The regulation of expression of ytkD, a gene that encodes the first functional antimutator 8-oxo-dGTPase activity of B . subtilis, was studied here . A ytkD-lacZ fusion integrated into the ytkDlocus of wild-type B . subtilis 168 revealed that this gene isexpressed during both vegetative growth and early stages of sporulation . In agreement with this result, ytkD mRNAs were detected by both Northern blotting and reverse transcription-PCR during both developmental stages . These results suggested that ytkD is transcribed by the sequential action of RNA polymerases containing the sigma factors ^A and ^F, respectively . In agreementwith this suggestion, the spore-associated expression was almostcompletely abolished in a sigF genetic background but not ina B . subtilis strain lacking a functional sigG gene . Primerextension analysis mapped transcriptional start sites on mRNAsamples isolated from vegetative and early sporulating cellsof B . subtilis. Inspection of the sequences lying upstream ofthe transcription start sites revealed the existence of typical ^A- and ^F-type promoters . These results support the conclusionthat ytkD expression is subjected to dual regulation and suggestthat the antimutator activity of YtkD is required not only duringvegetative growth but also during the early sporulation stagesand/or germination of B . subtilis. While ytkD expression obeyed a dual pattern of temporal expression, specific stress induction of the transcription of this gene does not appear to occur, since neither oxidative damage [following either treatment with paraquat or hydrogen peroxide] nor mitomycin C treatment or ^B general stress inducers [sodium chloride, ethanol, or heat]affected the levels of the gene product produced.

 
Reactive oxygen species [ROS] such as the superoxide radical, hydrogen peroxide, and the hydroxyl radical, generated as by-products of cellular metabolism [56, 57], have the potential to reactwith proteins, lipids, and DNA [48, 50, 55] . Oxidative damageto DNA can result in the generation of apurynic/apirimidinic[AP] sites, several types of base modification, sugar damageand single- and double-strand breaks [22] . Furthermore, the intracellular deoxyribonucleotide and ribonucleotide pools arealso potential targets of oxidative damage [30, 59] . Thus, asa consequence of ROS action, dGTP and GTP can be converted to8-oxo-dGTP and 8-oxo-GTP, respectively [59] . The former canbe incorporated into nascent DNA strands opposite adenine, thusplaying a significant role in mutagenesis, aging, and cancer[38] . On the other hand, it has been proposed that 8-oxo-GTPhas the potential of being incorporated into mRNAs, generatingtranscriptional errors [59] . To counteract the potential mutageniceffects of 8-oxo-dGTP and transcriptional errors induced by8-oxo-GTP, cells rely on the protein MutT, which degrades theoxidized nucleotides to the corresponding monophosphate forms.These chemical changes prevent the incorporation of the alterednucleotides into either DNA or mRNA, respectively [59] . Proteinswith 8-oxo-dGTPase activity have been described and isolatedfrom bacteria [6, 11, 29] as well as from mammalian cells [28, 42, 58].

Multiple mechanisms act together to prevent or repair oxidative damage to DNA in the gram-positive spore-forming bacterium Bacillus subtilis, and the genes that encode such mechanisms have shown to be temporally regulated during vegetative growth and postexponential differentiation . For instance, yqfS, which codes for a type IV AP endonuclease and is a component of the base excision repair pathway, is expressed in the forespore compartment of the sporulating bacteria under the control of ^G RNA polymerase [E[sig]G] [60].On the other hand, katA, katB, and katX encode catalases ofB . subtilis, and these genes, while also subjected to differential regulation, are not always transcribed in response to various developmental stages . For instance, katA is expressed during vegetative growth and following bacterial treatment with H₂O₂ [7], while the expression of katB and katX is controlled bythe stress-regulated ^B [19] and the spore-specific ^F [4], respectively.An extremely important reactive component of cells is the superoxideradical . B . subtilis seems to possesses a single superoxidedismutase [SOD] gene called sodA [12, 27], and this gene is expressed in all phases of growth and during sporulation from different promoters [12, 27].

As mentioned above, the product 8-oxo-dGTP is known to be problematic with respect to mutagenesis and survival in living cells . Therefore, it is not surprising that the genome of B . subtilis [33] possessesthe genes yqkG [nudF], mutT, yvcI, yjhV, and ytkD, whose readingframes encode potential homologs of the Escherichia coli MutTprotein, the archetype of the 8-oxo-dGTPases [1] . A recent studyrevealed that nudF encodes a nucleotide diphosphohydrolase [Nudix]with specificity to split ADP-ribose into AMP and ribose-5-phosphate[18] . Thus, the product of the nudF gene is not believed tobe associated with conferring protection to B . subtilis against the mutagenic effects of the oxidized nucleotide 8-oxo-dGTP. Accordingly, the identification and characterization of theproteins involved in protecting B . subtilis from the mutageniceffects of oxidized nucleotide pools generated by ROS actionremain to be established.

YtkD possesses a 23-amino-acid-long sequence that contains 9of the 10 absolutely conserved residues in the Nudix amino acid signature of all proteins that have been shown to hydrolyze 8-oxo-dGTP [5, 23] . In this communication, we report that ytkDnot only encodes the first reported 8-oxo-dGTPase of B . subtilisbut also possessed the ability to complement the mutator phenotypeof an E . coli mutT mutant . Further evidence provided here demonstratedthat while the transcription of ytkD followed a dual patternof temporal expression controlled by the sequential action of ^A- and ^F-containing RNA polymerases, the transcription of thisgene was not stimulated by oxidative damage or by inducers ofthe SOS or ^B general stress responses

 
Bacterial strains, plasmids, and growth conditions. The B . subtilis and E . coli strains and the plasmids used inthis study are shown in Table 1 . Difco sporulation medium [DSM] [52] and Luria-Bertani medium [LB] [41] were used to propagateB . subtilis and E . coli strains, respectively . When required,antibiotics were added to media at the following final concentrations:chloramphenicol, 3 µg/ml; ampicillin, 50 µg/ml;and kanamycin, 10 µg/ml . Liquid cultures were shaken at 250 rpm at 37°C . Cultures on solid medium were grown at37°C . The optical density [OD] of liquid cultures was monitoredwith a Pharmacia Ultrospec 2000 spectrophotometer set at 600nm.

