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From the
The Bacillus subtilis dlt operon (D-alanyl-lipoteichoic acid) is responsible for D-alanine esterification of both lipoteichoic acid (LTA) and
wall teichoic acid (WTA). The dlt operon contains five genes, dltA-dltE. Insertional inactivation of dltA-dltD results in complete absence of D-alanine from both LTA and WTA. Based on protein sequence
similarity with the Lactobacillus casei dlt gene products
(Heaton, M. P., and Neuhaus, F. C.(1992) J.Bacteriol. 174, 4707-4717), we propose that
[Abstract]
dltA encodes the D-alanine-D-alanyl carrier
protein ligase (Dcl) and dltC the D-alanyl carrier
protein (Dcp). We further hypothesize that the products of dltB and dltD are concerned with the transport of
activated D-alanine through the membrane and the final
incorporation of D-alanine into LTA. The hydropathy profiles
of the dltB and dltD gene products suggest a
transmembrane location for the former and an amino-terminal signal
peptide for the latter. The incorporation of D-alanine into
LTA and WTA did not separate in any of the mutants studied which
indicates that either one and the same enzyme is responsible for D-alanine incorporation into both polymers or a separate
enzyme, encoded outside the dlt operon, transfers the D-alanyl residues from LTA to WTA (Haas, R., Koch, H.-U., and
Fischer, W.(1984) FEMS Microbiol. Lett. 21, 27-31).
Inactivation of dltE has no effect on D-alanine ester
content of both LTA and WTA, and at present we cannot propose any
function for its gene product. Transcription analysis shows that the dlt operon is transcribed from a
Teichoic acids (TAs)
The poly(glycerophosphate)
chains of LTA and WTA are generally synthesized by separate enzyme
systems and contain enantiomeric glycerophosphate residues. For the
biosynthesis of the glycosylated WTA-linkage unit complex, CDP-glycerol
and nucleotide-activated sugars are used(3, 4) , which
suggests that it occurs on the cytosolic site of the membrane, where
the respective enzymes have access to their water-soluble substrates.
In contrast, the biosynthesis of LTA uses lipid substrates and is
thought to be located on the outer layer of the cytoplasmic
membrane(5, 6) .
The incorporation of D-alanine into LTA has been studied intensively in Lactobacillus casei and suggested to involve two enzymes and a D-alanyl carrier protein (Dcp)(1, 9) . D-Alanine, activated via D-alanyl AMP, is linked to
Dcp and is used, possibly via a putative undecaprenol phosphate
derivative, for the alanylation of membrane-associated LTA. Earlier
experiments with Staphylococcus aureus revealed that the D-alanine ester of LTA is subject to a rapid turnover: part is
lost by spontaneous hydrolysis, another part serves as donor for the D-alanylation of WTA(2) . The loss of D-alanine ester from LTA is continuously compensated for by
re-alanylation(10) , which requires activated D-alanine
on the outer membrane layer and may therefore be accomplished by the
same enzyme as the denovo incorporation into the
growing chain.
Essential roles for teichoic acids in bacterial
physiology have repeatedly been suggested but it was only recently that
insertional mutations documented that WTA is indeed required for growth
of Bacillus subtilis(11) . In contrast to B.subtilis, L.casei is devoid of WTA and
contains only LTA(12) . In L.casei, mutants
that loose the ability to synthesize LTA have not been observed and are
apparently lethal. Mutants defective in D-alanylation of LTA
displayed defective cell separation and aberrant morphology but
definite proof that both phenotypes are caused by the same mutation is
still lacking(13) .
Here we report the characterization of an
operon responsible for the D-alanylation of both LTA and WTA
in B.subtilis. This operon was identified in the
framework of the European sequencing project of the B.subtilis chromosome(14) , and we now describe the
deduced gene products and promoter region, the latter suggesting a
complex transcriptional regulation of the operon.
Transformation of B.subtilis strains by plasmid or chromosomal DNA was performed by standard
procedures(21) . Selections for antibiotic resistance were done
at the following concentrations: chloramphenicol, 5 µg/ml;
kanamycin, 2 µg/ml; erythromycin and lincomycin (MLS
Escherichia
coli DH5
WTA constituents were released from purified walls by
hydrolysis with 48% (by mass) HF, 2 °C, 36 h. After drying in
vacuum at 2 °C, the hydrolysate was suspended in 0.01 M lithium acetate, pH 4.7, and WTA constituents were separated from
dephosphorylated walls by centrifugation (12,000
LTA was hydrolyzed in 2 M HCl
at 100 °C for 2.5 h and analyzed for phosphorus, D-alanine, glucose, and glycerol, the latter being measured
after further treatment with phosphomonoesterase. Glucosamine was
quantified after hydrolysis of LTA in 4 M HCl, 100 °C, 18
h (33). Glc(
The total alanine of LTA and WTA was
ester-linked, as shown by release through mild alkaline treatment (0.1 M NaOH, 37 °C, 1 h).
