the cryptic competence pathway in streptococcus pyogenes ... · 99 activate the competence pathway...
TRANSCRIPT
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The Cryptic Competence Pathway in 4
Streptococcus pyogenes is Controlled by a 5
Peptide Pheromone 6
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Lauren Mashburn-Warren1, Donald A. Morrison2, and Michael J. Federle1,3# 9
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1Center for Pharmaceutical Biotechnology, College of Pharmacy, 2Laboratory for Molecular 11 Biology, Department of Biological Sciences, College of Liberal Arts and Sciences, 3Department 12
of Medicinal Chemistry and Pharmacognacy, College of Pharmacy, University of Illinois at 13 Chicago, Chicago, IL 60607 14
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16 Running title: XIP pheromone induces SigX and its targets in GAS 17 18 Keywords: GAS, ComRS, quorum sensing, transformation 19 20 21 22 23 #Corresponding author: 24 Michael Federle 25 Molecular Biology Research Building, Room 3152 26 900 S. Ashland Ave. (MC 870) 27 Chicago, IL 60607 28 Phone: 312-413-0213 29 Fax: 312-413-9303 30 E-mail:[email protected] 31 32
Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.00830-12 JB Accepts, published online ahead of print on 22 June 2012
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Abstract 34
Horizontal gene transfer is an important means of bacterial evolution facilitated by transduction, 35
conjugation, and natural genetic transformation. Transformation occurs after bacterial cells enter 36
a state of competence, where naked DNA is acquired from the extracellular environment. 37
Induction of the competent state relies on signals that activate master regulators, causing the 38
expression of genes involved in DNA uptake, processing, and recombination. All streptococcal 39
species contain the master regulator SigX and SigX-dependent effector genes required for 40
natural genetic transformation; yet not all streptococcal species have been shown to be 41
naturally competent. We recently demonstrated that competence development in Streptococcus 42
mutans requires the Type II ComRS quorum sensing circuit, comprising an Rgg transcriptional 43
activator and a novel peptide pheromone. The Type II ComRS system is shared by the 44
pyogenic, mutans, and bovis streptococci, including the clinically relevant pathogen, 45
Streptococcus pyogenes. Here we describe the activation of sigX by a small peptide pheromone 46
and an Rgg regulator of the Type II ComRS class. We confirm previous reports that SigX is 47
functional and able to activate sigX-dependent gene expression within the competence regulon, 48
and that SigX stability is influenced by the cytoplasmic protease ClpP. Genomic analyses of 49
available S. pyogenes genomes revealed the presence of intact genes within the competence 50
regulon. While this is the first report to show natural induction of sigX, S. pyogenes remained 51
non-transformable under laboratory conditions. Using radiolabeled DNA, we demonstrate that 52
transformation is blocked at the stage of DNA uptake. 53
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Acquisition of exogenous DNA via conjugation, transduction, and natural transformation 59
exists in both Bacteria and Archaea and these means of horizontal gene transfer have likely 60
contributed to their survival and evolution (29, 31). Over 70 bacterial species are capable of 61
natural genetic transformation, which transpires when bacterial cells enter a state termed 62
competence (24). In the competent state, a large number of genes encoding the DNA uptake 63
apparatus and DNA recombination proteins are expressed, facilitating the import and 64
chromosomal insertion of exogenous DNA. In many bacterial species the competent state is 65
constitutive, whereas in others competence is tightly regulated and depends on specific factors 66
or conditions such as pheromones, nutrient availability, and stress. Once sensed by the 67
bacterial cells, these conditions and factors stimulate the expression of a master regulator(s) 68
that in turn activates transcription of the genes for DNA uptake and recombination (for a review 69
see (24)). 70
Natural genetic transformation in Gram-positive bacteria is especially well studied in 71
Bacillus subtilis and Streptococcus pneumoniae, and a conserved set of genes found to be 72
required for the transformation process is conserved between them (7, 10, 21, 33, 39, 47). 73
Within the streptococci, the early genes of competence development include a quorum sensing 74
system that activates the master regulator of competence, the alternative sigma factor sigX (47). 75
SigX recognizes a conserved DNA sequence present upstream of the late competence genes 76
necessary for DNA uptake and recombination, known as a CIN-box. Together with the core 77
subunits of RNA polymerase, SigX binds to the CIN-box and promotes transcription of the late 78
genes (40). 79
In S. pneumoniae and other members of the anginosis and mitis groups of streptococci, 80
induction of sigX itself requires the secretion and detection of a peptide pheromone known as 81
the competence-stimulating peptide (CSP) (19). CSP, a cell-cell signaling peptide from the 82
double-glycine class, is encoded by comC, secreted by the dedicated ABC transporter ComAB, 83
and sensed by a two-component transduction system (ComDE). ComE, in turn, activates 84
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expression of sigX and ultimately switches on the competent state (for a review see (25)). In the 85
mutans, pyogenic, and bovis groups of streptococci, it appears that different cell-cell signaling 86
mechanisms control the competence regulon. We recently discovered that the competence 87
pathway in Streptococcus mutans depends on a quorum sensing mechanism wherein a novel 88
sigX-inducing peptide (XIP) interacts with the Rgg-like transcriptional regulator ComR (34). 89
Members of the Rgg family are stand-alone regulators found within a majority of Gram-positive 90
bacteria and are thought to interact with small peptides to modulate their activities (34, 36, 49, 91
52, 56). Collectively, ComR and XIP activate sigX and comS expression, leading to induction of 92
late competence genes and creating a positive auto-induction loop of the signaling system. (Fig. 93
1). The promoter regions of sigX and comS contain identical unique DNA sequence patterns 94
designated as P1, and we hypothesize that these patterns are the sites at which ComR/XIP bind 95
to activate transcription. Homologues of comR and the XIP gene, comS, exist in S. pyogenes 96
and in all other pyogenic, bovis and mutans species of streptococci with sequenced genomes. 97
Due to this conservation, we hypothesized that comRS may be the component necessary to 98
activate the competence pathway in species like S. pyogenes that have been refractory to 99
natural genetic transformation in the laboratory (34). 100
S. pyogenes, also known as Group A Streptococcus (GAS), is a human pathogen 101
capable of causing a variety of diseases. Such diseases range from acute noninvasive 102
infections such as pharyngitis (strep throat) and impetigo to invasive life-threatening diseases 103
like necrotizing fasciitis and bacteremia (11). Genome sequencing has revealed that horizontal 104
gene transfer is frequent among strains of S. pyogenes, as these strains are highly diverse and 105
non-clonal (4, 37). Although transduction has been suggested as a primary mechanism for 106
horizontal gene transfer in GAS, it seems likely, especially considering the high rates of 107
recombination among GAS strains (4, 18), that other means of horizontal gene transfer are 108
utilized, such as natural transformation. 109
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In addition to the principal and essential sigma factor σA, S. pyogenes contains a non-110