 
Genetic and molecular biology techniques. Preparation of competent E . coli or B . subtilis cells and their transformation with DNA were performed as previously described[8, 49] . Chromosomal DNA from B . subtilis was purified accordingto the protocol of Cutting and Vander Horn [16] . Small-scalepreparation of plasmid DNA from E . coli cells, enzymatic manipulations,and agarose gel electrophoresis were performed by standard techniques[49] . Medium-scale preparation and purification of plasmid DNAwere accomplished by using commercial ion-exchange columns accordingto the instructions of the supplier [Qiagen, Inc., Valencia,Calif.] . Nucleic acid sequencing by dideoxynucleotide chaintermination was performed with the Thermo Sequenase radiolabeledterminator cycle sequencing kit [U.S . Biochemical Corporation,Cleveland, Ohio] . Sequencing products were analyzed by autoradiographyafter electrophoresis through a 6% polyacrylamide sequencinggel.

Complementation of an E . coli mutT mutant by ytkD expression. E . coli strain SB3 [kindly provided by M . J . Bessman, The JohnHopkins University], lacking a functional MutT protein, wastransformed with either pTrc99A or pPERM426 [pTrc99A-ytkD] [Table1] . The resulting strains PERM425 and PERM426 were grown for24 h in LB medium containing 100-µg/ml ampicillin and1 mM isopropyl-ß-D-thiogalactopyranoside [IPTG] . Additionally,strains E . coli SB3 and E . coli JM83 were grown in LB mediumin the absence of antibiotics . Mutation frequencies were determinedby plating aliquots on LB plates containing nalidixic acid at20 µg/ml . The mutant colonies were counted after 1 dayof incubation at 37°C to estimate mutation frequencies.

Design of a plasmid to overexpress ytkD and purify a His₆-YtkD protein. The open reading frame [ORF] of ytkD lacking the first codonand extending 90 bp downstream of the stop codon was amplified by PCR utilizing Vent DNA polymerase [New England Biolabs, Beverly, Mass.] and oligonucleotide primers that inserted BamHI restriction sites into the cloned DNA . The PCR DNA fragment was first ligated into HincII-treated pUC18 and replicated into E . coli SURE [Stratagene,La Jolla, Calif.] . The resulting construct [pPERM279] was cutwith BamHI, and the 564-bp ytkD insert was cloned in-frame intoBamHI-treated pQE30 to generate pPERM427, which was transformedinto E . coli XL-10 Gold Kan [Stratagene].

Purification of His₆-YtkD. E . coli PERM427 was grown at 37°C in 50 ml of LB mediumsupplemented with ampicillin to an OD of 0.5 . Expression ofthe ytkD gene was induced during 4 h of incubation at 28°Cby the addition of IPTG [0.5 mM] . Cells were collected by centrifugationand washed two times with 10 ml of 50 mM Tris-HCl [pH 7.5] mixedwith 300 mM NaCl [buffer A] . The cells were frozen at -70°Cfor 12 h and disrupted by defrosting at room temperature . Thecell homogenate was resuspended in 10 ml of buffer A containing1% [vol/vol] Triton X-100, incubated for 30 min on ice, andthen subjected to centrifugation [27,200 x g for 10 min] to eliminate undisrupted cells and cell debris, and the supernatantwas applied to a 3-ml Ni-nitrilotriacetic acid [NTA]-agarose[Qiagen] column, previously equilibrated with buffer A . Thecolumn was washed with 100 ml of buffer A containing 20 mM imidazoleplus 100 ml of buffer A containing 30 mM imidazole, and theprotein bound to the resin was eluted with 10 ml of buffer Acontaining 100 mM imidazole; 2-ml fractions were collected duringthis step.

Assay of enzymatic activity. A colorimetric assay [described below] was used to measure therelative rates of hydrolysis of dGTP, GTP [purchased from Roche,Mannheim, Germany], 8-oxo-dGTP, and 8-oxo-GTP [purchased fromJENA Bioscience, Jena, Germany] . The nucleoside triphosphatase[NTP] activity of YtkD was measured in a 50-µl reactionmixture containing the following components: 40 mM Tris-HCl[pH 8.0], 8 mM MgCl₂, 4 mM the appropriate NTP, 10 mM dithiothreitol[DTT], and 0.5 U of yeast inorganic pyrophosphatase . A unitof pyrophophatase hydrolyzes 1 µmol of PP_i to P_i per minat 25°C . The reaction was allowed to proceed for 15 minat 37°C before being terminated by the addition of 50 µl of a 4:1 mixture of a 20% suspension of Norit A and 7% perchloric acid . This was mixed and allowed to stand for 5 min on ice before centrifugation to sediment the Norit A and absorbed nucleotides. An aliquot of the supernatant was then used to determine the amount of free inorganic orthophosphate by the method of Amesand Dubin [2] . A unit of enzyme activity hydrolyzes 1 µmol of substrate per min.

Construction of a B . subtilis strain containing a ytkD-lacZ gene fusion. Construction of an in-frame translational fusion between theytkD gene and the E . coli lacZ gene was carried out in the integrativeplasmid pJF751 [20] by inserting a 338-bp EcoRI-AviI fragmentfrom plasmid pPERM254 into pJF751 previously digested with EcoRIand SmaI . The resulting construct, containing the ytkD-lacZ fusion and designated pPERM274, was propagated into E . coli XL1-Blue . Plasmid pPERM274 was introduced by transformationinto competent cells of B . subtilis strains 168, 1S86 [sigF mutant], and WN118 [sigG mutant], and transformants were selected on solid DSM containing chloramphenicol.

Cell growth and enzymatic assays. B . subtilis strains carrying the ytkD-lacZ fusion were grownand allowed to sporulate in liquid DSM containing chloramphenicol.Samples of 1.5 ml were collected during vegetative growth andthroughout sporulation . Cells were washed with cold 0.1 M Tris-HCl[pH 7.5], and the cell pellets were stored at -20°C untildetermination of ß-galactosidase activity [43, 46].Briefly, washed cell samples were first disrupted with lysozymeand subjected to centrifugation . ß-Galactosidase activitypresent in the supernatant was measured and assigned to themother cell fraction [which actually consisted of mother cellsplus lysozyme-sensitive forespores] . The pellet, which consistedof lysozyme-resistant forespores containing spore coats, wassubjected to spore coat removal [43] and washed in 50 mM Tris-HCl[pH 7.5] buffer, and a second round of lysozyme treatment wasassigned to the forespore fraction for determination of ß-galactosidaseactivities [39, 43].