The product of the fourth gene, dltD, does not have any significant similarity to known
proteins. Amino acid composition and hydropathy profile (Fig. 3)
suggest the presence of an amino-terminal signal peptide: 3 positively
charged residues followed by a core of hydrophobic residues
(MKKRFFGPIILAFILFAGAIA). Therefore DltD could be a secreted protein,
and the amino-terminal sequence could anchor the protein to the outer
face of the cell membrane. The rest of the protein is hydrophilic and
highly positively charged (45 lysine, 10 arginine, and 8 histidine
residues versus 15 aspartic acid and 25 glutamic acid
residues). The positively charged residues are mostly clustered in
three regions at position 164 (KKKMMKRMLRFK), at position 268
(KKLKPKVPKLKGKNKGR) and at position 328 (KKGRTDYYKVNKQUIRAK).
The
product of the last gene of the operon is homologous to a large family
of oxidoreductases, including the E. coli 3-ketoacyl-ACP
reductase (40) (Fig. 2C). The deduced protein
sequence of DltE suggests a cytoplasmic localization.
None of the
constructed mutants showed a defective phenotype for cell division or
morphology as observed by phase-contrast microscopy with cultures grown
on agar plates or in liquid media. Unaltered cell morphology and
septation were confirmed by electron microscopy.
In a first series of experiments, bacteria were grown
in medium A. As shown in , the LTA of the wild type strain
JH642 contained on average 29 glycerophosphate residues/chain.
Forty-four percent of the glycerophosphate residues were substituted
with D-alanine ester, 10% with N-acetyl-
Insertional inactivation of dltA (pDLT65A), dltB
(pDLT72), dltC (pDLT77) and dltD (pDLT74A) each
caused complete absence of D-alanine ester from both LTA and
WTA (). However D-alanine incorporation into LTA
and WTA, to an extent comparable with that of the wild type strain, was
seen when dltE was inactivated (pDLT76). It should be noted
that, compared with the parent strain, the content of WTA and LTA was
unchanged in all mutant strains: per gram of bacterial dry mass it
amounted to 354 ± 24 µmol and 63 ± 3 µmol
phosphorus, respectively, and contributed 32.1 ± 1.5% and 5.7
± 0.3% to the total cellular phosphorus (data not shown). Also,
in the mutant strains there was no systematic change either in the
chain length of LTA or in the extent of glucosylation of WTA (). It was only the substitution of LTA-glycerol with N-acetylglucosaminyl residues that increased from 10 to 19%
when D-alanine ester was not incorporated.
In order to
verify that dltE is actually not involved in D-alanine incorporation into WTA, bacteria were grown in the
low salt medium B. Under these conditions, the alanine substitution of
WTA-glycerol increased from 10 to 25% in the wild type strain, and this
increase was paralleled by the alanine content of WTA in the mutant
strain pDLT76 in which dltE is inactive. Growth of the mutant
strain in the presence of chloramphenicol (5 µg/ml), which confirms
the presence of the chloramphenicol resistance-carrying insertion, did
not affect this result. The same results were obtained with a strain
inactivated in the dltE gene by kanamycin insertion via a
double crossover (data not shown).
In all mutant strains the
cellular content of total phosphorus (1150 ± 70 µmol/g dry
cells) and lipid phosphorus (38 ± 4 µmol/g dry cells) was
unaffected in the mutant strains. They also displayed no alteration in
the fatty acid composition nor in the TLC pattern of their polar lipids
(data not shown). Concerning logarithmic growth, there was no
difference between the wild type and the mutant strains, the doubling
times being 95 ± 1 min and 106 ± 4 min in high and low
salt medium, respectively.
Biochemical studies carried out on B. subtilis mutants in the dlt operon demonstrate that the gene
products of dltA through dltD are involved in the
incorporation of D-alanine into LTA and WTA, whereas the dltE gene product is not (). Insertional
inactivation of dltA-dltD results in complete absence
of D-alanine ester from both LTA and WTA. The first two genes, dltA and dltB, are highly similar to ORF1 and ORF2 of
the dlt operon in L. casei, and the product of dltC is homologous to the D-alanyl carrier protein of L. casei (1, 9). This allows us to propose that dltA
encodes the D-alanine-D-alanyl carrier protein ligase
(Dcl) and dltC the D-alanyl carrier protein (Dcp). In
a two-step reaction the ligase forms a high energy D-alanyl
AMP intermediate and transfers the alanyl residues from AMP to
Dcp(9) .
