title: Systematic production of inactivating and non-inactivating suppressor mutations at the relA locus that compensate the detrimental effects of complete spot loss and affect glycogen content in Escherichia coli.
abstract: In Escherichia coli, ppGpp is a major determinant of growth and glycogen accumulation. Levels of this signaling nucleotide are controlled by the balanced activities of the ppGpp RelA synthetase and the dual-function hydrolase/synthetase SpoT. Here we report the construction of spoT null (ΔspoT) mutants obtained by transducing a ΔspoT allele from ΔrelAΔspoT double mutants into relA+ cells. Iodine staining of randomly selected transductants cultured on a rich complex medium revealed differences in glycogen content among them. Sequence and biochemical analyses of 8 ΔspoT clones displaying glycogen-deficient phenotypes revealed different inactivating mutations in relA and no detectable ppGpp when cells were cultured on a rich complex medium. Remarkably, although the co-existence of ΔspoT with relA proficient alleles has generally been considered synthetically lethal, we found that 11 ΔspoT clones displaying high glycogen phenotypes possessed relA mutant alleles with non-inactivating mutations that encoded stable RelA proteins and ppGpp contents reaching 45-85% of those of wild type cells. None of the ΔspoT clones, however, could grow on M9-glucose minimal medium. Both Sanger sequencing of specific genes and high-throughput genome sequencing of the ΔspoT clones revealed that suppressor mutations were restricted to the relA locus. The overall results (a) defined in around 4 nmoles ppGpp/g dry weight the threshold cellular levels that suffice to trigger net glycogen accumulation, (b) showed that mutations in relA, but not necessarily inactivating mutations, can be selected to compensate total SpoT function(s) loss, and (c) provided useful tools for studies of the in vivo regulation of E. coli RelA ppGpp synthetase.
authors.affiliation: Instituto de Agrobiotecnología, UPNA/CSIC/Gobierno de Navarra, Mutiloabeti, Nafarroa, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Montero
authors.affiliation: Instituto de Agrobiotecnología, UPNA/CSIC/Gobierno de Navarra, Mutiloabeti, Nafarroa, Spain.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Rahimpour
authors.affiliation: Instituto de Agrobiotecnología, UPNA/CSIC/Gobierno de Navarra, Mutiloabeti, Nafarroa, Spain; Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET), Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: Alejandro M
authors.initials: AM
authors.lastname: Viale
authors.affiliation: Instituto de Agrobiotecnología, UPNA/CSIC/Gobierno de Navarra, Mutiloabeti, Nafarroa, Spain.
authors.forename: Goizeder
authors.initials: G
authors.lastname: Almagro
authors.affiliation: Instituto de Agrobiotecnología, UPNA/CSIC/Gobierno de Navarra, Mutiloabeti, Nafarroa, Spain.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Eydallin
authors.affiliation: Departamento de Bioquímica y Biología Molecular e Inmunología, Facultad de Química, Campus de Excelencia Internacional Regional "Campus Mare Nostrum", Universidad de Murcia, Murcia, Spain.
authors.forename: Ángel
authors.initials: Á
authors.lastname: Sevilla
authors.affiliation: Departamento de Bioquímica y Biología Molecular e Inmunología, Facultad de Química, Campus de Excelencia Internacional Regional "Campus Mare Nostrum", Universidad de Murcia, Murcia, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Cánovas
authors.affiliation: Departamento de Bioquímica y Biología Molecular e Inmunología, Facultad de Química, Campus de Excelencia Internacional Regional "Campus Mare Nostrum", Universidad de Murcia, Murcia, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Bernal
authors.affiliation: Departamento de Bioquímica y Biología Molecular e Inmunología, Facultad de Química, Campus de Excelencia Internacional Regional "Campus Mare Nostrum", Universidad de Murcia, Murcia, Spain.
authors.forename: Ana Belén
authors.initials: AB
authors.lastname: Lozano
authors.affiliation: Instituto de Agrobiotecnología, UPNA/CSIC/Gobierno de Navarra, Mutiloabeti, Nafarroa, Spain.
authors.forename: Francisco José
authors.initials: FJ
authors.lastname: Muñoz
authors.affiliation: Instituto de Agrobiotecnología, UPNA/CSIC/Gobierno de Navarra, Mutiloabeti, Nafarroa, Spain.
authors.forename: Edurne
authors.initials: E
authors.lastname: Baroja-Fernández
authors.affiliation: Instituto de Agrobiotecnología, UPNA/CSIC/Gobierno de Navarra, Mutiloabeti, Nafarroa, Spain.
authors.forename: Abdellatif
authors.initials: A
authors.lastname: Bahaji
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara, Japan.
authors.forename: Hirotada
authors.initials: H
authors.lastname: Mori
authors.affiliation: Lifesequencing S.L., Paterna, Valencia, Spain.
authors.forename: Francisco M
authors.initials: FM
authors.lastname: Codoñer
authors.affiliation: Instituto de Agrobiotecnología, UPNA/CSIC/Gobierno de Navarra, Mutiloabeti, Nafarroa, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Pozueta-Romero
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D002999:Clone Cells; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D056426:Genetic Loci; D005838:Genotype; D006003:Glycogen / Q000235:genetics* / Q000378:metabolism; D008025:Ligases / Q000172:deficiency / Q000235:genetics*; D008969:Molecular Sequence Data; D010641:Phenotype; D011755:Pyrophosphatases / Q000172:deficiency / Q000235:genetics*; D016415:Sequence Alignment; D013489:Suppression, Genetic; D014161:Transduction, Genetic
chemical_list: D006003:Glycogen; C027627:guanosine-3',5'-bis(diphosphate) 3'-pyrophosphatase; D011755:Pyrophosphatases; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases