Expanding the genetic toolbox for the obligate human pathogen Streptococcus pyogenes

Genetic tools form the basis for the study of molecular mechanisms. Despite many recent advances in the field of genetic engineering in bacteria, genetic toolsets remain scarce for non-model organisms, such as the obligatory human pathogen Streptococcus pyogenes. To overcome this limitation and enable the straightforward investigation of gene functions in S. pyogenes, we have developed a comprehensive genetic toolset. By adapting and combining different tools previously applied in other Gram-positive bacteria, we have created new replicative and integrative plasmids for gene expression and genetic manipulation, constitutive and inducible promoters as well as fluorescence reporters for S. pyogenes. The new replicative plasmids feature low- and high-copy replicons combined with different resistance cassettes and a standardized multiple cloning site for rapid cloning procedures. We designed site-specific integrative plasmids and verified their integration by nanopore sequencing. To minimize the effect of plasmid integration on bacterial physiology, we screened publicly available RNA-sequencing datasets for transcriptionally silent sites. We validated this approach by designing the integrative plasmid pSpy0K6 targeting the transcriptionally silent gene SPy_1078. Analysis of the activity of different constitutive promoters indicated a wide variety of strengths, with the lactococcal promoter P 23 showing the strongest activity and the synthetic promoter P xylS2 showing the weakest activity. Further, we assessed the functionality of three inducible regulatory elements including a zinc- and an IPTG-inducible promoter as well as an erythromycin-inducible riboswitch that showed low-to-no background expression and high inducibility. Additionally, we demonstrated the applicability of two codon-optimized fluorescent proteins, mNeongreen and mKate2, as reporters in S. pyogenes. We therefore adapted the chemically defined medium called RPMI4Spy that showed reduced autofluorescence and enabled efficient signal detection in plate reader assays and fluorescence microscopy. Finally, we developed a plasmid-based system for genome engineering in S. pyogenes featuring the counterselection marker pheS*, which enabled the scarless deletion of the sagB gene. This new toolbox simplifies previously laborious genetic manipulation procedures and lays the foundation for new methodologies to study gene functions in S. pyogenes, leading to a better understanding of its virulence mechanisms and physiology.

