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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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V5B-DEPENDENT PROMOTER OF BACILLUS SUBTILIS gtaB 3965
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
J. BACTERIOL.
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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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3968 VARON ET AL.
+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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,B-DEPENDENT PROMOTER OF BACILLUS SUBTILIS gtaB 3969
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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3970 VAR6N ET AL.
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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