Induction experiments. Experiments were performed to analyze whether paraquat, H₂O₂,ethanol, NaCl, mitomycin C, or heat [48°C] induced the expressionof the ytkD-lacZ fusion of the strain B . subtilis PERM276 . Eachcompound was tested independently as follows . Cells were grownin LB medium [lacking NaCl] to an OD at 600 nm [OD₆₀₀] of 0.5.The culture was divided into two subcultures of equal volume,and each of the compounds described above was added to one subcultureto the following final concentrations: paraquat, 10 µM;H₂O₂, 200 µM; NaCl, 4%; ethanol, 4%; and mitomycin C,0.5 µg/ml . The second subculture served as a control.Induction by heat was carried out by incubating the experimentalculture, with aeration, at 48°C . Cells were harvested after1 h of induction and assayed for ß-galactosidase activity.

Northern blot and primer extension experiments. The total RNA from vegetative and sporulating cells of strainsB . subtilis 168 and 1S86 was isolated as previously described[39] . RNA samples [40 µg] were separated by electrophoresisthrough a 1% agarose-formamide gel and transferred to a high-boundnylon membrane . The membrane containing the transferred RNAwas hybridized at 60°C with a 680-bp EcoRI-BamHI fragmentfrom pPERM279 encompassing the entire ytkD sequence . The probewas labeled by random priming with [-³²P]dCTP by using the RediprimeII DNA labeling system according to the instructions of theprovider [Amersham Bioscience, Buckinghamshire, England] . Detectionof hybrids was performed by autoradiography following exposureof the membranes to Kodak X-Omat films . The size of the hybridswas estimated by using RNA markers [Promega, Madison, Wis.]of 281, 623, 955, 1,383, 1,517, 1,908, 2,604, 3,638, and 4,981 nucleotides [nt], respectively.

The 5' ends of ytkD were mapped by primer extension [40] ofytkD transcripts produced during both vegetative growth and sporulation . To this end, total RNA was isolated [39] from vegetativeand sporulating cells [stage T₅ [5 h after the end of log-phasegrowth]] of B . subtilis 168 . The total RNA [40 µg fromeach sample] was hybridized with the 23-mer oligonucleotide5'-CCAGACATGCTTCGGGCTGTCCG-3', which was complementary to theytkD mRNA from nt 66 to 88 downstream from the putative ytkDtranslational start codon . The oligonucleotide was labeled onits 5' end with [-³²P]ATP and T4 polynucleotide kinase . Theprimer was extended with Moloney murine leukemia virus reversetranscriptase [Promega], and the extended products were separatedby electrophoresis through a 6% polyacrylamide DNA sequencinggel . The position of the extended products was determined byrunning a sequencing reaction generated with the same 23-baseprimer and as template DNA a 1,409-bp PCR product [PCR] extendingfrom 866 bp upstream and 543 bp downstream of the start codonof ytkD.

RT-PCR experiments. Total RNA from vegetative or sporulating B . subtilis 168 cellsgrown in DSM was isolated by using the Tri reagent [MolecularResearch Center, Inc.] . Reverse transcription-PCRs [RT-PCRs]were performed with the RNA samples and the Master AMP RT-PCRkit [Epicentre Technologies] according to the instructions ofthe provider . The primers used for RT-PCRs were 5'-GCTCTAGAGGGATAAACATGTACGAG-3'[forward] and 5'-CTTCTGCGCACTCCATCGGCTCTAG-3' [reverse] to generatea 204-bp RT-PCR product extending from 17 bp upstream from thestart codon of ytkD to 187 bp downstream of this point . As acontrol, in each experiment, the absence of chromosomal DNAin the RNA samples was assessed by PCRs carried out with VentDNA polymerase [New England Biolabs] and the set of primersdescribed above . The size of the RT-PCR product was assessedby utilizing the 1-kbp-plus DNA ladder [Life Technologies, Rockville,Md.].

 
Enzyme activity of YtkD. The antimutator effects of MutT homologs rely on the abilityof these proteins to catalyze the conversion of 8-oxo-dGTP intoits monophosphate form [38] . As shown in Fig . 1B, the five potentialMutT homologs from B . subtilis conserve a 23-amino-acid-long signature termed the MutT or Nudix box [5, 23], which keepsa high level of identity [47.8 to 56.5%] with the MutT box ofthe E . coli MutT protein . However, not all of the proteins thatconserve such a signature have been shown to be capable of hydrolyzing8-oxo-dGTP or complementing the mutator phenotype of a mutT-deficientE . coli strain [21, 45] . Therefore, we first investigated whetherYtkD catalyzed the hydrolysis of 8-oxo-dGTP . To this end, theORF of ytkD was expressed from the IPTG-inducible T₅ promoter of plasmid pQE30 in order to generate a recombinant protein containing an N-terminal His₆ tag . The His₆-YtkD protein wassuccessfully synthesized in E . coli cells and purified to apparenthomogeneity by metal chelate affinity on an Ni-NTA-agarose column[Fig . 1A] . As shown in Table 2, the purified His₆-YtkD proteinsuccessfully catalyzed the degradation of 8-oxo-dGTP . The resultsindicated that YtkD catalyzed the hydrolysis of 8-oxo-dGTP witha specific activity 413 times higher than that exhibited againstdGTP . It has been demonstrated that MutT from E . coli also possessesenzymatic activity against 8-oxo-GTP [59] . Therefore, we investigated whether YtkD utilized this oxidized mRNA precursor as a substrate. The results shown in Table 2 demonstrated that 8-oxo-GTP is indeed a much better substrate for YtkD hydrolysis than GTP. The specific activity of YtkD during degradation of oxidizedGTP was around 460 times higher than that obtained against GTP[Table 2] . These results demonstrated that YtkD is a protein with the enzyme properties required to preferentially catalyze the hydrolysis of 8-oxo-dGTP and 8-oxo-GTP . Such catalytic activity strongly suggests that this protein's potential physiologicalrole is to counteract the mutagenic effects of these oxidizednucleotides . In agreement with these ideas, a ytkD B . subtilisstrain that was recently constructed in our laboratory was demonstratedto be 1 order of magnitude more mutagenic than its parentalstrain [F . X . Castellanos-Juárez and M . Pedraza-Reyes,unpublished results].