On-line formulae not verified for accuracy REACTION 1a
On-line formulae not verified for accuracy REACTION 1bdltB is homologous to ORF2 of the dlt operon of L.
casei which is suggested to code for an enzyme that transfers the
alanyl residues from Dcp to undecaprenol phosphate (C
On-line formulae not verified for accuracy REACTION 2This hypothesis is supported by (i) the sensitivity of the incorporation
system to amphomycin and tunicamycin and (ii) the similarity of the
deduced amino acid sequence of this protein with undecaprenol phosphate
transferases(9) . Our results for the first time provide
evidence that a fourth gene product is required for the incorporation
of D-alanine into LTA and WTA. Given the involvement of dltA-dltC gene products of the B. subtilis operon in the activation of D-alanine, the gene product
of dltD is proposed to catalyze, as a final step, the transfer
of D-alanine residues from the proposed
undecaprenolphospho-derivative to the poly(glycerophosphate) chains.
The fact that the dltD gene product does not have any homology
with known proteins would be consistent with the unique structure of
its putative donor and acceptor substrates.
It is noteworthy that
the incorporation of D-alanine into LTA and WTA did not
separate in any of the mutants studied. This result indicates that the
activation sequence is identical for both processes. However, for the
final step, the incorporation of D-alanine into LTA and WTA,
two alternatives have to be considered: either one and the same enzyme
catalyses the incorporation into each polymer, or, as suggested earlier
for S. aureus(2) , D-alanyl-LTA is the donor
for the alanylation of WTA. The selective response of the D-alanine content of WTA to a change in the growth medium () may be taken as a preliminary hint favoring the second
possibility. In this case, the postulated additional enzyme must be
encoded by a gene which, on the basis of the present results, is
located outside the dlt operon.
The fifth gene, dltE, although separated from dltD by 90 base pairs,
was considered part of the dlt operon due to the observation
that no transcription terminator nor putative promoter regions were
identified in this intergenic region. However its inactivation was
without any effect on the D-alanine ester content of both LTA
and WTA (). The product of this gene belongs to a large
family of oxidoreductases including E. coli 3-ketoacyl-ACP
reductase, the product of fabG, which precedes and is
cotranscribed with acpP, the structural gene for the E.
coli acyl carrier protein, Acp(40) . At present we cannot
propose any function for the dltE gene product, but its
hydrophilic characteristics suggest that it is a cytosolic enzyme.
From the deduced protein sequences of the dltA-dltD gene products we can predict features
regarding their cellular location. The ligase enzyme Dcl, the product
of the dltA gene, is a hydrophilic protein consistent with the
water-soluble nature of its substrates, D-Ala, ATP, and the
acidic Dcp protein encoded by dltC. The protein encoded by the dltB gene is essentially hydrophobic, suggesting a
transmembrane location. This is in agreement with the proposed role of
transferring D-alanine from Dcp to C
These results
suggest that the action of the Spo0A and AbrB proteins is required to
turn off dlt expression before the transition phase. The rapid
loss of D-alanine ester from LTA and WTA observed during the
early stages of sporulation is explained by the lack of incorporation
and the spontaneous hydrolysis of the labile alanine ester(2) .
It is interesting to note that LTA synthesis is slowed down toward the
end of vegetative growth and reactivated 2 h after the transition
state(43) . Therefore, the spore membrane is apparently endowed
with newly synthesized LTA and this LTA is demonstrably free of D-alanine ester. It has been well established that
alanine-free purely anionic LTA binds strongly to positively charged
autolysins and so prevents their access to the cell wall
substrate(49, 50) . Autolysins are required for
germination, and the D-alanine-free LTA of spores may serve to
fix them in an inactive state near the outer surface of the cytoplasmic
membrane.
The question regarding the role of D-alanine
substituents of LTA and WTA during vegetative growth cannot yet be
answered. B. subtilis dlt mutants displayed normal growth in
complex media and showed no biochemical changes in the cell wall
membrane complex. The cellular content of WTA-, LTA-, and
lipid-phosphorus, the content of peptidoglycan, and the membrane lipid
composition remained unaltered. Electron microscopic studies revealed
unaltered cell shape and wall structure and normal septation during
cell separation.
M. P. thanks Alessandro Galizzi for helpful
discussion. W. F. and K. L. thank Barbara Orlicz-Welcz and Christian
Emilius for reliable technical assistance.
Volume 270,
Number 26,
Issue of June 30, pp. 15598-15606, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
IDENTIFICATION OF GENES AND REGULATION (*)
ABSTRACT
INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-dependent
promoter and follows the pattern of transcription of genes belonging to
the regulon. However, the turn off of transcription
observed before sporulation starts seems to be dependent on the Spo0A
and AbrB sporulation proteins and results in a D-alanine-free
purely anionic LTA in the spore membrane. The dlt operon is
dispensable for cell growth; its inactivation does not affect cell
growth or morphology as described for L.casei.
