化膿性鏈球菌 (Streptococcus pyogenes) 是感染人類的重要病原菌，其感染時可在含氧量充足的宿主環境進一步導致侵入性及高危險的疾病發生，如壞死性筋膜炎 (Necrotizing Fasciitis)、鏈球菌中毒性休克綜合症(Streptococcal Toxic Shock Syndrome)。在臨床檢驗及早期研究上皆指出了，不具有‎過氧化氫酶 (catalase) 的S. pyogenes確有抵禦由reactive oxygen species (ROS) 所衍生氧化壓力 (Oxidative stress) 的能力，使其得以存活於高氧化壓力的環境中。另外，在動物感染模式研究下亦證明了，氧化壓力與細胞內外金屬離子的恆定對於S. pyogenes的毒力因子表現及致病機轉有著關鍵性影響。故充分了解不具過氧化氫酶的S. pyogenes如何在感染過程中調控氧化壓力平衡而維持自身生長是一個有趣的研究方向。本篇主要以小角度X-ray散射技術 (Small-Angle X-ray Scattering) 探討氧化壓力調控因子PerR在溶液中的構形，進而了解其分子調控機制。PerR是一種需金屬離子協助的轉錄調控因子，有研究證明了在過度金屬離子和hydrogen peroxide stress的環境下，PerR可以直接調控Dpr蛋白的表現，來幫助S. pyogenes抵禦氧化壓力，故可透過Dpr promoter region來觀測PerR結合DNA的能力。在實驗室先前研究中我們確認了S. pyogenes PerR在溶液中具有結合DNA的能力且成功的解構了S. pyogenes PerR，但比較目前研究較深入的B. subtilis PerR後，我們發現S. pyogenes PerR與B. subtilis PerR在inactive狀態下的結構相似，為了釐清是否在結晶過程中造成了這個矛盾，我們利用了小角度X-ray散射技術，透過這項技術可以探知蛋白質在溶液狀態下的構形。從小角度X-ray散射的結果指出，S. pyogenes PerR的晶體結構和溶液中的構形是一致的，進一步證明了先前晶體結構下的S. pyogenes PerR具有結合DNA的能力。另一方面，我們也發現在後續由M9 medium表現出的S. pyogenes PerR在溶液下不具有結合DNA的能力，但當環境存在鋅離子時，S. pyogenes PerR可以恢復其DNA結合能力。綜合上述實驗結果，我們相信S. pyogenes PerR擁有和B. subtilis PerR一樣的活化模式，需要藉由結合金屬離子來活化其結合DNA的能力，而由M9 medium表現出的S. pyogenes PerR為inactive的狀態，當結合環境金屬離子後，會產生構形轉變而成為active的狀態。

Streptococcus pyogenes is a strictly human pathogen that causes severe and invasive diseases in oxygen-rich host environment. However, previous studies indicate S. pyogenes does not produce catalase but has a robust ability to resist killing by reactive oxygen species (ROS). Also, the survival and virulence of S. pyogenes correlates with the regulation of oxidative stress and metal homeostasis. Therefore, how S. pyogenes adapts to ROS stress during infection is an interesting question. The peroxide response regulator, PerR, is a metal-binding transcriptional regulator that controls gene expression of GAS for fighting multiple stresses. Dpr, an iron-binding protein, which is required for oxygen tolerance for S. pyogenes, is known to be repressed by PerR. Studies have shown that the expression of Dpr is induced by millimolar levels of zinc, nickel, or hydrogen peroxide. Aim to understand the molecular mechanism of how PerR regulates the expression of Dpr, the crystal structure of PerR has been solved in this lab. The structure reveals that PerR is a homodimer and coordinates a zinc atom at regulatory site of each monomer. In contrast with Bacillus subtilis PerR, the crystal structure of S. pyogenes PerR is similar to the inactive conformation of the B. subtilis PerR that does not possess DNA-binding ability. However, the PerR protein is active in solution tested by electrophoretic mobility shift assay. For the purpose of clarifying the effect of crystallization process causes this contradiction. We performed the small angle X-ray scattering (SAXS) to understand the solution conformation of PerR. SAXS has become an increasingly important technique and is widely used to study the low-resolution structure of biological macromolecules in solution. Surprisingly, the model of SAXS suggests the conformation of crystal structure is identical with that in solution. Preliminary of S. pyogenes PerR expressed for M9 medium indicates an inactive form. And the DNA-binding ability is rescued by addition of excess zinc ions. Taken together, we got an active conformation of S. pyogenes PerR which was able to recognize its specific DNA binding region. Following the mode of Fur protein, we speculate that S. pyogenes PerR is able to sense the excess metal ions from environment and act as a repressor dependent on conformational change.

