Functional Characterization of a Small Alarmone Hydrolase in Corynebacterium glutamicum

Abstract

The (pp)pGpp metabolism is an important component of bacterial physiology as it is involved in various stress responses and mechanisms of cell homeostasis, e.g., the regulation of growth. However, in order to better understand the (pp)pGpp associated regulation, it is crucial to study the molecular mechanisms of (pp)pGpp metabolism. In recent years, bioinformatic analyses of the RelA/SpoT homolog (RSH) superfamily have led to the discovery of small monofunctional RSH derivatives in addition to the well-known bifunctional Rel proteins. These are also referred to as small alarmone synthetases (SASs) or small alarmone hydrolases (SAHs). In this study, the ORF cg1485 from C. glutamicum was identified as a putative SAH encoding gene, based on a high similarity of the corresponding amino acid sequence with the (pp)pGpp hydrolysis domain. The characterization of its gene product, designated as RelH_Cg, represents the first functional investigation of a bacterial representative of the SAH subfamily. The predicted pyrophosphohydrolase activity was demonstrated in vivo by expression in two E. coli strains, characterized by different alarmone basal levels, as well as by in vitro analysis of the purified protein. During the assay-based analysis of hydrolysis activity in relation to the three known alarmone species, both RelH_Cg and the bifunctional RSH enzyme Rel_Cg were found to exhibit a pronounced substrate inhibition for alarmone concentrations of more than 0.75 mM. This characteristic of (pp)pGpp hydrolases could be an important mechanism for realizing the bistable character of the (pp)pGpp metabolism between a (pp)pGpp basal level and stress-associated alarmone production. The deletion of relH_Cg caused only a minor effect on growth behavior in both wild-type background and deletion mutants with deletion of (pp)pGpp synthetases. Based on this observation, the protein is probably only present or active under specific environmental conditions. The independent loss of the corresponding gene in numerous representatives of the genus Corynebacterium, which was found by bioinformatic analyses, also supports this hypothesis. Furthermore, growth analysis of all possible deletion combinations of the three active C. glutamicum RSH genes revealed interesting functional relationships which will have to be investigated in more detail in the future.

Keywords: (p)ppGpp; (pp)pGpp; Mesh1-L; RelH; SAH; alarmone; pGpp; stringent response.

FIGURE 1

Comparative structural analysis of enzymes involved in (pp)pGpp metabolism. True-to-scale representation of domain architecture and conserved sequence motives of stringent response associated enzymes of C. glutamicum ATCC 13032 (enclosed by dashed blue lines) and further selected species. These include the native Rel enzyme of Streptococcus equisimilis Rel_Seq, as well as the shortened and only hydrolytically active variant Rel_Seq D264G (Hogg et al., 2004; Sun et al., 2010) and representatives of the six other SAH subgroups (Atkinson et al., 2011): Mesh (Homo sapiens), paSpo (Erwinia tasmaniensis), pbcSpo (S. pneumoniae), pbcSpo2 (Desulfotalea psychrophila), rickSpo (Rickettsia bellii), and divSpo (Photobacterium profundum). Hydrolase domain HD4 (pfam13328) and ppGpp synthetase catalytic domain YjbM (COG2357) are indicated in green and orange, respectively. Additional C-terminal elements of long RSH enzymes, which are likely to have regulatory functions and are classified as TGS (ThrRS, GTPase, and SpoT) and ACT (aspartate kinase, chorismate mutase and TyrA) domains (Wolf et al., 1999; Chipman and Shaanan, 2001), are also indicated. Matches with conserved synthetase and hydrolase motifs are represented by black lines (Bag et al., 2014; Steinchen and Bange, 2016). Minor deviations from the conserved sequence motifs are shown by dashed black lines. The amino acid exchange of the protein Rel_Seq D264G is represented by a red dashed line and the resulting non-functional YjbM domain is faded out.

FIGURE 2

Effect of the deletion of putative (pp)pGpp metabolism associated genes on the growth of C. glutamicum CR099 in complex CASO broth (A) and minimal CGXII medium (B). To preclude possible enrichment of suppressor mutants all cultures were inoculated from precultures in complex CASO broth. An initial OD₆₀₀ of 0.2 was used for the cultivation in CASO broth. For the growth analysis in CGXII minimal medium, the precultures were washed in the main culture medium and then diluted to an OD₆₀₀ of 0.5. Mean values and standard deviations shown were calculated from three biological replicates. ^∗In the minimal medium cultivation of the strain CR099 ΔrelS_CgΔrelH_Cg, only two of the three replicas could be evaluated, because the third one showed a deviating growth behavior which did not correspond to the preliminary experiments and was therefore considered to be the result of a pseudoreversion.

