Gene, 109 (1991) 31-37 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0378.1119/91/$03.50

31

GENE 06210

The isolation, cloning and identification of a vegetative catalase gene from B a c i l l u s s u b t i l i s (Recombinant DNA; Campbell-like integration; transposon; TrO17::lacZ fusion; H202; catalase)

David K. Bol and Ronald E. Yasbin Department of Biological Sciences, Program in Molecular and Cellular Biology, University of Maryland, Baltimore County, MD 21228 (U.S.A.) Received by I. Smith: 8 August 1991 Revised/Accepted: 30 August/3 September 1991 Received at publishers: 30 September 1991

SUMMARY

A Bacillus subtilis library of Tn917::iacZ insertions was screened for mutants that were unable to grow in the presence of normally sublethal concentrations of hydrogen peroxide. The identification and subsequent analysis of one mutant strain, YB2003, which carried the mutation designated kat-19, revealed that this strain was deficient in the expression of a vegetative catalase. Regions ofthe chromosome both 5' and 3' to the site of the Tn917 insertion, as well as the gene without the insertion (kat-19 +) were cloned. The presence of the functional kat-19 + gene on a high-copy plasmid restored catalase activity to the kat-19::Tn917 strain as well as to strains orB. subtilis that carried the katA 1 mutation. While the katA + locus is believed to represent the structural gene for the vegetative catalase of B. subtilis [Loewen and Switala, J. Bacteriol. 169 (1987) 5848-5851], the sequence analysis of the cloned kat-19 + DNA fragments revealed an open reading frame that showed significant homology between the deduced amino acid sequence of this gene product and that of known eukaryotic catalases.

INTRODUCTION

The Gram +, aerobic, spore-forming bacterium B. subtilis has been used as a model for studying gene regulation. This bacterium is also rapidly emerging as a paradigm for the study ofiateractions between developmental processes and responses of organisms to environmental stresses. Recent results have demonstrated that several global regulatory

Correspondenceto: Dr. R.E. Yasbin, Department of Biological Sciences, 5401 Wilkens Ave., Baltimore, MD 21228 (U.S.A.) Tel. (301)455-3668; Fax (301)455-3875. Abbreviations: aa, amino acid(s); amy, gene encoding a-amylase ofB. licheniformis; Ap, ampicillin; B., Bacillus; bp, base pair(s); Cm, chloramphenicol; Er, erythromycin; kat-19 + , gene encoding catalase activity; Km, kanamycin; Lm, lincomycin; MLS, macrolides, lincosamides and streptogramin B; ORF, open reading frame; ori, origin of plasmid replication; R, resistance; SOB, DNA damage-inducible system in B. subtilis; SOS, DNA damage-inducible system in E. coli;Tn, transposon; ::, novel joint (insertion); [ ], denotes plasmid-carrier state; (), denotes plasmid integrated into host genome.

networks are activated when this organism achieves particular developmental states, and in response to specific types of environmental stress (Love etal., 1985). For example, during the development of the competent state (a differentiated stage in which a portion of the cells in a culture are able to take up exogenous DNA) there is the concurrent induction of another global regulatory network, the SOS-like or SOB system. Furthermore, the induction of the SOB system during competence actually depends on the action of two different regulatory networks (Love et al., 1985). Resistance ofB. subtilis to oxidative stress is achieved by the function of at least two independent regulatory networks (Bol and Yasbin, 1990). First, exposure of exponentially growing cells to oxygen radicals (related to H202) has been shown to induce the SOB system. This induction requires the regulatory function of the RecA protein as well as the enzymatic DNA repair function of this molecule (Love and Yasbin, 1984). Second, resistance to all concentrations of H202 depends upon the expression of a functional katA + gene (Bol and Yasbin, 1990). This gene

