JOURNAL OF BACTERIOLOGY, July 1993, p. 3964-39710021-9193/93/133964-08$02.00/0Copyright © 1993, American Society for Microbiology

Vol. 175, No. 13

Bacillus subtilis gtaB Encodes UDP-GlucosePyrophosphorylase and Is Controlled by Stationary-Phase

Transcription Factor &r`DEBORA VAR6N,1 SHARON A. BOYLAN,1 KATHLEEN OKAMOTO,2 AND CHESTER W.PRICEI*

Department ofFood Science and Technology, University of California, Davis, California 95616,1and Syva Company, Palo Alto, California 943032Received 16 February 1993/Accepted 29 April 1993

Transcription factor rB of Bacillus subtilis controls a large stationary-phase regulon, but in no case has thephysiological function of any gene in this regulon been identified. Here we show that transcription ofgtaB ispartly dependent on &B in vivo and thatgtaB encodes UDP-glucose pyrophosphorylase. ThegtaB reading framewas initially identified by a &B-dependent Tn9l7lacZ fusion, csb42. We cloned the region surrounding the csb42insertion, identified the reading frame containing the transposon, and found that this frame encoded apredicted 292-residue product that shared 45% identical residues with the UDP-glucose pyrophosphorylase ofAcetobacter xylinum. The identified reading frame appeared to lie in a monocistronic transcriptional unit.Primer extension and promoter activity experiments identified tandem promoters, one &rB dependent and theother oM' independent, immediately upstream from the proposed coding region. A sequence resembling afactor-independent terminator closely followed the coding region. By polymerase chain reaction amplificationof a B. subtilis genomic library carried in yeast artificial chromosomes, we located the UDP-glucosepyrophosphorylase coding region near gtaB, mutations in which confer phage resistance due to decreasedglycosylation of cell wall teichoic acids. Restriction mapping showed that the coding region overlapped theknown location ofgtaB. Sequence analysis of a strain carrying thegtaB290 allele found an alteration that wouldchange the proposed initiation codon from AUG to AUA, and an insertion-deletion mutation in this frameconferred phage resistance indistinguishable from that elicited by thegtaB290 mutation. We conclude thatgtaBencodes UDP-glucose pyrophosphorylase and is partly controlled by &rB. Because this enzyme is important forthermotolerance and osmotolerance in stationary-phase Escherichia coli cells, our results suggest that somegenes controlled by &rB may play a role in stationary-phase survival of B. subtilis.

How bacteria control stationary-phase gene expression inresponse to external and internal signals is an important butpoorly understood area of prokaryotic physiology. Bacillussubtilis, which carries out a wide variety of stationary-phaseactivities (44), provides an excellent system to study hownongrowing cells integrate environmental and cellular sig-nals to yield the appropriate response. One way that bacteriarespond to environmental change is by the association ofalternative a factors with the RNA polymerase core, thusreprogramming the promoter recognition specificity of theenzyme to activate genes needed under the new condition(16, 18, 30). The alternative transcription factor &e of B.subtilis appears to be an important element for the transmis-sion of stationary-phase signals to the transcriptional ma-chinery (6). Three transcription units whose stationary-phase expression is directly controlled by cB have beenidentified: the ctc gene (22, 23, 33), the csbA gene (7), andthe &e operon itself (27). However, the functions of ctc andcsbA have yet to be established, and null &e mutants have noobvious growth or sporulation phenotype (4, 14, 23, 27), sothe physiological role of ae remains mysterious.Our approach toward understanding the physiological role

of &e is to identify and characterize additional genes in the&e regulon (7). In the accompanying article (5), Boylan et al.report the isolation and initial characterization of six newoperon fusions that are directly or indirectly controlled bycr (csb fusions). Here we demonstrate that the gene identi-

* Corresponding author.

fled by one such fusion, csb42, is transcribed from a e-dependent promoter in vivo and encodes UDP-glucose py-rophosphorylase. We further demonstrate that the csb42fusion is an allele ofgtaB, mutations in which confer phageresistance as a result of their decreased glycosylation of cellwall teichoic acids (48). From the known role of UDP-glucose pyrophosphorylase in stationary-phase survival ofenteric bacteria (15, 19), we speculate that some of the genescontrolled by might respond to stress in the stationarygrowth phase.

