Identification and Functional Characterization of Small Alarmone Synthetases in Corynebacterium glutamicum

Abstract

The hyperphosphorylated guanosine derivatives ppGpp and pppGpp represent global regulators of the bacterial stress response, as they act as central elements of the stringent response system. Although it was assumed that both, (p)ppGpp synthesis and hydrolysis, are catalyzed by one bifunctional RSH-protein in the actinobacterial model organism Corynebacterium glutamicum ATCC 13032, two putative short alarmone synthetases (SASs) were identified by bioinformatic analyses. The predicted sequences of both enzymes, designated as RelP^*_Cg and RelS_Cg, exhibit high similarities to the conserved (p)ppGpp synthetase catalytic domain. In the context of sequence analysis, significant differences were found between the RelP variants of different C. glutamicum isolates. In contrast to the bifunctional RelA/SpoT homolog (RSH) protein Rel_Cg, whose gene deletion results in a reduced growth rate, no change in growth characteristics were observed for deletion mutants of the putative SAS proteins under standard growth conditions. The growth deficit of the Δrel strain could be restored by the additional deletion of the gene encoding RelS_Cg, which clearly indicates a functional relationship between both enzymes. The predicted pyrophosphokinase activity of RelS_Cg was demonstrated by means of genetic complementation of an Escherichia coli ΔrelAΔspoT strain. For the expression of RelP^*_Cg , as well as the slightly differing variant RelP_Cg from C. glutamicum AS1.542, no complementation was observed, concluding that both RelP versions possess no significant pyrophosphokinase activity in vivo. The results were confirmed by in vitro characterization of the corresponding proteins. In the course of this investigation, the additional conversion of GMP to pGpp was determined for the enzyme RelS_Cg. Since the SAS species analyzed extend both the network of stringent response related enzymes and the number of substances involved, the study of this class of enzymes is an important component in understanding the bacterial stress response. In addition to the comprehension of important biological processes, such as growth rate regulation and the survival of pathogenic species in the host organism, SAS enzymes can be used to produce novel hyperphosphorylated nucleotide species, such as pGpp.

Keywords: (p)ppGpp; ActRel; RelP; RelS; alarmone; pGpp; phylogeny; stringent response.

Figure 1

C. glutamicum enzymes involved in (p)ppGpp synthesis. (A) True-to-scale representation of domain architecture and conserved sequence motives of stringent control-associated enzymes of C. glutamicum ATCC 13032. Hydrolase domain (pfam01966) and ppGpp synthetase catalytic domain YjbM (COG2357) are indicated in green and orange, respectively. Matches with conserved synthetase motifs are represented by black lines (Bag et al., ; Steinchen and Bange, 2016). Minor deviations from the conserved sequence motifs are shown by dashed black lines. (B) Sequence alignment of the C-termini of RelP variants from C. glutamicum ATCC 13032 (Cg ATCC13032) and C. glutamicum R (Cg R) with the synthetase catalytic domain consensus sequence YjbM (COG2357). Identical residues among all three sequences are indicated by dark shading and matches between two sequences by light shading, respectively. Amino acid positions are shown at both sequence ends. (C) Alignment of the nucleotide sequences of the relP variants from C. glutamicum ATCC 13032 and C. glutamicum R. Codons and the corresponding amino acid residues are indicated below the nucleotide sequences with stop codons highlighted in red. Nucleotide positions are shown at both sequence ends. Since the relP sequence of C. glutamicum ATCC 13032 ends before the end of the alignment, no positional information can be given. Matches between the nucleotide and protein sequences are represented by gray shadings. The relationship between the nucleotide sequences and the corresponding amino acid sequences is illustrated by a dashed red line.

Figure 2

Effect of the deletion of genes with a putative (p)ppGpp synthetase motive 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.

Figure 3

Growth complementation of E. coli ΔrelAΔspoT on minimal medium plates by heterologous expression of C. glutamicum genes rel_Cg, relS_Cg, relP^*_Cg or relP_Cg. The parental strain E. coli MG1655 and E. coli ΔrelAΔspoT each containing the empty vector pBAD24 were used as a positive and negative control, respectively. All strains were cultivated using initial OD₆₀₀ values of 0.05 in LB-Medium containing 0.001% arabinose 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 minimal medium plates with 0.4% glycerol as carbon source and different arabinose concentrations. Incubation was carried out at 37°C for 48 h.

Figure 4

In vitro activity characterization of the putative (p)ppGpp synthetases RelP^*_Cg and RelS_Cg, as well as the enzyme Rel_Cg. Graphical representation of the HPLC analysis of 50 μL assay reactions containing 500 ng of the corresponding enzyme and ATP+GTP (A), ATP+GDP (B) and ATP+GMP (C) as substrate combinations with a concentration of 4 mM each. HPLC separation was performed using a SeQuant ZIC-pHILIC column and isocratic elution with 38% of 10 mM ammonium bicarbonate buffer (pH 9.3) and 62% acetonitrile. The reaction products were identified by their specific masses and retention times. Their relative abundance is given as the peak area of the UV signal (252 nm) per mg of the respective enzyme per hour. The results shown were corrected by the values determined for enzyme-free controls. For some measurements pppGpp (^*) or pGpp (^**) were detected by their characteristic MS signals, but the UV detection limits were not reached.

Figure 5

Kinetics of RelS_Cg with respect to the concentrations of GMP, GDP, and GTP as substrates. Specific activity was determined for different substrate concentrations by in vitro analysis and given in μmol per minute per milligram of RelS_Cg. In order to illustrate the data and the graphical determination of the kinetic parameters K_m and v_max, a polynomial fit was performed for all substrates and displayed as a dashed line. K_m and v_max values for GDP and GTP are represented as vertical and horizontal dashed lines, respectively. For GMP the parameters could not be determined due to the untypical activity profile. Mean values and standard deviations shown were calculated from three replicates.

Figure 6

Analysis of relevant reaction parameters on the activity of RelS_Cg and Rel_Cg. Specific pyrophosphokinase activities were determined by in vitro analysis under variation of pH-value (A), temperature (B), and MgCl₂ concentration (C). The initial reaction mixture contained 50 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 4 mM ATP, 4 mM GTP as well as 500 ng RelS_Cg (257 nM) or Rel_Cg (118 nM), respectively and was incubated for 4 h at 30°C. The various buffer systems (50 mM each) used to vary the pH value are illustrated by different icons: square: citrate buffer; circle: phosphate buffer; triangle: TRIS buffer; hexagon: ammonia buffer. Mean values and standard deviations shown were calculated from three replicates.