Supplementary Figure 3. S. pyogenes growth (solid lines) and luminescence (dotted lines) signal of the luciferase reporter strains harboring different constitutive promoters (blue) in comparison to the control without promoter (grey).(A) Growth (shown in OD620nm) of S. pyogenes strains harboring either the control plasmid (pSpy1C-ffluc) or pSpy1C featuring the fusions of different constitutive promoters to the luciferase reporter ffluc (e.g.pSpy1C-P23_ffluc).(B) Growth, shown in OD620nm (top panel), and promoter activities, shown in relative luminescence units normalized by optical density (RLU/OD620nm) (bottom panel) of strains harboring luciferase reporter plasmids with different variants of the Pveg promoter (1)(2)(3).(C) DNA sequences of the native Pveg promoter and its derivatives (1-3) including the -35 region, the spacer and the -10 region.The respective changes to the spacer sequence to obtain Pveg(1) (G-to-A and T-to-G mutation), Pveg(2) (insertion of two adenines) and Pveg(3) (deletion of two thymines) are highlighted in apricot.(D) Comparison of the mean promoter activities (RLU/OD620nm) of the native Pveg promoter and its derivatives Pveg (1), Pveg (2), and Pveg(3) after 4 hours of growth in THY.Statistical significance was analyzed using a Brown-Forsythe and Welch ANOVA test for multiple comparisons (n.s.= not significant).Experiments were performed in biological triplicates and measurements in technical duplicates.Supplementary Figure 4. Growth (solid lines) and luminescence signal (dotted lines) of the S. pyogenes reporter strains featuring different inducible promoters.(A) Growth of the reporter plasmids harboring the inducible promoter systems Ptet, PnisA, Plac(Spn) and Ptre is shown in OD620nm over time in the top row, while the luminescence signal is shown in relative luminescence units normalized by optical density (RLU/OD620nm) in the bottom row.Cultures were induced with the respective inducer compounds or the control at the beginning of growth (t=0) (Ptet: 10 ng/mL anhydrotetracycline, PnisA: 0.75 µg/mL nisin, Ptre: 0.4% trehalose, Plac(Spn): 1 mM IPTG, distilled water served as negative control).Data are shown for the uninduced condition (data points with white fill) and the induced condition (data points with red fill).(B) Growth of the strains harboring reporter plasmids with the inducible regulatory systems PZn and PgyrA(Sag)_ermBL-ermB' shown as OD620nm over time in the absence or presence of the respective inducer compounds (PZn: 80 µM ZnSO4 and PgyrA(Sag)_ermBL-ermB': 3 ng/mL erythromycin).Data are shown for the uninduced condition (data points with white fill) and the induced condition (data points with red fill).Distilled water served as a negative control for PZn induction and ethanol was used as a negative control for PgyrA(Sag)_ermBL-ermB'.Experiments were performed in biological triplicates and measurements in technical duplicates.Supplementary Figure 5. Growth (solid lines) and luminescence signal (dotted lines) of the S. pyogenes control strains harboring pSpy1C-ffluc (no promoter) in the presence (data points with grey fill) and absence (data points with white fill) of different inducer compounds (Ptet: 10 ng/mL anhydrotetracycline, PnisA: 0.75 µg/mL nisin, Ptre: 0.4% trehalose, PZn: 80 µM ZnSO4, Plac(Spn): 1 mM IPTG, PgyrA(Sag)_ermBL-ermB': 3 ng/mL erythromycin).Distilled water (dH2O) or ethanol were used as negative controls for induction.Growth is shown in OD620nm over time in the first and third row.The luminescence signal is shown in relative luminescence units normalized by optical density (RLU/OD620nm) in the second and fourth row.Experiments were performed in biological triplicates and measurements in technical duplicates.Supplementary Figure 6.Promoter activities of the metal-inducible promoters PcopA and PmntE in the presence or absence of CuSO4 and MnSO4.Growth in OD620nm (top row and solid lines) and luminescence signal in relative luminescence units normalized by optical density (RLU/OD620nm) (bottom row and dotted lines) of the S. pyogenes reporter strains harboring the metal-inducible promoters PcopA or PmntE or the control (no promoter) in the presence (data points with red or grey fill) and absence (data points with white fill) of different inducer compounds (80 µM MnSO4 and 80 µM CuSO4).Distilled water (dH2O) was used as a negative control for induction.Experiments were performed in biological triplicates and measurements in technical duplicates.Supplementary Figure 7. Activity of IPTG-inducible Plac(Spn) promoters harboring different modifications within the translation initiation region in a LacI-expressing S. pyogenes strain.(A) S. pyogenes growth (solid lines) and luminescence signal (dotted lines) of the reporter strains harboring different metal-inducible promoters and the control (no promoter) in the presence (data points with red fill) and absence (data points with white fill) of 1 mM IPTG.Distilled water (dH2O) was used as a negative control for induction.Growth is shown in OD620nm over time (top row), while the luminescence signal is shown in relative luminescence units normalized by optical density (RLU/OD620nm) (bottom row).(B) Growth shown in OD620nm (left) and luminescence signal depicted in RLU/OD620nm (right) for the control strain harboring the reporter plasmid without a promoter in the presence (data points with grey fill) and absence (data points with white fill) of 1 mM IPTG.(C) Growth curves of the LacI expression strain (EC3732) compared to S. pyogenes wildtype (EC2514) and S. pyogenes containing the empty p7INT plasmid (EC3241).Experiments were performed in biological triplicates and measurements in technical duplicates.
