Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Jul;178(13):3869–3876. doi: 10.1128/jb.178.13.3869-3876.1996

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

D V Debabov 1, M P Heaton 1, Q Zhang 1, K D Stewart 1, R H Lambalot 1, F C Neuhaus 1
PMCID: PMC232648  PMID: 8682792

Abstract

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 (493.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Archibald A. R., 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. doi: 10.1016/0005-2736(73)90468-9. [DOI] [PubMed] [Google Scholar]
  3. BADDILEY J., NEUHAUS F. C. The enzymic activation of D-alanine. Biochem J. 1960 Jun;75:579–587. doi: 10.1042/bj0750579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brautigan V. M., Childs W. C., 3rd, Neuhaus F. C. Biosynthesis of D-alanyl-lipoteichoic acid in Lactobacillus casei: D-alanyl-lipophilic compounds as intermediates. J Bacteriol. 1981 Apr;146(1):239–250. doi: 10.1128/jb.146.1.239-250.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Childs W. C., 3rd, Neuhaus F. C. 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. doi: 10.1128/jb.143.1.293-301.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cunningham B. C., Jhurani P., Ng P., Wells J. A. Receptor and antibody epitopes in human growth hormone identified by homolog-scanning mutagenesis. Science. 1989 Mar 10;243(4896):1330–1336. doi: 10.1126/science.2466339. [DOI] [PubMed] [Google Scholar]
  7. 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. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dunny G. M., Leonard B. A., Hedberg P. J. Pheromone-inducible conjugation in Enterococcus faecalis: interbacterial and host-parasite chemical communication. J Bacteriol. 1995 Feb;177(4):871–876. doi: 10.1128/jb.177.4.871-876.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Edman P., Begg G. A protein sequenator. Eur J Biochem. 1967 Mar;1(1):80–91. doi: 10.1007/978-3-662-25813-2_14. [DOI] [PubMed] [Google Scholar]
  10. Fenn J. B., Mann M., Meng C. K., Wong S. F., Whitehouse C. M. Electrospray ionization for mass spectrometry of large biomolecules. Science. 1989 Oct 6;246(4926):64–71. doi: 10.1126/science.2675315. [DOI] [PubMed] [Google Scholar]
  11. Fischer W., Rösel P., Koch H. U. 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. doi: 10.1128/jb.146.2.467-475.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Geiger O., Spaink H. P., Kennedy E. P. Isolation of the Rhizobium leguminosarum NodF nodulation protein: NodF carries a 4'-phosphopantetheine prosthetic group. J Bacteriol. 1991 May;173(9):2872–2878. doi: 10.1128/jb.173.9.2872-2878.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Glaser P., Kunst F., Arnaud M., Coudart M. P., Gonzales W., Hullo M. F., Ionescu M., Lubochinsky B., Marcelino L., Moszer I. 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] [Google Scholar]
  14. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  15. Heaton M. P., Neuhaus F. C. 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. doi: 10.1128/jb.174.14.4707-4717.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Heaton M. P., Neuhaus F. C. Role of the D-alanyl carrier protein in the biosynthesis of D-alanyl-lipoteichoic acid. J Bacteriol. 1994 Feb;176(3):681–690. doi: 10.1128/jb.176.3.681-690.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Heptinstall S., Archibald A. R., Baddiley J. Teichoic acids and membrane function in bacteria. Nature. 1970 Feb 7;225(5232):519–521. doi: 10.1038/225519a0. [DOI] [PubMed] [Google Scholar]
  18. Hill R. B., MacKenzie K. R., Flanagan J. M., Cronan J. E., Jr, Prestegard J. H. Overexpression, purification, and characterization of Escherichia coli acyl carrier protein and two mutant proteins. Protein Expr Purif. 1995 Aug;6(4):394–400. doi: 10.1006/prep.1995.1052. [DOI] [PubMed] [Google Scholar]
  19. Hirel P. H., Schmitter M. J., 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. doi: 10.1073/pnas.86.21.8247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Holak T. A., Nilges M., Prestegard J. H., Gronenborn A. M., Clore G. M. 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. doi: 10.1111/j.1432-1033.1988.tb14159.x. [DOI] [PubMed] [Google Scholar]
  21. Hughes A. H., Hancock I. C., Baddiley J. The function of teichoic acids in cation control in bacterial membranes. Biochem J. 1973 Jan;132(1):83–93. doi: 10.1042/bj1320083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hughes C., Stanley P., Koronakis V. E. coli hemolysin interactions with prokaryotic and eukaryotic cell membranes. Bioessays. 1992 Aug;14(8):519–525. doi: 10.1002/bies.950140804. [DOI] [PubMed] [Google Scholar]
  23. Jones T. A., Thirup S. Using known substructures in protein model building and crystallography. EMBO J. 1986 Apr;5(4):819–822. doi: 10.1002/j.1460-2075.1986.tb04287.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Keating D. H., Carey M. R., Cronan J. E., 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. doi: 10.1074/jbc.270.38.22229. [DOI] [PubMed] [Google Scholar]
