L. Decatur, P. N. Rather, C. P. Moran, Jr., and RLosick 1994. Bacillus subtilis Lon protease prevents inappropriatetranscription of genes under the control of the sporulation tran-scription factor c0. J. Bacteriol. 176:6528-6537.

52. Schoemaker, J. M., R C. Gayda, and A. Markovitz. 1984. Regu-lation of cell division in Escherichia coli: SOS induction andcellular location of the SulA protein, a key to lon-associatedfilamentation and death. J. Bacteriol. 158:551-561.

53. Stout, V., A. Torres-Cabassa, M. R Maurizi, D. Gutnick, and S.Gottesman. 1991. RcsA, an unstable positive regulator of capsularpolysaccharide synthesis. J. Bacteriol. 173:1738-1747.

54. Studier, F. W., and B. A. Mofatt. 1986. Use of bacteriophage T7RNA polymerase to direct selective high-level expression of clonedgenes. J. Mol. Biol. 189:113-130.

55. Stulke, J., RK Hanschke, and M. Hecker. 1993. Temporal activationof IP-glucanase synthesis in Bacillus subtilis is mediated by theGTP-pool. J. Gen. Microbiol. 39:2041-2045.

56. Thomas, C. D., J. Modha, T. M. Razzaq, P. M. Cullis, and A. J.Rivett. 1993. Controlled high-level expression of the ion gene ofEscherichia coli allows overproduction of Lon protease. Gene136:237-242.

57. Tojo, N., S. Inouye, and T. Komano. 1993. Cloning and nucleotidesequence of the Myxococcus xanthus ion gene-indispensability ofion for vegetative growth. J. Bacteriol. 175:2271-2277.

58. Tojo, N., S. Inouye, and T. Komano. 1993. The lonD gene ishomologous to the Ion gene encoding an ATP-dependent proteaseand is essential for the development of Myxococcus xanthus. J.Bacteriol. 175:4545-4549.

59. Torres-Cabassa, A. S., and S. Gottesman. 1987. Capsule synthesisin Escherichia coli K-12 is regulated by proteolysis. J. Bacteriol.169:981-989.

60. Vandyck, L., D. A. Pearce, and F. Sherman. 1994. PIM1 encodes a

J. BACTERIOL.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


mitochondrial ATP-dependent protease that is required for mito-chondrial function in the yeast Saccharomyces cerevisiae. J. Biol.Chem. 269:238-242.

61. Varon, D., S. A. Boylan, K. Okamoto, and C. W. Price. 1993.Bacillus subtilis gtaB encodes UDP-glucose pyrophosphorylase andis controlled by stationary-phase transcription factor-(&B. J. Bacte-riol. 175:3964-3971.

62. Volker, U., S. Engelmann, B. Maul, S. Riethdorf, A. Volker, R.Schmid, H. Mach, and M. Hecker. 1994. Analysis of the inductionof general stress proteins of Bacillus subtilis. Microbiology 140:741-752.

63. Volker, U., H. Mach, R. Schmid, and M. Hecker. 1992. Stressproteins and cross-protection by heat shock and salt stress inBacillus subtilis. J. Gen. Microbiol. 138:2125-2135.

64. Volker, U., S. Riethdorf, A. Winkler, B. Weigend, P. Fortnagel, andM. Hecker. 1993. Cloning and characterization of heat-induciblepromoters of Bacillus subtilis. FEMS Microbiol. Lett. 106:287-294.

65. Wang, N., S. Gottesman, M. C. Willingham, M. M. Gottesman,and M. R. Maurizi. 1993. A human mitochondrial ATP-dependent

protease that is highly homologous to bacterial Lon protease.Proc. Natl. Acad. Sci. USA 90:11247-11251.

66. Watabe, S., and T. Kimura. 1985. Adrenal cortex mitochondrialenzyme with ATP-dependent protease and protein-dependentATPase activities. J. Biol. Chem. 260:14498-14504.

67. Wetzstein, M., U. Volker, J. Dedio, S. L"bau, U. Zuber, M.Schiesswohl, C. Herget, M. Hecker, and W. Schumann. 1992.Cloning, sequencing, and molecular analysis of the dnaK locusfrom Bacillus subtilis. J. Bacteriol. 174:3300-3310.

68. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. ImprovedM13 phage cloning vectors and host strains: nucleotide sequencesof the M13mpl8 and pUC19 vectors. Gene 33:103-119.

69. Yu, A., V. A. Antonov, A. E. Gorbalenya, S. A. Kotova, T. V.Rotanova, and E. V. Shimbarevich. 1991. Site-directed mutagen-esis of La protease. A catalytically active serine residue. FEBSLett. 287:211-214.

70. Zuber, U., and W. Schumann. 1994. CIRCE, a novel heat shockelement involved in regulation of heat shock operon dnaK ofBacillus subtilis. J. Bacteriol. 176:1359-1363.

VOL. 176, 1994


http://jb.asm.org/

Dow

nloaded from
http://jb.asm.org/

nucleotide sequence, and expression ofthe bacillus subtilisthe gram-positive bacterium bacillus...

Documents