essential alternative sigma factor, σX, which is 40% identical to sigX of S. pneumoniae (41-42). 111
In vitro, recombinant SigX from GAS activates the transcription of genes containing CIN-boxes 112
within their promoter regions; however, in vivo production of SigX is restricted at two levels (41, 113
59). First, under laboratory conditions sigX expression remains very low. However, engineered 114
over-expression of sigX leads to the induction of a number of putative late competence genes, 115
indicating that sigX is functional in vivo. Second, SigX accumulation and stability is negatively 116
affected by the protease ClpP (Fig. 1) (41, 59). ClpP is a cytoplasmic protein that affects stability 117
of many bacterial proteins and negatively affects SigX stability and transformation in S. 118
pneumoniae (30, 51). 119
Although S. pyogenes is considered to be non-competent, there is one report suggesting 120
DNA transfer among GAS isolates. A locus important for invasive disease known as the 121
streptococcal invasion locus (sil) was discovered in GAS and has homology to the comABCDE 122
systems of S. pneumoniae and S. mutans (20). This locus is present in about 18% of GAS 123
isolates and contains genes which encode a cell-cell signaling peptide SilCR as well as an ABC 124
transporter (silDE) and a two-component signal transduction system (silAB). SilCR is part of the 125
double-glycine family and is part of a regulatory circuit that auto-induces transcription of the 126
peptide itself and silDE (13, 20). Hidalgo-Grass et al. reported sil-containing strains of GAS 127
were able to exchange exogenous DNA, although the frequency of DNA transfer was very low 128
at ~10-8 (20). While the mechanism of such DNA transfer is undefined, it has since been 129
demonstrated that SilCR does not influence expression of sigX and it appears the sil locus is 130
responsible for regulating the expression of bacteriocins (1, 13). 131
In addition to sigX, GAS contains all of the late genes known to be required for 132
competence in B. subtilis and S. pneumoniae, but the genes necessary for activation of sigX 133
have not been defined (59). Here we report on the mechanism of sigX induction in S. pyogenes 134
and demonstrate that the competence regulon is activated by a small peptide pheromone. 135
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Materials and Methods 136
Bacterial strains, media, and plasmids. Strains MGAS315 and UA159 were kind gifts from 137
Michael Chaussee (University of South Dakota) and Lin Tao (UIC Dental College), respectively. 138
Strain MGAS8232 was obtained from the ATCC (BAA-572). Other strains and plasmids are 139
described in Table 1. Oligonucleotide primers are listed in Table 2. Cultures of GAS were grown 140
in closed tubes at 37°C in chemically defined medium (CDM) (8, 58) or Todd Hewitt Broth (THB) 141
and stored at -80°C in the same medium supplemented with 10% glycerol. For many 142
experiments, mid-logarithmic frozen stocks were prepared by diluting overnight cultures into 143
fresh medium, followed by logarithmic growth to an OD600 ~0.4, at which time samples were 144
supplemented with glycerol and quickly frozen in culture tubes placed in a dry-ice/ethanol bath. 145
Mid-logarithmic stock cultures used to inoculate fresh medium consistently demonstrated 146
reproducible growth rates with a short lag phase. For selection, cells were plated onto THY agar 147
(Todd Hewett Broth + 0.2% yeast extract + 1.5% agar) with the appropriate antibiotic and 148
incubated for 24h at 37°C in 5% CO2. Selective levels of antibiotics for S. pyogenes were 1 149
µg/ml erythromycin, 100 µg/ml spectinomycin, and 4 µg/ml chloramphenicol. For E. coli, 150
antibiotics used were 500 µg/ml erythromycin, 100 µg/ml spectinomycin, and 10 µg/ml 151
chloramphenicol. 152
Construction of mutants. comR and clpP mutants were prepared according to Lau et al. (28), 153
using primers and plasmids listed in Table 1 and 2. The comR mutant was constructed by 154
insertion of a chloramphenicol resistance cassette (cat) into the comR ORF. Fragments of 1 kb 155
flanking comR (comR-US and comR-DS) were amplified by PCR using the primer pairs 156
LMW30/31 and LMW32/33 and MGAS315 DNA as template. These primers created SalI sites 157
at the 3’ and 5’ ends of comR-US and comR-DS PCR fragments, respectively. ComR-US and 158
comR-DS fragments were fused together by in vitro ligation and then amplified using primer 159
pairs LMW30/33 creating the comR-US/DS fusion. These primers created NotI and XhoI sites at 160
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the 5’ and 3’ ends of comR-US/DS allowing insertion into pFED760 using the NotI and XhoI 161
sites. The cat cassette was amplified from pEVP3 using primers pairs LMW34/35, which created 162
SalI sites at both the 5’ and 3’ ends. cat was then ligated into pFED760 between comR-US and 163
comR-DS to create the comR knock out construct, pWAR195. The clpP mutant was constructed 164
by insertion of a spectinomycin cassette (aad9) into the clpP ORF. Fragments of 1 kb flanking 165
clpP (clpP-US and clpP-DS) were amplified by PCR using the primer pairs LMW36/37 and 166
LMW38/39 and MGAS315 template DNA. These primers created NotI and SpeI sites at the 5’ 167
and 3’ ends of clpP-US and PstI and XhoI sites at the 5’ and 3’ ends of clpP-DS. The 168
spectinomycin cassette aad9 was amplified from pLZ12-Spc using primers LMW40/41 creating 169
SpeI and PstI sites on the 5’ and 3’ ends. ClpP-US, aad9, and clpP-DS were each separately 170
inserted into pFED760 using their respective restrictions sites to create the clpP knock out 171
construct pWAR251. Both pWAR195 and pWAR251 knock-out constructs were transferred into 172
E. coli BH10C (22) separately by electroporation and selection on Luria-Bertani agar containing 173
either 10 µg/ml chloramphenicol or 100 µg/ml spectinomycin. To create the comR and clpP 174
mutants pWAR195 and pWAR251 were transferred into S. pyogenes MGAS315 and selected 175
and confirmed as described previously by Chang et al. (8). 176
Construction of reporter plasmids. The PsigX-luxAB reporter plasmid was constructed by 177
amplifying the sigX promoter region using primers LMW26/27 and MGAS315 DNA template. 178
The sigX reporter region (561 bp) was then inserted into pWAR303 using the SalI and PstI sites 179
to create pWAR200. The PssbB-luxAB reporter plasmid was prepared by amplifying the ssbB 180
promoter region (362 bp) using primers LMW28/29 using MGAS315 DNA, followed by insertion 181
into the SalI and PstI sites of pWAR303 to create pWAR205. Both reporter constructs were 182
transferred into S. pyogenes strains as described previously (8). 183
Preparation of synthetic peptides. Peptides were purchased from NEO-Peptide (Cambridge, 184
MA) at a 50-53% purity grade. Stock solutions were dissolved in DMSO at a concentration of 185
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500 µM based on their specific purity. Peptide sequences are as follows: M1 XIP:ComS24-31: 186
SAVDWWRL; M3 XIP:ComS25-32: EFDWWNLG; SilCR: DIFKLVIDHISMKARKK; S. mutans 187
XIP:ComS11-17: GLDWWSL. 188
Reporter assays. Optical densities were measured throughout growth in closed 20 mm screw-189
cap tubes using a Milton Roy Spectronic 20D spectrophotometer (Fisher Scientific, Pittsburg, 190
PA) and luminescence was assayed with a Wallac 1450 Microbeta Plus Liquid Scintillation 191
Counter (Perkin Elmer, Wathum, MA). Reporter strains were grown at 37°C in CDM or THB 192
after dilution from mid-logarithmic stocks to OD600 0.01. Cultures were grown to OD600 0.1 and 193
supplemented with either DMSO (vehicle) or exogenous peptides. Aliquots (100 µl) were placed 194
in a Falcon white flat-bottom 96-well plate (Becton Dickinson Labware, Franklin Lakes, NJ) and 195