1. Altuvia, S., Almiron, M., Huisman, G., Kolter, R., and
Storz, G. (1994). The dps promoter is activated by OxyR
during growth and by IHF and sigma S in stationary
phase. Mol Microbiol 13, 265-272.

2. Altuvia, S., Weinstein-Fischer, D., Zhang, A., Postow,
L., and Storz, G. (1997). A small, stable RNA induced
by oxidative stress: role as a pleiotropic regulator
and antimutator. Cell 90, 43-53.

3. Anjem, A., Varghese, S., and Imlay, J.A. (2009).
Manganese import is a key element of the OxyR response
to hydrogen peroxide in Escherichia coli. Mol Microbiol
72, 844-858. Epub 2009 Apr 2021.

4. Ardanuy, C., Domenech, A., Rolo, D., Calatayud, L.,
Tubau, F., Ayats, J., Martin, R., and Linares, J.
(2010). Molecular characterization of macrolide- and
multidrug-resistant Streptococcus pyogenes isolated
from adult patients in Barcelona, Spain (1993-2008). J
Antimicrob Chemother 65, 634-643.

5. Ashbaugh, C.D., Warren, H.B., Carey, V.J., and Wessels,
M.R. (1998). Molecular analysis of the role of the
group A streptococcal cysteine protease, hyaluronic
acid capsule, and M protein in a murine model of human
invasive soft-tissue infection. J Clin Invest 102, 550-
560.

6. Aslund, F., Zheng, M., Beckwith, J., and Storz, G.
(1999). Regulation of the OxyR transcription factor by
hydrogen peroxide and the cellular thiol-disulfide
status. Proc Natl Acad Sci U S A 96, 6161-6165.

7. Bagg, A., and Neilands, J.B. (1987). Ferric uptake
regulation protein acts as a repressor, employing iron
(II) as a cofactor to bind the operator of an iron
transport operon in Escherichia coli. Biochemistry
26, 5471-5477.

8. Beall, B., Facklam, R., Hoenes, T., and Schwartz, B.
(1997). Survey of emm gene sequences and T-antigen
types from systemic Streptococcus pyogenes infection
isolates collected in San Francisco, California;
Atlanta, Georgia; and Connecticut in 1994 and 1995. J
Clin Microbiol 35, 1231-1235.

9. Beall, B., Facklam, R., and Thompson, T. (1996).
Sequencing emm-specific PCR products for routine and
accurate typing of group A streptococci. J Clin
Microbiol 34, 953-958.

10.Beres, S.B., Sylva, G.L., Barbian, K.D., Lei, B., Hoff,
J.S., Mammarella, N.D., Liu, M.Y., Smoot, J.C.,
Porcella, S.F., Parkins, L.D., et al. (2002). Genome
sequence of a serotype M3 strain of group A
Streptococcus: phage-encoded toxins, the high-virulence
phenotype, and clone emergence. Proc Natl Acad Sci U S
A 99, 10078-10083. Epub 12002 Jul 10016.

11.Berlett, B.S., Chock, P.B., Yim, M.B., and Stadtman,
E.R. (1990). Manganese(II) catalyzes the
bicarbonate-dependent oxidation of amino acids by
hydrogen peroxide and the amino acid-facilitated
dismutation of hydrogen peroxide. Proc Natl Acad Sci U
S A 87, 389-393.

12.Brenot, A., King, K.Y., and Caparon, M.G. (2005). The
PerR regulon in peroxide resistance and virulence of
Streptococcus pyogenes. Mol Microbiol 55, 221-234.

13.Brenot, A., Weston, B.F., and Caparon, M.G. (2007). A
PerR-regulated metal transporter(PmtA) is an interface
between oxidative stress and metal homeostasis in
Streptococcus pyogenes. Mol Microbiol 63, 1185-1196.