32 is suspected to code for a protein that has catalase activity (Loewen and Switala, 1987c). Furthermore, changes in katA + expression itself are in response to two different cellular signals. Specifically, katA + expression responds to H202 concentrations (Loewen and Switala, 1987a). Interestingly, regulation of this gene also appears to be related to the developmental growth cycle, since vegetative catalase production in B. subtilis is characterized by elevated levels of this enzyme during entry into the stationary phase of the growth cycle (Loewen and Switala, 1987a). Analysis of the responses of B. subtilis to H202 demonstrated that bacteria pretreated with sublethal concentrations of H202 were more resistant to subsequent challenge with higher levels of H202 than were nonpretreated cells. This characteristic of the stress response to H202 was termed induced protection, and was shown to be independent of the function of the recA + gene (and the SOB system), but to require a functional katA + gene (Bol and Yasbin, 1990). We have identified a mutant of B. subtilis carrying a Tn917: :lacZ insertion that is characterized by (i) increased sensitivity to H202; (ii)reduced catalase expression; and (iii) an absence of induced protection. This mutant phenotype would suggest that the locus identified by the transposition event should either be the structural gene for catalase or a regulatory gene required for catalase expression. To distinguish between these two possibilities, we have sequenced the gene interrupted by the Tn917::iacZ insertion. Results reported here indicate that this gene codes for a protein that has catalase activity and may be encoded by the katA + locus.

RESULTS AND DISCUSSION

(a) Cloning the kat-19 + locus Screening the library of Tn917: :iacZ insertions resulted in the identification of one H202 sensitive mutant, strain YB2002. Further characterization ofthis mutant strain subsequently revealed a deficiency in the production of catalase. Further transposition of Tn917 in this mutant strain (YB2002) was prevented by the method of Youngman et ai. (1984). Specifically, strain YB2002 was transformed with pTV21J2. The result of this procedure ~s that MLS resistance within the original transposon in the chromosome is exchanged for Cm a, pBR322 sequences, as well as Ap g determinants. Additionally, this genetic exchange results in the deletion of sequences necessary for transposition (Youngman et al., 1984). To clone chromosomal regions flanking the Tn917 insertion, chromosomal DNA from one of these Cm R, MLS-sensitive transformants, strain YB2003, was then

isolated and manipulated as described by Youngman et al. (1984). Specifically, chromosomal DNA from strain YB2003 was isolated and digested to completion with HindIII. This digested DNA (1 #g) was then ligated in a volume of 100 #l. This intramolecular ligation results in the formation of plasmids from the region of the chromosome containing the recombinant transposon insertion. The iigation mixture was then used to transform E. coil strain LE392 to obtain Ap R colonies. Plasmid pDKB10 DNA isolated from these colonies consists of the region of the B. subtilis chromosome 5' to the insertion site of the transposon, the left arm of Tngl 7, pBR322 sequences, and the Ap s and Cm s genes. Sequence analysis of B. subtilis chromosomal DNA cloned in pDKB10 indicated that the gene into which Tn917 had been inserted in strain YB2002 coded for a protein that was homologous to known catalase enzymes (Table I; Fig. 2). To complete the sequence of the ORF encoding this protein, it was necessary to obtain the portions of the B. subtilis chromosome that were 3' to the site of the Tn917 insertion. Plasmid rescue procedures that resulted in the cloning of the entire ORF were achieved using the Gram + integration vector pRQ200 (Lane et al., 1991). This vector contains a Gram- ori, and Cm R and MLS R determinants. Additionally, this vector contains a promoterless amy gene that is preceded by several convenient restriction sites. For Cm R Gram + colonies to arise, this vector must be integrated into the chromosome by Campbell-like integration. This is achieved by inserting B. subtilis chromosomal DNA upstream from the promoterless amy gene and integrating this plasmid into the B. subtilis chromosome by homology with that insert. The resulting genomic construct generates a merodiploid region of the chromosome from which a new recombinant plasmid may be formed. To generate such a merodiploid strain a 2.5-kb EcoRI fragment of B. subtilis DNA from pDKB10 was isolated by gel purification (Maniatis et ai., 1982). This fragment of B. subtilis DNA located upstream from the Tn917 integration site in strain YB2003 was then ligated into the EcoRI site of pRQ200, adjacent to the amy gene. This ligation mixture was used to transform E. coil strain JM 109 to Ap R. The orientation of this fragment with respect to the promoterless amy gene in pRQ200 was determined by restriction mapping of the recombinant plasmids isolated from E. coll. The proximity of a Pstl restriction site in the 5' end of the amy gene to an Xmn I restriction site within the kat-19 + gene suggested that transcription within the kat- 19 + structural gene would continue into the amy gene. This orientation is the same as that preceding the Tn917::lacZ fusion in the chromosome of strain YB2003. One plasmid, pDKB20, was found to contain the 2.5-kb EcoRI fragment in an orientation such that transcription from the kat-19 + promoter would tran-