Supplementary Figure 8. Growth and luminescence signal of the three inducible systems PZn, PgyrA(Sag)_ermBL-ermB' and Plac (1) in response to increasing inducer concentrations.The legend to the right of each panel shows the applied inducer concentrations.Growth and luminescence signals are shown in OD620nm or relative luminescence units normalized by optical density (RLU/OD620nm) on the y-axis, while the time post induction (p.i.) is indicated on the x-axis.(A) S. pyogenes growth (top row and solid lines) and luminescence signal (bottom row and dotted lines) of the strain harboring either the control plasmid without promoter (left) or the zinc-inducible promoter (right).(B) S. pyogenes growth (top row and solid lines) and luminescence signal (bottom row and dotted lines) of the strain harboring either the control plasmid without promoter (left) or the erythromycin-inducible riboswitch (right).(C) S. pyogenes growth (top row and solid lines) and luminescence signal (bottom row and dotted lines) of the strain harboring either the control plasmid without promoter (left) or the reporter plasmid with the IPTG-inducible Plac(1) promoter (right).Experiments were performed in biological triplicates and measurements in technical duplicates.Supplementary Figure 10.Growth of strains harboring either the mKate2 reporter (P23_mKate2, EC3391) or the negative control without a promoter (no promoter, EC3480) integrated at different genomic locations compared to the S. pyogenes wildtype (EC2514).The reporter was integrated at the tmRNA locus (attB(T12)) using p7INT (left), into the sagB gene using pSpy0C4 (center) and into a transcriptionally silent open reading frame (SPy_1078) using pSpy0K6 (right).The growth is shown in OD620nm on the y-axis, while the time in hours is indicated on the x-axis.Experiments were performed in biological triplicates and each measurement in technical duplicates.Modifications to the original sequences are highlighted in red.The start codons (ATG) as well as the sequence part of the erythromycininducible riboswitch are underlined.The -10 and -35 motifs of the promoter sequences were predicted using BPROM and are highlighted in italics (12).The truncated ermB' coding sequence that is translationally fused to the downstream reporter is marked in yellow.For the constitutive promoters, we added the optimized 5'UTR of pLZ12Km2-P23R:TA:ffluc that is colored in grey.Putative binding sites of regulatory proteins are highlighted in bold.The ssrA tag sequence attached to the fluorescent reporter genes is marked in blue.PgyrA(Sag) -PgyrA promoter from Streptococcus agalactiae.  5. Details of the construction of reporter plasmids featuring different constitutive promoters upstream of the firefly luciferase reporter ffluc.The first column ('Plasmid') indicates the name of the plasmid that was constructed.The second column ('Assembly method') defines which assembly strategy was used.The amplicons or fragments required for plasmid assembly were obtained by PCR and the amplicons, respective oligonucleotides and the templates used are specified in the following columns.Abbreviations: TA = Toxin-Antitoxin Supplementary Table 6.Details of the construction of reporter plasmids featuring different inducible promoters upstream of the firefly luciferase reporter ffluc.The first column ('Plasmid') indicates the name of the plasmid that was constructed.The second column ('Assembly method') defines which assembly strategy was used to ligate the fragments together to create the plasmid.The amplicons or fragments required for plasmid assembly were obtained by PCR and the amplicons, respective oligonucleotides and the templates used are specified in following columns.Symbols: (~) indicates translational fusions; TA = Toxin-Antitoxin The start indicates the start coordinate of the transcriptionally silent region in the genome of the target strain, while the end respectively marks the last coordinate of this region.While the target strain shows the strain in which the transcriptionally silent region was identified, the reference strain is the strain to be compared against.For each region, the average read coverage is listed.Regions that are homologous between reference and target strain are indicated by 'true', while non-homologous sequences are indicated by 'false'.Conserved expression of the regions is marked by 'true', while differences in expression are indicated by 'false'.