 
Genetic complementation of the E . coli mutT mutation by ytkD. To further support the contention that ytkD encodes a physiologically functional 8-oxo-dGTPase, we investigated whether the expression of ytkD complemented the mutator phenotype of E . coli cells lacking a functional mutT gene . It has been demonstrated that the occurrence of A:T-to-G:C transversion is several ordersof magnitude greater in E . coli strains deficient in MutT activity than the numbers seen in isogenic wild-type bacteria [22, 38].Accordingly, the ORF of ytkD was cloned in pTRC99A, and theresulting plasmid, pPERM426, was introduced by transformationinto the mutT mutant strain in order to examine the effectsof ytkD expression on the levels of the spontaneous mutationfrequency for nalidixic acid resistance . As shown below, themutation frequency of the bacteria lacking a functional mutTgene and expressing ytkD was significantly lower than that ofthe mutT mutant bacteria carrying only the pTRC99A vector . ForE . coli strains JM83 [wild type], SB3 [mutT mutant], PERM425[SB3 + pTrc99A], and PERM426 [SB3 + pTrc99A-ytkD], the mutationfrequencies [Nal^r] per 10⁸ cells were 0.01 ± 0.009, 8.30± 1.21, 7.10 ± 1.33, and 0.819 ± 0.22,respectively . [Mean values of mutation frequencies were calculatedfrom at least four independent experiments.] A similar resultwas previously obtained when the cDNA encoding the MutT humanhomolog was used in this complementation experiment [58] . Takentogether, these results demonstrate that YtkD not only degrades 8-oxo-dGTP and 8-oxo-GTP but also genetically complements the mutagenic effects of the mutT deficiency in E . coli.

Expression of ytkD during growth and sporulation. To analyze the pattern of expression of ytkD, the B . subtilis strain PERM276 harboring a single copy of the ytkD-lacZ fusionwas induced to sporulate in DSM . The ytkD-directed ß-galactosidaseactivity was detected during both vegetative growth and sporulation[Fig . 2] . In the nonspore fraction, the activity was found tobe present during growth and then began to increase as the cellsentered stationary phase, followed by a marked decrease betweenstages T₄ and T₅ [Fig . 2] . This pattern of expression suggesteda compartmental expression of the ytkD gene into the forespore. As shown in Fig . 2, ß-galactosidase activity was indeed detected in the forespore fraction from sporulation stages T₄ to T₅ and continued to accumulate until at least stage T₈.

 
The dual pattern of temporal expression of ytkD was further corroborated as follows . First, Northern blot experiments were performed with total RNA isolated from cells of the strain B. subtilis 168 collected before and after the onset of sporulation. The results shown in Fig . 3A indicated that ytkD mRNA appearedas a major 0.5-kb band during both vegetative growth and sporulation[T₀ to T₅] . A minor hybridization band was also observed in this experiment, which could correspond to a degradation product from the major band, because the former was not observed inthe Northern blot experiments performed with mRNAs isolatedfrom a sigF B . subtilis strain [Fig . 3C].

 
In addition, following RT-PCR experiments with total RNA samples isolated from both vegetative and sporulating cells [Fig . 3B], the resulting amplification products both had the expected molecular size of 204 bp . However, the 204-bp RT-PCR product was more abundant when RNA samples from growth stages T₃ to T₅ of sporulationwere utilized in the assays . Taken together, these results areconsistent with the existence of a double level of regulationof ytkD transcription: one associated with vegetative growthand a second putative forespore mechanism that controls theexpression of ytkD during sporulation.

Sigma factor dependence of ytkD expression. The transcription of genes in the forespore compartment of B.subtilis is carried out through the sequential action of twotemporally expressed RNA polymerases containing either the ^F or ^G factors [24, 31, 32] . However as shown in Fig . 2, the patternof expression of the ytkD-directed ß-galactosidaseactivity during sporulation suggested that the transcriptionof ytkD might be under control of the E[sig]F form of the RNApolymerase . To better investigate this notion, a single copyof the ytkD-lacZ fusion was introduced, by transformation, intostrains B . subtilis 1S86 and WN118, which lack a functionalSigF or SigG, respectively . Our results demonstrated that amutation in the spoIIAC gene, which encodes the forespore-specific ^F [34, 51], drastically reduced the expression of the ytkD-lacZfusion during sporulation but not during vegetative growth [Fig.4A] . On the other hand, the expression of the ytkD-directed ß-galactosidase activity during either vegetativegrowth or sporulation [Fig . 4B] was not affected in the strain that lacked a functional SigG activity . Furthermore, when Northern blot experiments were performed with RNA isolated from vegetative and stationary-phase cells of the SigF-deficient strain grown in liquid DSM, only a strong hybridization signal of the expected size was observed with RNA samples isolated from vegetativelygrowing cells [Fig . 4C, lane 1] and not with those isolated from the stationary phase of growth [Fig . 4C, lanes 2 to 6]. Therefore, we conclude that the transcription of the ytkD gene associated with sporulation is dependent on the E[sig]F formof the RNA polymerase

 
Mapping the transcriptional start sites of ytkD. The reported genome of B . subtilis [http://genolist.pasteur.fr/SubtiList/] demonstrates that the ytkD gene is flanked upstream by ytkC, which encodes a putative protein of unknown function, similar to an autolytic amidase . Despite the existence of a 208-bp-long intergenic region between ytkC and ytkD, no potential transcriptionalterminators are reported [http://genolist.pasteur.fr/SubtiList/] until the end of ytkD . This gene arrangement suggests that ytkC and ytkD might form part of a bicistronic operon . To investigate whether ytkC and ytkD are coexpressed as part of the same mRNA,the 5' ends of ytkD were mapped in vivo by primer extension[40] . Accordingly, primer extension analysis was performed withtotal RNA samples isolated from B . subtilis 168 cells duringvegetative growth, at the point at which the culture ceasedexponential growth [T₀] and 5 h later [T₅] . As shown in Fig. 6B, when total RNA isolated from vegetative cells was used asa template, a major extension product was detected, located31 to 32 bp upstream of the ytkD start codon [Fig . 5B; lane 3] . A smaller amount of this product was also detected in RNA samples of stage T₀ [Fig . 5B, lane 2] . Analysis of the regionslying upstream of this ytkD transcription start site revealedthe existence of sequences with good homology to promoters of ^A-dependent genes [Fig . 6A].

 
Interestingly, as shown in Fig . 5A, a second major extension product of ytkD was obtained with RNA samples from T₅ [Fig.5A, lane 1]; such a product was also detected at lower levelsin reactions performed with RNA isolated from cells at T₀ [Fig.5A, lane 2] . The second 5' end of ytkD started 80 to 81 bp upstreamof the first codon of ytkD . Inspection of the sequences located upstream of this transcription start site allowed the identification of a second promoter that has conserved homology with thosereported for ^F-dependent genes [Fig . 6B] . These results againsupport the conclusion that ytkD is expressed during both vegetative growth and sporulation from the ^A and ^F promoters, respectively.

