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Jenny A. Herbert, Andrea M. Mitchell, Ryan Ritchie, Jiangtao Ma, Kirsty Ross-Hutchinson, Timothy J. Mitchell
Expression of the lux genes in Streptococcus pneumoniae modulates pilus expression and virulence
www.plosone.org
www.plosone.org
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3.6906 0 Td
(and)Tj
1.7348 0 Td
(with)Tj
-33.5449 -1.2983 Td
(a)Tj
0.6519 0 Td
(12h)Tj
1.7008 0 Td
(light/dark)Tj
4.1839 0 Td
(cycle.)Tj
2.4151 0 Td
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1.3946 0 Td
(mice)Tj
2.1544 0 Td
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(this)Tj
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(study)Tj
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(MF1)Tj
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(\(Harlan,)Tj
3.5887 0 Td
(UK\))Tj
1.9672 0 Td
(68)Tj
1.6894 0 Td
(week)Tj
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(old)Tj
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(out-)Tj
-34.8942 -1.2983 Td
(bred)Tj
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(female)Tj
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(mice)Tj
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(\(Harlan,)Tj
3.5887 0 Td
(UK\).)Tj
2.194 0 Td
(Mice)Tj
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(sacrificed)Tj
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0.9525 0 Td
(a)Tj
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(well)Tj
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(defined)Tj
3.2258 0 Td
(clinical)Tj
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(endpoint)Tj
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1.0261 0 Td
(reduce)Tj
2.914 0 Td
(suffering.)Tj
3.9855 0 Td
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3.5943 0 Td
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(performed)Tj
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(cervical)Tj
3.2598 0 Td
(dislocation)Tj
4.6091 0 Td
(while)Tj
2.3698 0 Td
(under)Tj
2.6192 0 Td
(terminal)Tj
-31.7308 -1.2982 Td
(anaesthesia.)Tj
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1.1962 -1.2983 Td
(Pneumonia)Tj
5.0343 0 Td
(infection)Tj
3.9571 0 Td
(model.)Tj
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3.668 0 Td
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3.2201 0 Td
(of)Tj
1.0148 0 Td
(twenty)Tj
2.9254 0 Td
(mice)Tj
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Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 218.948 481.4929 cm
BT
/F8 1 Tf
1 TL