Figure 9 .
Growth of S. pyogenes SF370 in different formulations of the RPMI4Spy chemically defined medium.The growth is shown in OD620nm on the y-axis, while the time in hours is indicated on the x-axis.(A) Initial test experiment using RPMI1640 cell culture medium supplemented with glutathione, fetal bovine serum, RPMI non-essential amino acid solution or RPMI amino acid solution or combinations thereof.Abbreviations: GSH = Glutathione, NEAA = non-essential amino acids, AA = amino acids, FBS = fetal bovine serum (B) Test experiments with different RPMI4Spy medium formulations supplemented with either the commercial RPMI amino acid solution (V1) or a self-prepared amino acid mix (V2).Both basic formulations were further supplemented with different vitamin solutions (BME Vitamin Solution, MEM Vitamin Solution or RPMI Vitamin Solution) and two different concentrations of niacinamide (NAD).Experiments were performed in biological triplicates.Abbreviations: RPMImod = modified RPMI1640.

Table 1 .
Bacterial strains used and created in this study.'Code' refers to the internal strain numbering system used in our laboratory.Symbols: (~) indicates translational fusions; (*) indicates an introduced stop codon (TAA)

Table 2 .
Oligonucleotides used in this study.Overhangs used for cloning purposes are underlined.'Code' refers to the internal oligo numbering system used in our laboratory.

Table 3 .
Plasmids used in this study.Information on all plasmids used either for cloning purposes or for in vivo experiments including the plasmid names ('designation'), their encoded resistance genes ('marker') and a short description of their most important characteristics ('features').'Code' refers to the internal plasmid numbering system used in our laboratory.Symbols: (~) indicates translational fusions; TA = Toxin-Antitoxin; (*) indicates stop codon (TAA), except for pheS*, where (*) highlights the introduction of two mutations.

Table 4 .
Information on the DNA sequence of the reporters, promoters and other genetic parts used in this study.

Table 7 .
Details of the construction of reporter plasmids featuring the fluorescent reporter genes mKate2 and mNeongreen.The first column ('Plasmid') indicates the name of the plasmid that was constructed.The second column ('Assembly method') defines which assembly strategy was used to ligate the fragments together to create the plasmid.The amplicons or fragments required for plasmid assembly were obtained by PCR and the amplicons, respective oligonucleotides and the templates used are specified in following columns.Symbols: (~) indicates translational fusions; (*) indicates stop codon (TAA)

Table 8 .
Initial compositions for the development of an RPMI1640-based chemically defined medium.Specification of the components and their concentrations supplemented to the RPMI4Spy interim media RPMImod version 1 (V1) and RPMImod version 2 (V2).The recipe in the third column of RPMImod V1 resembles the final RPMI4Spy recipe (with 1x Niacinamide and 1x BME vitamin solution).

Table 9 .
Transcriptionally silent sites in the genome of S. pyogenes SF370.The start indicates the start coordinate of the transcriptionally silent region in the genome of the target strain, while the end respectively marks the last coordinate of this region.The different region types are differentiated as follows: (1) not_expressed_genes: regions annotated as genes including the promoter region upstream, but not expressed above threshold and not within annotated prophage region; (2) not_expressed_intergenic: regions not annotated as genes, not expressed above threshold and not within annotated prophage region.For each region, the average normalized read coverage is listed.

Table 11 .
Identification of transcriptionally silent sites showing homology between S. pyogenes SF370 and M1T1 5448.

Table 12 .
Nucleotide sequence conservation of the amyA locus between different serotypes of S. pyogenes.For this analysis, we selected the genomic region of S. pyogenes SF370 including the regions of homologous recombination as well as the sequence that is deleted by allelic replacement (coordinates used: start 1,078,523, end: 1,082,749) and performed a multiple sequence alignment.Sequence identity shown in % was analyzed using the integrated MUSLE alignment tool using standard parameters within the geneious software.

Table 13 .
Nucleotide sequence conservation of the sagB locus between different serotypes of S. pyogenes.For this analysis, we selected the genomic region of S. pyogenes SF370 including the regions of homologous recombination as well as the sequence that is deleted by allelic replacement (coordinates used: start 598,115, end: 599,750) and performed a multiple sequence alignment.Sequence identity shown in % was analyzed using the integrated MUSLE alignment tool using standard parameters within the geneious software.

Table 14 .
Nucleotide sequence conservation of the Spy_1078 locus between different serotypes of S. pyogenes.For this analysis, we selected the genomic region of S. pyogenes SF370 including the regions of homologous recombination as well as the sequence that is deleted by allelic replacement (coordinates used: start 884,129, end: 886,197) and performed a multiple sequence alignment.Sequence identity shown in % was analyzed using the integrated MUSLE alignment tool, using standard parameters within the geneious software.