MATERIALS AND METHODS

Bacteria, phage, and genetic methods. Escherichia coliDH5a (Bethesda Research Laboratories) was the host for allplasmid constructions. B. subtilis strains used are shown inTable 1. B. subtilis PB2 and its derivatives were recipientsfor natural transformations (13) with linear and plasmidDNA, both for strain constructions and for genetic crossesto map the chromosomal locus of the csb42 fusion. We madethe gtaBA1::ery insertion-deletion mutation in strain PB319by removing the 622-nucleotide (nt) SnaBI-BglII fragmentfrom within the coding region identified by csb42 and replac-ing it with a 1.4-kb fragment carrying the macrolide-lincosa-mide-streptogramin B (ery) resistance gene from pE194 (21).Transformation selections for drug-resistant B. subtilisstrains were done on tryptose blood agar plates (DifcoLaboratories) containing 5 jig of chloramphenicol per ml (forcat), 5 jxg of kanamycin per ml (for aph), or 0.5 Fg oferythromycin per ml and 12.5 pug of lincomycin per ml (for

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TABLE 1. B. subtilis strains

Strain Genotypea Description, reference, orconstructionb

PB2 trpC2 Wild-type Marburg strain (40)PB105 sigBAl trpC2 27PB209 sigBA2::cat trpC2 7PB271 csb42::Tn9l7lacZ socBl pheAl trpC2 5PB272 csb42::Tn917lacZ trpC2 5PB313 csb42::Tn9I7lacZ sigBA1 trpC2 PB272- PB105PB314 csb42::Tn9l7lacZ::pDV5 socBI pheA1 trpC2 pDV5--PB271PB315 csb42::Tn917lacZ::pDV6 socBl pheAl trpC2 pDV6--+PB271PB316 csb42::Tn917lacZ::pDV5 sigBAl trpC2 pDV5-+PB313PB317 gtaB290 trpC2 BGSCC lA105PB318 gtaB290::pDV3 trpC2 pDV3--PB317PB319 gtaBA&1::ery trpC2 This studyQB4238 A(degS degU)::aphA3 trpC2 34KUS1033 rodDC::cat pTV21A2--KUS1025 (20)

a Because the csb42::Tn9171acZ fusion is an allele of gtaB, in the future we will call this fusion gtaB42::Tn9171acZ.b Arrows indicate transformation from donor to recipient.c BGSC, Bacillus Genetic Stock Center, Ohio State University.

efy and the erm resistance of Tn917). Luria broth (LB) wasdescribed by Davis et al. (11), and Schaeffer's 2xSG sporu-lation medium was described by Leighton and Doi (29).Sporulation frequency was determined by counting the num-ber of chloroform-resistant CFU present after 24 h of growthin 2xSG sporulation medium. Resistance to B. subtilisbacteriophage 429 was tested by spotting a high-titer lysateonto freshly streaked strains, as described by Karamata etal. (28).DNA methods. All standard recombinant DNA methods

were performed as described by Davis et al. (11), andpolymerase chain reaction (PCR) experiments were done byestablished protocols (25). DNA sequencing was done by thedideoxynucleotide chain termination method with sequenc-ing reactions primed on double-stranded DNA templates, aspreviously described (7). We sequenced 1.3 kb of the csb42(gtaB) region on both strands and through all restrictionendpoints used in fragment subcloning. Direct sequencing ofPCR products with the fmol DNA sequencing system(Promega) was used to analyze the csb42 region in strainPB317, which carries the gtaB290 allele. PCR methods werealso applied to physically locate the csb42 fusion on the B.subtilis chromosome, using an ordered yeast artificial chro-mosome (YAC) library ofB. subtilis DNA as the template (2,36). Primers used were 5'-CCTAAAGGTCTCGGACATGC,identical to nt 438 to 457 in Fig. 3, and 5'-GAATGGCGTCTGTGAGCTGA, complementary to nt 803 to 822 (OperonTechnologies). B. subtilis and Saccharomyces cerevisiaechromosomal DNAs were the positive and negative controlsfor the mapping reactions.

Isolation of the gtaB region by plasmid excision. We usedthe plasmid method of Youngman (49) to isolate the chro-mosomal region adjacent to the csb42 insertion. StrainPB272 (csb42::Tn9171acZ trpC2) was transformed with theintegrative plasmid pLTV1 (49) to place a ColEl origin ofreplication, a bla gene, and a polylinker region within theTn9171acZ chromosomal locus. Chromosomal DNA wasextracted from the resultant integrant, cut with EcoRI tocleave both within the integrated plasmid and upstream fromthe Tn9171acZ insertion, and then treated with DNA ligaseto promote self-ligation of the fragment. The ligation mixturewas transformed into E. coli, with selection for the ampicillinresistance of the expected replicon. Several identical plas-mids which bore the hybrid plasmid-transposon sequences