-0.0053 Tc
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(6)Tj
ET
Q
q
1 j
1 J
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10 0 0 10 224.674 477.0141 cm
BT
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1 TL
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(colony)Tj
2.9083 0 Td
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(50)Tj
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[()]TJ
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2.1543 0 Td
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1.0148 0 Td
(either)Tj
2.5285 0 Td
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3.0217 0 Td
(TIGR4)Tj
2.982 0 Td
(or)Tj
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(T4P2.)Tj
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1.8936 0 Td
(mice)Tj
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-34.3613 -1.2982 Td
(each)Tj
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(group)Tj
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4.0081 0 Td
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0.9525 0 Td
(24hr,)Tj
2.296 0 Td
(48hr)Tj
2.0693 0 Td
(or)Tj
1.0998 0 Td
(72hr)Tj
2.0693 0 Td
(post)Tj
1.9049 0 Td
(infection)Tj
3.787 0 Td
(\(PI\))Tj
1.8029 0 Td
(and)Tj
1.7291 0 Td
(five)Tj
1.6554 0 Td
(mice)Tj
2.1487 0 Td
(were)Tj
2.1259 0 Td
(moni-)Tj
-34.4066 -1.2983 Td
(tored)Tj
2.3471 0 Td
(for)Tj
1.3832 0 Td
(survival.)Tj
3.566 0 Td
(Mice)Tj
2.228 0 Td
(were)Tj
2.1203 0 Td
(sacrificed)Tj
4.0081 0 Td
(at)Tj
0.9525 0 Td
(the)Tj
1.4683 0 Td
(designated)Tj
4.4957 0 Td
(time)Tj
2.0296 0 Td
(point)Tj
2.3584 0 Td
(and)Tj
1.7292 0 Td
(organs)Tj
2.9083 0 Td
(were)Tj
2.1203 0 Td
(removed)Tj
-33.715 -1.2982 Td
(\(lungs,)Tj
2.9309 0 Td
(liver,)Tj
2.211 0 Td
(spleen,)Tj
2.9651 0 Td
(brain\),)Tj
2.914 0 Td
(nasal)Tj
2.245 0 Td
(wash)Tj
2.2223 0 Td
(and)Tj
1.7348 0 Td
(blood)Tj
2.5115 0 Td
(taken)Tj
2.4208 0 Td
(and)Tj
1.7291 0 Td
(viable)Tj
2.5568 0 Td
(counts)Tj
2.8857 0 Td
(performed)Tj
4.4674 0 Td
(using)Tj
2.3867 0 Td
(the)Tj
-36.1811 -1.304 Td
(Miles)Tj
2.4095 0 Td
(and)Tj
1.729 0 Td
(Misra)Tj
2.5455 0 Td
(method)Tj
3.3278 0 Td
([)Tj
0.83 0.64 0.02 0 k
(17)Tj
0 g
(].)Tj
2.075 0 Td
(Tail)Tj
1.7801 0 Td
(vein)Tj
1.9106 0 Td
(bleeds)Tj
2.7099 0 Td
(were)Tj
2.1203 0 Td
(also)Tj
1.7688 0 Td
(taken)Tj
2.4207 0 Td
(throughout)Tj
4.7792 0 Td
(and)Tj
1.7292 0 Td
(animal)Tj
2.9706 0 Td
(weights)Tj
-34.2762 -1.2982 Td
(and)Tj
1.7291 0 Td
(clinical)Tj
3.0784 0 Td
(scores)Tj
2.6645 0 Td
(were)Tj
2.1203 0 Td
(recorded.)Tj
4.0252 0 Td