14.Brosnahan, A.J., Mantz, M.J., Squier, C.A., Peterson,
M.L., and Schlievert, P.M. (2009). Cytolysins augment
superantigen penetration of stratified mucosa. J
Immunol 182, 2364-2373.

15.Bsat, N., Chen, L., and Helmann, J.D. (1996). Mutation
of the Bacillus subtilis alkyl hydroperoxide reductase
(ahpCF) operon reveals compensatory interactions among
hydrogen peroxide stress genes. J Bacteriol 178, 6579-
6586.

16.Bsat, N., and Helmann, J.D. (1999). Interaction of
Bacillus subtilis Fur (ferric uptake repressor)
with the dhb operator in vitro and in vivo. J Bacteriol
181, 4299-4307.

17.Burns, E.H., Jr., Marciel, A.M., and Musser, J.M.
(1996). Activation of a 66-kilodalton human
endothelial cell matrix metalloprotease by
Streptococcus pyogenes extracellular cysteine
protease. Infect Immun 64, 4744-4750.


18.Carapetis, J.R., Steer, A.C., Mulholland, E.K., and
Weber, M. (2005). The global burden of group A
streptococcal diseases. Lancet Infect Dis 5, 685-694.

19.Chang, H., Shen, X., Fu, Z., Liu, L., Shen, Y., Liu,
X., Yu, S., Yao, K., Zhao, C., and Yang, Y.
(2010). Antibiotic resistance and molecular analysis of
Streptococcus pyogenes isolated from healthy
schoolchildren in China. Scand J Infect Dis 42, 84-89.

20.Chao, S.-Y. (2010). Structural studies of the stress
response regulator PerR from
Streptococcus pyogenes. In Department of Microbiology
and Immunology,National Cheng Kung University, Tainan,
Taiwan

21.Chen, L., and Helmann, J.D. (1995). Bacillus subtilis
MrgA is a Dps(PexB) homologue: evidence for
metalloregulation of an oxidative-stress gene. Mol
Microbiol 18, 295-300.

22.Chen, L., James, L.P., and Helmann, J.D. (1993).
Metalloregulation in Bacillus subtilis: isolation and
characterization of two genes differentially repressed
by metal ions. J Bacteriol 175, 5428-5437.

23.Chen, L., Keramati, L., and Helmann, J.D. (1995).
Coordinate regulation of Bacillus subtilis
peroxide stress genes by hydrogen peroxide and metal
ions. Proc Natl Acad Sci U S A 92, 8190-8194.

24.Chen, Z., Lewis, K.A., Shultzaberger, R.K., Lyakhov,
I.G., Zheng, M., Doan, B., Storz, G., and Schneider,
T.D. (2007). Discovery of Fur binding site clusters in
Escherichia coli by information theory models. Nucleic
Acids Res 35, 6762-6777.

25.Christman, M.F., Morgan, R.W., Jacobson, F.S., and
Ames, B.N. (1985). Positive control of a regulon for
defenses against oxidative stress and some heat-shock
proteins in Salmonella typhimurium. Cell 41, 753-762.

26.Clancy, J., Petitpas, J., Dib-Hajj, F., Yuan, W.,
Cronan, M., Kamath, A.V., Bergeron, J., and
Retsema, J.A. (1996). Molecular cloning and functional
analysis of a novel macrolide-resistance determinant,
mefA, from Streptococcus pyogenes. Mol Microbiol 22,
867-879.

27.Cleary, P.P., Prahbu, U., Dale, J.B., Wexler, D.E., and
Handley, J. (1992). Streptococcal C5a peptidase is a
highly specific endopeptidase. Infect Immun 60, 5219-
5223.

28.Cone, L.A., Stone, R.A., Schlievert, P.M., Sneider,
R.A., Rubin, A.M., Jesser, K., and Renker, S.W. (2006).
An early favorable outcome of streptococcal toxic shock
syndrome may require a combination of antimicrobial and
intravenous gamma globulin therapy together with
activated protein C. Scand J Infect Dis 38, 960-963.

29.Cunningham, L., Gruer, M.J., and Guest, J.R. (1997).
Transcriptional regulation of the aconitase genes (acnA
and acnB) of Escherichia coli. Microbiology 143, 3795-
3805.

30.D'Autreaux, B., and Toledano, M.B. (2007). ROS as
signalling molecules: mechanisms that generate
specificity in ROS homeostasis. Nat Rev Mol Cell Biol
8, 813-824.