()are components of
the cell wall-membrane complex in a large number of Gram-positive
bacteria. They are named after the phosphate groups they contain in
diester bonds and are classified in two groups: wall teichoic acids
(WTA), which are phosphodiester-linked via a linkage unit to muramic
acid residues of peptidoglycan(3, 4) , and lipoteichoic
acids (LTA), which are macroamphiphiles being anchored hydrophobically
through the fatty acid residues of their glycolipid component in the
outer layer of the cytoplasmic membrane(5, 6) . In Bacillus subtilis 168, from which the strains studied here are
derived, LTA and WTA possess a poly(glycerophosphate) chain that is
substituted with D-alanine ester(7, 8) . The
LTA is further substituted with N-acetyl-
-D-glucosaminyl, the WTA with
-D-glucopyranosyl residues.
Sequence Analysis
DNA sequences were analyzed
using DNA Strider 1.1 software(15) . Protein sequence analysis
was carried out with the FASTA (16) (in Swissprot release 29)
and the BLAST (17) programs. Searches using the BLAST program
were performed in the nonredundant protein library at the National
Center for Biotechnology Information. Sequences were compared using the
Wisconsin Genetics Computer Group sequence analysis software package,
version 6.0 (University of Wisconsin Biotechnology Center, Madison,
WI).
Bacterial Strains, Growth Conditions, and
Transformation
Isogenic B.subtilis strains
derivative of JH642 (trpC2, phe-1) used in this study
were: JH646 (spo0A12), JH703abr4 (spo0A677, abrB4), JH642::pLM5 (sigD::pLM5cat)(18) . B.subtilis strains were grown in Schaeffer's sporulation medium (19) or in PY broth (Difco antibiotic medium 3). For biochemical
analysis, bacteria were grown in two different media (A and B) that
contained (per liter) the following: medium A: casein hydrolysate, 10
g; yeast extract, 0.5 g; glucose, 5 g (sterilized separately);
NH
Cl, 2 g; K
HPO, 18.5 g;
KHPO, 2.5 g; trisodium citrate, 1 g;
MgSO
7HO, 0.4 g;
FeSOHO, 0.01 g;
MnSOHO, 0.015 g; the pH was adjusted to
7.5 with NaOH and medium B: casein hydrolysate, 5 g; meat extract, 1.5
g; yeast extract, 1.5 g; glucose, 2 g (sterilized separately);
KHPO, 3.68 g; KHPO,
1.32 g; the pH was adjusted to 7.4 with NaOH. For batch growth,
overnight cultures were diluted 40-fold and grown with vigorous
aeration on a rotary shaker at 32 °C to an A of approximately 4 and 2 in medium A and B, respectively.
Cultures were rapidly cooled to 0 °C, harvested by centrifugation
(3600 rpm, 20 min) at 4 °C and washed with cold 0.1 M sodium acetate, pH 4.7 (buffer A), containing 9 g of NaCl/liter.
Sporulation assays were carried out with cells grown for 24 h in
Schaeffer's broth. Serial dilutions were then plated on
Schaeffer's agar plates before and after treatment with
CHCl
. Motility was assessed on semisolid agar
plates(20) .
(macrolide, lincosamide, and streptogramin B resistance)) 1 and
25 µg/ml, respectively. The lacZ fusion plasmid pDLT68 was
used in the circular form to transform strain JH642 (selecting for
Km) to obtain strain JH642::pDLT68. The correct integration
into the dlt locus was checked by PBS1 transduction and the Km
resistance was found to be linked to the sacA marker at
335° on the chromosomal map. The fusion was transferred to
different mutant strains using 0.1 µg of chromosomal DNA extracted
from JH642::pDLT68 and selecting for Km.
used for plasmid construction and propagation was
grown in LB medium supplemented with ampicillin at 100 µg/ml.
DNA Manipulations
Chromosomal DNA from B.subtilis was prepared by the method of Marmur (22) with some modifications. Plasmid DNA from E. coli was purified by the boiling method of Holmes and
Quigley(23) . The plasmids described in this study were
constructed in the integrative vector pJM103 that carries a
chloramphenicol resistance marker selectable in B.subtilis(24) . The lacZ fusion plasmid
pDLT68 was constructed in pJM115 that derives from pDH32 by replacement
of the chloramphenicol resistance gene with a Km resistance gene.
Plasmid pDLT71 was constructed in the chloramphenicol cassette vector
pJM105A (24) in two steps. First, the 400-base pair BamHI-BglII fragment from pDLT52 was placed
downstream of the cat gene. Then, the 1150-base pair fragment PvuI-BamHI from pDLT55 was placed upstream of the cat gene. In order to construct plasmid pDLT72, pDLT55 was
digested with BclI, and the ends were blunted with Klenow
polymerase. The 750-base pair central fragment was discarded, whereas
the 5-kb portion carrying the vector was purified and ligated with a
fragment containing the ermG gene obtained from the
erythromycin cassette vector pJM109B (24) after digestion with BamHI and SalI. All the ends were first made blunt
using Klenow polymerase. Plasmid pDLT74A was obtained by first cloning
the SphI-SalI fragment (Fig. 1) in pJM103 and
then inserting the fragment with the km gene from the Km
cassette vector pJM114 (24) in the EcoRV site.