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3.4469 0 Td
(group)Tj
2.6135 0 Td
(mice)Tj
2.1543 0 Td
(were)Tj
2.1203 0 Td
(sacrificed)Tj
4.0082 0 Td
(once)Tj
2.1203 0 Td
(clinical)Tj
3.0784 0 Td
(symptoms)Tj
-33.1594 -1.2983 Td
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1.7065 0 Td
(reached)Tj
3.3504 0 Td
(a)Tj
0.6576 0 Td
(designated)Tj
4.4958 0 Td
(clinical)Tj
3.0784 0 Td
(end)Tj
1.7177 0 Td
(point.)Tj
2.5852 0 Td
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3.6964 0 Td
(load)Tj
1.9332 0 Td
(in)Tj
1.0375 0 Td
(organs)Tj
2.9083 0 Td
(and)Tj
1.7291 0 Td
(blood)Tj
2.5115 0 Td
(were)Tj
2.126 0 Td
(compared)Tj
-33.5336 -1.3039 Td
(between)Tj
3.5205 0 Td
(mice)Tj
2.143 0 Td
(infected)Tj
3.4129 0 Td
(with)Tj
1.9842 0 Td
(Xen35)Tj
2.7893 0 Td
(and)Tj
1.7235 0 Td
(TIGR4)Tj
2.9763 0 Td
(at)Tj
0.9411 0 Td
(varying)Tj
3.2088 0 Td
(time)Tj
2.0183 0 Td
(points.)Tj
2.9367 0 Td
(Graphical)Tj
4.1442 0 Td
(representation)Tj
-31.7988 -1.2983 Td
(and)Tj
1.7291 0 Td
(statistical)Tj
3.9061 0 Td
(analysis)Tj
3.3278 0 Td
(was)Tj
1.7008 0 Td
(performed)Tj
4.4674 0 Td
(in)Tj
1.0374 0 Td
(Prism)Tj
2.5966 0 Td
(version)Tj
3.1521 0 Td
(4.0b)Tj
1.9048 0 Td
(\(GraphPad)Tj
4.6488 0 Td
(Software\).)Tj
4.2633 0 Td
(Statistical)Tj
-32.7342 -1.2982 Td
(analysis)Tj
3.3278 0 Td
(was)Tj
1.6951 0 Td
(performed)Tj
4.473 0 Td
(using)Tj
2.3868 0 Td
(a)Tj
0.6519 0 Td
(non-parametric)Tj
6.5367 0 Td
(Mann-Whitney)Tj
6.48 0 Td
(two)Tj
1.7121 0 Td
(sample)Tj
3.0217 0 Td
(rank)Tj
2.0693 0 Td
(test,)Tj
1.8255 0 Td
(signifi-)Tj
-34.1799 -1.304 Td
(cance)Tj
2.4717 0 Td
(P)Tj
/F17 1 Tf
0 Tc
(<)Tj
/F8 1 Tf
-0.004 Tc
(0.05.)Tj
3.4299 0 Td
(This)Tj
1.9843 0 Td
(statistical)Tj
3.9117 0 Td
(test)Tj
1.5988 0 Td
(was)Tj
1.7008 0 Td
(also)Tj
1.7688 0 Td
(used)Tj
2.0579 0 Td
(for)Tj
1.3833 0 Td
(comparison)Tj
4.9776 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.4683 0 Td
(percentage)Tj
4.5525 0 Td
(weight)Tj
2.8856 0 Td
(loss)Tj
-35.206 -1.2982 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.4683 0 Td
(mice)Tj
2.1543 0 Td
(over)Tj
1.9785 0 Td
(time.)Tj
2.2507 0 Td
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3.4526 0 Td
(of)Tj
1.0148 0 Td
(mice)Tj
2.1543 0 Td
(infected)Tj
3.4186 0 Td
(with)Tj
1.9956 0 Td
(different)Tj
3.6566 0 Td
(strains)Tj
2.863 0 Td
(was)Tj
1.7008 0 Td
(compared)Tj
4.2406 0 Td
(using)Tj
2.3924 0 Td
(a)Tj
-35.7559 -1.2983 Td
(Kaplain)Tj
3.3391 0 Td
(Meier)Tj
2.5965 0 Td
(survival)Tj
3.3392 0 Td
(curve)Tj
2.4208 0 Td
(and)Tj
1.7291 0 Td
(analysed)Tj
3.651 0 Td
(using)Tj
2.3868 0 Td
(a)Tj
0.6576 0 Td
(Log-rank)Tj
3.9288 0 Td
(Test)Tj
1.9162 0 Td
(\(P)Tj
/F17 1 Tf
0 Tc
(<)Tj
/F8 1 Tf
-0.004 Tc
(0.05\).)Tj
4.1046 0 Td
(No)Tj
1.4683 0 Td
(statistical)Tj
3.9061 0 Td
(anal-)Tj
-35.4441 -1.3039 Td
(ysis)Tj
1.6611 0 Td
(was)Tj
1.7007 0 Td
(performed)Tj
4.4674 0 Td
(on)Tj
1.2755 0 Td
(data)Tj
1.9163 0 Td
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0.7766 0 Td
(group)Tj
2.6079 0 Td
(sizes)Tj
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(were)Tj
2.126 0 Td
(below)Tj
2.5852 0 Td
(5.)Tj
-19.9614 -1.2983 Td
(Competition)Tj
5.3404 0 Td
(experiments)Tj
5.1363 0 Td
(were)Tj
2.126 0 Td
(done)Tj
2.2223 0 Td
(by)Tj
1.1849 0 Td
(inoculating)Tj
4.7509 0 Td
(5)Tj
0.6973 0 Td
(MF1)Tj
2.1146 0 Td
(mice)Tj
2.1543 0 Td
(as)Tj
1.0148 0 Td
(above)Tj
2.5455 0 Td
(intraperitoneally)Tj
-30.4835 -1.2982 Td
(\(IP\))Tj
1.7972 0 Td
(with)Tj
1.9955 0 Td
(1x10)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 256.8755 286.4692 cm
BT
/F8 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(4)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 260.4472 282.0472 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(CFU)Tj
2.1486 0 Td
(of)Tj
1.0148 0 Td
(Xen35)Tj
2.7949 0 Td
(and)Tj
1.7292 0 Td
(TIGR4)Tj
2.982 0 Td
(in)Tj
1.0375 0 Td
(200)Tj
/F15 1 Tf
[()]TJ
/F8 1 Tf
(l)Tj
2.3981 0 Td
(given)Tj
2.381 0 Td
(in)Tj
1.0375 0 Td
(a)Tj
0.652 0 Td
(1:1)Tj
1.4003 0 Td
(ratio.)Tj
2.3187 0 Td
(Mice)Tj
2.228 0 Td
(were)Tj
2.1203 0 Td
(sacrificed)Tj
4.0082 0 Td
(at)Tj
-36.2945 -1.2982 Td
(24hr)Tj
2.0693 0 Td
(PI)Tj
1.1168 0 Td
(and)Tj
1.7348 0 Td
(bacterial)Tj
3.6226 0 Td
(count)Tj
2.5228 0 Td
(enumerated)Tj
5.0173 0 Td
(from)Tj
2.211 0 Td
(organs,)Tj
3.1294 0 Td
(blood)Tj
2.5172 0 Td
(and)Tj
1.7291 0 Td
(nasal)Tj
2.245 0 Td
(wash.)Tj
2.4492 0 Td
(Mice)Tj
2.228 0 Td
(were)Tj
2.1203 0 Td
(pro-)Tj
-34.7128 -1.3039 Td
(cessed)Tj
2.7383 0 Td
(as)Tj
1.0148 0 Td
(above)Tj
2.5454 0 Td
(except)Tj
2.7722 0 Td
(that)Tj
1.7802 0 Td
(organs,)Tj
3.1294 0 Td
(blood)Tj
2.5172 0 Td
(and)Tj
1.7291 0 Td
(nasal)Tj
2.245 0 Td
(wash)Tj
2.228 0 Td
(were)Tj
2.1203 0 Td
(differentially)Tj
5.3064 0 Td
(plated)Tj
2.6646 0 Td
(onto)Tj
2.0806 0 Td
(BAB)Tj
-34.8715 -1.2983 Td
(only)Tj
1.9729 0 Td
(and)Tj
1.7348 0 Td
(BAB)Tj
2.0636 0 Td
(with)Tj
1.9955 0 Td
(kanamycin)Tj
4.6488 0 Td
(to)Tj
1.0205 0 Td
(evaluate)Tj
3.4639 0 Td
(the)Tj
1.4683 0 Td
(ratio)Tj
2.0976 0 Td
(of)Tj
1.0148 0 Td
(TIGR4)Tj
2.9877 0 Td
(to)Tj
1.0205 0 Td
(Xen35.)Tj
3.0217 0 Td
(Statistical)Tj
4.0252 0 Td
(analysis)Tj
3.3278 0 Td
(was)Tj
-35.8636 -1.2983 Td
(performed)Tj
4.4674 0 Td
(as)Tj
