Search tips
Search criteria 


Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. 1996 July; 178(13): 3869–3876.
PMCID: PMC232648

The D-Alanyl carrier protein in Lactobacillus casei: cloning, sequencing, and expression of dltC.


The incorporation of D-alanine into membrane-associated D-alanyl-lipoteichoic acid in Lactobacillus casei requires the 56-kDa D-alanine-D-alanyl carrier protein ligase (Dcl) and the 8.9-kDa D-alanyl carrier protein (Dcp). To identify and isolate the gene encoding Dcp, we have cloned and sequenced a 4.3-kb chromosomal fragment that contains dcl (dltA). In addition to this gene, the fragment contains three other genes, dltB, d1tC, and a partial dltD gene. dltC (246 nucleotides) was subcloned from this region and expressed in Escherichia coli. The product was identified as apo-Dcp lacking the N-terminal methionine (8,787.9 Da). The in vitro conversion of the recombinant apo-Dcp to holo-Dcp by recombinant E. coli holo-ACP synthase provided Dcp which accepts activated D-alanine in the reaction catalyzed by Bcl. The recombinant D-alanyl-Dcp was functionally identical to native D-alanyl-Dcp in the incorporation of D-alanine into lipoteichoic acid. L. casei Dcp is 46% identical to the putative product of dltC in the Bacillus subtilis dlt operon (M. Perego, P. Glaser, A. Minutello, M. A. Strauch, K. Leopold, and W. Fischer, J. Biol. Chem. 270:15598-15606, 1995), and therefore, this gene also encodes Dcp. Comparisons of the primary sequences and predicted secondary structures of the L. casei and B. subtilis Dcps with that of the E. coli acyl carrier protein (ACP) were undertaken together with homology modeling to identify the functional determinants of the donor and acceptor specificities of Dcp. In the region of the phospho-pantetheine attachment site, significant similarity between Dcps and ACPs was observed. This similarity may account for the relaxed acceptor specificity of the Dcps and ACPs in the ligation Of D-alanine catalyzed by Dcl. In contrast, two Dcp consensus sequences, KXXVLDXLA and DXVKXNXD, share little identity with the rest of the ACP family and, thus, may determine the donor specificity of D-alanyl-Dcp in the D-alanylation of membrane-associated D-alanyl-lipoteichoic acid.

