title: Diaminopimelic Acid Amidation in Corynebacteriales: NEW INSIGHTS INTO THE ROLE OF LtsA IN PEPTIDOGLYCAN MODIFICATION.
abstract: A gene named ltsA was earlier identified in Rhodococcus and Corynebacterium species while screening for mutations leading to increased cell susceptibility to lysozyme. The encoded protein belonged to a huge family of glutamine amidotransferases whose members catalyze amide nitrogen transfer from glutamine to various specific acceptor substrates. We here describe detailed physiological and biochemical investigations demonstrating the specific role of LtsA protein from Corynebacterium glutamicum (LtsACg) in the modification by amidation of cell wall peptidoglycan diaminopimelic acid (DAP) residues. A morphologically altered but viable ΔltsA mutant was generated, which displays a high susceptibility to lysozyme and β-lactam antibiotics. Analysis of its peptidoglycan structure revealed a total loss of DAP amidation, a modification that was found in 80% of DAP residues in the wild-type polymer. The cell peptidoglycan content and cross-linking were otherwise not modified in the mutant. Heterologous expression of LtsACg in Escherichia coli yielded a massive and toxic incorporation of amidated DAP into the peptidoglycan that ultimately led to cell lysis. In vitro assays confirmed the amidotransferase activity of LtsACg and showed that this enzyme used the peptidoglycan lipid intermediates I and II but not, or only marginally, the UDP-MurNAc pentapeptide nucleotide precursor as acceptor substrates. As is generally the case for glutamine amidotransferases, either glutamine or NH4(+) could serve as the donor substrate for LtsACg. The enzyme did not amidate tripeptide- and tetrapeptide-truncated versions of lipid I, indicating a strict specificity for a pentapeptide chain length.
authors.affiliation: From the Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris Sud, F-91198 Gif-sur-Yvette, France.
authors.forename: Marjorie
authors.initials: M
authors.lastname: Levefaudes
authors.affiliation: From the Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris Sud, F-91198 Gif-sur-Yvette, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Patin
authors.affiliation: From the Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris Sud, F-91198 Gif-sur-Yvette, France.
authors.forename: Célia
authors.initials: C
authors.lastname: de Sousa-d'Auria
authors.affiliation: the Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, CH-4058 Basel, Switzerland.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Chami
authors.affiliation: From the Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris Sud, F-91198 Gif-sur-Yvette, France.
authors.forename: Didier
authors.initials: D
authors.lastname: Blanot
authors.affiliation: From the Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris Sud, F-91198 Gif-sur-Yvette, France.
authors.forename: Mireille
authors.initials: M
authors.lastname: Hervé
authors.affiliation: INSERM, UMR S1138, Centre de Recherche des Cordeliers, Equipe 12, F-75006 Paris, France, the Sorbonne Universités, UPMC Université Paris 06, UMR S1138, Centre de Recherche des Cordeliers, F-75006 Paris, France, and the Université Paris-Descartes, Sorbonne Paris Cité, UMR S1138, Centre de Recherche des Cordeliers, F-75006, Paris, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Arthur
authors.affiliation: From the Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris Sud, F-91198 Gif-sur-Yvette, France, christine.houssin@u-psud.fr.
authors.forename: Christine
authors.initials: C
authors.lastname: Houssin
authors.affiliation: From the Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris Sud, F-91198 Gif-sur-Yvette, France, dominique.mengin-lecreulx@u-psud.fr.
authors.forename: Dominique
authors.initials: D
authors.lastname: Mengin-Lecreulx
mesh_terms: D000577:Amides / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002473:Cell Wall / Q000378:metabolism; D002478:Cells, Cultured; D003352:Corynebacterium / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D003960:Diaminopimelic Acid / Q000737:chemistry* / Q000378:metabolism; D007124:Immunoenzyme Techniques; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D009113:Muramidase / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010457:Peptidoglycan / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000637:Transaminases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000577:Amides; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D010457:Peptidoglycan; D012333:RNA, Messenger; D003960:Diaminopimelic Acid; D000637:Transaminases; C020656:glutamine-pyruvate aminotransferase; D009113:Muramidase
keywords: Corynebacteriales; DAP amidation; antibiotics; bacterial metabolism; cell wall; enzyme; gene knockout; glutaminase; lysozyme; peptidoglycan