33 TABLE I Strains and plasmids Strain-~

Relevant genotype

Parent strain/vector

Reference

YB2001 YB2002 YB2003 YB2004 YB2005

trpC2, katA 1 metB 5, trpC2, kat-19, MLS R metB5, trpC2, kat-19, CmR metB5, trpC2, amy +, CmR metB5, trpC2, CmR, KmR

YB886 ::Tng17 ::lacZ

Bol and Yasbin (1990) present study present study present study present stud~,-

YB2002[pTV21A2] YB886[pDKB20] YB2003[pDKB30]

Plasmidb

Parental vector

Insert

pDKB i 0 pDKB20 pDKB21Aa pDKB30

pTV2i 32 pRQ200 pRQ200 pMK3

3.4-kb HindllI-Tn917 (5' end kat-19+) 2.5-kb EcoRI (5' end kat-19+) 4.0-kb HindIII (kat-19+) 3.l-kb EcoRV-HindIII (kat-19+)

All genomic constructs were isolated in the background of the DNA repair-proficient,'prophage-free' B. subtilis laboratory parent strain YB886(Yasbin et al., 1980). To identify loci required for resistance orB. ~ubtilis to H202, chromosomal DNA isolated from a B. subtilis Tn917::lacZ insertion library (Youngman et al., 1983)was used to transform strain YB886 to MLS resistance. Transformants were selected for by plating on TBAB medium (Difco) containing 25/~g Lm/mi, and 1 #g Er/ml (Sigma Chemical Co.). H,O2-sensitive mutants were identified by replica-plating these MLS-resistant colonies onto freshly made (less than 12 h old) TBAB medium containing I mM H202 in addition to Er + Lm, as well as onto TBAB that contained only the antibiotics. The isolation of critical strains and plasmids is described in section a. Catalase activityof appropriate strains was detected by the evolution ofoxygen followingthe application of 8M H202to freshlygrown colonies. When necessary, ~-amylaseactivitywas detected by a zone of hydrolysisaround a colony grown on TBAB with 2% Baker starch. b Enzymesused and specifications for their use were as suggestedby Promega(Madison, WI). E. coli transformations into E. coli strains LE392or JM 109 were performed by electroporation as recommended by the Bio-Rad gene pulser apparatus manual. B. subtilis transformations (Yasbin et al., 1980), as well as isolation of chromosomal (Maniatis et al., 1982) and plasmid DNA (Maniatis et al., 1982) were done as described.

scribe the a m y gene in pRQ200. Plasmid pDKB20 was then transformed into B. subtilis strain YB886, creating strain YB2004, which was merodiploid for the 2.5-kb E c o R I fragment in pDKB20, and Cm R (Fig. 1). The resulting merodiploid region of the chromosome (in a 5 ' - 3 ' direction) in strain YB2004 contained a transcriptional fusion of the k a t - 1 9 + promoter to the a m y gene, the vector pRQ200, and the regenerated, intact k a t - 1 9 + gene (Fig. 1). Transcriptional fusion of the k a t - 1 9 + promoter to the a m y gene was confirmed by the production of amylase from strain YB2004. Transformation of strain YB886 with recombinant plasmids from E. coli which contained the 2.5-kb E c o R I fragment in the reverse orientation from that found in pDKB20 did not result in B. subtilis strains that produced amylase. The methods used to obtain the region of the chromosome adjacent to pDKB20 in strain YB2004 were similar to those used to clone the 5' end of the gene using the transposon-like vector described earlier. This procedure makes use of a unique H i n d I I I site in pRQ200 that is located at the 3' end of the a m y gene. By digesting chromosomal D N A from strain YB2004 with H i n d I I I , ligating in high volume, and transforming E. coli, plasmids were isolated that contained Er R and Cm R genes, a G r a m - ori from pRQ200, as well as regions of the B. subtilis chromo-