exposed for 30 seconds to vapors that result from 50 µl of decyl aldehyde spread on the micro-196
titer plate lid. Plate lids were removed and luciferase activity (counts per minute) of each sample 197
was measured. A 100 µl aliquot of cell-free culture medium served as control to measure 198
background levels of luciferase, and was then subtracted from the luciferase activity detected in 199
sample wells. 200
Immunoblotting. MGAS315 wild type and the clpP mutant strain were grown at 37°C in 10 ml 201
of CDM after dilution from mid-logarithmic stocks to OD600 0.01. Cultures were grown to OD600 202
0.4 and supplemented with 1 µM M3 XIP or DMSO (vehicle), followed by growth at 37°C for 30 203
or 60 minutes. After the indicated times, hyaluronidase (10 units/ml) was added and cells were 204
harvested by centrifugation at 4°C and suspended in 150 µl TE buffer. To the cell suspension, 1 205
µl Ready-Lyse Lysozyme (Epicentre, Madison, WI), mutanolysin (1 unit/ml, Sigma-Aldrich), 150 206
µl Gram-positive lysis solution (Epicentre, Madison, WI), and ~150 µl of glass beads (100 µm, 207
Sigma-Aldrich) were added and cells were lysed with a Disruptor Genie (Scientific Industries, 208
Bohemia, NY) for 5 minutes for a total of 3 times with incubation on ice in between disruptions. 209
Twenty µg of protein from total cell lysates were subjected to sodium dodecyl sulfate- 15% 210
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polyacrylamide gel electrophoresis, followed by protein transfer to a nitrocellulose membrane. 211
Protein transfer was performed for 60 min. at 20 volts using 1X Transfer Buffer (25 mM TRIS-212
HCl, pH 8, 190 mM glycine, 20% methanol) at 4°C. Following transfer, the blot was blocked 213
overnight in 1X Blocking Buffer (3% dry milk, 10 mM Tris-HCl, pH 8, 300 mM NaCl, 0.05% 214
Tween) at room temperature with agitation. The blot was exposed to a 1:5,000 dilution of rabbit 215
anti-σX antibody (41) and detected with a 1:1,000 dilution of horseradish peroxidase-conjugated 216
goat-anti-rabbit antibody with Super Signal West Duration Substrate (Thermo Scientific, 217
Rockford, IL). 218
Microarray Analysis. Six separate cultures of the MGAS315 wild type were grown at 37°C in 219
CDM after dilution from mid-logarithmic stocks to OD600 0.01. At OD600 0.4, three of the cultures 220
were treated with 200 nM M3 XIP peptide and the other three with the peptide vehicle only 221
(0.04% DMSO). After 60 min. at 37°C, hyaluronidase (10 units/ml) was added to degrade 222
capsule and cells were harvested by centrifugation at 4°C. RNA was extracted using a 223
RiboPure-bacteria kit (Ambion, Austin, TX) according to the manufacturer’s instructions, 224
including the DNase treatment. RNA quality was analyzed by agarose gel electrophoresis and 225
concentrations were determined using a NanoDrop 1000 Spectrophotometer (Thermo Scientific, 226
Rockford, IL). Equal amounts of RNA from each of the three separate cultures were pooled 227
together and served as the templates for cDNA synthesis. cDNA generation and labeling, and 228
microarray hybridization, scanning, and normalization analysis were performed by the W.M. 229
Keck Center for Comparative and Functional Genomics at the University of Illinois at Urbana-230
Champaign following NimbleGen protocols as described previously (27). Microarrays were 231
designed based on the NZ131 genomic sequence (NC_011375.1), and contained an average of 232
20 different 60 nucleotide oligo probes per gene unit. Each probe was replicated four times per 233
array and standard deviations presented in Table 4 are technical replicates of hybridization 234
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values among probe replicates. Microarray data has been deposited with the Gene Expression 235
Omnibus (GEO) www.ncbi.nlm.nih.gov/projects/geo (accession # GSE37974). 236
Transformation assays. Unless designated otherwise, bacterial cultures were grown at 37°C in 237
the indicated culture medium after dilution from mid-logarithmic stocks. At the indicated times, 1 238
µg exogenous DNA harboring an antibiotic resistance marker was added followed by incubation 239
at 37°C. For assays using the Biolog Phenotypic MicroArrays (PM1-PM10, Hayword, CA) for 240
Microbial Cells, MGAS8232 was grown in CDM to OD600 0.1 followed by the addition of 1 µM 241
XIP and 1 µg genomic DNA harboring a spectinomycin resistance cassette. 150 µL of the 242
bacterial mixture was added to each well of the Phenotypic Microarray and allowed to grow at 243
37°C for 24 hours. Cells were then patched to THY agar plates containing 100 µg/mL 244
spectinomycin and incubated at 37°C in 5% CO2 for 24 hours. 245
Radiolabeling of genomic DNA. Genomic DNA was labeled by incorporation of 2,8-3H-246
adenine. MGAS8232 and UA159 were grown in CDM containing half the normal amount of 247
adenine. 200 µCi of 2,8-3H-adenine was added and cultures were grown overnight at 37°C in 248
5% CO2. Genomic DNA was prepared using the Master Pure Gram-Positive DNA Purification 249
Kit (Epicentre, Madison, WI) according to the manufacturer’s instructions. Free nucleotides were 250
removed by adding to the DNA preparation 1/10 volume of 5 M NaCl, followed by two volumes 251
of ice cold 95% ethanol and incubated at -80°C for 30 min. The precipitant DNA was collected at 252
16k x g for 15 minutes at 4°C in a microfuge. The resulting DNA pellet was rinsed twice with 253
70% ethanol, air dried for 15 minutes and dissolved in 100 µl H2O. DNA concentrations were 254
determined using a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Rockford, IL). 255
DNA uptake assays. MGAS315, MW151, UA159, and MW02 were grown at 37°C in CDM after 256
dilution from mid-logarithmic stocks to OD600 0.01. Cultures were grown to OD600 0.1 and 257
supplemented with 1 µM XIP (M3 or UA159) and allowed to grow for 60 min. at 37°C. After 60 258
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min., hyaluronidase (10 units/ml) was added and cells were harvested by centrifugation and 259
washed 1 time with CDM. Cells were suspended in CDM and hyaluronidase and 1 µM XIP 260
peptide was added and incubated at 37°C for 15 min. 300 ng of 3H-genomic DNA was added to 261
1 mL aliquots of each strain. At the indicated times, aliquots were chilled and harvested by 262
centrifugation at 4°C. DNA remaining in cell-free supernatants were subject to standard ethanol 263
precipitation; intact, linear DNA is able to precipitate, whereas free nucleotides remain in 264
solution. Cells were washed twice with cold CDM and suspended in 100 µl SEDS lysis buffer 265
(0.15 M sodium citrate, 0.15 M NaCl, 0.1% Triton X-100, and 0.01% sodium dodecyl sulfate) 266
(53). Cells were lysed for 10 min. at 37°C and mixed with 400 µl H2O; all samples received 10 267
ml EcoLume Scintillation fluid (MP Biomedicals, Solon, OH) and measured with a Beckman 268
6000IC scintillation counter. 269
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Results 271
S. pyogenes contains early and late genes for competence development. The goal of this 272
study was to identify the roles comR and comS play in competence development, and to better 273
characterize the regulation of putative competence genes in S. pyogenes. Since no 274
comprehensive analyses of competence genes in GAS have been reported, we took advantage 275
of the 14 available GAS genomes to determine the conservation of such genes among strains 276
isolated from a variety of geographical and anatomical sites. It is important to determine if these 277
genes have acquired mutations, as this could cause loss of function and might explain why no 278
descriptions of natural transformation of GAS are available. Two allelic forms of the comRS 279
locus (M1 and M3) were found among the 14 GAS genomes (Table 3), always located between 280
purB and ruvB. All M1 comR genes have >99% identity at the nucleotide level with the 281