together with an additional 4.9 kb of B. subtilis DNA wererecovered. This B. subtilis DNA extended from the site oftransposon integration to an EcoRI site in the chromosomalregion promoter proximal to the csb42 insertion (see Fig. 1).The plasmid bearing this upstream region was called pDV1.To obtain DNA extending downstream from the isolated

chromosomal fragment, a 753-bp HindIII-BglII fragmentinternal to the upstream fragment was subcloned into theHindIII and BamHI sites of the pGEM-3Zf(+)cat-1 integra-tion plasmid (49). The resulting pDV2 plasmid was inte-grated into a wild-type PB2 strain containing no Tn9171acZelement. The chromosomal DNA was extracted, cut withEcoRI, ligated, and transformed as described above into E.coli. We found several identical plasmids which carriedDNA extending from the HindIII-BglII fragment to anEcoRI site 3.6 kb downstream (see Fig. 1). The plasmidbearing this downstream region was called pDV3.Use of plasmid integration to locate csb42 (gtaB) promoter

activity. The plasmid integration method of Piggot et al. (38)was used to locate regions important for csb42 promoteractivity, modified for the fusion system as previously de-scribed (7). Fusion-bearing strains PB271 (csb42::Tn9171acZsocBI pheAl trpC2) and PB313 (csb42::Tn9171acZ sigBAltrpC2) were transformation recipients for derivatives ofintegration vector pCP115 (40), which carried fragmentsfrom the putative promoter-containing region. These frag-ments were derived from pDV1, the plasmid used to isolatethe region upstream of the csb42 insertion. As shown in Fig.1, pDV5 carried the 1,375-nt HindIII-SalI fragment frompDV1 subcloned into the HindIII and SalI sites of pCP115and pDV6 carried the 473-nt XmnI-XmnI fragment sub-cloned into the EcoRV site.Mapping of the 5' ends of csb42 (gB) mRNA by primer

extension. RNA was prepared essentially by the method ofIgo and Losick (24), with the following modifications. PB2cells were grown in 50 ml of LB containing 5% glucose and0.2% glutamine and harvested 1.5 h after the end of expo-nential growth. Cells were resuspended in 3 ml of LETSbuffer (24) containing 0.4 ml of Vanadyl RibonucleosideComplex (Bethesda Research Laboratories) and were dis-rupted by sonication for 1 min with a Fisher model 300 sonicdismembrator. RNA was extracted as described previously(24) and precipitated by overnight incubation at -20°C with2 volumes of ethanol. For primer extension reactions, a

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3966 VAR6N ET AL.

A.

pDV1

8 8.2kb

ol C: C CrMB.

B. 0

I

m'

E coCO

pDV5

pDV6

B-gal ActivitysoCB1 JigB+++ +

- ND

FIG. 1. Physical map of the csb42 (gtaB) region. (A) The location of the Tn9171acZ element in strain PB272 (csb42::Tn9171acZ) is indicatedby the solid triangle at 4.9 kb. The map is derived from restriction analysis of B. subtilis genomic fragments carried by pDV1 and pDV3 (top).pDV1 is a pLTV1 derivative isolated by integrating pLTV1 into the Tn9171acZ element, as described in Materials and Methods. The opentriangle indicates the Tn9171acZ sequences carried by pDV1. pDV3 is a pGEM-3Zf(+)cat-1 derivative isolated by integrating pDV2 (carryingthe 753-bp HindIII-BglII fragment) into the csb42 (gtaB) region of wild-type strain PB2, as described in Materials and Methods. (B) Thelocation of thegtaB open reading frame identified by the csb42 fusion is indicated by the open box above the restriction map, the tandem csb42(gtaB) promoters are shown by the arrows labeled A and B, and the predicted transcription terminator is represented by the stem-loop. The1.2-kb SacI-BglII fragment that containsgtaB transforming activity (31) is indicated by the double-headed arrow, shown above the map. Thesite of integration for the Tn9171acZ element in strain PB272 is symbolized by the solid triangle at the right of the map; the SalI site withinTn917 is in parentheses. The horizontal lines beneath the map show the two fragments subcloned from pDV1 into integration vector pCP115to locate csb42 (gtaB) promoter activity. The open triangle at the right of the larger fragment represents 374 bp from the left end of Tn9171acZ.The table indicates relative 13-galactosidase (13-gal) activity resulting from integration of each plasmid into the PB271 (sigB+ csb42::Tn9171acZsocBl) and PB313 (csb42::Tn917lacZ sigBAl) recipients, assayed on tryptose blood agar base plates containing 5-bromo-4-chloro-3-indolylp-D-galactoside. ND, not determined.