31.D'Autreaux, B., Touati, D., Bersch, B., Latour, J.M.,
and Michaud-Soret, I. (2002). Direct inhibition by
nitric oxide of the transcriptional ferric uptake
regulation protein via nitrosylation of the iron. Proc
Natl Acad Sci U S A 99, 16619-16624.

32.Delany, I., Rappuoli, R., and Scarlato, V. (2004). Fur
functions as an activator and as a repressor of
putative virulence genes in Neisseria meningitidis. Mol
Microbiol 52, 1081-1090.

33.Delany, I., Spohn, G., Rappuoli, R., and Scarlato, V.
(2001). The Fur repressor controls transcription of iron-
activated and -repressed genes in Helicobacter pylori.
Mol Microbiol 42, 1297-1309.

34.Dintilhac, A., Alloing, G., Granadel, C., and Claverys,
J.P. (1997). Competence and virulence of Streptococcus
pneumoniae: Adc and PsaA mutants exhibit a requirement
for Zn and Mn resulting from inactivation of putative
ABC metal permeases. Mol Microbiol 25, 727-739.

35.Duarte, V., and Latour, J.M. (2010). PerR vs OhrR:
selective peroxide sensing in Bacillus subtilis. Mol
Biosyst 6, 316-323.

36.Facklam, R., Beall, B., Efstratiou, A., Fischetti, V.,
Johnson, D., Kaplan, E., Kriz, P., Lovgren, M., Martin,
D., Schwartz, B., et al. (1999). emm typing and
validation of provisional M types for group A
streptococci. Emerg Infect Dis 5, 247-253.

37.Faulkner, M.J., and Helmann, J.D. (2011). Peroxide
stress elicits adaptive changes in bacterial
metal ion homeostasis. Antioxid Redox Signal 15, 175-
189. Epub 2011 Apr 2010.

38.Ferretti, J.J., McShan, W.M., Ajdic, D., Savic, D.J.,
Savic, G., Lyon, K., Primeaux, C., Sezate, S., Suvorov,
A.N., Kenton, S., et al. (2001). Complete genome
sequence of an M1 strain of Streptococcus pyogenes.
Proc Natl Acad Sci U S A 98, 4658-4663.

39.Friedman, Y.E., and O'Brian, M.R. (2004). The ferric
uptake regulator (Fur) protein from Bradyrhizobium
japonicum is an iron-responsive transcriptional
repressor in vitro. J Biol Chem 279, 32100-32105.

40.Fuangthong, M., Herbig, A.F., Bsat, N., and Helmann,
J.D. (2002). Regulation of the Bacillus subtilis fur
and perR genes by PerR: not all members of the PerR
regulon are peroxide inducible. J Bacteriol 184, 3276-
3286.

41.Gaballa, A., and Helmann, J.D. (2002). A peroxide-
induced zinc uptake system plays an important role in
protection against oxidative stress in Bacillus
subtilis. Mol Microbiol 45, 997-1005.

42.Giedroc, D.P. (2009). Hydrogen peroxide sensing in
Bacillus subtilis: it is all about the (metallo)
regulator. Mol Microbiol 73, 1-4.

43.Greenberg, J.T., and Demple, B. (1988). Overproduction
of peroxide-scavenging enzymes in Escherichia coli
suppresses spontaneous mutagenesis and sensitivity to
redox-cycling agents in oxyR-mutants. Embo J 7, 2611-
2617.

44.Gryllos, I., Grifantini, R., Colaprico, A., Cary, M.E.,
Hakansson, A., Carey, D.W., Suarez-Chavez, M., Kalish,
L.A., Mitchell, P.D., White, G.L., et al. (2008). PerR
confers phagocytic killing resistance and allows
pharyngeal colonization by group A Streptococcus.
PLoS Pathog 4, e1000145.

45.Guinier, A. (1938). The diffusion of x-rays under the
extremely weak angles applied to the study of fine
particles and colloidal suspension. C R Hebd Seances
Acad Sci 206, 1374–1376.

46.Hantke, K. (1981). Regulation of ferric iron transport
in Escherichia coli K12: isolation of a
constitutive mutant. Mol Gen Genet 182, 288-292.