1.0148 0 Td
(noted)Tj
2.5284 0 Td
(above.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 203.9811 cm
BT
/F14 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Whole)Tj
2.9716 0 Td
(genome)Tj
3.4867 0 Td
(sequencing)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 186.9732 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
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3.9231 0 Td
(DNA)Tj
2.3754 0 Td
(was)Tj
1.7008 0 Td
(isolated)Tj
3.2825 0 Td
(from)Tj
2.2053 0 Td
(mid)Tj
1.8368 0 Td
(log)Tj
1.44 0 Td
(phase)Tj
2.4832 0 Td
(cultures)Tj
3.3959 0 Td
(using)Tj
2.3924 0 Td
(a)Tj
0.6519 0 Td
(phenol)Tj
2.9934 0 Td
(chloroform)Tj
4.7849 0 Td
(extraction)Tj
-33.4656 -1.2982 Td
(method.)Tj
3.5489 0 Td
(Pre-sequencing)Tj
6.4176 0 Td
(DNA)Tj
2.3754 0 Td
(was)Tj
1.7008 0 Td
(fragmented)Tj
4.8189 0 Td
(using)Tj
2.3924 0 Td
(a)Tj
0.652 0 Td
(Bioruptor)Tj
4.1669 0 Td
(sonicator)Tj
3.9401 0 Td
(and)Tj
1.7291 0 Td
(processed)Tj
-31.7421 -1.2983 Td
(using)Tj
2.3867 0 Td
(a)Tj
0.6576 0 Td
(TruSeq)Tj
3.1351 0 Td
(DNA)Tj
2.3755 0 Td
(library)Tj
2.8743 0 Td
(preparation)Tj
4.9095 0 Td
(kit)Tj
1.2813 0 Td
(\(Illumina,)Tj
4.2123 0 Td
(USA\).)Tj
2.6815 0 Td
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3.1975 0 Td
(and)Tj
1.7291 0 Td
(quantity)Tj
3.5546 0 Td
(was)Tj
1.7008 0 Td
(mea-)Tj
-34.6958 -1.2982 Td
(sured)Tj
2.4321 0 Td
(using)Tj
2.3867 0 Td
(the)Tj
1.4684 0 Td
(bioanalyser)Tj
4.7735 0 Td
(and)Tj
1.7348 0 Td
(Kapa)Tj
2.279 0 Td
(library)Tj
2.8686 0 Td
(quantification)Tj
5.8394 0 Td
(kits)Tj
1.6384 0 Td
(\(KAPA)Tj
3.1634 0 Td
(Biosystems,)Tj
4.8756 0 Td
(UK\))Tj
-33.4599 -1.304 Td
(respectively.)Tj
5.1079 0 Td
(Samples)Tj
3.498 0 Td
(were)Tj
2.1203 0 Td
(loaded)Tj
2.8856 0 Td
(onto)Tj
2.0863 0 Td
(the)Tj
1.4684 0 Td
(flow)Tj
1.9445 0 Td
(cells)Tj
1.9219 0 Td
(at)Tj
0.9524 0 Td
(12pM)Tj
2.5795 0 Td
(on)Tj
1.2756 0 Td
(the)Tj
1.4684 0 Td
(Illumina)Tj
3.651 0 Td
(Cluster)Tj
3.1124 0 Td
(station)Tj
-34.0722 -1.2982 Td
(and)Tj
1.7291 0 Td
(sequenced)Tj
4.388 0 Td
(using)Tj
2.3867 0 Td
(the)Tj
1.4684 0 Td
(IlluminaGAII)Tj
5.7316 0 Td
(\(Xen35\))Tj
3.4752 0 Td
(or)Tj
1.0999 0 Td
(IlluminaGAIIx)Tj
6.1908 0 Td
(\(T4P2\).)Tj
3.2485 0 Td
(Output)Tj
3.1351 0 Td
(files)Tj
1.8085 0 Td
(were)Tj
-34.6618 -1.2983 Td
(processed)Tj
4.1328 0 Td
(with)Tj
2.0013 0 Td
(CASAVA)Tj
4.1045 0 Td
(and)Tj
1.7291 0 Td
(fastQ)Tj
2.3471 0 Td
(files)Tj
1.8085 0 Td
(were)Tj
2.1203 0 Td
(generated.)Tj
-17.0474 -1.3039 Td
(Data)Tj
2.1259 0 Td
(analysis)Tj
3.3279 0 Td
(was)Tj
1.6951 0 Td
(performed)Tj
4.473 0 Td
(in)Tj