17-mer oligonucleotide primer (5'-GGl7lJlATCAACGATAGG, complementary to nt 210 to 226 in Fig. 3) was 5' endlabeled with [(y-32P]dATP (3,000 Ci/mmol; Amersham) andT4 polynucleotide kinase (Promega). Annealing and primerextension were performed by using the Promega primerextension kit according to the manufacturer's instructions,except that 50 ,ug of RNA and 5 ng of end-labeled primerwere used in a 20-,ul reaction volume.Computer analysis. The protein sequence alignments were

done with the FASTA programs of Pearson and Lipman (37),using the National Biomedical Research Foundation ProteinIdentification Resource data base and a VAX computer.Related sequences usually have an optimized alignmentscore above 100.

Nucleotide sequence accession number. The nucleotidesequence data shown in Fig. 3 have been assigned GenBankaccession number L12272.

RESULTS

csb42 has tandem promoters. The Tn9171acZ fusion csb42defined one of six new loci identified in a screen for genesdependent on the stationary-phase transcription factor &B(5). As described in the accompanying article, preliminarycharacterization of csb42 expression showed that fusionactivity increased in the stationary growth phase when cells

were grown in LB supplemented with glucose and glutamineand that most of this increase was dependent on &e. Becauseof the nature of the genetic screen used to identify csb42, itwas possible that the &3-dependent component of csb42expression required &e only indirectly (5).

In order to address this question, we first used the plasmidintegration and excision method of Youngman (49) to isolatethe csb42 promoter region from strain PB272 (csb42::Tn9171acZ). As described in Materials and Methods, weused one forward chromosomal walk to isolate 4.9 kb ofDNA upstream from the site of Tn9171acZ insertion (Fig. 1).With this upstream DNA as a starting point, a second,backward chromosomal walk in wild-type strain PB2 al-lowed us to isolate an additional 3.25 kb of DNA down-stream from the site of transposon insertion. The restrictionmaps of the upstream and downstream fragments overlappedand were consistent with the chromosomal restriction mapdetermined by Southern analysis (data not shown).The isolated DNA upstream from the csb42 fusion allowed

us to locate promoter activities by the standard plasmidintegration method of mapping 5' boundaries of promoterelements (38). We subcloned into the pCP115 integrationvector appropriate fragments from plasmid pDV1, whichcontained hybrid plasmid-transposon sequences and up-stream chromosomal DNA from the csb42 region (Fig. 1).We then transformed strain PB271 (csb42::Tn9171acZ

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V1r-DEPENDENT PROMOTER OF BACILLUS SUBTILIS gtaB

socBl) with the various circular plasmids, selecting forchloramphenicol resistance to force plasmid integration intothe csb42::Tn9171acZ region of homology on the recipientchromosome. In the transformed strains, &B-dependent pro-moter activity was enhanced by the socBl mutation in thersbX negative regulator of &rB (23, 27). This enhanced activ-ity facilitated finding the &3-dependent promoter element ofthe csb42 fusion. If the 5' end of the chromosomal insertcarried by the integrative plasmid lay upstream of an impor-tant csb42 promoter element, csb42::Tn9171acZ expressionwould be unaffected by the integration, whereas if the 5' endlay downstream of the element, expression would be re-duced or eliminated.As shown in Fig. 1, integration of plasmid pDV5 bearing

the 1,375-bp HindIII-SalI fragment still allowed fusion ex-pression, strongly suggesting that at least one promoterelement lay on this fragment. In contrast, integration ofplasmid pDV6 bearing the 473-bp XmnI-XmnI fragmentcompletely blocked fusion expression, indicating that anypromoter activity in the region, whether orB dependent orindependent, must lie further upstream. Thus, at least onepromoter element must lie between the HindIII site at nt 2and the XmnI site at nt 205 (see Fig. 1 and 3).To determine whether the csb42 promoter activity in the

HindIII-XmnI interval was dependent on or independentof U", we compared fusion activity in PB316 (csb42::Tn9171acZ::pDV5 sigBAl), a strain which lacks detectableU" activity, with that in strain PB314 (csb42::Tn9171acZ::pDV5 socBl), in which U" activity is enhanced. In order topermit direct comparison of the promoter activities in theHindIII-XmnI intervals of these two strains, each strain wastransformed with the pDV5 integrative plasmid to block anytranscription originating upstream of the HindIII site. Theexpression of the csb42 fusion was greatly diminished butnot abolished in PB316, the strain bearing sigBAl. The sumof these results suggests that the 204-bp HindIII-XmnIregion contains two promoter activities, one dependent oncr' and the other independent (Fig. 1).The primer extension experiment shown in Fig. 2 revealed