47.Herr, M., Grabein, B., Palm, H.G., Efinger, K.,
Riesner, H.J., Friemert, B., and Willy, C. (2011).
[Necrotizing fasciitis. 2011 update]. Unfallchirurg
114, 197-216.

48.Hollingshead, S.K., Simecka, J.W., and Michalek, S.M.
(1993). Role of M protein in pharyngeal colonization by
group A streptococci in rats. Infect Immun 61, 2277-
2283.

49.Horsburgh, M.J., Wharton, S.J., Cox, A.G., Ingham, E.,
Peacock, S., and Foster, S.J. (2002). MntR modulates
expression of the PerR regulon and superoxide
resistance in Staphylococcus aureus through control of
manganese uptake. Mol Microbiol 44, 1269-1286.

50.Hytonen, J., Haataja, S., Gerlach, D., Podbielski, A.,
and Finne, J. (2001). The SpeB virulence
factor of Streptococcus pyogenes, a multifunctional
secreted and cell surface molecule with
strepadhesin, laminin-binding and cysteine protease
activity. Mol Microbiol 39, 512-519.

51.Imlay, J.A. (2003). Pathways of oxidative damage. Annu
Rev Microbiol 57, 395-418.

52.Imlay, J.A. (2008). Cellular defenses against
superoxide and hydrogen peroxide. Annu Rev Biochem 77,
755-776.

53.Jacquamet, L., Traore, D.A., Ferrer, J.L., Proux, O.,
Testemale, D., Hazemann, J.L., Nazarenko, E., El
Ghazouani, A., Caux-Thang, C., Duarte, V., et al.
(2009). Structural characterization of the active form
of PerR: insights into the metal-induced activation of
PerR and Fur proteins for DNA binding. Mol Microbiol
73, 20-31. Epub 2009 Jun 2008.

54.Jacques, D.A., and Trewhella, J. (2010). Small-angle
scattering for structural biology--expanding the
frontier while avoiding the pitfalls. Protein Sci 19,
642-657.

55.Janulczyk, R., Ricci, S., and Bjorck, L. (2003). MtsABC
is important for manganese and iron transport,
oxidative stress resistance, and virulence of
Streptococcus pyogenes. Infect Immun
71, 2656-2664.

56.Johansson, L., Thulin, P., Low, D.E., and Norrby-
Teglund, A. (2010). Getting under the skin:
the immunopathogenesis of Streptococcus pyogenes deep
tissue infections. Clin Infect Dis 51, 58-65.

57.Johnson, D.R., and Kaplan, E.L. (1993). A review of the
correlation of T-agglutination patterns
and M-protein typing and opacity factor production in
the identification of group A streptococci.
J Med Microbiol 38, 311-315.

58.Kapur, V., Topouzis, S., Majesky, M.W., Li, L.L.,
Hamrick, M.R., Hamill, R.J., Patti, J.M., and
Musser, J.M. (1993). A conserved Streptococcus pyogenes
extracellular cysteine protease cleaves human
fibronectin and degrades vitronectin. Microb Pathog 15,
327-346.

59.Kaul, R., McGeer, A., Low, D.E., Green, K., and
Schwartz, B. (1997). Population-based surveillance for
group A streptococcal necrotizing fasciitis: Clinical
features, prognostic indicators, and microbiologic
analysis of seventy-seven cases. Ontario Group A
Streptococcal Study. Am J Med 103, 18-24.

60.Kehres, D.G., Zaharik, M.L., Finlay, B.B., and Maguire,
M.E. (2000). The NRAMP proteins of Salmonella
typhimurium and Escherichia coli are selective
manganese transporters involved in the response to
reactive oxygen. Mol Microbiol 36, 1085-1100.

61.King, K.Y., Horenstein, J.A., and Caparon, M.G. (2000).
Aerotolerance and peroxide resistance in peroxidase and
PerR mutants of Streptococcus pyogenes. J Bacteriol
182, 5290-5299.

62.Kliegman, J.I., Griner, S.L., Helmann, J.D., Brennan,
R.G., and Glasfeld, A. (2006). Structural basis for the
metal-selective activation of the manganese transport
regulator of Bacillus subtilis.
Biochemistry 45, 3493-3505.