some 3' to the site of plasmid integration (Fig. 1). The portion of the B. subtilis chromosome in strain YB2004 adjacent to the integrated vector contains regions that are duplicated by integration of the 2.5-kb E c o R I insert in pDKB20. The integrated vector is flanked on one side by the 2.5-kb E c o R I insert, and on the other side by the intact k a t - 1 9 + gene, which consists of the insert plus the D N A immediately 3' to the E c o R I site in the kat-19 + ORF. The resulting plasmid, pDKB213a, isolated from E~coli contains a region ofthe chromosome of strain YB2004 that was thought to contain the intact kat-19 + gene. Specifically, integration ofthe recombinant pDKB20 via homology with the 5' end of the k a t - 1 9 + gene that immediately precedes the a m y gene results in the introduction of the vector upstream from the k a t - 1 9 + gene. The location of the H i n d I I I restriction site at the end of the a m y gene, and the absence of H i n d I I I restriction sites in the k a t - 1 9 + gene, enable the cloning of the k a t - 1 9 + gene from the chromosome of strain YB2004 by including the Cm R, Er R, and ori from pRQ200 on the same H i n d I I I restriction fragment as the k a t - 1 9 + gene. Restriction analysis of the insert in pDKB21Aa, and comparison with the known restriction map of the 5' end of the k a t - 1 9 ÷ gene suggested that the 5' end of the k a t - 1 9 + gene was cloned with additional D N A 3' of the E c o R I site within the kat-19 + ORF (Fig. 1).

34 Om on',

a

X R

R

R

R

I

I

H Cm

Er

R

b

R

been digested with HindIII + SmaI. This ligation mixture was used to transform E. coil strain LE392 for Ap R, and plasmids from selected transformants were screened by restriction analysis for recombinants that contained the appropriate insert. One such plasmid, pDKB30, was subsequently transformed into strain YB2003 (selecting for Km R) and returned this strain to catalase proficiency, as determined by the evolution of oxygen following exposure of Km R transformants to 8 M H202. That pDKB30 encoded the structural gene for catalase was confirmed by the transformation of E. coli catalase-deficient mutant strain UM262 (Loewen et al., 1990). Plasmid pDKB30 restored catalase production to this catalase-deficient strain. B. subtilis strain YB2003 was complemented for all ofthe phenotypes associated with catalase deficiency when transformed with pDKB30 (Fig. 3). Integration of pDKB21Aa, generating a merodiploid, and introducing the cloned gene in single copy also resulted in a catalase-positive phenotype. Similarly, strain YB2001 (katA 1) was restored to catalasepositive phenotype by the presence of pDKB30, rhese results indicate that mutations katA 1 and kat-19 + may be alleles of the same gene, however, the function of the gene identified by the katA l mutation is uncertain at the present time.

H

O Fig. 1. Campbell-likeintegration ofE. coli pDKB20 (map a; Table I) into the B. subtilis chromosome (map b) via homologywith the 2.5-kb EcoRl (R) insert in the parent vector, pRQ200 (Lane et al., 1991). The chromosome of the resultant merodiploid strain YB2004 (map e) was digested with Hindlll (H) and ligated to generate the recombinant plasmid pDKB21Aa in E. coli (Table I and section a).