FIGURE 3

Growth characterization of E. coli MG1655 and E. coli MG1655 ΔrelA, each heterologously expressing the C. glutamicum genes rel_Cg, relH_Cg or relS_Cg, on different solid medium plates. The empty vector pZMP was used as control. All strains were cultivated using initial OD₆₀₀ values of 0.05 in LB-medium for 3 h. The cells were washed and serially diluted in PBS-buffer. 5 μL aliquots of every strain and dilution stage were spotted on LB medium plates (LB), minimal medium plates (MM) and minimal medium plates containing each 1 mM L-serine, L-methionine, and L-glycine (MM+SMG). All solid media plates contained 50 μg mL^-1 kanamycin and 4 g L^-1 glycerol was used as C source for all minimal medium plates. Incubation was carried out at 37°C for 24 h in the case of LB solid medium and 48 h for MM and MM+SMG plates.

FIGURE 4

In vitro characterization of Rel_Cg and RelH_Cg with respect to divalent ion concentration and pH. The specific pyrophosphohydrolase activities of the purified enzymes were determined by (pp)pGpp pyrophosphohydrolase assays and subsequent HPLC analysis (see Material and Methods) under variation of MgCl₂/MnCl₂ concentration (A) and pH-value (B). The reaction mixture for determining the dependence on divalent ions contained 50 mM HEPES-Na (pH 8.0), 200 mM NaCl, 0.5 mM ppGpp as well as 52.3 nM RelH_Cg or 98.2 nM Rel_Cg respectively, and was incubated at 30°C. In order to avoid oxidation of Mn²⁺, the corresponding solutions were saturated with nitrogen before the addition of substrates and enzyme to remove dissolved oxygen. The incubation time was 25 min for RelH_Cg and 45 min for Rel_Cg containing approaches. Since comparatively low activities were measured using magnesium, 10 times higher enzyme concentrations were used. The pH dependency was determined with identical reaction components and experimental parameters, whereby different buffer systems and a defined MnCl₂ concentration of 1 mM were used. The various buffer systems (50 mM each) are illustrated by different icons: square: citrate buffer; triangle: phosphate buffer; rhombus: HEPES-buffer; circle: TRIS buffer; hexagon: ammonia buffer. Mean values and standard deviations shown were calculated from three replicates. ^∗ Enzyme free controls were carried out at the highest and lowest pH values of 5 and 9.5 and did not yield any unspecific production of GDP or other components, so that the substrate ppGpp is considered to be stable at the pH conditions used.

FIGURE 5

Kinetics of RelH_Cg (A) and Rel_Cg (B) using pGpp, ppGpp and pppGpp as substrates. Specific activities were determined for different substrate concentrations by in vitro analysis and given in kat per mol of the respective enzyme. The reaction mixtures contained 50 mM HEPES-Na (pH 8.0), 200 mM NaCl, 1 mM MnCl₂ as well as 52.3 nM RelH_Cg or 98.2 nM Rel_Cg, respectively. The incubation times of the different enzyme-substrate combinations were adapted to the values determined in preliminary tests and ranged from 20 to 45 min. The incubation temperature was 30°C. Mean values and standard deviations shown were calculated from three replicates.

FIGURE 6

Phylogenetic tree of the genus Corynebacterium with additional species-specific information about the occurrence of a relH_Cg ortholog, the respective isolation site and the biosafety level. The phylogenetic tree was generated on the basis of the respective 16S rDNA sequences using the RDP database (Cole et al., 2014). The analysis of available genomes for the presence of a relH_Cg ortholog was based on a Hidden Markov model (see Materials and Methods). Species with a respective gene are shown in green, species without relH gene in red and species without an available genome in black. The genomes of species printed in bolt type are available in databases, whereas the other genomes are our unpublished data. The first box after the species name represents the isolation site, where green illustrates environmental isolates, yellow those isolated from animals, and red human isolates. The second box corresponds to the biosafety classification (according to German law); green: level one; red: level two; no box: no classification available.