Figure 1:
Restriction map of the B.
subtilis chromosomal region containing the dlt operon.
The location of the five open reading frames encoding for the DltA-DltE
proteins is indicated by the arrows. The fragments subcloned
in integrative vectors are indicated by horizontal lines. Broken
lines stand for regions deleted and replaced by an antibiotic
resistance gene whose direction of transcription is indicated by a small arrow. Restriction sites: B = BamHI; Bc = BclI; Bg = BglII; D = DraI; EV = EcoRV; Hc = HincII; P = PvuII; Pv = PvuI; S = Sau3A; Sp = SphI.
The restriction sites shown are the ones relevant for plasmid
construction, but not all sites are
indicated.
Chemical Analyses
Bacteria (5 g, wet mass) were
suspended in buffer A (12.5 ml) and disrupted in a Braun disintegrator
with glass beads under cooling with CO(25) . Glass
beads were removed by filtration in a sintered glass funnel. Samples of
the homogenate were taken in triplicate for the determination of dry
mass (1 ml), phosphorus (0.025 ml), isolation of LTA (2 ml),
preparation of cell walls (0.25 ml), and lipid extraction (2 ml).
Operational steps were performed at pH 4.7 and, as possible, at
2-4 °C. LTA was extracted from the homogenate by hot
phenol/water and isolated from the aqueous layer by hydrophobic
chromatography on octyl-Sepharose using a downscaled centrifugation
procedure(26) . For cell wall preparation, the homogenate was
diluted fourfold with SDS in buffer A to a final concentration of 2%
(mass/volume). The mixture was sonicated for 15 min, then vigorously
shaken at 65 °C for 1 h, followed by centrifugation and washing of
the pellet five times with buffer A (1 ml each). Lipids were extracted
by a modified Bligh Dyer procedure (27) using buffer A instead
of water.
g).
The supernatant was subjected to hydrolysis with 2 M HCl, 100
°C, 2.5 h and then analyzed for phosphorus(28) , D-glucose(29) , D-alanine (30), and
glycerol(31) . Glc(
1-2)Gro was identified in HF
hydrolysates as trifluoroacetate by gas liquid
chromatography(34) . Galactosamine, a constituent of the minor
WTA species(37) , was measured after hydrolysis with 4 M HCl (100 °C, 18 h).
1-2)Gro, GlcNAc(1-2)Gro, and
Glc(
1-6)Glc(1-3)Gro were identified in the HF
hydrolysate of deacylated LTA as trifluoroacetates by gas liquid
chromatography(34) . The chain length of LTA was calculated from
molar amounts of phosphorus and glucose by the formula: phosphorus/0.5
glucose multiplied by 1.1 for correction of chain
Glc(1-2)Gro.
Cultures for
-Galactosidase Assays-galactosidase assays were grown in Schaeffer's sporulation
medium. The assay was carried out as described previously(35) ,
and the units were calculated according to Miller(36) .
DNase I Footprinting Experiments
DNase I
footprinting experiments were performed using plasmid pDLT62. The
fragment carrying the dlt promoter was digested with HindIII (which is in the multiple cloning site adjacent to the PvuII) site, end-labeled using Klenow polymerase and
[-P]dATP (Amersham Corp.), and excised with BamHI digestion. DNase I protection experiments with Spo0A and
AbrB were performed as described previously(37) . The labeled
fragment was also subjected to Maxam and Gilbert A + G and C
+ T sequencing reactions (38) to generate a reference
ladder.
The dlt Operon Gene Products
The deduced amino
acid sequences of the five genes from the dlt operon were
analyzed using the programs described under ``Material and
Methods.'' The dltA gene product is highly similar to L.casei Dcl, the dltA gene product, and
also, to a lower extent, to peptidyl antibiotic synthetase domains. The
alignment of the B.subtilis DltA protein sequence
with its counterpart from L.casei and the synthetase
domain from Bacillus brevis gramicidin synthase (GrsB) (39) is presented in Fig. 2A. The region
surrounding the putative phosphate binding loop
GXXGXPKG as well as the two other regions proposed by
Heaton and Neuhaus (1) as essential for the formation of the
acyl adenylate are particularly well conserved in these three proteins.
Analysis of the hydropathy profile of DltA (data not shown) suggests a
cytoplasmic localization.
