title: Co-Inactivation of GlnR and CodY Regulators Impacts Pneumococcal Cell Wall Physiology.
abstract: CodY, a nutritional regulator highly conserved in low G+C Gram-positive bacteria, is essential in Streptococcus pneumoniae (the pneumococcus). A published codY mutant possessed suppressing mutations inactivating the fatC and amiC genes, respectively belonging to iron (Fat/Fec) and oligopeptide (Ami) ABC permease operons, which are directly repressed by CodY. Here we analyzed two additional published codY mutants to further explore the essentiality of CodY. We show that one, in which the regulator of glutamine/glutamate metabolism glnR had been inactivated by design, had only a suppressor in fecE (a gene in the fat/fec operon), while the other possessed both fecE and amiC mutations. Independent isolation of three different fat/fec suppressors thus establishes that reduction of iron import is crucial for survival without CodY. We refer to these as primary suppressors, while inactivation of ami, which is not essential for survival of codY mutants and acquired after initial fat/fec inactivation, can be regarded as a secondary suppressor. The availability of codY- ami+ cells allowed us to establish that CodY activates competence for genetic transformation indirectly, presumably by repressing ami which is known to antagonize competence. The glnR codY fecE mutant was then found to be only partially viable on solid medium and hypersensitive to peptidoglycan (PG) targeting agents such as the antibiotic cefotaxime and the muramidase lysozyme. While analysis of PG and teichoic acid composition uncovered no alteration in the glnR codY fecE mutant compared to wildtype, electron microscopy revealed altered ultrastructure of the cell wall in the mutant, establishing that co-inactivation of GlnR and CodY regulators impacts pneumococcal cell wall physiology. In light of rising levels of resistance to PG-targeting antibiotics of natural pneumococcal isolates, GlnR and CodY constitute potential alternative therapeutic targets to combat this debilitating pathogen, as co-inactivation of these regulators renders pneumococci sensitive to iron and PG-targeting agents.
authors.affiliation: Centre National de la Recherche Scientifique, LMGM-UMR5100, F-31000 Toulouse, France; Université de Toulouse, UPS, Laboratoire de Microbiologie et Génétique Moléculaires, F-31000 Toulouse, France.
authors.forename: Calum
authors.initials: C
authors.lastname: Johnston
authors.affiliation: Laboratory of Pediatric Infectious Diseases, Radboud University Medical Centre, 6500 HB Nijmegen, The Netherlands.
authors.forename: Hester J
authors.initials: HJ
authors.lastname: Bootsma
authors.affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Richardson Road, Newcastle upon Tyne NE2 4AX, United Kingdom.
authors.forename: Christine
authors.initials: C
authors.lastname: Aldridge
authors.affiliation: Bases Moléculaires et Structurales des Systèmes Infectieux, UMR 5086, Université Lyon 1, CNRS, IBCP, F-69007 Lyon, France.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Manuse
authors.affiliation: Research Center Borstel, Leibniz-Center for Medicine and Biosciences, 23845 Borstel, Germany.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Gisch
authors.affiliation: Research Center Borstel, Leibniz-Center for Medicine and Biosciences, 23845 Borstel, Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Schwudke
authors.affiliation: Laboratory of Pediatric Infectious Diseases, Radboud University Medical Centre, 6500 HB Nijmegen, The Netherlands.
authors.forename: Peter W M
authors.initials: PW
authors.lastname: Hermans
authors.affiliation: Bases Moléculaires et Structurales des Systèmes Infectieux, UMR 5086, Université Lyon 1, CNRS, IBCP, F-69007 Lyon, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Grangeasse
authors.affiliation: Centre National de la Recherche Scientifique, LMGM-UMR5100, F-31000 Toulouse, France; Université de Toulouse, UPS, Laboratoire de Microbiologie et Génétique Moléculaires, F-31000 Toulouse, France.
authors.forename: Patrice
authors.initials: P
authors.lastname: Polard
authors.affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Richardson Road, Newcastle upon Tyne NE2 4AX, United Kingdom.
authors.forename: Waldemar
authors.initials: W
authors.lastname: Vollmer
authors.affiliation: Centre National de la Recherche Scientifique, LMGM-UMR5100, F-31000 Toulouse, France; Université de Toulouse, UPS, Laboratoire de Microbiologie et Génétique Moléculaires, F-31000 Toulouse, France.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Claverys
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002439:Cefotaxime / Q000494:pharmacology; D002473:Cell Wall / Q000187:drug effects / Q000378:metabolism*; D006868:Hydrolysis; D009154:Mutation; D013296:Streptococcus pneumoniae / Q000166:cytology* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D001426:Bacterial Proteins; D000581:Amidohydrolases; C019665:amidase; D002439:Cefotaxime