63.Kotb, M., Norrby-Teglund, A., McGeer, A., El-Sherbini,
H., Dorak, M.T., Khurshid, A., Green, K.,
Peeples, J., Wade, J., Thomson, G., et al. (2002). An
immunogenetic and molecular basis for differences in
outcomes of invasive group A streptococcal infections.
Nat Med 8, 1398-1404. Epub 2002 Nov 1318.

64.Kozin, M.B., and Svergun, D.I. Automated matching of
high- and low-resolution structural models.

65.Lamagni, T.L., Efstratiou, A., Vuopio-Varkila, J.,
Jasir, A., and Schalen, C. (2005). The
epidemiology of severe Streptococcus pyogenes
associated disease in Europe. Euro Surveill
10, 179-184.

66.Lancefield, R.C. (1928). THE ANTIGENIC COMPLEX OF
STREPTOCOCCUS HAEMOLYTICUS : V. ANAPHYLAXIS WITH THE
TYPE-SPECIFIC SUBSTANCE. J Exp Med 47, 857-875.

67.Lancefield, R.C. (1962). Current knowledge of type-
specific M antigens of group A streptococci.
J Immunol 89, 307-313.

68.Lee, J.W., and Helmann, J.D. (2006). The PerR
transcription factor senses H2O2 by
metal-catalysed histidine oxidation. Nature 440, 363-
367.

69.Lee, J.W., and Helmann, J.D. (2007). Functional
specialization within the Fur family of
metalloregulators. Biometals 20, 485-499.

70.Lewin, A.C., Doughty, P.A., Flegg, L., Moore, G.R., and
Spiro, S. (2002). The ferric uptake regulator of
Pseudomonas aeruginosa has no essential cysteine
residues and does not contain a structural zinc ion.
Microbiology 148, 2449-2456.

71.Li, J.M., Su, Y.L., Gao, X.L., He, J., Liu, S.S., and
Wang, X.W. (2011). Molecular characterization and
oxidative stress response of an intracellular Cu/Zn
superoxide dismutase (CuZnSOD) of the whitefly, Bemisia
tabaci. Arch Insect Biochem Physiol 3, 20428.

72.Ligeza, A., Tikhonov, A.N., Hyde, J.S., and Subczynski,
W.K. (1998). Oxygen permeability of thylakoid
membranes: electron paramagnetic resonance spin
labeling study. Biochim Biophys Acta 1365, 453-463.

73.Liochev, S.I., and Fridovich, I. (1992). Fumarase C,
the stable fumarase of Escherichia coli, is
controlled by the soxRS regulon. Proc Natl Acad Sci U S
A 89, 5892-5896.

74.Luca-Harari, B., Darenberg, J., Neal, S., Siljander,
T., Strakova, L., Tanna, A., Creti, R., Ekelund, K.,
Koliou, M., Tassios, P.T., et al. (2009). Clinical and
microbiological characteristics of severe Streptococcus
pyogenes disease in Europe. J Clin Microbiol 47, 1155-
1165. Epub 2009 Jan 1121.

75.Lynskey, N.N., Lawrenson, R.A., and Sriskandan, S.
(2011). New understandings in Streptococcus pyogenes.
Curr Opin Infect Dis 24, 196-202.

76.Ma, Z., Lee, J.W., and Helmann, J.D. (2011).
Identification of altered function alleles that affect
Bacillus subtilis PerR metal ion selectivity. Nucleic
Acids Res.

77.Masse, E., and Gottesman, S. (2002). A small RNA
regulates the expression of genes involved
in iron metabolism in Escherichia coli. Proc Natl Acad
Sci U S A 99, 4620-4625.

78.Meisal, R., Andreasson, I.K., Hoiby, E.A., Aaberge,
I.S., Michaelsen, T.E., and Caugant, D.A.
(2010). Streptococcus pyogenes isolates causing severe
infections in Norway in 2006 to 2007: emm types,
multilocus sequence types, and superantigen profiles. J
Clin Microbiol 48, 842-851. Epub 2009 Dec 2030.

79.Moore, C.M., and Helmann, J.D. (2005). Metal ion
homeostasis in Bacillus subtilis. Curr Opin
Microbiol 8, 188-195.

80.Morantes, M.C., and Lipsky, B.A. (1995). "Flesh-eating
bacteria": return of an old nemesis. Int
J Dermatol 34, 461-463.