(b) Complementation of the catalase-deficient phenotype of strain YB2003 Strain YB2003 is sensitive to I mM H 2 0 2 in freshly made agar plates, and it shows no evolution of oxygen upon contact with 8 M H 2 0 2. This strain is much more sensitive to concentrations of H 2 0 2 during exponential growth than is the parent strain YB886. Additionally, strain YB2003 failed to achieve increased resistance to H 2 0 2 by pretreatment with 50 #M H 2 0 2. This phenotype is similar to that of the isogenic katA l mutant strain YB2001 (Loewen et al., 1987; Bol and Yasbin, 1990). The presence of the complete kat-19 + gene was confirmed by complementation of the catalase deficiency of strain YB2003. This complementation was achieved by subcloning a 3.5-kb fragment of B. subtilis DNA from pDKB21Aa into the shuttle vector pMK3 (Sullivan et al., 1984). Specifically, an EcoRV-Hindlll fragment from pDKB21Aa was isolated and ligated into pMK3 that had

(c) Sequence analysis The nt sequence of 1813 bp of B. subtilis DNA from pDKB30 was determined from subclones generated in pUCI9. The predicted translation of a possible transcript from this region revealed the presence of an ORF 1461 nt long (Fig. 2), and corresponded to a 65.8-kDa protein of 483 aa. Identification of the possible function of the protein encoded by this ORF was achieved by submitting the deduced aa sequence to GenBank in a Word Search Command file (Devereux et al., 1984). Homology to several known catalase enzymes was found to exist. The most homologous of these were the Rattus norvegicus (Norway rat; Furuta et al., 1986) liver catalase, the human (Bell et al., 1986) kidney catalase, and the Bos primigenius taurus (bovine: Schroeder et al., 1982) liver catalase. Gap alignment to aa sequences for catalases from rat liver, human kidney, and bovine liver showed the catalase enzyme encoded by the kat-19 + gene to be 57.3?/0, 55.8?/0, and 56.2% identical, respectively. These same proteins were 75.2~, 73 %, and 72.8 % similar to our deduced catalase. Additionally, the degree of homology is most strongly conserved among these enzymes within the N-terminal half of the aa sequence. Interestingly, no other bacterial aa sequences found in GenBank were as similar to this putative B. subtilis catalase as these eukaryotic proteins. As mentioned above, the kat-19 + clone on pDKB30 restores catalase production to the catalase-deficient E. coii

35 1905/1 ATG AGT TCA AAT AAA CTG A C A ACT AGC TGG GGC GCT CCG GTT GGA GAT AAT CAA AAC TCA Met set ser asn lys leu thr thr ser trp gl¥ ala pro val gly asp asn gin asn ser

1965121 ATG ACT met thr 2025/41 GCC CAT ala his

GCC GGT TCT COO GGA CCA ACT TTA A~T CAA GAT GTA CAT TTA CTC GAA AAA TTG ala gly ser arg gly pro thr leu ile g l n asp val his leu leu glu lys leu TTC AAC CGA G A A CGT GTT CCT GAA CGT GTT GTT CAC GCC AAA GGA GCA GGC GCA phe asn arg glu arg val pro glu arg val val his ala lys glY ala gly ala

2085/61 CAC GGA his g1¥ 2145/81 GAA GTC glu val

TAT TTT GAA GTG A C A AAC GAC GTA ACA A A A TAC ACG AAA GCC GeT TTC CTT TCT tyr phe glu val thr ash asp val thr lys tyr thr lys ala ala phe leu ser GGC AAA CGC A C A CCG TTG TTC ATC CGT TTC TCA ACA GTT GCC GGT G A A CTT GGC gl¥ lys arg thr pro leu phe ile arg phe se~" thr val ala gl¥ glu leu gly

2205/101 TCT GCT GAC ACA GTT COO GAC CCG CGC GGA TTT GCT GTT AAA TTT TAT ACT GAA GAA GGA set ala asp thr val arg asp pro arg gly phe ala val lys phe tyr thr glu glu gly

2265/121 AAC TAC OAC ATC GTC GGC AAC AAT ACG CCT GTA TTC TTT ATC CGC GAT GCG A T T AAG TTC ash tyr asp ile val gly ash ash thr pro val phe phe ile arg asp ala ile lys phe

