title: From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis.
abstract: The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. In Enterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (RelEf) and the small alarmone synthetase (SAS) RelQEf. Although Rel is the main enzyme responsible for SR activation in Firmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQEf synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQEf also efficiently utilized GMP to form GMP 3'-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQEf was activated only by ppGpp. Furthermore, enzymatic activity of RelQEf is insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of "long" RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp.
  
  
  OBJECTIVE
  Accumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ of Enterococcus faecalis (RelQEf), we found that, in addition to (p)ppGpp, RelQEf is an efficient producer of pGpp (GMP 3'-diphosphate). In vitro analysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQEf and suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones.
authors.affiliation: Center for Oral Biology and Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, USA.
authors.forename: Anthony O
authors.initials: AO
authors.lastname: Gaca
authors.affiliation: University of Tartu, Institute of Technology, Tartu, Estonia.
authors.forename: Pavel
authors.initials: P
authors.lastname: Kudrin
authors.affiliation: Center for Oral Biology and Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, USA.
authors.forename: Cristina
authors.initials: C
authors.lastname: Colomer-Winter
authors.affiliation: University of Tartu, Institute of Technology, Tartu, Estonia.
authors.forename: Jelena
authors.initials: J
authors.lastname: Beljantseva
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Kuanqing
authors.initials: K
authors.lastname: Liu
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Brent
authors.initials: B
authors.lastname: Anderson
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Jue D
authors.initials: JD
authors.lastname: Wang
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic v.v.i., Prague, Czech Republic.
authors.forename: Dominik
authors.initials: D
authors.lastname: Rejman
authors.affiliation: Department of Molecular Biology, University of Gdańsk, Gdańsk, Poland Section on Molecular Regulation, Program in Genomics of Development, Eunice Kennedy Shriver NICHD, NIH, Bethesda, Maryland, USA.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Potrykus
authors.affiliation: Section on Molecular Regulation, Program in Genomics of Development, Eunice Kennedy Shriver NICHD, NIH, Bethesda, Maryland, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Cashel
authors.affiliation: University of Tartu, Institute of Technology, Tartu, Estonia Department of Molecular Biology and Laboratory for Molecular Infection Medicine Sweden, Umeå University, University Hospital Area, Umeå, Sweden vasili.hauryliuk@umu.se jose_lemos@urmc.rochester.edu.
authors.forename: Vasili
authors.initials: V
authors.lastname: Hauryliuk
authors.affiliation: Center for Oral Biology and Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, USA vasili.hauryliuk@umu.se jose_lemos@urmc.rochester.edu.
authors.forename: José A
authors.initials: JA
authors.lastname: Lemos
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000737:chemistry; D004151:Dipeptides / Q000096:biosynthesis / Q000737:chemistry; D013293:Enterococcus faecalis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006153:Guanosine Diphosphate / Q000378:metabolism; D006158:Guanosine Pentaphosphate / Q000378:metabolism*; D006159:Guanosine Tetraphosphate / Q000096:biosynthesis*; D006160:Guanosine Triphosphate / Q000378:metabolism; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium; D015394:Molecular Structure; D013312:Stress, Physiological; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004151:Dipeptides; C580565:relacin; D006153:Guanosine Diphosphate; D006159:Guanosine Tetraphosphate; D006158:Guanosine Pentaphosphate; D006160:Guanosine Triphosphate; D008025:Ligases; D003849:Deoxyguanosine; D008274:Magnesium