81.Mostertz, J., Scharf, C., Hecker, M., and Homuth, G.
(2004). Transcriptome and proteome analysis of Bacillus
subtilis gene expression in response to superoxide and
peroxide stress. Microbiology 150, 497-512.

82.O'Loughlin, R.E., Roberson, A., Cieslak, P.R.,
Lynfield, R., Gershman, K., Craig, A., Albanese,
B.A., Farley, M.M., Barrett, N.L., Spina, N.L., et al.
(2007). The epidemiology of invasive group A
streptococcal infection and potential vaccine
implications: United States, 2000-2004. Clin
Infect Dis 45, 853-862. Epub 2007 Aug 2029.

83.Olsen, R.J., Sitkiewicz, I., Ayeras, A.A., Gonulal,
V.E., Cantu, C., Beres, S.B., Green, N.M., Lei,
B., Humbird, T., Greaver, J., et al. (2010). Decreased
necrotizing fasciitis capacity caused by a single
nucleotide mutation that alters a multiple gene
virulence axis. Proc Natl Acad Sci U S A
107, 888-893. Epub 2010 Jan 2014.

84.Outten, F.W., Djaman, O., and Storz, G. (2004). A suf
operon requirement for Fe-S cluster assembly during
iron starvation in Escherichia coli. Mol Microbiol 52,
861-872.

85.P. V. Konarev, M.V.P.V.V.V., and Svergun, D.I. ATSAS
2.1, a program package for small-angle
scattering data analysis.

86.Passali, D., Lauriello, M., Passali, G.C., Passali,
F.M., and Bellussi, L. (2007). Group A
streptococcus and its antibiotic resistance. Acta
Otorhinolaryngol Ital 27, 27-32.

87.Pohl, E., Haller, J.C., Mijovilovich, A., Meyer-
Klaucke, W., Garman, E., and Vasil, M.L. (2003).
Architecture of a protein central to iron homeostasis:
crystal structure and spectroscopic analysis of the
ferric uptake regulator. Mol Microbiol 47, 903-915.

88.Powell, S.R. (2000). The antioxidant properties of
zinc. J Nutr 130, 1447S-1454S.

89.Rada, B., and Leto, T.L. (2008). Oxidative innate
immune defenses by Nox/Duox family NADPH oxidases.
Contrib Microbiol 15, 164-187.

90.Ricci, S., Janulczyk, R., and Bjorck, L. (2002). The
regulator PerR is involved in oxidative stress response
and iron homeostasis and is necessary for full
virulence of Streptococcus pyogenes. Infect Immun 70,
4968-4976.

91.Rizzo, A.M., Berselli, P., Zava, S., Montorfano, G.,
Negroni, M., Corsetto, P., and Berra, B. (2011).
Endogenous Antioxidants and Radical Scavengers. Adv Exp
Med Biol 698, 52-67.

92.Safo, M.K., Zhao, Q., Ko, T.P., Musayev, F.N.,
Robinson, H., Scarsdale, N., Wang, A.H., and
Archer, G.L. (2005). Crystal structures of the BlaI
repressor from Staphylococcus aureus and

93.its complex with DNA: insights into transcriptional
regulation of the bla and mec operons. J Bacteriol 187,
1833-1844.

94.Schöneich, C. (2000). Mechanisms of metal-catalyzed
oxidation of histidine to 2-oxo-histidine in peptides
and proteins. Journal of Pharmaceutical and Biomedical
Analysis 21, 1093-1097.

95.Shelburne, S.A., 3rd, Keith, D., Horstmann, N., Sumby,
P., Davenport, M.T., Graviss, E.A., Brennan, R.G., and
Musser, J.M. (2008). A direct link between carbohydrate
utilization and virulence in the major human pathogen
group A Streptococcus. Proc Natl Acad Sci U S A 105,
1698-1703. Epub 2008 Jan 1629.

96.Shulman, S.T., Tanz, R.R., Dale, J.B., Beall, B.,
Kabat, W., Kabat, K., Cederlund, E., Patel, D.,
Rippe, J., Li, Z., et al. (2009). Seven-year
surveillance of north american pediatric group a
streptococcal pharyngitis isolates. Clin Infect Dis 49,
78-84.

