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• W2087965877 abstract "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 σD-dependent promoter and follows the pattern of transcription of genes belonging to the σD 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. 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 σD-dependent promoter and follows the pattern of transcription of genes belonging to the σD 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. Teichoic acids (TAs)1( 1The abbreviations used are: TAsteichoic acidsLTAlipoteichoic acidWTAwall teichoic aciddltD-alanyl-lipoteichoic acidKmkanamycinACPacyl carrier proteinGlcD-glucopyranosylGlcNAc2-acetamido-2-deoxy-D-glucopyranosyl. )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(3Ward J.B. Microbiol. Rev. 1981; 45: 211-243Crossref PubMed Google Scholar, 4Hancock I.C. Baddiley J. Martonosi A.N. The Enzymes of Biological Membranes. Plenum Press, New York1985: 279-307Crossref Google Scholar), 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(5Fischer W. Handb. Lipid Res. 1990; 6: 123-234Google Scholar, 6Fischer W. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall, New Comprehensive Biochemistry. Elsevier Science Publishers B. V., Amsterdam1994: 199-215Google Scholar). 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(7Glaser L. Burger M.M. J. Biol. Chem. 1964; 239: 3187-3191Abstract Full Text PDF PubMed Google Scholar, 8Iwasaki H. Shimada A. Ito E. J. Bacteriol. 1986; 167: 508-516Crossref PubMed Google Scholar). The LTA is further substituted with N-acetyl-α-D-glucosaminyl, the WTA with α-D-glucopyranosyl residues. teichoic acids lipoteichoic acid wall teichoic acid D-alanyl-lipoteichoic acid kanamycin acyl carrier protein D-glucopyranosyl 2-acetamido-2-deoxy-D-glucopyranosyl. 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(3Ward J.B. Microbiol. Rev. 1981; 45: 211-243Crossref PubMed Google Scholar, 4Hancock I.C. Baddiley J. Martonosi A.N. The Enzymes of Biological Membranes. Plenum Press, New York1985: 279-307Crossref Google Scholar), 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(5Fischer W. Handb. Lipid Res. 1990; 6: 123-234Google Scholar, 6Fischer W. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall, New Comprehensive Biochemistry. Elsevier Science Publishers B. V., Amsterdam1994: 199-215Google Scholar). 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)(1Heaton M.P. Neuhaus F.C. J. Bacteriol. 1992; 174: 4707-4717Crossref PubMed Google Scholar, 9Heaton M.P. Neuhaus F.C. J. Bacteriol. 1994; 176: 681-690Crossref PubMed Google Scholar). 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(2Haas R. Koch H.U. Fischer W. FEMS Microbiol. Lett. 1984; 21: 27-31Crossref Scopus (32) Google Scholar). The loss of D-alanine ester from LTA is continuously compensated for by re-alanylation(10Koch H.U. Döker R. Fischer W. J. Bacteriol. 1985; 164: 1211-1217Crossref PubMed Google Scholar), which requires activated D-alanine on the outer membrane layer and may therefore be accomplished by the same enzyme as the de novo 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(11Maul C. Young M. Margot P. Karamata D. Mol. & Gen. Genet. 1989; 215: 388-394Crossref PubMed Scopus (59) Google Scholar). In contrast to B. subtilis, L. casei is devoid of WTA and contains only LTA(12Kelemen M.V. Baddiley J. Biochem. J. 1961; 80: 246-254Crossref PubMed Scopus (59) Google Scholar). 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(13Ntamere A.S. Taron D.J. Neuhaus F.C. J. Bacteriol. 1987; 169: 1702-1711Crossref PubMed Google Scholar). 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(14Glaser P. Kunst F. Arnaud M. Coudart M.-P. Gonzales W. Hullo M.-F. Ionescu M. Lubochinsky B. Marcelino M. Moszer I. Presecan I. Santana M. Schneider E. Schweizer J. Vertes A. Rapoport G. Danchin A. Mol. Microbiol. 1993; 10: 371-384Crossref PubMed Scopus (167) Google Scholar), and we now describe the deduced gene products and promoter region, the latter suggesting a complex transcriptional regulation of the operon. DNA sequences were analyzed using DNA Strider 1.1 software(15Marck C. Nucleic Acids Res. 1988; 16: 1829-1836Crossref PubMed Scopus (815) Google Scholar). Protein sequence analysis was carried out with the FASTA (16Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Crossref PubMed Scopus (9328) Google Scholar) (in Swissprot release 29) and the BLAST (17Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (68368) Google Scholar) 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). Isogenic B. subtilis strains derivative of JH642 (trpC2, phe-1) used in this study were: JH646 (spo0A12), JH703abr4 (spo0A677, abrB4), JH642::pLM5 (sigD::pLM5cat)(18Helmann J.D. Marquez L.M. Chamberlin M.J. J. Bacteriol. 