two 5' ends of csb42 message in the HindIII-XmnI interval.The locations of these 5' ends are indicated on the nucleotidesequence of the csb42 region shown in Fig. 3. The 5' endmapping to the guanine at nt 42, labeled +1A, is U" inde-pendent and is preceded by the sequence TTGllTT-17bp-TATCAT (nt 5 to 33). This sequence closely matches theconsensus recognized by an RNA polymerase holoenzymecontaining the major a factor of B. subtilis, crA (18). The 5'end mapping to the adenine at nt 75, labeled +1B, is U"dependent and is preceded by the sequence ATGTGTAA-14bp-GGGTAA (nt 38 to 65). This sequence and spacingcombination is very similar to elements found in other.B-dependent promoters (7, 27, 33), suggesting that the

csb42 fusion is directly transcribed by a U"-containingholoenzyme. Because the U"-dependent and U"-independentpromoter activities were mapped to the HindIII-XmnI frag-ment by plasmid integration, and because +1A and +1Brepresent the predominant 5' ends in this region, we con-clude that +1A and +1B are the initiation sites for tandempromoters.The reading frame identified by the csb42 fusion encodes a

predicted product similar to bacterial UDP-glucose pyrophos-phorylases. Immediately following the tandem promoter re-gion is a sequence resembling a B. subtilis ribosomal bindingsite (nt 104 to 109 in Fig. 3), appropriately spaced from apotential AUG initiation codon (17). This initiation codonbegins an open reading frame encoding a hypothetical 292-

ACGT 1 2 A- ~~~~~A

AA

-.*-+1A-GA ~~~~~T

G0 ~ ~~~Tini ~~~~A

A

G

w .I. __ _ A

A

G_~~~~~

* P~~~~A

FIG. 2. Mapping of the 5' ends of csb42 (gtaB) message byprimer extension. The 17-mer oligonucleotide synthetic primer5'-GGTITlATCAACGATAGG was complementary to nt 210 to 226,a sequence 135 bp downstream from the cr"-dependent transcriptionstart site (+1B at nt 75 in Fig. 3). Lane 1, end-labeled primerannealed with RNA isolated from the wild-type strain PB2 (sigB+) instationary phase and extended with reverse transcriptase. Lane 2 isthe same as lane 1 but with RNA isolated from the orB null strainPB105 (sigBAI). A sequencing ladder with the same primer isshown. The letters A, C, G, and T above the lanes indicate thedideoxynucleotide used to terminate the reaction. The sequencesindicated on the right are from the nontranscribed strand and are thecomplement of the sequence that can be read from the sequencingladder. The probable 5' end of the major &'-dependent csb42 (gtaB)message is indicated by +1B at adenine 75, because the primer-extended product was phosphorylated and ran slightly faster thanthe unphosphorylated ladder fragments. The probable 5' end of theo33-independent csb42 message is indicated by +1A at guanine 42.The faint 5' end at guanine 62 is likely an extension artifact or aprocessed form of the +1A message.

residue product that is strikingly similar to the UDP-glucosepyrophosphorylase ofAcetobacterxylinum (9), sharing 45%identical residues with an optimized alignment score of 638(37). The inference that the gene identified by the csb42fusion codes for B. subtilis UDP-glucose pyrophosphorylaseis strongly supported by the mapping of its chromosomallocus, described in the following section.

Physical and genetic mapping establish that csb42 is an alleleof gtaB. To determine whether the function of this readingframe had been previously identified on the basis of anotherphenotype, we mapped the chromosomal locus of the csb42fusion. Transductional mapping with PBS1 phage was un-successful because of an inability of strain PB272 (csb42::Tn9171acZ) to produce an effective transducing lysate (5).The reason for this inability was not clear, because theTn917 element carried by PB272 had inserted into theputative transcription terminator following the identifiedreading frame and thus should be phenotypically silent (seeFig. 3). We therefore turned to a physical mapping method(36) that took advantage of a library of B. subtilis genomicDNA carried in 59 ordered, overlapping YAC clones (2).As described in Materials and Methods, we chose two

PCR primers from the sequence of the csb42 region which

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+1A-4 +1B-4

1 AAGC AAATGGTTTATCCG AAAAAGGA AACATATTGAAAA TAGAGAATAGTTTAACCATAAATTTTTTCGATCAHindIII

M K K V R K A I I P A A G L G T R F L P A T K A M P K101 TAAaQAAiaaTGCCTTTTAAATGAAAAAAGTACGTAAAGCCATAATTCCAGCAGCAGGCTTAGGAACACGTTTTCTTCCGGCTACGAAAGCAATGCCGAAA

gtaB290t SnaBI

E M L P I V D K P T I Q Y I I E E A V E A G I E D I I I V T G K S K201 GAAATGCTTCCTATCGTTGATAAACCTACCATTCAATACATAATTGAAGAAGCTGTTGAAGCCGGTATTGAAGATATTATTATCGTAACAGGAAAAAGCA

