[en] Lipoate is an essential cofactor for key enzymes of oxidative and one-carbon metabolism. It is covalently attached to E2 subunits of dehydrogenase complexes and GcvH, the H subunit of the glycine cleavage system. Bacillus subtilis possess two protein lipoylation pathways: biosynthesis and scavenging. The former requires octanoylation of GcvH, insertion of sulfur atoms and amidotransfer of the lipoate to E2s, catalyzed by LipL. Lipoate scavenging is mediated by a lipoyl protein ligase (LplJ) that catalyzes a classical two-step ATP-dependent reaction. Although these pathways were thought to be redundant, a ∆lipL mutant, in which the endogenous lipoylation pathway of E2 subunits is blocked, showed growth defects in minimal media even when supplemented with lipoate and despite the presence of a functional LplJ. In this study, we demonstrate that LipL is essential to modify E2 subunits of branched chain ketoacid and pyruvate dehydrogenases during lipoate scavenging. The crucial role of LipL during lipoate utilization relies on the strict substrate specificity of LplJ, determined by charge complementarity between the ligase and the lipoylable subunits. This new lipoyl-relay required for lipoate scavenging highlights the relevance of the amidotransferase as a valid target for the design of new antimicrobial agents among Gram-positive pathogens.
Disciplines :
Microbiology
Author, co-author :
Rasetto, Natali Belen ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques ; Instituto de Biología Molecular y Celular de Rosario (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas, and Departamento de Microbiología Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario. Ocampo y Esmeralda, Predio CONICET, Rosario, S2000FHQ, Argentina
Lavatelli, Antonela; Instituto de Biología Molecular y Celular de Rosario (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas, and Departamento de Microbiología Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario. Ocampo y Esmeralda, Predio CONICET, Rosario, S2000FHQ, Argentina
Martin, Natalia; Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, 48824, USA
Mansilla, María Cecilia ; Instituto de Biología Molecular y Celular de Rosario (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas, and Departamento de Microbiología Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario. Ocampo y Esmeralda, Predio CONICET, Rosario, S2000FHQ, Argentina
Language :
English
Title :
Unravelling the lipoyl-relay of exogenous lipoate utilization in Bacillus subtilis.
CONICET - Consejo Nacional de Investigaciones Científicas y Técnicas MCTeIP - Ministerio de Ciencia Tecnología e Innovación Productiva
Funding text :
We gratefully acknowledge Marta Perego for the gift of plasmid pJM134, and John E. Cronan, Jr., for the gift of pQC079. We acknowledge Marina Avecilla and Viviana Villalba for technical support. N. Rasetto was a fellow of Consejo Interuniversitario Nacional (CIN), A. Lavatelli is a Fellow of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), N. Martin was a fellow of CONICET and M.C. Mansilla is a Career Investigator of the same institution. This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (PICT 2012-1341), CONICET (P-UE 2016-IBR) and Ministerio de Ciencia, Tecnología e Innovación Productiva (EULACH 16/T02-0161).
Altschul, S.F. and Lipman, D.J. (1990) Protein database searches for multiple alignments. Proceedings of the National Academy of Sciences, 87(14), 5509–5513.
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. et al. (1987) Current Protocol in Molecular Biology. New York: John Wiley & Sons.
Bachmann, B.J. (1996) Derivations and genotypes of some mutant derivatives of Escherichia coli K-12. In: Neidhart, F.C., Curtiss III, R., Ingraham, J.L., Lin, E.C.C, Low, K.B., Magasanik, B et al. (Eds.) Escherichia coli and Salmonella: Cellular and Molecular Biology. Washington, DC: ASM Press, pp. 2460–2488.
Braakman, R. and Smith, E. (2014) Metabolic evolution of a deep-branching hyperthermophilic chemoautotrophic bacterium. PLoS ONE, 9(2), e87950.
Cao, X., Hong, Y., Zhu, L., Hu, Y. and Cronan, J.E. (2017) Development and retention of a primordial moonlighting pathway of protein modification in the absence of selection presents a puzzle. Proceedings of the National Academy of Sciences, 115(4), 647–655.
Cao, X., Zhu, L., Song, X., Hu, Z. and Cronan, J.E. (2018) Protein moonlighting elucidates the essential human pathway catalyzing lipoic acid assembly on its cognate enzymes. Proceedings of the National Academy of Sciences, 155(30), 7063–7072.
