SUPPLEMENTARY MATERIAL
Supplementary Methods
Microarray experiments: The GAS oligonucleotide array (1)was used with RNA isolated, labeled and hybridized as described (1). RNA was harvested from triplicate cultures in late exponential phase (80 Klett units with the red filter) and two hours into stationary phase (Fig. S2). cDNA was generated from each RNA sample, labeled with Cy3 and pooled prior to hybridization to two separate arrays. This was repeated in triplicate from three independent cultures (i.e., for each growth phase, Cy3-labeled cDNA was generated from nine RNA preparations and pooled into three groups of three. Each pool was hybridized to two separate microarrays). For each set of experiments, a reference cDNA sample consisted of pooled Cy5-labeled cDNA generated from all 18 RNA samples to be examined. Arrays were hybridized, washed, scanned and filtered as described previously (1).
For each array, spots with signals below a threshold (consisting of the mean background level of the whole experiment plus one standard deviation) were adjusted to the threshold level. The ratios of signals from the test (Cy3) and reference (Cy5) channels were calculated and data for each gene from each of three individual spots on the array were averaged and treated as a single value for the experiment. The data were then filtered to remove values for genes that were not present in strain MGAS315.
Following data filtration and ratio of signal calculations, we compared the relative abundance of the transcript in one phase to its relative abundance in the other phase using two normalization steps. First, an average for the total signal of the six arrays from each growth phase was calculated (1256 was the average in exponential phase and 389 was the average in stationary phase). To correct for signal variations between replicate arrays, the value for each gene was adjusted by a factor so that the total signal for each array was equal to the average for the six in that growth phase. Second, the value for each gene adjusted for differences between replicates was divided by the average total signal in exponential phase. Normalized data was then log2 transformed, and statistical analysis and fold changes in expression in stationary phase versus exponential phase were obtained using Significance Analysis of Microarrays (SAM) software (4). The SAM plot for this analysis is shown in Fig. S3. Data are deposited in the Gene Expression Omnibus site (GEO: www.ncbi.nlm.nih.gov/geo; series record GSE5179).
Construction of ribonuclease mutant strains:
For pnpA, a region encompassing the first 63 bp of pnpA and 1543 bp upstream was amplified by PCR using primers pnp2 and pnp3 (Fig. S1A). Similarly, a region encompassing the last 54 bp of pnpA and 1534 bp downstream was amplified using primers pnp6 and pnp7. The gel-purified PCR products were used as templates and fused together in a second-round PCR, using primers pnp2 and pnp7, by virtue of their overlapping sequences. The resulting PCR fragment has a region complementary to the 5' end of pnpA and upstream sequences, followed by a stop codon, a SalI restriction site, a small stuffer region, a NdeI restriction site, and a region complementary to the 3' end of pnpA and downstream sequences (Fig. S1B). The resulting 3.2 kb PCR fragment was gel-purified and cloned into pCR-BluntII TOPO vector (Invitrogen) to create pJRS1286.
A similar procedure was used to construct mutations in rnr (pJRS1390) and yhaM (pJRS1381) using primers rnr2 and rnr3 (5' rnr), rnr6 and rnr7 (3' rnr), yha2 and yha3 (5' yhaM), and yha6 and yha7 (3' yhaM). The promoterless aad9 gene was amplified by PCR from plasmid pUCSpec using primers aad1 and aad4 (Fig. S1C), digested with SalI and NdeI and cloned into SalI-NdeI-digested plasmid pJRS1386 to create pJRS1392 (Fig. S1D). The same procedure was used to clone the promoterless aad9 gene into pJRS1390 and pJRS1381, to create plasmids pJRS1394 and pJRS1391, respectively. Approximately 50 mg of each plasmid was linearized by digestion with SmaI and introduced into MGAS315 by electroporation followed by selection for spectinomycin-resistant colonies.
Allelic replacement of each ribonuclease gene was confirmed by a series of PCR reactions. For pnpA, the 5' and 3' junctions were amplified using primers pnp1 and aad3 (5' junction), and aad2 and pnp8 (3' junction; Fig. S1D), and deletion of pnpA was confirmed by the inability to amplify an internal portion of pnpA using primers pnp4 and pnp5 (Fig. S1A). A similar procedure was used to verify the deletions in rnr and yhaM using primers rnr1 and aad3 (5' junction rnr), aad2 and rnr8 (3' junction rnr ), rnr4 and rnr5 (deletion rnr), yha1 and aad3 (5' junction yhaM), aad2 and yha8 (3' junction yhaM), yha4 and yha5 (deletion yhaM). The ribonuclease mutants of MGAS315 were designated JRS1392 (pnpA), JRS1394 (rnr) and JRS1391 (yhaM).