A ytkD-lacZ fusion is not induced by oxidative stress or during the SOS or ^B general stress responses. The strain B . subtilis PERM276 containing the ytkD-lacZ fusionintegrated at the ytkD locus was used to investigate whetherthe ytkD gene is induced as part of an oxidative stress regulon.Accordingly, strain B . subtilis PERM276 was grown to the mid-exponential phase and treated for 1 h with either paraquat [10 µM]or hydrogen peroxide [200 µM] . The results [data not shown]revealed that no transcription induction occurred followingthe treatment of the bacteria with these two oxidative stress-inducingchemicals.

Similarly, it has been shown that the expression of severalgenes whose products are putatively involved in mounting a generalcellular response to conditions that promote a nongrowing orstarving state is under the control of the ^B stress regulon[24, 25, 61]. ^B-dependent stress genes are strongly inducedby heat, salt, acid, or ethanol as well as by energy depletion[24] . We investigated whether ytkD is part of the ^B stress regulonby treating exponentially growing cells of the strain B . subtilisPERM276 with either sodium chloride [4%] or ethanol [4%] orheating the culture to 48°C . The results demonstrated thatnone of the stress conditions utilized was able to induce [duringa period from 15 to 120 min] expression of the ytkD-lacZ fusion[data not shown], suggesting that ytkD is not part of the ^B stress regulon . In agreement with this conclusion, our results showed that in a B . subtilis sigB mutant, the ytkD-lacZ fusionfollowed a temporal pattern of expression similar to that observedin B . subtilis PERM276 [data not shown], reinforcing the ideathat the transcription of ytkD is not regulated by E[sig]B.

Additionally, we investigated whether ytkD is part of the global SOS response [35], a gene circuitry that controls the expressionof genes involved in DNA repair such as the uvrA,C [also termeddinA] and recA genes [36, 47] . The SOS response in B . subtilisis induced following the introduction of certain types of damageinto the chromosomal DNA and by the development of the physiologicalstate of competence [37] . Thus, the B . subtilis strain PERM276was grown to the exponential phase and then treated with the DNA-damaging agent mitomycin C to a final concentration of 0.5 µg/ml . After 1 h of mitomycin C treatment, the levelsof ß-galactosidase of the strain B . subtilis PERM276 were not significantly raised above those expressed by theuntreated control . On the contrary, the levels of ß-galactosidaseof a recA-lacZ fusion-containing B . subtilis strain were inducedby mitomycin C treatment around 11 times above those expressedby the untreated control [data not shown].

 
Proteins containing the MutT motif are widely distributed among several species; however, not all of them catalyze the splittingof 8-oxo-dGTP [21, 45] . This is best exemplified by yqkG [nudF]of B . subtilis, which despite encoding a MutT homolog, showsspecificity to hydrolyze the diphosphate linkage of ADP-ribose[18] . Therefore, the demonstration that a purified His₆-YtkDprotein possessed the ability to catalyze the degradation ofboth 8-oxo-dGTP and 8-oxo-GTP [Table 2] revealed for the firsttime that B . subtilis possesses this type of antimutator proteinto sanitize its nucleotide pools . In support of this conclusion,ytkD was able to genetically complement the mutator phenotypeof an E . coli mutT mutant . It is relevant to point out thatthe level of complementation of the E . coli mutT strain withytkD was similar to that obtained when the mutator phenotypeof this strain was complemented with the cDNA of the human MutThomolog [MTH1] [58] . As mentioned above, there is a high levelof identity between YtkD, MutT, and MTHI in the absolutely conserved residues of the MutT box [Fig . 1B] [23] . Therefore, the variationsof the level of complementation of E . coli SB3 with either mutT,ytkD, or MTH1 most probably are the result of amino acid divergenceslying out of the MutT boxes of these proteins . Thus, the abilityof ytkD to genetically complement the DNA repair deficiencyof the mutT E . coli mutant strongly suggests that its productconfers protection to cells against the mutagenic effects of8-oxo-dGTP and 8-oxo-GTP . Therefore, YtkD represents the firstMutT homolog of B . subtilis with a demonstrated biochemicaland physiological function, and as such, we propose to nameit MutTA . It remains to be investigated whether MutT, YvcI,and YjhV, the other putative B . subtilis MutT homologs, encodetrue 8-oxo-dGTPases.

Upon finding that YtkD of B . subtilis possesses an activity involved in sanitizing the oxidized nucleotide pools, it wasof interest to determine how the expression of ytkD is regulated by B . subtilis during vegetative growth and sporulation . Thus, a ytkD-lacZ fusion integrated by Campbell-type recombination into the ytkD locus of B . subtilis revealed that the transcriptionof ytkD is activated not only during vegetative growth but alsoduring the first steps of sporulation . ytkD mRNAs were detectedduring both developmental stages, suggesting that ytkD is transcribedby the sequential action of RNA polymerases containing the ^A and ^F factors, respectively . In agreement with this suggestion,the spore-associated expression was almost completely abolishedin a sigF genetic background but was not in a B . subtilis strain lacking a functional sigG gene.

In vivo mapping of the 5' ends of ytkD messengers expressed during vegetative growth allowed the identification of a putative promoter which showed to hold a significant degree of conserved homology with ^A/⁷⁰-type promoters supporting the conclusionthat the vegetative growth-associated transcription of ytkDoccurs from a promoter that is recognized by ^A RNA polymerase.

While there are a number of genes that belong to the ^G regulon[24, 44, 53], there are many fewer genes that are known to beunder ^F control [4, 24] . Consequently, the characteristics ofthe promoters that control the expression of ^F-dependent genesare less well known . Experimental evidence detailed above indicatedthat the sporulation-associated expression of ytkD was dependenton ^F RNA polymerase . A major extension product located 80 to81 bp upstream of the putative ytkD start codon was amplifiedfrom RNA samples obtained from cells at T₅ . The sequences that preceded this putative transcriptional start site revealed the existence of a promoter with homology to the suggested consensus sequence of ^F promoters [24] . Although the -10 region was relatively far from the mapped 5' end of ytkD, it demonstrated conservation of 7 of the 9 consensus bases, including 2 characteristic guanines exclusively found in true ^F-dependent promoters [4, 24] . Furthermore, the -35 region that conserved 3 of the 5 ^F consensus bases wasshown to be separated from the -10 region by a 15-bp-long spacer,which is typical in these promoters [4, 24] . Therefore, theseresults, together with the demonstrated forespore-specific expressionand ^F dependence of ytkD, strongly support the conclusion that ytkD is a new member of the ^F regulon.