1988; 170: 1568-1574Crossref PubMed Scopus (85) Google Scholar). B. subtilis strains were grown in Schaeffer's sporulation medium (19Schaeffer P. Millet J. Aubert J.P. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 704-711Crossref PubMed Scopus (864) Google Scholar) 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); NH4Cl, 2 g; K2HPO4, 18.5 g; KH2PO4, 2.5 g; trisodium citrate, 1 g; MgSO4.7H2O, 0.4 g; FeSO4.H2O, 0.01 g; MnSO4.H2O, 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); K2HPO4, 3.68 g; KH2PO4, 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 A578 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 CHCl3. Motility was assessed on semisolid agar plates(20Albertini A.M. Caramori T. Crabb W.D. Scoffone F. Galizzi A. J. Bacteriol. 1991; 173: 3573-3579Crossref PubMed Google Scholar). Transformation of B. subtilis strains by plasmid or chromosomal DNA was performed by standard procedures(21Anagnostopoulos C. Spizizen J. J. Bacteriol. 1961; 81: 741-746Crossref PubMed Google Scholar). Selections for antibiotic resistance were done at the following concentrations: chloramphenicol, 5 μg/ml; kanamycin, 2 μg/ml; erythromycin and lincomycin (MLSR (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 KmR) 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 KmR. Escherichia coli DH5α used for plasmid construction and propagation was grown in LB medium supplemented with ampicillin at 100 μg/ml. Chromosomal DNA from B. subtilis was prepared by the method of Marmur (22Marmur J. J. Mol. Biol. 1961; 3: 208-218Crossref Scopus (8945) Google Scholar) with some modifications. Plasmid DNA from E. coli was purified by the boiling method of Holmes and Quigley(23Holmes D.S. Quigley M. Anal. Biochem. 1981; 114: 193-197Crossref PubMed Scopus (2013) Google Scholar). The plasmids described in this study were constructed in the integrative vector pJM103 that carries a chloramphenicol resistance marker selectable in B. subtilis(24Perego M. Sonenshein A.L. Hoch J.A. Losick R. Bacillus subtilis and Other Gram-positive Bacteria. American Society for Microbiology, Washington, D. C.1993: 615-624Google Scholar). 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 (24Perego M. Sonenshein A.L. Hoch J.A. Losick R. Bacillus subtilis and Other Gram-positive Bacteria. American Society for Microbiology, Washington, D. C.1993: 615-624Google Scholar) 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 (24Perego M. Sonenshein A.L. Hoch J.A. Losick R. Bacillus subtilis and Other Gram-positive Bacteria. American Society for Microbiology, Washington, D. C.1993: 615-624Google Scholar) 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 (24Perego M. Sonenshein A.L. Hoch J.A. Losick R. Bacillus subtilis and Other Gram-positive Bacteria. American Society for Microbiology, Washington, D. C.1993: 615-624Google Scholar) in the EcoRV site. 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 CO2(25Fischer W. Koch H.U. Haas R. Eur. J. Biochem. 1983; 133: 523-530Crossref PubMed Scopus (154) Google Scholar). 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(26Koch H.U. Haas R. Fischer W. Eur. J. Biochem. 1984; 138: 357-363Crossref PubMed Scopus (101) Google Scholar). 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 (27Kates M. Techniques in Lipidology. Elsevier Science Publishing Co., Inc., New York1994Google Scholar) using buffer A instead of water. 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 x g). The supernatant was subjected to hydrolysis with 2 M HCl, 100°C, 2.5 h and then analyzed for phosphorus(28Schnitger H. Papenberg K. Ganse E. Czok R. Bücher T. Adam H. Biochem. J. 1959; 332: 167-185Google Scholar), D-glucose(29Kunst A. Draeger B. Ziegenhorn J. Bermeyer H.U. Bergmeyer J. Grassl M. Methods of Enzymatic Analysis. Verlag Chemie, Weinheim, Germany1984: 163-172Google Scholar), D-alanine (30Grassl M. Supp M. Bermeyer H.U. Bergmeyer J. Grassl M. Methods of Enzymatic Analysis. Verlag Chemie, Weinheim, Germany1985: 336-340Google Scholar), and glycerol(31Nägele U. Wahlefeld A.W. Ziegenhorn J. Bermeyer H.U. Bergmeyer J. Grassl M. Methods of Enzymatic Analysis. Verlag Chemie, Weinheim, Germany1985: 2-12Google Scholar). Glc(α1-2)Gro was identified in HF hydrolysates as trifluoroacetate by gas liquid chromatography(34Leopold K. Fischer W. Eur. J. Biochem. 