Figure 2:
Protein sequences alignments. A,
alignments of the deduced amino acid sequences of B. subtilis DltA, L. casei Dcl (1), and the first synthetase domain
of B.brevis GrsB (39). The putative phosphate
binding loop is marked by asterisks. The three conserved
regions putatively involved in the formation of the acyl adenylate are underlined. B, alignments of the amino acid sequences
of B. subtilis Dcp and ACPs from E. coli (EC) (40), R. meliloti (RM) (51), Saccharomyces cerevisae (SC) (52), and Arabidopsis thaliana (AT) (53). The amino-terminal
sequence of the L. casei Dcp protein (LC) (9) is also
shown. The asterisk indicates the position of the serine
residue to which the 4`-phosphopanthoteine prosthetic group is linked. C, alignment of the deduced amino acid sequences of B.
subtilis DltE, Streptomyces violaceoruber granaticin
synthase putative ketoacyl reductase 1 (Dhk1) (54), and E.
coli 3-ketoacyl-acyl carrier protein reductase (FabG)
(40). Multiple alignments were first performed with the CLUSTAL method
(55) and then refined manually.
Protein sequence comparison of the B.
subtilis DltB protein with the first 188 codons of L.casei dltB gene product (1) (not shown) reveals an
high percentage of identical residues (50%). The hydropathy profile of
DltB from B.subtilis shows the presence of
hydrophobic domains which suggest a transmembrane localization (Fig. 3). The amino acid composition of this protein also shows a
strong predominance of positively charged versus negatively
charged residues (24 lysine, 12 arginine, and 17 histidine residues versus 8 aspartic acid and 7 glutamic acid residues).
Figure 3:
Hydropathy profiles of the B. subtilis
dltB and dltD gene products. The Kyte and Doolittle index
was used to calculate the profiles with a window of 11 (56). S.P. = signal peptide.
The dltC gene product is a putative D-alanine carrier
protein (Dcp) based on similarity to acyl carrier proteins (ACPs) of
fatty acid biosynthesis. Amino acid sequence alignments of the B.subtilis DltC with ACPs from different origins and the
amino-terminal sequence of L.casei Dcp (9) are shown in Fig. 2B. The sequence
surrounding the serine residue to which the 4`-phosphopanthoteine
prosthetic group is linked is an highly conserved region. Like the
other ACPs, the B.subtilis Dcp is a negatively
charged protein (8 aspartic and 10 glutamic acid residues for a
78-amino acid-long protein).
Gene Inactivation
In order to define the
physiological role for each of the five genes in the B.subtilis dlt operon, we constructed a series of mutant
strains using the insertional mutagenesis technique with integrational
vectors(24) . Two strategies were followed. 1) B.subtilis competent cells were transformed with an
integrative vector containing a DNA fragment internal to the dlt operon. Chromosomal integration of such a plasmid by a
Campbell-type single crossover event results in the disruption of the dlt transcriptional unit. In this way, we obtained: (i)
integration of plasmid pDLT65A that resulted in interruption of
transcription at the BamHI site internal to dltA;
(ii) integration of pDLT77 that stopped transcription at the DraI site in dltC but allowed the synthesis of
complete dltA and dltB gene products; iii)
integration of pDLT76 that gave rise to a mutant that was missing only
the dltE gene product (Fig. 1). 2) Two plasmids were
constructed, pDLT71 and pDLT72 (Fig. 1) which, upon linearization
and integration in the B.subtilis chromosome by
double crossover gave rise to a deletion-gene replacement event.
Integration of pDLT71 resulted in the deletion of the promoter region, dltA and part of dltB (replaced by the
chloramphenicol resistance gene). The resulting strain was defective
for all the functions coded by the dlt operon. Integration of
pDLT72, on the other hand, deleted a portion of dltB (replaced
by the erythromycin resistance determinant), but left the dltA
gene intact. A third plasmid, pDLT74A (Fig. 1), after integration
by double crossover resulted in interruption of the transcriptional
unit at the EcoRV site in dltD due to the presence of
the km gene. Since transcription from the km promoter
occurs opposite to the transcription of dlt, no transcription
of dltE could occur. This resulted in a mutant strain
synthesizing a truncated dltD gene product and completely
defective for dltE but unaffected for the transcription of dltA, dltB, and dltC.
()However, dlt mutants were not motile
compared with the wild type strain, as assessed on semisolid agar
plates, although they were equally motile when cells were grown in
liquid medium and observed by phase-contrast microscopy.
D-Alanine Content in LTA and WTA and Other
Biochemical Parameters during Vegetative Growth
LTA was
extracted, and cell walls were prepared from mechanically disintegrated
bacteria under conditions that preserve the native substitution of LTA
and WTA with D-alanine ester(25, 41) . In B.subtilis JH642, LTA and WTA are composed of
poly(glycerophosphate) chains, as indicated by an equimolar ratio in
both polymers of glycerol and phosphorus (data not shown). A minor WTA
species composed of Glc(1-3)GalNAc-1-P repeats (32) did not contribute more than 9 ± 2% to total WTA
phosphorus, as was estimated by galactosamine measurement in WTA
hydrolysates.