In the chromosome of B . subtilis, ytkD is preceded by an ORF termed ytkC encoding a putative autolytic amidase, and both genes are separated by an intergenic region . The lack of a transcriptional terminator in the 298-bp-long intergenic region is suggestive that genes are cotranscribed . However, the results of primer extension experiments, in combination with the detection ofytkD messengers of around 0.5 kb, indicate that ytkD is transcribed in a temporal manner from two different promoters located inthe intergenic region between ytkC and ytkD.

B . subtilis displays an adaptive response to H₂O₂ that includesinduction of katA, ahpCF, mrgA, and the hemA operon [3, 7, 9,13, 17] . Furthermore, it has been demonstrated that this responseis regulated by PerR, a Fur homolog [10, 26] . While the functionof YtkD is associated with preventing the mutagenic effects of oxidized dNTPs, specific stress induction of the transcription of this gene does not appear to occur, since neither H₂O₂ norparaquat treatment affected the levels of the gene product produced.Moreover, experimental evidence described here revealed thattranscription of ytkD is not activated under conditions thatpromote the ^B stress response [24] . Likewise, the lack of induction of ß-galactosidase in the ytkD-lacZ fusion strain following treatment by the DNA-damaging agent mitomycin C revealed that ytkD is not under the control of the SOS regulon [14, 62].

In addition to the transcriptional regulation of ytkD described in this paper, results of a proteomic study revealed that the synthesis of YtkD was induced under anaerobic growth, by nitrate respiration [15] . The physiological relevance of these resultsremains to be established . In any case, the expression of the ytkD gene in the forespore raises the question as to what is the role played by this protein not only during developing of the sporulating cell but also in dormant spores . Clearly itis possible that a sanitizing role of YtkD would be inoperativein dormant spores, due in large part to their lack of metabolismand inability to perform DNA synthesis and transcription [54]. Moreover, most of the enzymes existing in the spore core are believed to be in an inactive state [12, 54] . On the other hand,it must be pointed out that production of ROS may well be exacerbatedin germinating spores as a result of hydration of the sporecores and the triggering of metabolism . Thus, spore-specificprotective enzymes might play an essential role in counteractingthe effects of oxidative stress during spore germination and/oroutgrowth . In support of this contention, the spore-specificcatalase KatX was demonstrated to be essential for H₂O₂ resistanceduring spore germination [4] . Accordingly, our laboratory isinvestigating the physiological role[s] played by YtkD in thesurvival of vegetative cells of B . subtilis as well as a possibleprotective role of this enzyme against oxidative stress in thedeveloping spore and/or during spore germination.

 
This work was supported by grant 31767-N from the Consejo Nacionalde Ciencia y Tecnología [CONACYT] of México toMario Pedraza-Reyes . Martha I . Ramírez and FranciscoX . Castellanos-Juárez were supported by doctoral fellowshipsfrom CONACYT . R.E.Y . was supported by MCB-9975140 from the NationalScience Foundation.

We wish to thank Jesús García for critical reviewof the manuscript and Norma Urtiz-Estrada and Eliel R . Romero-Garcíafor excellent technical assistance.

 
* Corresponding author . Mailing address: Institute of Investigation in Experimental Biology, Building L, Faculty of Chemistry, University of Guanajuato, Noria Alta S/N, Noria Alta, P.O . Box 187, Guanajuato Gto . 36050, Mexico . Phone: [473] 73 2 00 06, ext . 8161 . Fax: [473] 73 2 00 06, ext . 8153 . E-mail: pedrama@quijote.ugto.mx .