XmnI

R A I E D H F D Y S P E L E R N L E E K G K T E L L E K V K K A S301 AGCGTGCGATTGAGGATCATTTTGATTACTCTCCTGAGCTTGAAAGAAACCTAGAAGAAAAAGGAAAAACTGAGCTGCTTGAAAAAGTGAAAAAGGCTTC

N L A D I H Y I R Q K E P K G L G H A V W C A R N F I G D E P F A401 TAACCTGGCTGACATTCACTATATCCGCCAAAAAGAACCTAAAGGTCTCGGACATGCTGTCTGGTGCGCACGCAACTTTATCGGCGATGAGCCGTTTGCG

V L L G D D I V Q A E T P G L R Q L M D E Y E K T L S S I I G V Q Q501 GTACTGCTTGGTGACGATATTGTTCAGGCTGAAACTCCAGGGTTGCGCCAATTAATGGATGAATATGAAAAAACACTTTCTTCTATTATCGGTGTTCAGC

V P E E E T H R Y G I I D P L T S E G R R Y Q V K N F V E K P P K601 AGGTGCCCGAAGAAGAAACACACCGCTACGGCATTATTGACCCGCTGACAAGTGAAGGCCGCCGTTATCAGGTGAAAAACTTCGTTGAAAAACCGCCTAA

Xmn I

G T A P S N L A I L G R Y V F T P E I F M Y L E E Q Q V G A G G E701 AGGCACAGCACCTTCTAATCTTGCCATCTTAGGCCGTTACGTATTCACGCCTGAGATCTTCATGTATTTAGAAGAGCAGCAGGTTGGCGCCGGCGGAGAA

SnaBl BglII.

I Q L T D A I Q K L N E I Q R V F A Y D F E G K R Y D V G E K L G F801 ATTCAGCTCACAGACGCCATTCAAAAGCTGAATGAAATTCAAAGAGTGTTTGCTTACGATTTTGAAGGCAAGCGTTATGATGTTGGTGAAAAGCTCGGCT

I T T T L E F A M Q D K E L R D Q L V P F M E G L L N K E El *901 TTATCACAACAACTCTTGAATTTGCGATGCAGGATAAAGAGCTTCGCGATCAGCTCGTTCCATTTATGGAAGGTTTACTAAACAAAGAAGAAATCTAAAC

VTn9171acZ1001 AAAAAGGCTATTGGACATTCATCCAATAGCCTTTTTTTATTTCAACATCAAAGTCAATGTATGCTCTTCATTATCAACTGCGAAGACCTGATCAACGGCC

1101 TGCCGCAAAAATTAATAATCCCAGACAAACACACTGATTGTAGAGTTAAACAAACTAAATGAAAACATAAATACAAACATGCAGATTAAATAGTAAATAT

1201 CTTGTATTCAATGCAAGCTAAGTAAACACCACTTGCTAAAAGAACAATGACTAAACAACCAGCATTACAAAT

FIG. 3. Nucleotide sequence of the csb42 (gtaB) region. Nucleotides are numbered from the 5' end of the nontranscribed strand, withintervals of 20 bp marked by dots. The predicted amino acid sequence for the csb42 (gtaB) product is given in single-letter code above theDNA sequence; the probable ribosomal binding site is underlined. The G to A transition that alters the proposed initiation codon from AUGto AUA in strain PB317 (gtaB290) is indicated by an arrow at nt 122. The probable -35 and -10 recognition sequences for the &3-dependentcsb42 promoter (start site +1B at nt 75) are boxed. The proposed -35 and -10 recognition sequences for the ao-like csb42 promoter (startsite +1A at nt 40 to 42) are also boxed, and the inverted repeat for the proposed terminator structure (nt 1006 to 1132) is represented byconverging arrows. The site of the csb42::Tn9171acZ insertion into this terminator region is indicated by an inverted open triangle at nt 1114.