  25. Khosla C., Ebert-Khosla S., Hopwood D. A. Targeted gene replacements in a Streptomyces polyketide synthase gene cluster: role for the acyl carrier protein. Mol Microbiol. 1992 Nov;6(21):3237–3249. doi: 10.1111/j.1365-2958.1992.tb01778.x. [DOI] [PubMed] [Google Scholar]
  26. Kim Y., Prestegard J. H. Refinement of the NMR structures for acyl carrier protein with scalar coupling data. Proteins. 1990;8(4):377–385. doi: 10.1002/prot.340080411. [DOI] [PubMed] [Google Scholar]
  27. Lambalot R. H., Walsh C. T. Cloning, overproduction, and characterization of the Escherichia coli holo-acyl carrier protein synthase. J Biol Chem. 1995 Oct 20;270(42):24658–24661. doi: 10.1074/jbc.270.42.24658. [DOI] [PubMed] [Google Scholar]
  28. Lambert P. A., Hancock I. C., Baddiley J. Influence of alanyl ester residues on the binding of magnesium ions to teichoic acids. Biochem J. 1975 Dec;151(3):671–676. doi: 10.1042/bj1510671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Linzer R., Neuhaus F. C. Biosynthesis of membrane teichoic acid. A role of the D-alanine-activating enzyme. J Biol Chem. 1973 May 10;248(9):3196–3201. [PubMed] [Google Scholar]
  30. Morris S. A., Revill W. P., Staunton J., Leadlay P. F. 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. doi: 10.1042/bj2940521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nakai K., Kanehisa M. Expert system for predicting protein localization sites in gram-negative bacteria. Proteins. 1991;11(2):95–110. doi: 10.1002/prot.340110203. [DOI] [PubMed] [Google Scholar]
  32. Neuhaus F. C., Heaton M. P., Debabov D. V., 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. doi: 10.1089/mdr.1996.2.77. [DOI] [PubMed] [Google Scholar]
  33. Ntamere A. S., Taron D. J., Neuhaus F. C. 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. doi: 10.1128/jb.169.4.1702-1711.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Oppenheim D. S., Yanofsky C. Translational coupling during expression of the tryptophan operon of Escherichia coli. Genetics. 1980 Aug;95(4):785–795. doi: 10.1093/genetics/95.4.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ou L. T., Marquis R. E. Electromechanical interactions in cell walls of gram-positive cocci. J Bacteriol. 1970 Jan;101(1):92–101. doi: 10.1128/jb.101.1.92-101.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Perego M., Glaser P., Minutello A., Strauch M. A., 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. doi: 10.1074/jbc.270.26.15598. [DOI] [PubMed] [Google Scholar]
  37. Prescott D. J., Vagelos P. R. Acyl carrier protein. Adv Enzymol Relat Areas Mol Biol. 1972;36:269–311. doi: 10.1002/9780470122815.ch8. [DOI] [PubMed] [Google Scholar]
  38. Rawlings M., Cronan J. E., 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] [Google Scholar]
  39. Reusch V. M., Jr, Neuhaus F. C. D-Alanine: membrane acceptor ligase from Lactobacillus casei. J Biol Chem. 1971 Oct 25;246(20):6136–6143. [PubMed] [Google Scholar]
  40. Rock C. O., Cronan J. E., Jr Acyl carrier protein from Escherichia coli. Methods Enzymol. 1981;71(Pt 100):341–351. doi: 10.1016/0076-6879(81)71043-7. [DOI] [PubMed] [Google Scholar]
  41. Rock C. O., Cronan J. E., Jr Re-evaluation of the solution structure of acyl carrier protein. J Biol Chem. 1979 Oct 10;254(19):9778–9785. [PubMed] [Google Scholar]
  42. Rost B., Sander C. Combining evolutionary information and neural networks to predict protein secondary structure. Proteins. 1994 May;19(1):55–72. doi: 10.1002/prot.340190108. [DOI] [PubMed] [Google Scholar]
  43. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., 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. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  44. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Shen B., Summers R. G., Gramajo H., Bibb M. J., Hutchinson C. R. Purification and characterization of the acyl carrier protein of the Streptomyces glaucescens tetracenomycin C polyketide synthase. J Bacteriol. 1992 Jun;174(11):3818–3821. doi: 10.1128/jb.174.11.3818-3821.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Shen Z., Byers D. M. 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. doi: 10.1128/jb.178.2.571-573.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  48. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Therisod H., Kennedy E. P. 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. doi: 10.1073/pnas.84.23.8235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Vanden Boom T., Cronan J. E., Jr Genetics and regulation of bacterial lipid metabolism. Annu Rev Microbiol. 1989;43:317–343. doi: 10.1146/annurev.mi.43.100189.001533. [DOI] [PubMed] [Google Scholar]
  51. 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. doi: 10.1089/mdr.1996.2.123. [DOI] [PubMed] [Google Scholar]

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

RESOURCES