Christensen, Q.H. and Cronan, J.E. (2010) Lipoic acid synthesis: a new family of octanoyltransferases generally annotated as lipoate protein ligases. Biochemistry, 49(64), 10024–10036.
Christensen, Q.H., Hagar, J.A., O'Riordan, M. and Cronan, J.E. (2011a) A complex lipoate utilization pathway in Listeria monocytogenes. Journal of Biological Chemistry, 286(36), 31447–31456.
Christensen, Q.H., Martin, N., Mansilla, M.C., de Mendoza, D. and Cronan, J.E. (2011b) A novel amidotransferase required for lipoic acid cofactor assembly in Bacillus subtilis. Molecular Microbiology, 80(2), 350–363.
Cronan, J.E. (2016) Assembly of lipoic acid on its cognate enzymes: an extraordinary and essential biosynthetic pathway. Microbiology and Molecular Biology Review, 80(2), 429–450.
DeLano, W.L. (2002) The PyMOL User's Manual. San Carlos, CA: DeLano Scientific, 452 p.
Dubnau, D. and Davidoff-Abelson, R. (1971) Fate of transforming DNA following uptake by competent Bacillus subtilis: I. Formation and properties of the donor-recipient complex. Journal of Molecular Biology, 56(2), 209–221.
Fujiwara, K., Okamura-Ikeda, K. and Motokawa, Y. (1996) Lipoylation of acyltransferase components of α-ketoacid dehydrogenase complexes. Journal of Biological Chemistry, 271(22), 12932–12936.
Fujiwara, K., Toma, S., Okamura-Ikeda, K., Motokawa, Y., Nakagawa, A. and Taniguchi, H. (2005) Crystal structure of lipoate-protein ligase A from Escherichia coli. Determination of the lipoic acid-binding site. Journal of Biological Chemistry, 280(39), 33645–33651.
Fujiwara, K., Maita, N., Hosaka, H., Okamura-Ikeda, K., Nakagawa, A. and Taniguchi, H. (2010) Global conformational change associated with the two-step reaction catalyzed by Escherichia coli lipoate-protein ligase A. Journal of Biological Chemistry, 285(13), 9971–9980.
Grayczyk, J.P., Harvey, C.J., Laczkovich, I. and Alonzo, F. (2017) A lipoylated metabolic protein released by Staphylococcus aureus suppresses macrophage activation. Cell Host and Microbe, 22(5), 678–687.
Johansson, P. and Hederstedt, L. (1999) Organization of genes for tetrapyrrole biosynthesis in gram-positive bacteria. Microbiology, 145(3), 529–538.
Jordan, S.W. and Cronan, J.E. (2003) The Escherichia coli lipB gene encodes lipoyl (octanoyl)-acyl carrier protein: protein transferase. Journal of Bacteriology, 185(5), 1582–1589.
Karimova, G., Ullmann, A. and Ladant, D. (2000) A bacterial two-hybrid system that exploits a cAMP signaling cascade in Escherichia coli. Methods in enzymology, 328, 59–73.
Keeney, K.M., Stuckey, J.A. and O'Riordan, M. (2007) LplA1-dependent utilization of host lipoyl peptides enables Listeria cytosolic growth and virulence. Molecular Microbiology, 66(3), 758–770.
Kim, K.H., Lee, H.H., Lee, S.J., Ha, J.Y., Yoon, H.J. and Suh, S.W. (2005) Crystal structure of lipoate-protein ligase A bound with the activated intermediate. Insights into interaction with lipoyl domains. Journal of Biological Chemistry, 280(45), 38081–38089.
Laczkovich, I., Teoh, W.P., Flury, S., Grayczyk, J.P., Zorzoli, A. and Alonzo, F. III. (2018) Increased flexibility in the use of exogenous lipoic acid by Staphylococcus aureus. Molecular Microbiology 109(2), 150–168. Available at: https://doi.org/10.1111/mmi.13970.
Lewis, P.J. and Marston, A.L. (1999) GFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilis. Gene, 227(1), 101–109.
Ma, Q., Zhao, X., Eddine, A.N., Geerlof, A., Li, X. and Cronan, J.E. (2006) The Mycobacterium tuberculosis LipB enzyme functions as a cysteine/lysine dyad acyltransferase. Proceedings of the National Academy of Sciences, 103(23), 8662–8667.