would allow amplification of a unique 384-bp fragment fromany YAC clone bearing this particular region of the B.subtilis chromosome. By this strategy, we located csb42sequences on YAC clone 12-4SOS, which carries the gtaB-tag region and extends from about 3190 to 3230 on the B.subtilis genetic map (1, 2). This physical map location wascorroborated by transformational mapping experiments withthe degSU and rodDC markers which flank the gtaB locus(1). We found that the macrolide-lincosamide-streptograminB resistance of the csb42 fusion was 23% linked to theA(degS degU)::aphA3 marker of strain QB4238 and 36%linked to the cat marker adjacent to the rodDC operon ofstrain KUS1033.The physical map location was further supported by

comparison of the restriction map shown in Fig. 1 with the

restriction map of the gtaB-tag region developed by Maueland colleagues (31). This comparison showed that the read-ing frame identified by the csb42 fusion overlaps most of the1.2-kb SacI-BglII fragment that contains gtaB transformingactivity (31). gtaB mutations confer resistance to phage +29by reducing glycosylation of cell wall teichoic acids (48), andthis resistance is coupled with a loss of UDP-glucose pyro-phosphorylase activity (39).We used the +29 resistance conferred by an authentic

gtaB mutation to better locate the gtaB gene within thecloned region. The +29 resistance phenotype of strain PB317(gtaB290) was complemented by integration of pDV3 intothe csb42 (gtaB) region of the PB317 chromosome (Fig. 1).From this result, we conclude that gtaB complementationactivity lies downstream from the HindIII site (at nt 2 in Fig.

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3). Moreover, Mauel et al. (31) have shown that gtaBtransforming activity lies upstream of the BglII site at nt 754.Together these results genetically define the location ofgtaB, and the csb42 control region and reading frame fullyoverlap this location. We therefore used direct PCR se-quencing to analyze the csb42 region of strain PB317(gtaB290). A single change from the wild-type sequence wasfound between nt 1 and 1040, a G to A transition at nt 122which alters the proposed initiation codon from AUG toAUA (Fig. 3).To determine whether a loss of csb42 (gtaB) function

indeed conferred phage resistance, we made a nullgtaBAl::ery mutation in strain PB319 and found that thisstrain was resistant to killing by +29. The gtaBA1::erymutation also caused pleiotropic growth and sporulationphenotypes. The null mutant had a small colonial phenotypeon plates, its growth rate was about 50% slower than that ofthe wild type in both rich and minimal media at 370C, andsporulation frequency was reduced by 20- to 50-fold. Thusthe gtaB gene, identified by the csb42 fusion, has an impor-tant role in both exponential- and stationary-phase metabo-lism.

DISCUSSION

Our approach to understanding the physiological role ofcr3 in stationary-phase cells is to identify and characterizegenes in the regulon, with the expectation that the maplocations and predicted products of such genes would pro-vide important clues to eB function (5). Here we presentevidence that expression of gtaB is partly dependent on aeand that gtaB is the structural gene for UDP-glucose pyro-phosphorylase, the enzyme catalyzing the synthesis of UDPglucose.Three lines of evidence indicate that the &3-dependent

csb42 insertion is an allele of gtaB, a locus defined bymutations that reduce glycosylation of cell wall teichoicacids and confer resistance to phage 429 (48). First, thereading frame identified by the csb42 insertion lies on thesame 1.2-kb fragment known to carry gtaB transformingactivity (31), and a null mutation in this frame confers 429resistance. Second, a strain carrying thegtaB290 allele bearsan AUG to AUA alteration of the proposed csb42 (gtaB)initiation codon. Third, Pooley et al. (39) found that gtaBmutants (including one bearing the gtaB290 allele) lackUDP-glucose pyrophosphorylase activity, and we deter-mined that the predicted product of our reading frame sharessignificant sequence identity with the UDP-glucose pyro-phosphorylase of A. xylinum (9).

This last finding was important because it provided strongevidence that gtaB is the structural gene for UDP-glucosepyrophosphorylase in B. subtilis. Salmonella typhimuriumhas two loci that affect UDP-glucose pyrophosphorylaseactivity: galU is considered the structural gene for UDP-glucose pyrophosphorylase, and galF is thought to encodean activity that modifies thegalU product (35, 42). Thus, thedemonstration that B. subtilis gtaB mutations affect UDP-glucose pyrophosphorylase activity, while suggestive, doesnot provide unambiguous proof that gtaB is the structuralgene. However, in the case of A. xylinum, there is clearexperimental evidence that the reading frame analyzed doesencode UDP-glucose pyrophosphorylase activity (9). To-gether with the results of Pooley et al. (39), our finding thatthe predicted product of B. subtilis gtaB is similar to aknown UDP-glucose pyrophosphorylase is strong evidencethat gtaB is the structural gene.