97.Sitkiewicz, I., Nagiec, M.J., Sumby, P., Butler, S.D.,
Cywes-Bentley, C., and Musser, J.M. (2006). Emergence
of a bacterial clone with enhanced virulence by
acquisition of a phage encoding a secreted
phospholipase A2. Proc Natl Acad Sci U S A 103, 16009-
16014. Epub 12006 Oct 16016.

98.Smith, J.L. (2004). The physiological role of ferritin-
like compounds in bacteria. Crit Rev Microbiol 30, 173-
185.

99.Steer, A.C., Law, I., Matatolu, L., Beall, B.W., and
Carapetis, J.R. (2009). Global emm type distribution of
group A streptococci: systematic review and
implications for vaccine development. Lancet Infect Dis
9, 611-616.

100.Stevens, D.L. (1999). The flesh-eating bacterium:
what's next? J Infect Dis 179, S366-374.

101.Stevens, D.L., Gibbons, A.E., Bergstrom, R., and Winn,
V. (1988). The Eagle effect revisited: efficacy of
clindamycin, erythromycin, and penicillin in the
treatment of streptococcal myositis. J Infect Dis 158,
23-28.

102.Stone, J.R. (2004). An assessment of proposed
mechanisms for sensing hydrogen peroxide in
mammalian systems. Arch Biochem Biophys 422, 119-124.

103.Sun, Q.Y., Ding, L.W., He, L.L., Sun, Y.B., Shao,
J.L., Luo, M., and Xu, Z.F. (2009). Culture of
Escherichia coli in SOC medium improves the cloning
efficiency of toxic protein genes. Anal Biochem 394,
144-146.

104.Tamayo, E., Montes, M., Garcia-Medina, G., Garcia-
Arenzana, J.M., and Perez-Trallero, E. (2010). Spread
of a highly mucoid Streptococcus pyogenes emm3/ST15
clone. BMC Infect Dis 10, 233.

105.Traore, D.A., El Ghazouani, A., Ilango, S., Dupuy, J.,
Jacquamet, L., Ferrer, J.L., Caux-Thang, C., Duarte,
V., and Latour, J.M. (2006). Crystal structure of the
apo-PerR-Zn protein from Bacillus subtilis. Mol
Microbiol 61, 1211-1219.

106.Traore, D.A., El Ghazouani, A., Jacquamet, L., Borel,
F., Ferrer, J.L., Lascoux, D., Ravanat,
J.L., Jaquinod, M., Blondin, G., Caux-Thang, C., et
al. (2009). Structural and functional characterization
of 2-oxo-histidine in oxidized PerR protein. Nat Chem
Biol 5, 53-59. Epub 2008 Dec 2014.

107.Tsou, C.C., Chiang-Ni, C., Lin, Y.S., Chuang, W.J.,
Lin, M.T., Liu, C.C., and Wu, J.J. (2008). An iron-
binding protein, Dpr, decreases hydrogen peroxide
stress and protects Streptococcus pyogenes against
multiple stresses. Infect Immun 76, 4038-4045.

108.Tsou, C.C., Chiang-Ni, C., Lin, Y.S., Chuang, W.J.,
Lin, M.T., Liu, C.C., and Wu, J.J. (2010). Oxidative
stress and metal ions regulate a ferritin-like gene,
dpr, in Streptococcus pyogenes. Int J Med Microbiol
300, 259-264. Epub 2009 Oct 2029.

109.Varghese, S., Wu, A., Park, S., Imlay, K.R., and
Imlay, J.A. (2007). Submicromolar hydrogen
peroxide disrupts the ability of Fur protein to
control free-iron levels in Escherichia coli. Mol
Microbiol 64, 822-830.

110.Volkov, V.V., and Svergun, D.I. Uniqueness of ab
initio shape determination in small-angle
scattering.

111.Wang, M., Su, X., Li, Y., Jun, Z., and Li, T. (2010).
Cloning and expression of the Mn-SOD gene from
Phascolosoma esculenta. Fish Shellfish Immunol 29, 759-
764. Epub 2010 Jul 2021.

112.Weiss, K.A., and Laverdiere, M. (1997). Group A
Streptococcus invasive infections: a review.
Can J Surg 40, 18-25.

113.Zheng, M., Aslund, F., and Storz, G. (1998).
Activation of the OxyR transcription factor by
reversible disulfide bond formation. Science 279, 1718-
1721.