-D-glucosaminyl, and less than 1% with
-D-glucopyranosyl residues. In WTA 9% of the
glycerophosphate moieties were substituted with D-alanine
ester and 64% with -D-glucopyranosyl residues.
Gene Expression and Regulation
Transcriptional
regulation of the dlt operon was investigated using the dlt-lacZ transcriptional fusion plasmid pDLT68 (Fig. 1).
The timing of dlt expression in a wild type strain (JH642)
grown in sporulation conditions (Schaeffer's medium) showed that
transcription occurs maximally during the exponential phase of growth,
and it decreases drastically approximately 1 h before transition to
stationary phase, T
(Fig. 4). In
nonsporulating conditions (PY medium), expression from the dlt promoter appears to be constitutive during the growth cycle (data
not shown), indicating that the turn off at T in Schaeffer's medium is associated with the transition
state that begins the sporulation process.
Figure 4:
-Galactosidase activity of the dlt-lacZ fusion in the sigD mutant strain.
Plasmid pDLT68 was integrated in the dlt locus. Cells were
grown in Schaeffer's sporulation medium. Closed circle,
parental strain JH642; open square, sigD mutant
JH642::pLM5. 0 = T, transition from
vegetative growth to sporulation.
The presence of putative
-35 and -10 consensus sequences for
-containing RNA polymerase
(TTCA-N-GCCGATAT) (Fig. 5) in the dlt promoter region prompted us to analyze dlt transcription
in a sigD mutant strain. The results showed a 3-fold reduction
of dlt transcription in the sigD mutant (JH642::pLM5)
compared with the wild type strain (JH642) (Fig. 4). The residual
activity may be accounted for by transcription from an additional
promoter. In fact, two putative
promoters were
identified whose -35 and -10 consensus sequences
(TTGACT-N-TATTAT and TTCACA-N
-TATATT) are
localized upstream and downstream of the
promoter,
respectively (Fig. 5), but their activity in vivo has
not been determined.
Figure 5:
Nucleotide sequence of the dlt promoter region. -35 and -10 recognition sequences for
- and -containing RNA polymerase
and the 0A box for Spo0A binding are indicated by bold
letters. The region protected by the Spo0A protein is indicate by
square parentheses. The first nucleotide reported correspond to
nucleotide number 5670 in the sequence deposited in the
EMBL/GenBank/DDBJ Nucleotide Sequence Data Libraries under
accession number X73124. Asterisks are positioned every 30
nucleotides. The ribosome binding site for dlt is underlined. The first codon of the dltA gene is
reported in lowercase letters.
The possibility of a direct involvement of
sporulation-specific proteins in regulating dlt expression was
suggested by the presence of a Spo0A DNA binding recognition sequence
(``0A box'' TGTCGAA) (Fig. 5) (42) in the dlt promoter region. A DNase I footprinting assay was
performed on the chromosomal fragment carried by plasmid pDLT62 using
purified Spo0A protein. The Spo0A protein was found to protect a region
of 30 nucleotides containing the 0A box (Fig. 6), supporting a
direct role in regulating dlt transcription. In vivo transcription analysis using the dlt-lacZ fusion
plasmid pDLT68 showed a 2-fold reduction of dlt expression in
the spo0A mutant strain JH646 (Fig. 7). The repression
observed was released by an abrB mutation that also caused an
higher level of transcription during the first hours of the stationary
phase (Fig. 7). A DNase I footprinting assay carried out with
purified AbrB protein did not show any binding to the dlt promoter region (data not shown). The transfer of an abrB
mutation into the sigD mutant strain JH642::pLM5 resulted in a
2-fold increase in dlt transcription compared with the strain
carrying the sigD mutation alone (data not shown). This
indicated that the target of AbrB repression, although apparently
indirect, must be exerted on the transcription generated at one of the
putative -dependent promoters.
Figure 6:
DNase I protection of the dlt promoter region by the Spo0A protein. The results were obtained
with the end-labeled nontemplate strand. Binding of Spo0A at 5
µM (lane 3), 3 µM (lane 4),
1 µM (lane 5). No Spo0A was added to lanes 1,
2, 6, and 7. The Maxam and Gilbert purine (R)
and pyrimidine reactions (Y) are shown for references. Arrows show the region protected by the Spo0A
binding.
Figure 7:
-Galactosidase activity of the dlt-lacZ fusion in spo0A and abrB
mutants. Plasmid pDLT68 was integrated in the dlt locus.