exception of the M49 strain NZ131, which has a duplication resulting in the addition of three-282
amino acids within the C-terminal portion of the protein. Likewise, all four M3 comR alleles are 283
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identical. comS is located immediately downstream of comR, and the two comS alleles encode 284
proteins of 31 (M1) or 32 (M3) amino acids containing a double tryptophan motif within the 285
active C-terminal portion of the peptide (Table 3). As in S. pneumoniae, two copies of sigX are 286
situated apart from one another within the GAS chromosome (42). The conserved P1 promoter 287
pattern of comS is present upstream of each copy of sigX (Fig. 2) (34). All sigX ORFs are intact 288
and share >99% identityity among GAS strains, with the exceptions of MGAS2096 and NZ131, 289
where indels in sigX.2 result in respective frame shifts after amino acid 19 or 78 (Fig. 2). 290
The late genes of competence in S. pneumoniae encode the machinery needed for DNA 291
binding, uptake of single-stranded DNA, and recombination within the chromosome (Fig. 2). A 292
number of GAS genomes were found to contain mutations in one or more of the genes 293
necessary for the DNA uptake complex that render them predictably nonfunctional. In contrast 294
no mutations were discovered in the genes needed for DNA processing and recombination (Fig. 295
2). Although publically available genome sequence data indicate that half of the GAS genomes 296
contain one or two mutations in genes of the putative competence regulon, an equal number of 297
genomes appear to have completely intact sigX-dependent competence systems (M1 SF370, 298
MGAS5005, MGAS8232, MGAS9429, ATCC 10782, Manfredo, and MGAS10270) (Fig. 2). 299
300
XIP induces sigX expression in S. pyogenes. We previously demonstrated that comRS is 301
required for sigX activation in S. mutans, and we hypothesized that this same regulation exists 302
for S. pyogenes (34). To test this idea, we constructed a luciferase transcriptional reporter 303
containing the P1 promoter region of S. pyogenes sigX linked to luxAB, and transferred it into 304
wild type strains of S. pyogenes MGAS315 and MGAS8232, which contain the M3 and M1 305
comRS alleles, respectively (Table 3). To test for sigX induction by the ComS peptide, these 306
strains were grown in CDM at 37°C and cell density and light production were monitored 307
throughout growth. Using synthetic ComS analogues of different lengths, we found that the last 308
eight amino acids of both comS alleles produced the highest induction of sigX in GAS (data not 309
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shown). Therefore, we refer here to the active synthetic peptide containing the last eight amino 310
acids of ComS as XIP for sigX-inducing peptide. Without XIP addition, strains MW134 and 311
MW200 exhibited low activity that was slightly above background levels (cell-free medium), 312
indicating low expression from the sigX promoter. Addition of synthetic XIP caused an 313
immediate and robust induction of sigX expression that was dose dependent. Reporter activity 314
was detected with the addition of 0.5 nM XIP and was enhanced by the addition of increasing 315
amounts of peptide with saturation of sigX expression occurring when cultures were 316
supplemented with 100 nM XIP or higher (Fig. 3B and 3C). Unlike S. mutans, no growth 317
inhibition was observed in GAS cultures grown in the presence of XIP, even at 500 nM (Fig. 3A) 318
(12). Due to the similarity of M1 and M3 ComS peptides, we were interested in the possibility of 319
cross-talk between GAS allele types. To examine this, various concentrations of M1 and M3 XIP 320
were added to strains MW134 and MW200 respectively. As shown in Figure 3B, M1 XIP 321
induced sigX expression in MW134, but only when supplied at high concentration (500 nM). 322
Likewise, the addition of high concentrations (200-500 nM) of M3 XIP activated sigX expression 323
in MW200 (Fig. 3C).Thus, while cross-talk can be detected, the XIP receptor exhibited high 324
specificity. 325
To further understand the regulation of sigX by XIP, we transferred our PsigX::luxAB 326
reporter construct into various genetic backgrounds of MGAS315 and tested for sigX induction 327
by XIP. 200 nM XIP was chosen for further studies as this caused a strong induction of sigX 328
expression without affecting the growth rate of the culture (Fig. 3A). Luciferase activity in the 329
comR deletion strain MW151 indicated no induction of sigX by exogenous XIP pheromone, 330
confirming the prediction that comR is required for S. pyogenes response to XIP (Fig. 4A). 331
Although supplementation by peptide pheromone resulted in the induction of sigX transcription, 332
it is unknown whether the GAS SigX protein is stable and functional. To test this , we created a 333
luciferase reporter using the CIN-box-containing promoter region of ssbB, a late gene important 334
for natural transformation in S. pneumoniae (47). As illustrated in Fig. 4B, addition of exogenous 335
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pheromone led to induction of ssbB expression within 20 minutes, whereas no change was 336
seen with the vehicle control. The induction of ssbB was dependent on comR, as the low 337
luciferase activity observed in the ΔcomR strain MW256 was unchanged after addition of 338
exogenous XIP (Fig. 4B). We conclude that the SigX protein induced by XIP is sufficiently stable 339
to activate expression of at least one late competence gene. 340
Although SigX activated transcription of ssbB, we were interested in the effect of ClpP 341
on XIP-stimulated expression of sigX and ssbB. To determine if ClpP affects transcriptional 342
regulation of sigX or ssbB, we created a deletion mutant of clpP in MGAS315 and studied the 343
expression of sigX and ssbB using the same luciferase reporter constructs. Addition of XIP to 344
the clpP mutant caused detectable activation of sigX expression, although 10-fold less than in 345
the wild type background (Fig. 4A). In contrast, ssbB induction was slightly greater in the clpP 346
deletion strain compared to wild type. Interestingly, without the addition of the pheromone, the 347
ssbB reporter displayed a background activity that was already 10-fold higher in the clpP mutant 348
compared to the wild type (Fig. 4B). These data demonstrate that as in S. pneumoniae, ClpP 349
exerts negative effects on competence regulation in S. pyogenes. 350
To determine the accumulation and stability of SigX in response to peptide pheromone, 351
we used immunoblotting to assay crude whole cell lysates of MGAS315 wild type and the ΔclpP 352
strains prepared from cultures grown in CDM with or without XIP. Cells were allowed to grow to 353
mid-logarithmic phase and DMSO or XIP peptide was added and allowed to incubate for 30 or 354
60 minutes. SigX was not detected in the wild type strain, even in the presence of XIP (Fig. 5). 355
However, SigX was readily detected at both time points in the clpP mutant, but only after 356
pheromone addition, indicating that XIP induces the production of SigX protein, although its 357
stability depends on levels of ClpP protein within the cyotosol (Fig. 5). These results are 358
consistent with the idea that the ClpP protease participates in the degradation of SigX, thus 359
likely influencing transcription of genes downstream of sigX expression. 360
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Finally, the streptococcal invasion locus (sil) has been implicated as being important in 361
DNA transfer both in vitro and in vivo in S. pyogenes, although the reported DNA transfer 362
frequencies were very low (10-8). The sil locus is present in about 20% of S. pyogenes isolates 363
(13, 20) including MGAS8232, which contains intact copies of silAB, the two component system 364