Full Text

The Full Text of this article is available as a PDF (493K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. [PubMed]
  • Archibald AR, Baddiley J, Heptinstall S. The alanine ester content and magnesium binding capacity of walls of Staphylococcus aureus H grown at different pH values. Biochim Biophys Acta. 1973 Feb 16;291(3):629–634. [PubMed]
  • BADDILEY J, NEUHAUS FC. The enzymic activation of D-alanine. Biochem J. 1960 Jun;75:579–587. [PubMed]
  • Brautigan VM, Childs WC, 3rd, Neuhaus FC. Biosynthesis of D-alanyl-lipoteichoic acid in Lactobacillus casei: D-alanyl-lipophilic compounds as intermediates. J Bacteriol. 1981 Apr;146(1):239–250. [PMC free article] [PubMed]
  • Childs WC, 3rd, Neuhaus FC. Biosynthesis of D-alanyl-lipoteichoic acid: characterization of ester-linked D-alanine in the in vitro-synthesized product. J Bacteriol. 1980 Jul;143(1):293–301. [PMC free article] [PubMed]
  • Cunningham BC, Jhurani P, Ng P, Wells JA. Receptor and antibody epitopes in human growth hormone identified by homolog-scanning mutagenesis. Science. 1989 Mar 10;243(4896):1330–1336. [PubMed]
  • Devereux J, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. [PMC free article] [PubMed]
  • Dunny GM, Leonard BA, Hedberg PJ. Pheromone-inducible conjugation in Enterococcus faecalis: interbacterial and host-parasite chemical communication. J Bacteriol. 1995 Feb;177(4):871–876. [PMC free article] [PubMed]
  • Edman P, Begg G. A protein sequenator. Eur J Biochem. 1967 Mar;1(1):80–91. [PubMed]
  • Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM. Electrospray ionization for mass spectrometry of large biomolecules. Science. 1989 Oct 6;246(4926):64–71. [PubMed]
  • Fischer W, Rösel P, Koch HU. Effect of alanine ester substitution and other structural features of lipoteichoic acids on their inhibitory activity against autolysins of Staphylococcus aureus. J Bacteriol. 1981 May;146(2):467–475. [PMC free article] [PubMed]
  • Geiger O, Spaink HP, Kennedy EP. Isolation of the Rhizobium leguminosarum NodF nodulation protein: NodF carries a 4'-phosphopantetheine prosthetic group. J Bacteriol. 1991 May;173(9):2872–2878. [PMC free article] [PubMed]
  • Glaser P, Kunst F, Arnaud M, Coudart MP, Gonzales W, Hullo MF, Ionescu M, Lubochinsky B, Marcelino L, Moszer I, et al. Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325 degrees to 333 degrees. Mol Microbiol. 1993 Oct;10(2):371–384. [PubMed]
  • Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. [PubMed]
  • Heaton MP, Neuhaus FC. Biosynthesis of D-alanyl-lipoteichoic acid: cloning, nucleotide sequence, and expression of the Lactobacillus casei gene for the D-alanine-activating enzyme. J Bacteriol. 1992 Jul;174(14):4707–4717. [PMC free article] [PubMed]
  • Heaton MP, Neuhaus FC. Role of the D-alanyl carrier protein in the biosynthesis of D-alanyl-lipoteichoic acid. J Bacteriol. 1994 Feb;176(3):681–690. [PMC free article] [PubMed]
  • Heptinstall S, Archibald AR, Baddiley J. Teichoic acids and membrane function in bacteria. Nature. 1970 Feb 7;225(5232):519–521. [PubMed]
  • Hill RB, MacKenzie KR, Flanagan JM, Cronan JE, Jr, Prestegard JH. Overexpression, purification, and characterization of Escherichia coli acyl carrier protein and two mutant proteins. Protein Expr Purif. 1995 Aug;6(4):394–400. [PubMed]
  • Hirel PH, Schmitter MJ, Dessen P, Fayat G, Blanquet S. Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8247–8251. [PubMed]
  • Holak TA, Nilges M, Prestegard JH, Gronenborn AM, Clore GM. Three-dimensional structure of acyl carrier protein in solution determined by nuclear magnetic resonance and the combined use of dynamical simulated annealing and distance geometry. Eur J Biochem. 1988 Jul 15;175(1):9–15. [PubMed]
  • Hughes AH, Hancock IC, Baddiley J. The function of teichoic acids in cation control in bacterial membranes. Biochem J. 1973 Jan;132(1):83–93. [PubMed]
  • Hughes C, Stanley P, Koronakis V. E. coli hemolysin interactions with prokaryotic and eukaryotic cell membranes. Bioessays. 1992 Aug;14(8):519–525. [PubMed]
  • Jones TA, Thirup S. Using known substructures in protein model building and crystallography. EMBO J. 1986 Apr;5(4):819–822. [PubMed]
  • Keating DH, Carey MR, Cronan JE., Jr The unmodified (apo) form of Escherichia coli acyl carrier protein is a potent inhibitor of cell growth. J Biol Chem. 1995 Sep 22;270(38):22229–22235. [PubMed]