Martin, N., Lombardía, E., Altabe, S.G., de Mendoza, D. and Mansilla, M.C. (2009) A lipA (yutB) mutant, encoding lipoic acid synthase, provides insight into the interplay between branched-chain and unsaturated fatty acid biosynthesis in Bacillus subtilis. Journal of Bacteriology, 191(24), 7447–7455.
Martin, N., Christensen, Q.H., Mansilla, M.C., Cronan, J.E. and de Mendoza, D. (2011) A novel two-gene requirement for the octanoyltransfer reaction of Bacillus subtilis lipoic acid biosynthesis. Molecular Microbiology, 80(2), 335–349.
Middleton, R. and Hofmeister, A. (2004) New shuttle vectors for ectopic insertion of genes into Bacillus subtilis. Plasmid, 51(3), 238–245.
Morris, T.W., Reed, K.E. and Cronan, J.E. (1994) Identification of the gene encoding lipoate-protein ligase A of Escherichia coli. Molecular cloning and characterization of the lplA gene and gene product. Journal of Biological Chemistry, 269(23), 16091–16100.
Morris, T.W., Reed, K.E. and Cronan, J.E. (1995) Lipoic acid metabolism in Escherichia coli : the lplA and lipB genes define redundant pathways for ligation of lipoyl groups to apoprotein. Journal of Bacteriology, 177(1), 1–10.
Notredame, C., Higgins, D.G. and Heringa, J. (2000) T-coffee: a novel method for fast and accurate multiple sequence alignment. Journal Molecular of Biology, 302(1), 205–217.
O'Riordan, M., Moors, M.A. and Portnoy, D.A. (2003) Listeria intracellular growth and virulence require host-derived lipoic acid. Science, 302(5644), 462–465.
Perego, M. (1993) Integrational vectors for genetic manipulation in Bacillus subtilis. In: Sonenshein, J.A.H.A.L. and Losick, R. (Eds.) Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics. Washington, DC: American Society for Microbiology, pp. 615–624.
Perham, R.N. (2000) Swinging arms and swinging domains in multifunctional enzymes: catalytic machines for multistep reactions. Annual Review of Biochemistry, 69(1), 961–1004.
Rachman, H., Strong, M., Ulrichs, T., Grode, L., Schuchhardt, J. and Mollenkopf, H. (2006) Unique transcriptome signature of Mycobacterium tuberculosis in pulmonary tuberculosis. Infection and Immunity, 74(2), 1233–1242.
Reed, K.E. and Cronan, J.E. (1993) Lipoic acid metabolism in Escherichia coli: sequencing and functional characterization of the lipA and lipB genes. Journal of Bacteriology, 175(5), 1325–1336.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Schonauer, M.S., Kastaniotis, A.J., Kursu, V., Hiltunen, J.K. and Dieckmann, C.L. (2009) Lipoic acid synthesis and attachment in yeast mitochondria. Journal of Biological Chemistry, 284(35), 23234–23242.
Sekiguchi, J., Takada, N. and Okada, H. (1975) Genes affecting the productivity of α-amylase in Bacillus subtilis Marburg. Journal of Bacteriology, 121(2), 688–694.
Soreze, Y., Boutron, A., Habarou, F., Barnerias, C., Nonnenmacher, L. and Delpech, H. (2013) Mutations in human lipoyltransferase gene LIPT1 cause a Leigh disease with secondary deficiency for pyruvate and α-ketoglutarate dehydrogenase. Orphanet Journal of Rare Disease 8, 192.
Spizizen, J. (1958) Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proceedings of the National Academy of Sciences, 44(10), 1072–1078.
Zhang, Y. (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, 9(1), 40.
Zhao, X., Miller, J.R., Jiang, Y., Marletta, M.A. and Cronan, J.E. (2003) Assembly of the covalent linkage between lipoic acid and its cognate enzymes. Chemistry & Biology, 10(12), 1293–1302.
Zorzoli, A., Grayczyk, J.P. and Alonzo, F. (2016) Staphylococcus aureus tissue infection during sepsis is supported by differential use of bacterial or host-derived lipoic acid. PLoS Path 12(10), e1005933.