that detects and senses the signaling peptide SilCR, but contains truncations in silDE, the ABC 365
transporter of SilCR (1, 13). This strain should be capable of detecting SilCR, but unable to 366
secrete the peptide. Because sil resembles the cell-cell signaling system for expression of sigX 367
in S. pneumoniae, we were interested in whether SilCR might affect sigX expression in S. 368
pyogenes. To examine this, we added synthetic SilCR peptide to MW200 grown in CDM and 369
rich media (THB). SilCR did not influence luciferase activity alone or with exogenous XIP, 370
indicative that it has no effect on sigX transcription (Fig. 4C, data not shown). Although the 371
mechanism of DNA transfer provoked by sil is not known, it appears that this locus activates the 372
expression of bacteriocins, and whether a correlation exists between sil-mediated DNA transfer 373
and natural genetic transformation in GAS remains unknown. 374
375
Microarray analysis reveals induction of late gene expression by XIP. Using the PssbB-376
luxAB reporter strain, we observed induction of the late competence gene ssbB upon addition of 377
exogenous XIP (see above, Fig. 4B). To begin to understand which additional genes are 378
differentially regulated by this peptide pheromone, we performed transcriptome analysis of the 379
effect of XIP on gene expression in wild type GAS. Specifically we were interested in 380
determining which of the genes required for competence in other streptococcal species were 381
induced and how specific the effect was in response to XIP. To examine this, we performed 382
microarray analysis of MGAS315 wild type supplemented with XIP. Cells were grown in CDM at 383
37°C to mid-logarithmic growth phase (OD600 = 0.1) and 200 M3 nM XIP or DMSO was added. 384
Cultures were grown for an additional 60 min. at 37°C followed by RNA isolation. 385
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The microarray pattern revealed two-fold induction, or greater, of only 30 genes, most of 386
which are known to be involved in competence development in naturally transformable 387
streptococci (Table 4). No genes were down-regulated by more than 2-fold in response to XIP. 388
Table 4 lists all of the genes whose expression was increased at least 2-fold in response to XIP 389
(compared to the vehicle control). 21 genes containing CIN-boxes within their promoter regions 390
were up-regulated in response to XIP, including the comY and comE operons, involved in DNA 391
binding and transport. Transcription levels of ssbB and both copies of sigX were increased; 392
confirming our observations with the corresponding reporter constructs (Fig. 3 and 4). Several 393
genes necessary for DNA processing and recombination were also up-regulated, although not 394
as strongly as those encoding the DNA uptake machinery (Table 4). The genes cinA, dut, radA, 395
and recA, which have high levels of constitutive expression in GAS, did not exhibit high 396
induction ratios (Table 4 and unpublished results). Therefore, all induced genes known to be 397
required for genetic transformation in B. subtilis and S. pneumoniae were expressed when cells 398
were provided with XIP. We also detected the activation of murM2, a protein thought to be 399
important in antimicrobial resistance (3, 26, 37, 59). The promoter of murM2 contains a CIN-box 400
and was previously shown to be activated by over-expression of sigX in GAS (58), but whether 401
this protein is important for natural transformation is unknown (59). Nine genes which do not 402
possess obvious CIN-boxes, were also up-regulated, although not to the same extent as CIN-403
box-containing genes. These genes may be indirectly controlled by XIP/SigX, but their functions 404
in competence development, if any, remain undefined. Interestingly, rocA (spy1605), a histidine 405
kinase found previously to influence capsule regulation and the activity of the covR/S two-406
component signal transduction system (5), was the only other putative regulatory gene seen to 407
be under XIP control. 408
409
S. pyogenes remains non-competent after induction by XIP under laboratory conditions, 410
where natural transformation is blocked at DNA uptake. Since induction of the competence 411
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regulon in S. pyogenes was achieved by XIP stimulation, we predicted that GAS might also 412
become naturally transformable in the presence of this peptide pheromone. To test for 413
competence, MGAS315 wild type and the clpP mutant were grown in CDM at 37°C. Throughout 414
growth, cells were incubated in the presence of 1 µM XIP and 1 µg donor DNA harboring an 415
antibiotic resistance marker. Various forms of DNA were used for transformation assays, 416
including GAS genomic DNA, plasmid DNA, and linear DNA from PCR products. After 417
incubation at 37°C for 2h, cells were plated onto rich media with the appropriate antibiotics to 418
select for transformants. Despite numerous attempts using a number of additional GAS strains, 419
S. pyogenes yielded no transformants. 420
Because growth conditions and other factors within bacterial cultures can affect the 421
ability for bacterial cells to become naturally competent (12, 16-17, 34, 38, 50), we used a 422
variety of additional growth media, together with exogenous DNA, to test for transformation in S. 423
pyogenes. These media included rich broth formulations, such as Todd-Hewett Broth, Brain-424
Heart Infusion Broth, Tryptic Soy Broth, and C-medium (32). We also grew cultures in CDM (8, 425
58) with varying carbon sources including glucose, mannose, and fructose, with and without the 426
addition of exogenous XIP. To test a wide range of growth substrates for competence 427
development in S. pyogenes, we also employed the commercially available Phenotypic 428
MicroArrays from Biolog (plates PM1-10). These arrays consist of 10, 96-well plates containing 429
differing growth substrates, including carbon, nitrogen, phosphorous, and sulfur sources, as well 430
as nutrient supplements, osmolytes, and varying pH. To test for natural transformation with the 431
Biolog system, MGAS8232 was grown in CDM at 37°C to mid–logarithmic growth phase 432
followed by the addition of 1 µM XIP and 1 µg GAS donor DNA harboring the antibiotic 433
resistance cassette aad9. The cell mixture was then added to the phenotypic arrays and 434
incubated at 37°C for 24 hours. Cells were subsequently transferred to agar plates containing 435
spectinomycin and allowed to grow at 37° for an additional 24 hours to select for transformants. 436
Glucose was the chosen carbon source for CDM, except in the arrays where the carbon 437
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sources were substituted (plates PM-1 and PM-2); for these arrays, mid-logarithmic-grown cells 438
were diluted into CDM containing no carbon. Although we tested ~1000 different growth 439
substrates using the phenotypic microarrays, no transformants were detected (data not shown). 440
To gain insight into the step at which transformation is blocked in GAS expressing the 441
comR/sigX regulon, we tested the ability of XIP-treated cultures to transport radiolabeled DNA 442
into the cell. Cell cultures were grown to mid-logarithmic phase in CDM and XIP was added to a 443
final concentration of 1 µM and incubated for 60 min at 37°C to stimulate competence. Cells 444
were then washed to remove any extracellular DNases, suspended in CDM with 1 µM peptide 445
pheromone, and incubated for an additional 15 minutes. 300 ng of radiolabeled genomic DNA 446
was added and samples of cells were removed periodically after addition of DNA to measure 447
the amount of radioactivity within the cells. In cultures of S. mutans treated the same way as a 448
control, we observed a linear increase in DNA uptake over time, whereas the S. mutans comR 449
deletion strain was unable to transport DNA (Fig. 6). Interestingly, the S. pyogenes wild type 450
and comR mutant resembled the S. mutans comR mutant, where no DNA was transported (Fig. 451
6). The fate of the extracellular DNA was also observed; upon adding ethanol the DNA 452
precipitated, indicating that it remained largely intact in polymeric form, and not degraded to 453
single nucleotides. This suggests that the inability of DNA to be imported was not due to its 454
degradation by extracellular DNases during the assay period (data not shown). The same 455
results were also observed using strains MGAS5005 and MGAS8232. Taken together with 456
microarray and reporter studies, these data indicate that although the competence regulon is 457
induced in S. pyogenes, gene transfer is blocked at the stage of DNA uptake. 458
459
Discussion 460
The present study gives new insights into the regulation of competence within the 461
pyogenic streptococci, a group considered to be non-competent within the laboratory. An 462
important human pathogen within the pyogenic group is S. pyogenes that exhibits high levels of 463
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genetic recombination events between strains of this species (4, 18). The conservation of 464
competence genes described here suggests that natural transformation provides a mechanism 465
for this process. Although S. pyogenes contains the master competence regulator sigX and all 466
the late effector genes needed for the competent state in other streptococci, the signal to induce 467
sigX has remained unknown (41, 59). The goal of this study was to identify upstream regulators 468
of sigX in GAS, and test the hypothesis that they include a Type II ComRS system (34). The two 469
comR alleles (M1 and M3) found among GAS strains (Table 3) are identical for the first 120 470
amino acids, corresponding to the predicted DNA binding domain of the Rgg protein. However, 471
they differ in the C-terminal portion, which is the probable site of peptide-protein interaction. In 472
all cases of sequenced genomes, M1 and M3 comS peptide alleles are always located together 473
downstream of their respective M1 and M3 comR genes. We interpret this pattern to indicate 474
that M1 ComR proteins recognize their equivalent M1 ComS peptide, whereas the M3 ComR 475
regulators interact with their respective M3 ComS peptide. The two comS alleles encode 476
peptides that are 31 and 32 amino acids in length, but are most likely processed into the active 477
portion that exhibits activity. In other Rgg-peptide interactions, the mature peptide constitutes 478
the C-terminal fraction of the last 5-10 amino acids (8, 15-16, 34, 54). Here we report that in S. 479
pyogenes the last 8 amino acids of both ComS alleles exhibit activity, and like other predicted 480
Type II ComS peptides, the WW motif within ComS is conserved (Table 3) (34). The sigX 481
reporter studies indicate that both comR alleles in S. pyogenes are capable of activating the 482
transcription of the alternative sigma factor gene sigX upon addition of XIP. Using microarray 483
analysis and a transcriptional reporter for the sigX-dependent gene ssbB, we also demonstrated 484
that sigX is functional and able to activate late genes within the competence pathway important 485
for DNA uptake and recombination. Consistent with prior genomic analyses of competence 486
pathways in Gram-positive bacteria (10, 33), all genes known to be required for genetic 487
transformation in S. pneumoniae and B. subtilis were found to be present and intact in more 488
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than half of all sequenced GAS genomes, and data presented here find all these genes to be 489
expressed during treatment with XIP. 490
Also consistent with previous findings, SigX stability was affected by ClpP, a cytoplasmic 491
protease previously shown to be important in SigX accumulation (41, 59). In a clpP deletion 492
strain, SigX accumulation was detected, but only after the addition of exogenous XIP. This 493
strain also supported higher inductions of ssbB, indicating that ClpP has an effect on the 494
stability and function of SigX in vivo. Interestingly, we observed approximately 10-fold reduction 495
in expression from the sigX promoter in the clpP strain. Since accumulation of SigX was 496
increased in the clpP strain compared to the wild type, we suspect a negative feedback loop 497
directed at sigX transcription may exist, and since a CIN box was not identified upstream of the 498
sigX genes, an indirect regulatory mechanism seems more probable than SigX directly affecting 499
its own transcription. A gene encoding a histidine kinase, rocA, was found to be differentially 500
regulated by transcriptome profiling and thus may be a candidate for additional regulatory 501
control. 502
Similar to our transcriptome results, previous microarray studies in two naturally 503
transformable bacterial species, S. mutans and S. pneumoniae, revealed the induction of 504
competence genes by their respective competence-stimulating peptides. Although the 505
microarray studies presented here showed induction of putative competence genes in S. 506
pyogenes, the fold induction observed was lower than those reported in other species. One 507
reason for this could be the amount of peptide used. Here we used nM concentrations of XIP, 508
whereas other studies used µM concentrations of the respective peptides. Furthermore, the 509
timing of induction could have affected our results. Our sigX and ssbB reporter strains show 510
immediate activation of gene expression in response to XIP, although levels of expression 511
appear to decrease over time. RNA was harvested 60 minutes after supplementation with XIP, 512
which could explain why we observe lower induction of sigX and other putative competence 513
genes in the microarray than in our reporter studies (9-fold vs. 100-fold) (Table 4, Fig. 4). 514
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Despite the lower induction observed in our studies, this is the first report on the activation of 515
sigX and sigX-dependent genes without the need for genetic manipulations. These data indicate 516
that XIP activates putative competence genes in S. pyogenes and is dependent on the 517
transcriptional regulator ComR. 518
The growth medium for natural transformation can be a critical variable for competence 519
development (12), 15-16, 36, 48). For example, in S. mutans the standard method for attaining 520
competence is growth in rich medium with the addition of horse serum, although the factors that 521
promote competence within this medium remain unknown (43). However, it was recently 522
demonstrated that high frequencies of transformation in S. mutans are obtained in a chemically 523
defined medium after the addition of exogenous XIP pheromone (34). S. thermophilus and S. 524
salivarius do not become transformable in rich medium, but require a peptide-free, chemically 525
defined medium (16-17). These species require the Type I ComRS system mentioned above 526
and it is thought that peptides in rich media may compete with ComS for entry to the cytoplasm 527
via the Opp transporter, thus interfering with the signaling circuit. Vibrio cholerae is another 528
bacterial species that until recently was thought to be non-transformable. Meibom et al. reported 529
that the competent state in V. cholerae is induced by chitin, a polymer that is abundant in 530
marine environments (38). These examples illustrate that competence induction can be 531
influenced by specific and non-specific nutritional growth factors. 532
Because many other bacterial species require specific growth conditions for developing 533
competence for natural transformation, we were interested in testing a variety of growth factors 534