TABLE 2. Promoter regions of known or'-dependent genesa

Gene -35 region -10 region

gtaB ATGTGTAA GGGTAAcsbA GTGATTGA GGGTATsigB AGGTTTAA GGGTATctc AGGTTTAA GGGTAT

* *** *

a Bases important for ctc promoter function in vivo (41) and in vitro (45) aredenoted by asterisks. In each promoter, there is 14 nt between the -35 and-10 regions. In each promoter but ctc, there is 8 nt between the -10 regionand +1, the site of transcription initiation; in ctc, there is 9 nt in this interval.All four promoters contain the TAG sequence at +1, and the site oftranscription initiation is the internal adenosine for the gtaB (Fig. 2), csbA (7),and sigB (27) promoters and either the adenosine or guanosine for ctc (33).

The inference that B. subtilis gtaB encodes UDP-glucosepyrophosphorylase suggests the function of two unidentifiedopen reading frames in the enteric system. First, the pre-dicted gtaB product shares 45% identical residues with the33-kDa product of an open reading frame (orf33) at 27.5 minon the E. coli chromosome (46). E. coli galU encodesUDP-glucose pyrophosphorylase and maps in the sameregion (3), suggesting that orf33 and galU are equivalent.Second, the predicted gtaB product is 41% identical to thatof unidentified open reading frame 2.8 of S. typhimurium,which lies in a cluster of genes involved in 0-antigensynthesis (26). Open reading frame 2.8 has been proposed tobe coincident with galF, which maps at the same locus (26,42). Our results therefore suggest that galF of S. typhimu-rium might encode a form of UDP-glucose pyrophosphory-lase and not an enzyme that modifies UDP-glucose pyro-phosphorylase, as previously thought (35).We have shown that B. subtilis gtaB lies in a monocis-

tronic transcription unit headed by tandem promoters, one&B dependent and the other independent. As shown in Table2, the proposed recognition elements of the e'-dependentgtaB promoter are very similar in sequence and spacing tothose of other &3-dependent promoters, strongly suggestingthat gtaB is directly transcribed by a ce-containing holoen-zyme in vivo. The &3-dependent ctc promoter (23) has beenwell characterized by in vitro transcription studies and byDNA footprinting experiments (32, 33). Furthermore, ge-netic analysis has identified five bases within the ctc pro-moter that are important for promoter activity in vitro and invivo (41, 45). Notably, the &3-dependent gtaB promotermatches the ctc promoter in six of eight bases in theproposed -35 region and in five of six bases in the proposed-10 region, and the match is exact at the five positionscritical for ctc promoter function (Table 2).The &3-dependent component ofgtaB expression appears

early in the stationary phase in LB supplemented with highlevels of glucose and glutamine (5), conditions which pro-mote high activity of other &3-dependent promoters andwhich suppress both the sporulation process and the forma-tion of tricarboxylic acid cycle enzymes (5, 7, 24). Given theknown functions of UDP-glucose pyrophosphorylase, whatmight be the role ofgtaB in stationary-phase metabolism? Inaddition to its role in the cell wall metabolism of certaingram-positive bacteria, the product UDP-glucose is also aprecursor of trehalose (15). Trehalose is thought to protectbiological membranes against desiccation (10), and in E. colitrehalose is required for osmotolerance and thermotolerancein the stationary phase (15, 19).Although trehalose was not detected in the one available

study of osmoregulation in B. subtilis (47), the nature of the

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osmolytes accumulated in other bacteria is known to varyaccording to the strength of the hyperosmotic shock, thesolute that induces the shock, and the composition of thegrowth medium (12, 43). Whether B. subtifis synthesizestrehalose in response to osmotic shock therefore remains anunanswered question. Thus, one attractive hypothesis is thatgtaB function is important for response to stationary-phasestress in B. subtilis, particularly under conditions in whichsporulation is inhibited and &B is most active. In support ofthis notion, preliminary experiments have shown that boththe osmotolerance and thermotolerance of strain PB319(gtaBAJ::ery) are greatly diminished compared with those ofthe wild type (8). However, because the gtaBAJ::ery muta-tion has pleiotropic effects on growth and sporulation, thisfinding requires further investigation.

ACKNOWLEDGMENTS

We thank Bernard Reilly and Dwight Anderson for providing +29phage, Sue Fisher for strain QB4238, George Stewart for strainKUS1033, and the Bacillus Genetic Stock Center for strain lA105.We also thank Michele Igo for helpful comments on the manuscript.

This research was supported by Public Health Service grantGM42077 from the National Institute of General Medical Sciences.Ddbora Var6n was supported in part by a Graduate OpportunityFellowship from the University of California, and some costs of herresearch were borne by a Jastro-Shields Graduate Research Award.The PCR mapping of the gtaB (csb42) chromosomal locus, done byKathleen Okamoto, was supported by Public Health Service grantGM29231 from the National Institute of General Medical Sciences.

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