1. Abeygunawardana, C., D . J . Weber, A . G . Gittis, D . N . Frick, J . Lin, A . F . Miller, M . J . Bessman, and A . Mildvan. 1995 . Solution structure of the MutT enzyme a nucleoside triphosphate pyrophosphohydrolase . Biochemistry 34:14997-15005.
2. Ames, B . N., and D . T . Dubin. 1960 . The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid . J . Biol . Chem . 235:769-775.
3. Antelmann, H., S . Engelmann, R . Schmid, and M . Hecker. 1996 . General and oxidative stress responses in Bacillus subtilis: cloning, expression, and mutation of the alkyl hydroperoxide reductase operon . J . Bacteriol . 178:6571-6578.
4. Bagyan, I., L . Casillas-Martinez, and P . Setlow. 1998 . The katX gene, which codes for the catalase in spores of Bacillus subtilis, is a forespore-specific gene controlled by ^F, and KatX is essential for hydrogen peroxide resistance of the germinating spore . J . Bacteriol . 180:2057-2062 .
5. Bessman, M . J., D . N . Frick, and S . F . Handley. 1996 . The MutT proteins or "Nudix" hydrolases, a family of versatile, widely distributed, "housecleaning" enzymes . J . Biol . Chem . 271:25059-25062.
6. Bhatnagar, S . K., L . C . Bullions, and M . J . Bessman. 1991 . Characterization of the mutT nucleoside triphosphatase of Escherichia coli. J . Biol . Chem . 266:9050-9054 .
7. Bol, D . K., and R . E . Yasbin. 1994 . Analysis of the dual regulatory mechanisms controlling expression of the vegetative catalase gene of Bacillus subtilis. J . Bacteriol . 176:6744-6748.
8. Boylan, R . J., N . H . Mendelson, D . Brooks, and F . E . Young. 1972 . Regulation of the bacterial cell wall: analysis of a mutant of Bacillus subtilis defective in the biosynthesis of teichoic acid . J . Bacteriol . 110:281-290.
9. Bsat, N., L . Chen, and J . D . Helmann. 1996 . Mutation of the Bacillus subtilis alkyl hydroperoxide reductase [ahpCF] operon reveals compensatory interactions among hydrogen peroxide stress genes . J . Bacteriol . 178:6579-6586.
10. Bsat, N., and J . D . Helmann. 1999 . Interaction of Bacillus subtilis Fur [ferric uptake repressor] with the dhB operator in vitro and in vivo . J . Bacteriol . 181:4299-4307 .
11. Bullions, L . C., V . Mejean, J . P . Claverys, and M . J . Bessman. 1994 . Purification of the MutX protein of Streptococcus pneumoniae, a homologue of Escherichia coli MutT . Identification of a novel catalytic domain for nucleoside triphosphate pyrophosphohydrolase activity . J . Biol . Chem . 269:12339-12344 .
12. Casillas-Martinez, L., and P . Setlow. 1997 . Alkyl hydroperoxide reductase, catalase, MrgA, and superoxide dismutase are not involved in resistance of Bacillus subtilis spores to heat or oxidizing agents . J . Bacteriol . 179:7420-7425.
13. Chen, L., L . Keramati, and J . D . Helmann. 1995 . Coordinate regulation of Bacillus subtilis coordinate stress genes by hydrogen peroxide and metal ions . Proc . Natl . Acad . Sci . USA 92:8190-8194.
14. Cheo, D . L., K . W . Bayles, and R . E . Yasbin. 1991 . Cloning and characterization of DNA damage-inducible promoter regions from Bacillus subtilis. J . Bacteriol . 173:1696-1703.
15. Clements, L . D., U . N . Streips, and B . S . Miller. 2002 . Differential proteomic analysis of Bacillus subtilis nitrate respiration and fermentation in defined medium . Proteomics 2:1724-1734.
16. Cutting, S . M., and P . B . Vander Horn. 1990 . Genetic analysis, p . 27-74 . In C . R . Harwood and S . M . Cutting [ed.], Molecular biological methods for Bacillus. John Wiley and Sons, Sussex, United Kingdom.
17. Dowds, B . C . A. 1994 . The oxidative stress response in Bacillus subtilis. FEMS Microbiol . Lett . 124:255-264.
18. Dunn, C . A., S . O'Handley, D . N . Frick, and M . J . Bessman. 1999 . Studies on the ADP-ribose pyrophosphatase subfamily of the Nudix hydrolases and tentative identification of trgB, a gene associated with tellurite resistance . J . Biol . Chem . 274:32318-32324 .
19. Engelmann, S., C . Lindner, and M . Hecker. 1995 . Cloning, nucleotide sequence and regulation of katE encoding a ^B-dependent catalase in Bacillus subtilis. J . Bacteriol . 177:5598-5605.
20. Ferrari, E., S . M . H . Haward, and J . A . Hoch. 1985 . Effect of sporulation mutations on subtilisin expression, assayed using a subtilisin-ß-galactosidase gene fusion, p . 180-184 . In J . A . Hoch and P . Setlow [ed.], Molecular biology of microbial differentiation . American Society for Microbiology, Washington, D.C.
21. Frick, D . N., B . D . Townsend, and M . J . Bessman. 1995 . A novel GDP-mannose mannosyl hydrolase shares homology with the MutT family of enzymes . J . Biol . Chem . 270:24086-24091 .
22. Friedberg, E . C., G . C . Walker, and W . Siede. 1995 . DNA repair and mutagenesis . American Society for Microbiology, Washington, D.C.
23. Fujii, Y., H . Shimokawa, M . Sekiguchi, and Y . Nakabeppu. 1999 . Functional significance of the conserved residues for the 23-residue among MTH1 and MutT family proteins . J . Biol . Chem . 274:38251-38259 .
24. Haldenwang, W . G. 1995 . The sigma factors of Bacillus subtilis. Microbiol . Rev . 59:1-30.
25. Hecker, M., and U . Völker. 1998 . Non-specific, general and multiple stress resistance of growth restricted Bacillus subtilis cells by the expression of ^B regulon . Mol . Microbiol . 19:417-428.
26. Herbig, A . F., and J . D . Helmann. 2001 . Roles of metal ions and hydrogen peroxide in modulating the interaction of the Bacillus subtilis PerR peroxide regulon repressor with operator DNA . Mol . Microbiol . 41:849-859.
27. Inaoka, T., Y . Matsumura, and T . Tsuchido. 1998 . Molecular cloning and nucleotide sequence of the superoxide dismutase gene and characterization of its product from Bacillus subtilis . J . Bacteriol. 180:3697-3703 .
28. Kakuma, T., J . Nishida., T . Tsuzuki., and M . Sekiguchi. 1995 . Mouse MTH1 protein with 8-oxo-7, 8-dihydro-2'-deoxyguanosine5'-triphosphatase activity that prevents transversion mutation . J . Biol . Chem . 270:25942-25948 .
29. Kamath, A . V., and C . Yanofsky. 1993 . Sequence and characterization of mutT from Proteus vulgaris. Gene 134:99-102.
30. Kamiya, H., and H . Kasai. 1995 . Formation of 2-hydroxydeoxyadenosine triphosphate, an oxidatively damaged nucleotide, and its incorporation by DNA polymerases . Steady-state kinetics of the incorporation . J . Biol . Chem . 270:19446-19450 .
31. Kroos, L., and S . Cutting. 1994 . Intercellular and intercompartmental communication during Bacillus subtilis sporulation, p . 155-180 . In P . J . Piggot, C . P . Moran, Jr., and P . Youngman [ed.], Regulation of bacterial differentiation . American Society for Microbiology, Washington, D.C.
32. Kroos, L., B . Zhang, H . Ichikawa, and Y . T . Nicco-Yu. 1999 . Control of factor activity during Bacillus subtilis sporulation . Mol . Microbiol. 31:1285-1294.