Construction of JRS1288:
For sagA, the region from -1569 to -13 (numbering relative to the start of transcription of sagA) was amplified by PCR using primers sagA-US-F2 and DsagA-R. Similarly a region from +492 to +2082 was amplified using primers DsagA-F and sagB-R. Primers DsagA-F and DsagA-R contain 31 nucleotides of complementary sequence which allowed the first round PCR products to be fused together in a second round PCR using primers sagA-US-F2 and sagB-R. The resulting PCR product was cloned into pCR-XL-Topo (Invitrogen) to create pJRS1279. To allow selection of this mutation in GAS, a 3 BamHI WtetM fragment (Prentki and Krisch, 1984) from pJRS303 was cloned into the BglII site of pJRS1279, to create pJRS1285. pJRS1285 was linearized by digestion with SmaI, transformed into MGAS315 by electroporation, and tetracycline-resistant transformants were selected. Gene replacement of sagA was confirmed in the resulting strain (JRS1285) by PCR using primers RPAsagA-F2 and RPAsagA-R (to confirm loss of sagA) as well as a loss of hemolytic activity of sheep blood agar plates. A gene replacement mutation in covR was created in JRS1285 using pJRS1349 as described by Dalton et al. (2), to create JRS1288.
Table S1. Primers used in this study
aad1 (delRNase-aad9-F) / gcatgtcgacAAAATAGTGAGGAGGATATATTTGAATACATACG
aad2 (RPAaad9-F) /
GGTTACAAGAGCTTTATGAACAAGG
aad3 (RPAaad9-R) /CTTTTGGTATGATTTTACCCGTGTCC
aad4 (delRNase-aad9-R) / gcatcatatgtatctcctcctcaatttttttATAATTTTTTTAATCTGTTATTTAAATAGhasC-DS-XhoI-R / gcatctcgagTTTTGAAGTTCTTGTTAATTAGTTTTAACAAAAGC
inthas-S4 / cccacccAGCTTCAATGATGAGACAGTTTATG
inthas-A4 / gggtgggAGGAGGAATTCACCTAGGAATGTTTGATTTT
PhasM3-NotI-F / gcatgcggccgcGATTATACCATTTTTATTCAGGTTAGGTGAGTG
PhasM3-sag-F / tatattagaatattgaggataagaactagaTAGTTGTTGTGTTACAACAGTACAATTGAG
PhasM3-sag-R / tctagttcttatcctcaatattctaatataAATAAGAAATAATGTCAACTAGACATAGAG
pnp1 (Spy1946-F2) / GCCATTAAGAGTGGTCTTACCTTTGC
pnp2 (Spy1946-F) / CTTGATTCCAGCCTTCCAAGGTATTGC
pnp3 (delSpy1946-R) / catatgtatgcatgcgtcgacttaTTGACCAACTTCAACAACAAGGGGTTTCC
pnp4 (RPAspy1946-F) / cccacccGTTTCCTTATGCTATTCGTTTAGTTGC
pnp5 (RPAspy1946-R) / gggagggTTAGCTTGTGCAAGAGCTTCTTCTAGG
pnp6 (delSpy1946-F) / taagtcgacgcatgcatacatatgAAAGCTTTGATTCCACGCCCACCAAAA
pnp7 (Spy1946-R) / CACAATAGACTGTTTCTTCAGTCTAAGACTTGC
pnp8 (Spy1946-R2) / GATTTTTGAGACCTTAGGCTCAAAACTAAGG
pnpA-F4-Bam / gcatggatccCCTCTGAAAATTAAAAGGAGAATATATGTC
pnpA-R4-Xho / gcatctcgagCGAATATCTAATTCATTTGATTTAGTCATG
pREGaad9-F2-Bgl / gcatagatctCAACTCCTGATCCAAACATGTAAGTACCAATAAGG
pREGaad9-R2-Bgl / gcatagatctGTTAGCAGTTCGTAGTTATCTTGGAGAGAATATTG
Psag-has-F / ttactataataaaagtgcccttactttcAAGGAAATTAAAAAAGAAAGAG
Psag-has-R / gaaagtaagggcacttttattatagtaaAAAATGATTAATATGTAAACCC
Psag-NotI-F2 / gcatgcggccgcGGTATTACATATTTCAAAGTAGTATCTAGG
rnr1 (Spy503-F2) / GCTCTATATGTGGAAAATTTAGGTACTCC
rnr2 (Spy0503-F) / GCTTCAGCTTTATTTGCTCCTGTTTGG
rnr3 (delSpy0503-R) / catatgtatgcatgcgtcgacttaGGGAAAATGCTTAGCTCCAGCCATCTCC
rnr4 (RPAspy503-F) / cccaccCTAGCACCTTTGGTATTCACATTCAAGG
rnr5 (RPAspy503-R) / gggagggCTCAGCTTTTTTCATGGCTTCAACCACG
rnr6 (delSpy0503-F) / taagtcgacgcatgcatacatatgAAAGGGTCTAAAAAACCATTTTACAAAGAACAAGC
rnr7 (Spy0503-R) / CATTTGATGTATCAAGCCCTTTACTTAGTGG
rnr8 (Spy503-R2) / CCTATTCGTGCAGATGGTAAAGCAGC
RPAarcT-F / cccacccATTATATGTGCCATTAGACAGTGAATTGG
RPAarcT-R / gggagggTTTGAACAGCATCAGGGAAAAGG
RPAcat86-F / cccacccATCAAGTGAACGATGAACTTGG
RPAcat86-R / gggagggCATGATGTACTTGTATGGCAACG
RPAgyrA-F / cccacccTTGCTCAAGATGAATTTCGTGC
RPAgyrA-R / gggagggAATTTCGTAAGCTTTCAACC