Strains JH642 (parental (closed circle)), JH646 (spo0A12 (open circle)), and JH703abr4 (spo0A204, abrB4 (closed triangle)) were
grown in Schaeffer's sporulation medium. 0 = T, transition from vegetative growth to
sporulation.
Loss of D-Alanine Ester from LTA and WTA during
Sporulation
The turn off of dlt transcription in cells
entering the transition phase prompted us to analyze LTA and WTA of B. subtilis wild type during growth in Schaeffer's
sporulation medium(19) . The D-alanine/phosphorus molar
ratio of LTA decreased from 0.30 during vegetative growth (T) to 0.04 (T
), 0.01 (T
)
to 0 (T
) during the sporulation phase. The
respective values of WTA were 0.15, 0.06, 0.02, and 0. In all samples,
the length of LTA was unaltered and the ratio of LTA and WTA phosphorus
to total phosphorus remained constant at 0.33 ± 0.04 and 0.066
± 0.015. These findings are consistent with an earlier
observation that both polymers formed during vegetative growth are not
hydrolyzed until at least stage IV of sporulation(43) . In
isolated spores that lack WTA (44) and contain newly synthesized
LTA(43) , we were not able to detect D-alanine ester.
-P).
-P and
translocating the product from the inner to the outer layer of the cell
membrane(9, 45) . A typical signal peptide sequence was
identified in the amino-terminal end of the protein encoded by dltD. This suggests export of this protein and possible
anchoring to the membrane by the hydrophobic portion. This location
would allow the D-alanylation to occur on the outer layer of
the membrane, where LTA is thought to be
synthesized(5, 45) , and also enable the same enzyme to
accomplish the re-alanylation of completed LTA which is essential for
the maintenance of alanine substitution as demonstrated in S.
aureus(10) . The three clusters of positively charged amino
acids in the hydrophilic region of the dltD gene product may
serve to recognize nonalanylated, negatively charged regions of the
acceptor LTA. These observations complete and support the model for D-alanyl-LTA biosynthesis which was previously proposed (9, 45) and is depicted in Fig. 8.
Figure 8:
Model of D-alanyl-LTA
biosynthesis on the outer layer of the cytoplasmic membrane with the
nucleotide-requiring steps located on the cytosolic side (5, 6, 45). 1, D-alanine-D-alanyl carrier protein
ligase; 2 and 3, proposed D-alanyl transfer
from D-alanyl carrier protein to undecaprenol phosphate and
from the latter, after passage through the membrane, to LTA (see text).
Glycosylation of LTA via undecaprenol phosphate-activated sugars has
been established in Streptococcus sanguis and Bacillus
coagulans (57, 58). In the symbols used for structures, oxygen
atoms are omitted: , glycerol phosphate; , diacylglycerol; ,
undecaprenol phosphate.
The
availability of cloned genes allowed us to investigate the
transcription regulation of the genes responsible for the D-alanine esterification of TAs. Expression studies using a
promoter-lacZ fusion construct showed that the dlt operon is under control of a -dependent
promoter, and therefore it belongs to the regulon.
-Dependent genes belong to families of genes involved
in chemotaxis, motility, flagella assembly, and cell separation.
Expression of the structural gene for , sigD, and the regulon is activated in late
logarithmic growth during the transition to stationary
phase(46) . Peak expression of the dlt operon occurs
during logarithmic growth as well, but it stops before the beginning of
the transition state. Interestingly, temporal regulation of dlt transcription appears to be controlled by the Spo0A and AbrB
global regulators. The Spo0A protein controls the onset of sporulation
by regulating transcription of various genes in both positive and
negative manners depending on the promoters affected. Its activity is
dependent on the state of phosphorylation regulated by the
phosphorelay, a signal transduction system linking environmental
information to the activation of sporulation(47, 48) .
One function of the Spo0A protein is to negatively control the
transition state regulator abrB, which in turn controls the
expression of many genes associated with the initiation of
sporulation(42) . In a spo0A mutant we observed a
2-fold reduction of dlt expression, but this repression was
released by an abrB mutation. Spo0A binds to the dlt promoter in vitro, suggesting a direct role in
vivo, whereas AbrB does not bind. However an abrB
mutation can release the Spo0A dependence of dlt transcription, suggesting an indirect negative regulation by the
AbrB protein. The control by the Spo0A and AbrB proteins appears to be
exerted on the -dependent transcription of dlt, since an abrB mutation can increase dlt transcription in a sigD mutant strain.
However a slight reduction in sporulation
efficiency and a motility defect on semisolid agar plates were observed
(data not shown). These results indicate the dispensability of D-alanine ester for growth, but together with the dependence
of dlt transcription on -containing RNA
polymerase suggest some so far unknown role in cell envelope function.
Table: 0p4in
Growth in the absence and presence of
chloramphenicol (5 µg/ml), respectively.
ND,
not determined.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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