2325/141 CCT GAT TTC ATC CAT ACA CAA AAA AGA OAT CCA A A A ACA CAC CTG AAA AAC CCT ACG GCT

pro asp phe l i e h i s t h r g i n 1ys arg asp pro 1ys t h r h~s leu l y s asn pro t h r a l a 2385/161 GTA TGG OAT TTC TGG TeA CTT TCA CCA GAG TCA CTG CAC CAA GTG ACA ATC CTG ATG TCT val t r p asp phe t r p s e t l e u s e r pro g l u se,r l e u h i s g l n val t h r l l e l e u met s e t 2445/181 GAC CGC GGA ATT CCT GCG ACA CTT CGC CAC ATG CAC GGC TTC GGA AGC CAT A C A TTC AAA asp arg gly ile pro ala thr leu arg his met his gly phe gly ser his chr phe lys

2505/201 TGG ACA AAT GCC GAA CCC GAA GGC GTA TOG AqT A A A TAT CAC TTT AAA ACA GAA CAA GGC Crp t h r ash a l a g l u pro g l u gl¥ v a l t r p ile lys tyr his phe lys thr glu gln gly

2565/221 GTG AAA AAC val lys ash 2625/241 ACA GAA GAC thr glu asp

CTT CAT GTC AAT ACG GCA GCA AAA A T T GCC GGT GAA AAC CCT GAT TAC CAT leu asp val ash thr ala ala lys ile ala gly glu ash pro asp tyr his CTT TTC AAC GCA ATe GAA AAC GGT GAT TAT CCT GCA TGG AAA CTA TAT GTG leu phe ash ala ile glu ash gly asp tyr pro ala trp lys leu tyr val

2685/261 CAA ATC ATG gln ile met 2745/281 GTT TGG TCT val trp ser 2805/301 CCG GAA AAC pro glu ash 2865/321 GGT ATT GAT gi¥ ile asp

CCT TTA GAA GAT GCA AAT ACG TAC CGT TTC GAT CCG TTT GAT GTC ACA AAA pro leu glu asp ala ash Chr tyr arg phe asp pro phe asp val thr lys CAA AAA GAC TAC CCG TTA ATC GAG GTC GGA CGC ATG GTT CTA GAC AGA AAT gln lys asp Cy r pro leu ile glu val gly arg met val leu asp arg asn TAC TTT GCA GAG GTA GAA CAA GCG ACA TTT TeA CCT GGA ACC CTC GTG CCT tyr phe ala glu val glu gln ala thr phe set pro gly thr leu val pro GTT TCA CCG GAT AAA ATG CTT CAA GGT CGA CTT TTT GCT TAT CAT GAT GCA val ser pro asp lys met leu gln gly arg leu phe ala tyr his asp ala

2925/341 CAC CGC TAC CGT GTC GGT GCA AAC CAT CAA GCG CTG-CCA ATC AAC CGC GCA CGC AAC AAA his arg tyr arg val gl¥ ala ash his gln ala leu pro 11e ash arg ala arg ash lys

2985/361 GTA AAC AAT TAT CAG COT OAT GGG CAA ATG CGT TTT GAT GAT AAC GGC GGC GGA TCT GTG v a l asn asn t y r g i n arg asp gl¥ g i n met arg phe asp asp ash gly gly gly set val

3045/381 TAT TAC GAG CCT AAC AGC TTC GGC GGT CCA AAA GAG TCA CCT GAG GAT AAG CAA GCA GCA t y r t y r glu pro ash s e r phe gl¥ gl¥ pro lys glu set pro glu asp lys gln ala ala

3105/401 TAT CCG GTA CAA GGT ATC OCT CAC AGC GTA AGC TAC GAT CAC TAC GAT CAC TAC ACT CAA tyr pro val gin g1¥ ile ala asp ser val set tyr asp his Cyr asp his Cyr thr gln 3165/421 GCC GGC GAT ala gI¥ asp 3225/441 GTT AAT GCC val ash ala 3285/461 TAC AAA GCG tyr lys ala 3345/481 AAA GAT TCT