RPAsagA-F2 / cccacccattttagctactagtgtagC
RPAsagA-R / gggagggAGATTATTTACCTGGCGTA
RPAsda-F / cccacccGTCTTATGTCGGAGATTTCTGG
RPAsda-R / gggagggCTTGAGCTCTTTGTTCGGTATAGC
RPAslo-F / cccaccctttagtgcagctctaaaaggaacag
RPAslo-R / gggagggcactggtgtatgaaataggataagctgg
sagA+1-S / GATAAGAACTAGATAGTTGTTGTGTTACAAC
sagA-DS-Bam-R / gcatggatccTATCTGATGCTCCATCTCTAATTGATGC
sagA-DS-XhoI-R2 / gcatctcgagCAATTGCCTTGCTTTTTCAACAGTTATTGG
sagA-PE2 / GTTAACACAACAAGGACAAGGCTAGC
sda-PE / GCTGATACAAGAGCTACCATTGAAAATTTTACTAGC
T7sagA-R / taatacgactcactataggcGTAATTAGCAGGTACTGTCTAGTACCTGC
yha1 (Spy267-F2) / GCGTTTACGGATATAAAAGAGAAGGC
yha2 (Spy0267-F) / GGTATTGCTCCCTTTATGGCACAAATCG
yha3 (delSpy0267-R) / catatgtatgcatgcgtcgacttaGAACCCTTCAAAAAGCTGGTCTTTTTTCATTTG
yha4 (RPAspy267-F) / cccacccGAAAATGCAACGTGGCAACGAATTGTAAGG
yha5 (RPAspy267-R) / gggagggGAGAGAAATATGCCCAATCAAGTTACC
yha6 (delSpy0267-F) / taagtcgacgcatgcatacatatgAATCGGATTTTTGCCATGGATAATCGGTC
yha7 (Spy0267-R) / CAGTAGCTGTTAACTCTCTTTGATGTTCGG
yha8 (Spy267-R2) / GAAATGCTAGCTTTTTGTTCTGACAATGC
a lowercase letters indicate bases added to facilitate cloning or fusion of PCR fragments by overlap-extension; underlined letters are introduced restriction sites
SUPPLEMENTARY FIGURES
Figure S1. Strategy for the construction of a pnpA mutation in MGAS315. Details of this procedure are given in the Materials and Methods. A similar method was used to construct mutations in rnr and yhaM using primers listed in the Materials and Methods and Table S1.
Figure S2. Growth curve of GAS strain MGAS315 grown in THY broth at 37°C. Arrows indicate times at which samples were removed for late exponential (LE) and stationary (ST) phase. Growth was followed for each culture used to isolate RNA.
Figure S3. SAM plot. Scatter plot generated with SAM software (v1.21; (4)) for identification of genes with significant changes in expression in stationary phase relative to exponential phase. Red spots represent genes significantly upregulated in stationary phase. Green spots represent genes significantly down regulated in stationary phase.
Figure S4. Primer extension analysis of sagA and sda mRNA from late exponential (LE) and stationary (ST) phase cells. (A) 5 mg total RNA was used in each reaction. Primer extension analysis was performed as described by Opdyke et al. (3). Sequencing reactions were run alongside primer extension reactions. The 5' ends of each transcript are indicated with an arrow. (B) DNA sequence corresponding to the 5' end of the sagA and sda mRNAs from MGAS315. The transcriptional start sites are indicated with arrows.
Figure S5. Northern blot analysis of sagA mRNA decay in MGAS315 (wild type) and JRS1392 (pnpA). Northern blots were performed as described for Fig.3. The data for MGAS315 is the same as that shown in Fig.3.
REFERENCES
1. Dalton, T. L., J. T. Collins, T. C. Barnett, and J. R. Scott. 2006. RscA, a member of the MDR1 family of transporters, is repressed by CovR and required for growth of Streptococcus pyogenes under heat stress. J. Bacteriol. 188:77-85.
2. Dalton, T. L., R. I. Hobb, and J. R. Scott. 2006. Analysis of the role of CovR and CovS in the dissemination of Streptococcus pyogenes in invasive skin disease. Microb. Pathog. 40:221-7.
3. Opdyke, J. A., J. G. Kang, and G. Storz. 2004. GadY, a small-RNA regulator of acid response genes in Escherichia coli. J. Bacteriol. 186:6698-705.
4. Tusher, V. G., R. Tibshirani, and G. Chu. 2001. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. USA. 98:5116-21.