references.citation: Mol Microbiol. 2013 Jul;89(1):1-13
references.pmid: 23679048
references.citation: FEMS Microbiol Rev. 2008 Mar;32(2):149-67
references.pmid: 18194336
references.citation: FEMS Microbiol Rev. 2008 Mar;32(2):287-306
references.pmid: 18070068
references.citation: Biochemistry. 1999 Dec 7;38(49):16146-57
references.pmid: 10587437
references.citation: J Bacteriol. 2000 May;182(10):2696-701
references.pmid: 10781535
references.citation: Microbiology. 2001 May;147(Pt 5):1365-82
references.pmid: 11320139
references.citation: Glycobiology. 2001 Sep;11(9):107R-118R
references.pmid: 11555614
references.citation: J Biol Chem. 2003 Oct 24;278(43):41702-8
references.pmid: 12871942
references.citation: Proc Natl Acad Sci U S A. 2004 Jan 6;101(1):314-9
references.pmid: 14695899
references.citation: J Biol Chem. 2004 Jul 16;279(29):29974-80
references.pmid: 15131133
references.citation: J Biol Chem. 2004 Oct 1;279(40):41546-56
references.pmid: 15280360
references.citation: Biochem Biophys Res Commun. 1969 May 22;35(4):478-85
references.pmid: 5788504
references.citation: J Biol Chem. 1972 Feb 25;247(4):1180-7
references.pmid: 4621974
references.citation: Bacteriol Rev. 1972 Dec;36(4):407-77
references.pmid: 4568761
references.citation: J Mol Biol. 1973 Nov 15;80(4):575-99
references.pmid: 4204102
references.citation: Anal Biochem. 1976 May 7;72:248-54
references.pmid: 942051
references.citation: J Gen Microbiol. 1976 Sep;96(1):165-74
references.pmid: 10345
references.citation: Biochim Biophys Acta. 1978 Dec 8;527(2):414-24
references.pmid: 215212
references.citation: Gene. 1979 May;6(1):23-8
references.pmid: 383576
references.citation: J Bacteriol. 1980 Apr;142(1):212-20
references.pmid: 6102982
references.citation: J Bacteriol. 1982 Sep;151(3):1109-17
references.pmid: 6125497
references.citation: J Bacteriol. 2005 Apr;187(8):2747-57
references.pmid: 15805521
references.citation: J Bacteriol. 2005 Apr;187(8):2582-91
references.pmid: 15805504
references.citation: J Bacteriol. 1983 Jun;154(3):1284-90
references.pmid: 6222035
references.citation: Antimicrob Agents Chemother. 2006 Jan;50(1):250-5
references.pmid: 16377694
references.citation: J Biol Chem. 2006 Aug 11;281(32):22761-72
references.pmid: 16777846
references.citation: J Bacteriol. 2007 Jan;189(1):280-3
references.pmid: 17085565
references.citation: Can J Microbiol. 1984 May;30(5):553-9
references.pmid: 6430537
references.citation: J Bacteriol. 1985 Jul;163(1):208-12
references.pmid: 3891726
references.citation: Anal Biochem. 1988 Aug 1;172(2):451-64
references.pmid: 3056100
references.citation: FEMS Microbiol Lett. 1990 Jan 1;54(1-3):263-9
references.pmid: 2108897
references.citation: J Biol Chem. 1990 Oct 25;265(30):18200-6
references.pmid: 2211696
references.citation: J Bacteriol. 1991 Aug;173(15):4625-36
references.pmid: 1649817
references.citation: J Biol Chem. 1994 Mar 11;269(10):7450-7
references.pmid: 7907328
references.citation: Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3963-7
references.pmid: 18316738
references.citation: J Bacteriol. 2008 Jun;190(12):4360-6
references.pmid: 18408028
references.citation: J Bacteriol. 2008 Aug;190(16):5672-80
references.pmid: 18567661
references.citation: J Bacteriol. 2009 Jun;191(11):3752-7
references.pmid: 19329637
references.citation: J Bacteriol. 2009 Jun;191(11):3657-64
references.pmid: 19346308
references.citation: Mol Microbiol. 2009 Nov;74(3):650-61
references.pmid: 19807868
references.citation: Nat Med. 2010 Apr;16(4):466-9
references.pmid: 20305661
references.citation: J Bacteriol. 2011 Feb;193(3):778-82
references.pmid: 21097619
references.citation: J Bacteriol. 2011 Nov;193(22):6266-75
references.pmid: 21926230
references.citation: J Bacteriol. 2011 Nov;193(22):6323-30
references.pmid: 21949063
references.citation: J Bacteriol. 2012 Feb;194(3):587-97
references.pmid: 22123248
references.citation: PLoS Pathog. 2012 Jan;8(1):e1002509
references.pmid: 22291598
references.citation: PLoS Pathog. 2012 Jan;8(1):e1002508
references.pmid: 22303291
references.citation: Mol Microbiol. 2012 Oct;86(2):367-81
references.pmid: 22906310
references.citation: J Bacteriol. 1998 Sep;180(17):4603-12
references.pmid: 9721302
references.citation: Appl Environ Microbiol. 1999 Apr;65(4):1530-9
references.pmid: 10103247
references.citation: J Bacteriol. 1999 Jul;181(13):3956-66
references.pmid: 10383963
references.citation: Mol Microbiol. 2005 Feb;55(3):778-87
references.pmid: 15661003
references.citation: J Bacteriol. 2005 Apr;187(7):2341-7
references.pmid: 15774877
references.citation: J Bacteriol. 2008 Jan;190(2):655-61
references.pmid: 18024514
references.citation: Mol Microbiol. 2013 Aug;89(4):612-25
references.pmid: 23782343