in the transformation assays of S. pyogenes. A common assay for natural transformation in the 535
laboratory is to grow and incubate bacterial cells with exogenously-supplied DNA encoding a 536
selectable marker, such as antibiotic resistance, so that transformants can be selected. Using 537
several strains of S. pyogenes (MGAS315, MGAS5005, MGAS8232, HSC5) we tested for 538
natural transformation in a variety of different laboratory media, and examined conditions that 539
would mimic the natural environment of S. pyogenes, yet none of these growth conditions 540
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yielded transformants. To test a library of specific growth substrates for natural transformation, 541
we employed the Biolog Phenotypic MicroArray for Microbial Cells. These studies also yielded 542
no transformants. Furthermore, DNA uptake assays revealed that transformation is blocked at 543
the stage of DNA uptake even after sigX induction. We propose three interpretations. First, the 544
conserved competence genes may simply be remnants of an ancient competence system 545
retained from an ancestral species, but are no longer functional due to reasons that are 546
unknown. Secondly, it is possible the competence genes have acquired a new purpose whose 547
characteristics have not been discovered. Finally, active transformation may occur in nature, 548
but remain cryptic in laboratory conditions that do not sufficiently mimic conditions that support 549
natural transformation. We favor the third possibility since the complete set of genes under 550
consideration seems inconsequentially different from the functional systems of S. pneumoniae 551
and Bacillus subtilis. Consistent with this interpretation is that conditions favoring transformation 552
continue to be discovered among the streptococci. 553
Studies on “non-transformable” S. pyogenes have been performed for decades (35, 44-554
46), and one factor often proposed to contribute to incompetence is the production and 555
secretion of DNases by cells (35, 46). S. pyogenes produces at least four distinct DNases (48) 556
that have been associated with invasive diseases and are important for immune evasion by 557
degrading neutrophil extracellular traps (6). To address this possibility in the transformation 558
assays, we washed bacterial cells with fresh medium to rid the cultures of any secreted 559
DNases. The DNA uptake assays showed that during the time allotted exogenous DNA 560
remained precipitable, indicating that DNA was not extensively degraded, and therefore DNases 561
did not play a major role in the in the inability of GAS to become competent under tested 562
conditions. 563
There is a critical need in the field for methods that would allow rapid genetic 564
manipulations in several important pathogenic streptococci. Natural genetic transformation is 565
one such method currently used for studies of two clinically relevant pathogens, S. pneumoniae 566
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and S. mutans. Although we have been unsuccessful in demonstrating natural transformation in 567
S. pyogenes, we have taken a step forward in understanding the process by demonstrating 568
induction of the competence regulon without genetic manipulations. It seems possible that S. 569
pyogenes and other members of the pyogenic group are capable of natural transformation, but 570
the specific circumstances in which this occurs remain unknown. It is clear that normal 571
conditions within the laboratory are not conducive for competence development, yet natural 572
transformation appears to occur in the natural habitat for GAS, the human host. Future studies 573
should involve studying competence development in animal models and other situations that 574
more closely resemble the natural environment of GAS. Determining the circumstances in which 575
transformation occurs would advance development of a new genetic tool allowing an 576
acceleration of molecular studies of these important pathogens. 577
578
579
Acknowledgements 580
LMW is a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation. 581
This work was supported by an NIH grant AI091779. We thank Lin Tao and Michael Chaussee 582
for strains, and are grateful to Charles Moran for the kind gift of σx anti-sera. We are grateful to 583
Mark Band in the W.M. Keck Center for Comparative and Functional Genomics for microarray 584
processing. 585
586
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Table 1. Strains and plasmids used in this study.
Strain/plasmid Description Source
S. pyogenes MGAS315 S. pyogenes isolate (2)MW151 MGAS315 but ΔcomR::cat; CmR This studyMW211 MGAS315 but ΔclpP::spc; SpcR This studyMW134 MGAS315 with pWAR200; EmR This studyMW131 MGAS315 with pWAR205; EmR This studyMW161 MW151 with pWAR200; CmR, EmR This studyMW256 MW151 with pWAR205; CmR, EmR This studyMW219 MW211 with pWAR200; CmR, EmR This studyMW220 MW211 with pWAR205; CmR, EmR This studyMGAS8232 S. pyogenes isolate (55)MW200 MGAS8232 with pWAR200; EmR This studyS. mutans UA159 Transformable S. mutans isolate (57)MW02 UA159 but ΔcomR::spc; SpcR (34)Plasmid pFED760 Shuttle vector pGH9-ISS1 derivative deleted for ISS1
element, temperature-sensitive; 3752 bp; EmR (34)
pFED761 Heat-resistant pFED760 derivative containing wild-type repA allele; 3752 bp; EmR
(34)
pWAR303 pFED761 derivative carrying a 2222 bp fragment with luxAB inserted between PstI and NotI sites; 5946 bp; EmR
(34)
pWAR200 pWAR303 derivative carrying a 561 bp fragment with PsigX between the SalI and PstI sites; 6467 bp; EmR
This study
pWAR205 pWAR303 derivative carrying a 362 bp fragment with PssbB between the SalI and PstI sites; 6268 bp; EmR
This study
pWAR195 pFED760 derivative containing cat and DNA fragments flanking comR to create insertion mutant; see materials and methods; 6603 bp; CmR
This study
pWAR251 pFED760 derivative containing spc and DNA fragments flanking clpP to create insertion mutant; see materials and methods; 7065 bp; SpcR
This study
pEVP3 Plasmid encoding synthetic promoter and cat chloramphenicol resistance gene; 6302 bp; CmR
(9)
pLZ12-Spc Shuttle vector encoding spectinomycin resistance; pWV01 origin; 3733 bp; SpcR
(23)
Cm, chloramphenicol; Spc, spectinomycin; Em, erythromycin
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Table 2. Primers used in this study.
Primer Nucleotide sequence, 5’-3’ Description
LMW26 GCGTGGTCGACATACTAATAGCTCGAGGACT sigX promoter LMW27 GCGTGCTGCAGTTTGAGTCTCCTTTTCTTTT sigX promoter LMW28 GCGTGGTCGACGTTGCCTTACCAGGTGCTATAATGCCAAAT ssbB promoter LMW29 GCGTGCTGCAGACTGACCTCCTTACCTTATTATTCGTAAAA ssbB promoter LMW30 GCGTGGCGGCCGCAATGACCATCATGATGGGTCGTACCCA upstream of comR LMW31 GCGTGGTCGACGGTTTCTCTCCAACAACTGT upstream of comR LMW32 GCGTGGTCGACTAACAGGACAAAAATGTCAT downstream of comR LMW33 GCGTGCTCGAGTAAAATTTTCTGATCAATGT downstream of comR LMW34 GCGTGGTCGACGATGAAAATTTGTTTGATTT Cm cassette LMW35 GCGTGGTCGACTTATAAAAGCCAGTCATTAG Cm cassette LMW36 GCGTGGCGGCCGCTCCTCATTATTGGTGGCTTGGGAATGACGGTT upstream of clpP LMW37 GCGTGACTAGTCCACGGCTAGTTTGTTCAATAACAACAGGA upstream of clpP LMW38 GCGTGCTGCAGCGGTTTCATTGATGAAATCATGGAAAACAA downstream of clpP LMW39 GCGTGCTCGAGTTGTTTTAACAGTGGAATGAGTTGCTCTAA downstream of clpP LMW40 GCGTGACTAGTGTAACGTGACTGGCAAGAGATATTT Spc cassette LMW41 GCGTGCTGCAGAATAATAAAACAAAAAAATT Spc cassette
Cm, chloramphenicol; Spc, spectinomycin
Table 3. Full-length ComS sequences and comRS allele types in sequenced GAS genomes. Strains comR
allele
comS allele
ComS sequence
M1 SF370, MGAS8232, MGAS10394, MGAS6180, MGAS5005, MGAS9429, ATCC 10782, MGAS2096, MGAS10750, NZ131
M1 M1 MLKKYKYYFIFAALLSFKVVQELSAVDWWRL
Manfredo, MGAS10270, MGAS315, SSI-1
M3 M3 MLKKVKPFLLLAAVVAFKVARVMHEFDWWNLG
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Table 4. Genes induced 2-fold and above by XIP.