33. Kunst, F., N . Ogasawara, I . Moszer, A . M . Albertini, G . Alloni, V . Azevedo, M . G . Bertero, P . Bessieres, A . Bolotin, S . Borchert, R . Borriss, L . Boursier, A . Brans, M . Braun, S . C . Brignell, S . Bron, S . Brouillet, C . V . Bruschi, B . Caldwell, V . Capuano, N . M . Carter, S . K . Choi, J . J . Codani, I . F . Conerton, A . Danchin, et al. 1997 . The complete genome sequence of the Gram positive bacterium Bacillus subtilis. Nature 390:249-256.
34. Londoño-Vallejo, J.-A., C . Frehel, and P . Stragier. 1997 . spoIIO, a forespore expressed gene required for engulfment in Bacillus subtilis. Mol . Microbiol . 24:29-39.
35. Love, P . E., and R . E . Yasbin. 1984 . Genetic characterization of the inducible SOS-like system of Bacillus subtilis. J . Bacteriol . 160:910-920.
36. Love, P . E., M . S . Lyle, and R . E . Yasbin. 1985 . DNA damage inducible [din] loci are transcriptionally activated in competent Bacillus subtilis. Proc . Natl . Acad . Sci . USA 82:6201-6205.
37. Lovett, C . M., Jr., P . E . Love, and R . E . Yasbin. 1989 . Competence-specific induction of the Bacillus subtilis RecA protein analog: evidence for dual regulation of a recombination protein . J . Bacteriol. 171:2318-2322.
38. Maki, H., and M . Sekiguchi. 1992 . MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis . Nature 355:273-275.
39. Mason, J . M., R . H . Hackett, and P . Setlow. 1988 . Regulation of expression of genes coding for small, acid-soluble spore proteins of Bacillus subtilis spores: studies using lacZ gene fusions . J . Bacteriol . 170:239-244.
40. McKnight, S . L., and R . Kingsbury. 1982 . Transcription control signals of a eukaryotic protein-coding gene . Science 217:316-324.
41. Miller, J . H. 1972 . Experiments in molecular genetics . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
42. Mo, J., H . Maki, and M . Sekiguchi. 1992 . Hydrolytic elimination of a mutagenic nucleotide, 8-oxodGTP, by human 18-kilodalton protein: sanitization of nucleotide pool . Proc . Natl . Acad . Sci . USA 89:11021-11025.
43. Nicholson, W . L., and P . Setlow. 1990 . Sporulation, germination and outgrowth, p . 391-450 . In C . R . Harwood and S . M . Cutting [ed.], Molecular biological methods for Bacillus. John Wiley and Sons, Sussex, United Kingdom.
44. Nicholson, W . L., D . Sun, B . Setlow, and P . Setlow. 1989 . Promoter specificity of ^G-containing RNA polymerase from sporulating cells of Bacillus subtilis: identification of a group of forespore-specific promoters . J . Bacteriol . 171:2708-2718.
45. O'Handley, S . F., Dunn, C . A., and M . J . Bessman. 2001 . Orf135 from Escherichia coli is a Nudix hydrolase specific for CTP, dCTP and 5-methyl-dCTP . J . Biol . Chem . 276:5421-5426 .
46. Pedraza-Reyes, M., F . Gutiérrez-Corona, and W . L . Nicholson. 1994 . Temporal regulation and forespore-specific expression of the spore photoproduct lyase gene by sigma-G RNA polymerase during Bacillus subtilis sporulation . J . Bacteriol . 176:3983-3991.
47. Raymond-Denise, A., and N . Guillen. 1992 . Expression of the Bacillus subtilis dinR and recA genes after DNA damage and during competence . J . Bacteriol . 174:3171-3176.
48. Riley, P . A. 1994 . Free radicals in biology: oxidative stress and the effects of ionizing radiation . Int . J . Radiat . Biol . 65:27-37.
49. Sambrook, J., E . F . Fritsch, and T . Maniatis. 1989 . Molecular cloning: a laboratory manual, 2nd ed . Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
50. Saran, M., and W . Bors. 1990 . Radical reactions in vivo—an overview . Radiat . Environ . Biophys . 29:249-262.
51. Savva, D., and J . Mandelstam. 1984 . Cloning of the Bacillus subtilis spoIIA and spoVA loci in phage 105 DI:1t . J . Gen . Microbiol . 132:2137-2145.
52. Schaeffer, P., J . Millet, and J.-P . Aubert. 1965 . Catabolic repression of bacterial sporulation . Proc . Natl . Acad . Sci . USA 54:704-711.
53. Setlow, P. 1989 . Forespore-specific genes of Bacillus subtilis: function and regulation of expression, p . 211-221 . In I . Smith, R . Slepecky, and P . Setlow [ed.], Regulation of prokaryotic development: structural and functional analysis of bacterial sporulation and germination . American Society for Microbiology, Washington, D.C.
54. Setlow, P. 1994 . Mechanisms that contribute to the long-term survival of spores of Bacillus species . J . Appl . Bacteriol. 176[Suppl.]:49S-60S.
55. Simic, M . G., K . A . Taylor., J . F . Ward., and C . Von Sonntag [ed.]. 1988 . Oxygen radicals in biology and medicine . Basic life sciences, vol . 49 . Plenum Publishing Corp., New York, N.Y.
56. Storz, G., and J . A . Imlay. 1999 . Oxidative stress . Curr . Opin . Microbiol . 2:188-194.
57. Storz, G., and M . Zheng. 2000 . Oxidative stress, p . 47-59 . In G . Storz and R . Hengge-Aronis [ed.], Bacterial stress responses . American Society for Microbiology, Washington, D.C.
58. Sukumi, K., M . Furuichi, T . Tsusuki, T . Kakuma, S.-I . Kabawata, H . Maki, and M . Sekiguchi. 1993 . Cloning and expression of cDNA for a human enzyme that hydrolyzes 8-Oxo-dGTP, a mutagenic substrate for DNA synthesis . J . Biol . Chem . 268:23524-23530 .
59. Taddei, F., H . Hayakawa, M . F . Bouton, A . M . Cirinesi, L . Matic, M . Sekiguchi, and M . Radman. 1997 . Counteraction by MutT protein of transcriptional errors caused by oxidative damage . Science 278:128-130 .
60. Urtiz-Estrada, N., J . M . Salas-Pacheco, R . E . Yasbin, and M . Pedraza-Reyes. 2003 . Forespore-specific expression of Bacillus subtilis yqfS, which encodes type IV apurinic/apyrimidinic endonuclease, a component of the base excision repair pathway . J . Bacteriol . 185:340-348 .
61. Völker, U., B . Maul, and M . Hecker. 1999 . Expression of the ^B-dependent general stress regulon confers multiple stress resistance in Bacillus subtilis. J . Bacteriol . 181:3942-3948 .
62. Winterling, K . W., D . Chafin, J . J . Hayes, J . Sun., A . S . Levine, R . E . Yasbin, and R . Woodgate. 1998 . The Bacillus subtilis DinR binding site: redefinition of the consensus sequence . J . Bacteriol . 180:2201-2211 .

Free Online Full-text Article

What Is Yeast?, What Is Biotechnology?, What Is Rhizobia?, What Is Genetics?, What Is Cell Biology?, s, Bacteria, i, Bacterium, n, Microbes, n, Microorganism, r, Bacteriology, e, Pasteurella, i, Cell suspensions, s, Fermentations, e, Streptococcal, e, Bacteriological, a, Escherichia coli, s, Bacteriological, a, Pseudomonas aeruginosa, a, Bacillus, s, Yeasts, s, Escherichia coli, o, Bacillus, a, Bacillus, i, Bactericidal, a, Microbiological, c, Vibriosis, c, Gram negative, o, Pasteurella, e, Escherichia coli, r, Corynebacter, i, Haemophilus




 

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

Last modified: May 25, 2005