CTG TAT CGT TTA ATG AGT GAA GAT GAA CGT ACC CGC CTT GTT GAA AAT ATC leu tyr arg leu met ser glu asp glu arg thr arg leu val glu asn ile ATG AAG CCG GTA GAA AAA GAA GAA ATC AAG CTG CGC CAA AT(: GAG CAC TTC met lys pro val glu lys glu glu ile lys leu arg gln ile glu his phe GAT CCT GAA TAC GGA AAA CGC GTG GCA GAA GGC CTT GGA TTG CCG ATT AAA asp pro glu tyr gly lys a~g val ala glu gly leu gly leu pro ile lys TAA

lye asp ser OCH Fig. 2. Nucleotide sequence of the ~ t - 1 9 + gene. The deduced aa sequence used for protein homology comparisons with other known catalase proteins is indicated below the nt sequence. OCHdenotes termination of translation by an ochre stop codon. Sequences 5' to this ORF responsible for the initiation of transcription have not yet been identified. This ORF is immediately followed by a plausible transcription termination sequence (not shown). Numbers refer to nt/aa positions, aligned with the first digits of the first numbers.

36 REFERENCES 10"

--" m

I

.01

.001 .oooi

o

5

1o

15

20

25

DOSE, mM Ha02

Fig. 3. Hydrogenperoxideresistance of strains YB2003(kat.19 + ; boxed dots), and YB2005 pDKB30 (kat-19 +; blackened diamonds). Plasmid pDKB30 restored catalase activityand H202 resistance to strain YB2003 carrying the kat-19 mutation (Table I). Exponentiallygrowingcells were challengedwithvaryingconcentrations of H202 for 15 min before plating survivors. The data are representative of three individual experiments.

strain UM262 (data not shown). Also, the predicted size of the postulated catalase gone agrees with the observations of Loewen et al. (1987b). These results strongly suggest that the kat-19 + gone is the structural gone for catalase I of B. subtilis. The k a t - 1 9 + sequence is obtainable through GenBank under accession No. M80796.

(d) Conclusions (1) The results presented here clearly indicate distinct conservation between this possible B. subtilis catalase and that of catalases from eukaryotic organisms. Previous reports have also implied that the vegetative catalase enzyme of B. subtilis is not structurally similar to catalases isolated from other prokaryotic organisms (Loewen et al., 1987b). (2) Because of the protective nature of this enzyme this preservation of structure, and possibly function, may be an indication of tile relative levels of oxidative stress encountered I~y these aerobic organisms. (3) The gone disrupted by the T n 9 1 7 : : i a c Z insertion, designated k a t . 1 9 +, encodes the structural gone for catalasc. The relationship between/cat- 19 + and katA + has yet to be determined, and requires further attention.

ACKNOWLEDGEMENTS We wish to thank Dr. Peter Loewen for providing E. coil mutant strain UM262, and Dr. Ken Bayles for his editorial comments. This research was supported in part by NIH grant RO1DE08506, and is submitted in partial fulfilment o f the requirements for the Ph.D. degree for D.K.B.