Spy numbera Genea Descriptiona Fold change
Standard deviationb
spy0101 comYA ABC transporter subunit 14.4 1.3 spy0102 comYB ABC transporter subunit 16.8 1.8 spy0103 comYC competence protein 17.0 3.0 spy0104 pulG competence protein 13.1 1.2 spy0105 hypothetical protein 16.1 4.0 spy0106 comYD competence protein 15.0 0.5 spy0107 hypothetical protein 14.5 1.1 spyM3_0093c ssbB single-stranded binding protein 3.3 0.6 spy0300 sigX.1 alternative sigma factor 9.2 0.6 spyM3_0584c nucleoside diphosphate kinase 5.7 1.9 spy0593 hypothetical protein 2.4 0.4 spy0596 hydrolase 2.2 0.2 spy0600 predicted membrane protein 2.1 0.3 spy0798 predicted membrane protein 2.1 0.3 spy1163 smf DNA processing protein 9.2 0.7 spy1205 murM2 antimicrobial resistance protein 2.7 0.7 spy1395 coiA competence protein 1.7 0.3 spy1407 holA DNA polymerase III delta subunit 2.1 0.3 spy1408 comEC competence protein 9.5 0.7 spy1409 comEA competence protein 7.6 0.5 spyM3_1441c hypothetical protein 2.1 0.1 spy1532 cclA type IV prepillin peptidase 1.9 0.4 spy1605 rocA histidine kinase 2.8 0.2 spy1606 trmA RNA methyltransferase 6.3 0.7 spy1615 comFC competence protein 2.7 0.5 spy1616 comFA competence protein 2.0 0.3 spy1670 proA gamma-glutamyl phosphate reductase 2.1 0.3 spy1672 proB gamma-glutamyl kinase 2.3 0.2 spy1902 sigX.2 alternative sigma factor 8.9 0.8 spy2117 cinA competence/damage-inducible protein 1.7 0.3 aGene numbers, names, and descriptions based on the M1 genome SF370 (14). bStandard deviation, based on technical replicates from the array. cNot annotated in M1 SF370. Gene numbers based on the MGAS315 genome (2). Genes in bold indicate those containing CIN-boxes within their promoter regions.
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Figure Legends
Figure 1. Proposed model for competence gene regulation in S. pyogenes. Based on 754
previous findings in S. mutans (34), we hypothesize that ComRS regulates the expression of 755
sigX in GAS. ComS is secreted from the bacterial cell and processed by an unknown 756
mechanism to produce the mature sigX-inducing peptide (XIP). Importation of XIP depends on 757
the Opp transporter, and once in the cytosol, XIP interacts with ComR, proposed here as a 758
dimer. Together ComR/XIP binds the P1 promoter regions upstream of comS and sigX, 759
activating their transcription. SigX accumulation is dependent on the ClpP protease, but once 760
produced, SigX and RNA polymerase recognize and bind to CIN-box-containing promoter 761
regions upstream of late competence genes to activate transcription. 762
763
Figure 2. Characterization of the early and late competence genes in S. pyogenes. The
early genes comprise the quorum sensing system comRS and the alternative sigma factor sigX
which are required for activation of late competence genes. The P1 promoter pattern to which
ComR binds is designated as “P1.” P1 consensus sequence: AACAN GACA N4 TGTCN TGTT
N19 TATAAT (34). The genes listed as Late Genes are those known to be induced by ComX and
that are required for transformation in S. pneumoniae. These consist of genes involved in DNA
uptake and transport and those required for DNA processing and recombination. Binding sites
for SigX, known as CIN-boxes ("C"), have the consensus sequence: TACGAATA. *Gene
designations and spy numbers are based on the M1 SF370 genome. Solid lines within genes
represent stop codons, dashed lines represent frame shift mutations, and the double line in
comR indicates a 9 base-pair insertion. Mutations are based on publically available sequenced
genomes, but have not been substantiated except for those found in NZ131. Genomes: NZ131a,
MGAS2096b, MGAS6180c, MGAS10394d, MGAS10750e, MGAS315f, SSI-1g. ≠Synonym for sigX
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in S. pneumoniae, comX. +Synonyms for the comY operon in S. pneumoniae and Bacillus
subtilis, cgl, comG. ‡Synonym for the comE operon in S. pneumoniae, cel.
Figure 3. Induction of sigX is dose and XIP dependent. The dose dependent effects of XIP
on growth and sigX expression were determined in wild type strains containing the PsigX::luxAB
reporter construct. (A) Growth of MW134 (MGAS315 + PsigX::luxAB). Strains were diluted from
mid-logarithmic phase to OD600 = 0.01 and grown at 37°C until OD600 = 0.1, at which time the
indicated concentrations of XIP (nM) or the DMSO vehicle (V) were added (indicated by the
arrow). (B and C) Dose dependent induction of sigX by XIP. MW134 (MGAS315 + PsigX::luxAB)
and MW200 (MGAS8232 + PsigX::luxAB) were grown as described above. After XIP addition,
cultures were incubated at 37°C for 60 min at which time samples were taken to measure
luciferase activity. RLU (relative light units): counts per minute/ OD600. Error bars represent the
standard deviation of three separate experiments.
764
Figure 4. Induction of sigX and late gene expression requires exogenous peptide 765
pheromone and comR. Activity of the PsigX::luxAB and PssbB::luxAB reporter constructs in 766
various genetic backgrounds was determined in CDM after addition of exogenous synthetic 767
signaling peptides. S. pyogenes strains were diluted to OD600 = 0.01 and grown at 37°C until 768
OD600 = 0.1, at which time synthetic peptides (XIP or SilCR) or the DMSO vehicle (V) were 769
added to a final concentration of 200 nM (XIP) and/or 1 µM (SilCR). Samples were taken every 770
30 min. Data shown are representative of three similar experiments. RLU (relative light units): 771
counts per minute/OD600. (A) MGAS315, M3 XIP; (B) MGAS315, M3 XIP; (C) MGAS8232, M1 772
XIP, SilCR. 773
774
Figure 5. SigX accumulation is dependent on XIP and ClpP. Cultures of wild type and the 775
clpP deletion strain were grown at 37°C in CDM to mid-logarithmic phase and supplemented 776
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with DMSO (V) or XIP for 30 or 60 min. Whole cell lysates were subjected to SDS-PAGE 777
followed by immunoblotting with anti-SigX antiserum. Molecular weight standards (in kDa) are 778
shown to the left. Calculated molecular weight of SigX is 19.6 kDa. 779
780
Figure 6. Natural transformation in S. pyogenes is blocked at DNA uptake. DNA uptake by 781
S. mutans UA159 and S. pyogenes MGAS315 wild type and ΔcomR strains was determined by 782
incubation with radiolabeled DNA. Cultures were diluted to OD600 = 0.01 and grown in CDM at 783
37°C until OD600 = 0.1, at which time cells were supplemented with 1 µM XIP for 60 min. Cells 784
were then washed and suspended in CDM containing 1 µM XIP for 15 min. Radiolabeled DNA 785
was added to cell cultures and allowed to incubate at the indicated times when cells were 786
harvested and amount of radiolabeled DNA measured. Error bars represent the standard 787
deviation of three separate experiments. 788
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