1991; 196: 475-482Crossref PubMed Scopus (25) Google Scholar). Galactosamine, a constituent of the minor WTA species(37Strauch M.A. Spiegelman G.B. Perego M. Johnson W.C. Burbulys D. Hoch J.A. EMBO J. 1989; 8: 1615-1621Crossref PubMed Scopus (194) Google Scholar), was measured after hydrolysis with 4 M HCl (100°C, 18 h). 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 (33Fischer W. Behr T. Hartmann R. Peter-Katalinic J. Egge H. Eur. J. Biochem. 1993; 215: 851-857Crossref PubMed Scopus (166) Google Scholar). Glc(α1-2)Gro, GlcNAc(α1-2)Gro, and Glc(β1-6)Glc(β1-3)Gro were identified in the HF hydrolysate of deacylated LTA as trifluoroacetates by gas liquid chromatography(34Leopold K. Fischer W. Eur. J. Biochem. 1991; 196: 475-482Crossref PubMed Scopus (25) Google Scholar). 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(α1-2)Gro. 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). Cultures for β-galactosidase assays were grown in Schaeffer's sporulation medium. The assay was carried out as described previously(35Ferrari E. Henner D.J. Perego M. Hoch J.A. J. Bacteriol. 1988; 170: 289-295Crossref PubMed Google Scholar), and the units were calculated according to Miller(36Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 352-355Google Scholar). 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 [α-32P]dATP (Amersham Corp.), and excised with BamHI digestion. DNase I protection experiments with Spo0A and AbrB were performed as described previously(37Strauch M.A. Spiegelman G.B. Perego M. Johnson W.C. Burbulys D. Hoch J.A. EMBO J. 1989; 8: 1615-1621Crossref PubMed Scopus (194) Google Scholar). The labeled fragment was also subjected to Maxam and Gilbert A + G and C + T sequencing reactions (38Maxam A.M. Gilbert W. Methods Enzymol. 1980; 65: 499-560Crossref PubMed Scopus (8988) Google Scholar) to generate a reference ladder. 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) (39Turgay K. Krause M. Marahiel M.A. Mol. Microbiol. 1992; 6: 529-546Crossref PubMed Scopus (170) Google Scholar) 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 (1Heaton M.P. Neuhaus F.C. J. Bacteriol. 1992; 174: 4707-4717Crossref PubMed Google Scholar) 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. Protein sequence comparison of the B. subtilis DltB protein with the first 188 codons of L. casei dltB gene product (1Heaton M.P. Neuhaus F.C. J. Bacteriol. 1992; 174: 4707-4717Crossref PubMed Google Scholar) (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). 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 (9Heaton M.P. Neuhaus F.C. J. Bacteriol. 1994; 176: 681-690Crossref PubMed Google Scholar) 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). 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 (40Rawlings M. Cronan Jr., J.E. J. Biol. Chem. 1992; 267: 5751-5754Abstract Full Text PDF PubMed Google Scholar) (Fig. 2C). The deduced protein sequence of DltE suggests a cytoplasmic localization. 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(24Perego M. Sonenshein A.L. Hoch J.A. Losick R. Bacillus subtilis and Other Gram-positive Bacteria. American Society for Microbiology, Washington, D. C.1993: 615-624Google Scholar). 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. 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.2( 2J. Wecke, M. Perego, and W. Fischer, unpublished data. )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. 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(25Fischer W. Koch H.U. Haas R. Eur. J. Biochem. 1983; 133: 523-530Crossref PubMed Scopus (154) Google Scholar, 41Fischer W. Koch H.U. Rösel P. Fiedler F. J. Biol. Chem. 1980; 255: 4557-4562Abstract Full Text PDF PubMed Google Scholar). 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(β1-3)GalNAc-1-P repeats (32Shibaev V.N. Duckworth M. Archibald A.R. Baddiley J. Biochem. J. 1973; 135: 383-384Crossref PubMed Scopus (27) Google Scholar) did not contribute more than 9 ± 2% to total WTA phosphorus, as was estimated by galactosamine measurement in WTA hydrolysates. In a first series of experiments, bacteria were grown in medium A. As shown in Table I, 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-α-D-glucosaminyl, and less than 1% with α-D-glucopyranosyl residues. In WTA 9% of the glycerophosphate moieties were substituted with D-alanine ester and 64% with α-D-glucopyranosyl residues.Table I:LTA and WTA of B. subtilis wild type and dlt mutant strains during vegetative growth Open table in a new tab 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 (Table I). 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 (Table I). 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" @default.
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