Bell, G.I., Najaran, R.C., Mullenbach, G.T. and HalleweU,R.A.: cDNA sequen~-o coding for human kidney catalase. Nucleic Acids Res. 14 (1986) 5561-5562. Bol, D.K. and Yasbin, R.E.: Characterization of an inducible oxidative stress system in Bacillus subtilis. J. Bacterial. 172 (1990) 3503-3506. Devereux, J., Haeberli, P. and Smithies, O.: A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12 (1984) 387-395. Dowds, B.C., Murphy, P., McConnell,D.J. and Devine, K.M.: Relationship among oxidativestress, growth cycle,and sporulation in Bacillus subtilis. J. Bacterial. 169 (1987) 5771-5775. Dubnau, D.: Competence regulon of Bacillus subtiiis. In: Smith, I., Slepecky, R.A. and Setlow, P. (Eds.), Regulation of Prokaryotic Development; Structural and Functional Analysisof Bacterial Sporulation and Germination. American Society for Microbiology, Washington, DC, 1989. Furuta, S., Hayashi, H., Hujikata, M., Miyazawa, S., Osumi, T. and Hashimoto, T.: CompletenucleotidesequenceofcDNA and deduced amino acid sequenceofrat liver catalase. Proc. Natl. Acad. Sci. USA 83 (1986) 313-317. Haden, C.T. and Nester, E.W.: Purification of competent cells in the Bacillus subtilis transformation system. J. Bacterial. 95 (1968) 876-885. Lane, M.A., Bayles, K.W. and Yasbin, R.E.: Identification and initial characterization of glucose-repressible promoters of Streptococcus mutants. Gone 100 (1991) 225-229. Loewen, P.C. and Switala, J.: Multiple catalases in Bacillus subtilis. J. Bacterial. 169 (1987a) 3601-3607. Loewen, P.C. and Switala, J.: Purification and characterization of catalase I from Bacillus subtilis. Biochem. Cell Biol. 65 (1987b) 939-947. Loewen, P.C. and Switala, J.: Genetic mapping of katA, a locus that affects catalase 1 levels in Bacillus subtilis. J. Bacterial. 169 (1987c) 5848-5851. Loewen, P.C. and Switala,J.: Purification and characterization of sporespecificcat8lase-2 fromBacillus subtilis. Biochem.Cell Biol.66 (1988) 707-714. Loewen, P.C., Switala,J., Smolenski,M. and Triggs-Riane,B.L.: Molecular characterization of three mutations in katG affectingthe activity of hydroperoxidase I ofEscherichia coli. Biochem.Cell Biol.68 (1990) 1037-1044. Love, P.E. and Yasbin, R.E.: Genetic characterization of the inducible SOS-like system of Bacillus subtilis. J. Bacterial. 160 (1984) 910-920. Love, P.E., Lyle,MJ. and Yasbin, R.E.: DNA damage-inducibleloci are transcriptionally activated in competent Bacillus subtilis. Proc. Natl. Acad. Sci. USA 82 (1985) 6201-6205. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Murphy, P., Dowds, B.C,, McConnell,DJ. and Devine,K.M.: Oxidative stress and growth temperature in Bacillus subtilis. J. Bacterial. 169 (1987) 5766-5770. O'Kane, C., Stephens, M.A., and McConnell, D.: lntegrable a-amylase plasmid for generating random transcriptional fusions in Bacillus s,btilis. J. Bacterial. 168 (1986) 973-981. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencingwith chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Schroeder, W.A., Shelton,J.B., Robberson, B., Apell,G., Fang, R.S. and Bonaventura, J.: The complete amino acid sequence of bovine liver cr,talase and the partial sequence of bovine erythrocyte catalase. Arch. Biochem. Biophys.214 (1982) 397-421.

37 Sullivan, M., Yasbin, R.E. and Young, F.E.: New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection c¢ inserted fragments. Gene 29 (1984) 21-26. Yanisch-Perron, C., Vieira, J. and Messing, J.: Improved MI3 phage cloning vectors and host strains: nucleotide sequence~~ of the Ml3mpl8 and pUCl9 vectors. Gene 33 (1985) 103-119. Yasbin, R.E.: DNA repair in Bacillus subtilis, I. The presence of an inducible system. Mol. Gen. Genet. 1~3 (1977)211-218.

Yasbin, R.E., Fields, P.I. and Andersen, B.J.: Properties of Bacillus subtilis derivatives freed of their natural prophages. Gene 12 (1980) 155-157. Youngman, P3., Perkins, J.B. and Losick, R.: Genetic transposition and insertional mutagenesis in Bacillus subtilis with Streptococcusfaecalis transposon Tn917. Proc. Natl. Acad. Sci. USA 80 (1983) 2305-2309. Youngman, P., Perkins, J.B. and Losick, R.: A novel method for the rapid cloning in Escherichia coli of Bacillus subtilis chromosomal DNA adjacent to Tng17 insertions. Mol. Gen. Genet. 195 (1984) 424-433.

The isolation, cloning and identification of a vegetative catalase gene from Bacillus subtilis.

A Bacillus subtilis library of Tn917::lacZ insertions was screened for mutants that were unable to grow in the presence of normally sublethal concentr...
735KB Sizes 0 Downloads 0 Views