  • Khosla C, Ebert-Khosla S, Hopwood DA. Targeted gene replacements in a Streptomyces polyketide synthase gene cluster: role for the acyl carrier protein. Mol Microbiol. 1992 Nov;6(21):3237–3249. [PubMed]
  • Kim Y, Prestegard JH. Refinement of the NMR structures for acyl carrier protein with scalar coupling data. Proteins. 1990;8(4):377–385. [PubMed]
  • Lambalot RH, Walsh CT. Cloning, overproduction, and characterization of the Escherichia coli holo-acyl carrier protein synthase. J Biol Chem. 1995 Oct 20;270(42):24658–24661. [PubMed]
  • Lambert PA, Hancock IC, Baddiley J. Influence of alanyl ester residues on the binding of magnesium ions to teichoic acids. Biochem J. 1975 Dec;151(3):671–676. [PubMed]
  • Linzer R, Neuhaus FC. Biosynthesis of membrane teichoic acid. A role of the D-alanine-activating enzyme. J Biol Chem. 1973 May 10;248(9):3196–3201. [PubMed]
  • Morris SA, Revill WP, Staunton J, Leadlay PF. Purification and separation of holo- and apo-forms of Saccharopolyspora erythraea acyl-carrier protein released from recombinant Escherichia coli by freezing and thawing. Biochem J. 1993 Sep 1;294(Pt 2):521–527. [PubMed]
  • Nakai K, Kanehisa M. Expert system for predicting protein localization sites in gram-negative bacteria. Proteins. 1991;11(2):95–110. [PubMed]
  • Neuhaus FC, Heaton MP, Debabov DV, Zhang Q. The dlt operon in the biosynthesis of D-alanyl-lipoteichoic acid in Lactobacillus casei. Microb Drug Resist. 1996 Spring;2(1):77–84. [PubMed]
  • Ntamere AS, Taron DJ, Neuhaus FC. Assembly of D-alanyl-lipoteichoic acid in Lactobacillus casei: mutants deficient in the D-alanyl ester content of this amphiphile. J Bacteriol. 1987 Apr;169(4):1702–1711. [PMC free article] [PubMed]
  • Oppenheim DS, Yanofsky C. Translational coupling during expression of the tryptophan operon of Escherichia coli. Genetics. 1980 Aug;95(4):785–795. [PubMed]
  • Ou LT, Marquis RE. Electromechanical interactions in cell walls of gram-positive cocci. J Bacteriol. 1970 Jan;101(1):92–101. [PMC free article] [PubMed]
  • Perego M, Glaser P, Minutello A, Strauch MA, Leopold K, Fischer W. Incorporation of D-alanine into lipoteichoic acid and wall teichoic acid in Bacillus subtilis. Identification of genes and regulation. J Biol Chem. 1995 Jun 30;270(26):15598–15606. [PubMed]
  • Prescott DJ, Vagelos PR. Acyl carrier protein. Adv Enzymol Relat Areas Mol Biol. 1972;36:269–311. [PubMed]
  • Rawlings M, Cronan JE., Jr The gene encoding Escherichia coli acyl carrier protein lies within a cluster of fatty acid biosynthetic genes. J Biol Chem. 1992 Mar 25;267(9):5751–5754. [PubMed]
  • Reusch VM, Jr, Neuhaus FC. D-Alanine: membrane acceptor ligase from Lactobacillus casei. J Biol Chem. 1971 Oct 25;246(20):6136–6143. [PubMed]
  • Rock CO, Cronan JE., Jr Re-evaluation of the solution structure of acyl carrier protein. J Biol Chem. 1979 Oct 10;254(19):9778–9785. [PubMed]
  • Rock CO, Cronan JE., Jr Acyl carrier protein from Escherichia coli. Methods Enzymol. 1981;71(Pt 100):341–351. [PubMed]
  • Rost B, Sander C. Combining evolutionary information and neural networks to predict protein secondary structure. Proteins. 1994 May;19(1):55–72. [PubMed]
  • Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PubMed]
  • Shen B, Summers RG, Gramajo H, Bibb MJ, Hutchinson CR. Purification and characterization of the acyl carrier protein of the Streptomyces glaucescens tetracenomycin C polyketide synthase. J Bacteriol. 1992 Jun;174(11):3818–3821. [PMC free article] [PubMed]
  • Shen Z, Byers DM. Isolation of Vibrio harveyi acyl carrier protein and the fabG, acpP, and fabF genes involved in fatty acid biosynthesis. J Bacteriol. 1996 Jan;178(2):571–573. [PMC free article] [PubMed]
  • Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. [PubMed]
  • Tabor S, Richardson CC. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. [PubMed]
  • Therisod H, Kennedy EP. The function of acyl carrier protein in the synthesis of membrane-derived oligosaccharides does not require its phosphopantetheine prosthetic group. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8235–8238. [PubMed]
  • Vanden Boom T, Cronan JE., Jr Genetics and regulation of bacterial lipid metabolism. Annu Rev Microbiol. 1989;43:317–343. [PubMed]
  • Wecke J, Perego M, Fischer W. D-alanine deprivation of Bacillus subtilis teichoic acids is without effect on cell growth and morphology but affects the autolytic activity. Microb Drug Resist. 1996 Spring;2(1):123–129. [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)