1.0318 0 Td
(CLC)Tj
2.0863 0 Td
(Genomics)Tj
4.286 0 Td
(Workbench)Tj
4.9889 0 Td
(4.5.1)Tj
2.0977 0 Td
(\(CLC)Tj
2.4264 0 Td
(bio\).)Tj
2.0636 0 Td
(Xen35)Tj
-31.7988 -1.2983 Td
(genome)Tj
3.4072 0 Td
(sequence)Tj
3.8551 0 Td
(was)Tj
1.7007 0 Td
(reference)Tj
3.9175 0 Td
(assembled)Tj
4.32 0 Td
(to)Tj
1.0261 0 Td
(TIGR4)Tj
2.9877 0 Td
(genome)Tj
3.4072 0 Td
(sequence)Tj
3.8551 0 Td
(available)Tj
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-0.0033 Tc
0 0 Td
(Evaluation)Tj
4.7244 0 Td
(of)Tj
1.0677 0 Td
(bioluminescence)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 689.1023 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(1x10)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 218.948 693.5244 cm
BT
/F8 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(6)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 224.674 689.1023 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(CFU)Tj
2.1486 0 Td
(of)Tj
/F7 1 Tf
1.0148 0 Td
[()]TJ
/F8 1 Tf
(.)Tj
/F7 1 Tf
0.9014 0 Td
[()11()6()2()12()6()11()18()4( )6()]TJ
/F8 1 Tf
5.057 0 Td
(were)Tj
2.1203 0 Td
(added)Tj
2.6645 0 Td
(in)Tj
1.0318 0 Td
(20)Tj
/F15 1 Tf
[()]TJ
/F8 1 Tf
(l)Tj
1.9276 0 Td
(BHI)Tj
1.9049 0 Td
(to)Tj
1.0261 0 Td
(a)Tj
0.6576 0 Td
(black)Tj
2.3244 0 Td
(F96)Tj
1.7008 0 Td
(MicroWellTM)Tj
6.0661 0 Td
(plate)Tj
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(\(Nunc,)Tj
3.0216 0 Td
(Fisher)Tj
2.6986 0 Td
(Scientific,)Tj
4.1272 0 Td
(UK\))Tj
1.9673 0 Td
(containing)Tj
4.5297 0 Td
(180)Tj
/F15 1 Tf
[()]TJ
/F8 1 Tf
(l)Tj
2.3981 0 Td
(BHI,)Tj
2.1373 0 Td
(each)Tj
2.0239 0 Td
(strain)Tj
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(was)Tj
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(tested)Tj
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(triplicate.)Tj
3.9911 0 Td
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3.7927 0 Td
(was)Tj
1.7007 0 Td
(measured)Tj
4.1102 0 Td
(on)Tj
1.2756 0 Td
(a)Tj
0.652 0 Td
(FLUOstar)Tj
4.2349 0 Td
(OPTIMA)Tj
4.0535 0 Td
(\(BMG\))Tj
3.0841 0 Td
(plate)Tj
2.143 0 Td
(reader)Tj
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(taking)Tj
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(readings)Tj
3.617 0 Td
(every)Tj
2.3527 0 Td
(20)Tj
-36.5156 -1.2983 Td
(minutes)Tj
3.4638 0 Td
(for)Tj
1.389 0 Td
(10)Tj
1.1679 0 Td
(hours.)Tj
2.7439 0 Td
(Graphical)Tj
4.1556 0 Td
(presentation)Tj
5.2157 0 Td
(was)Tj
1.7007 0 Td
(performed)Tj
4.4674 0 Td
(in)Tj
1.0375 0 Td
(Prism)Tj
2.5965 0 Td
(version)Tj
3.1521 0 Td
(4.0b)Tj
1.9049 0 Td
(\(GraphPad)Tj
-32.995 -1.3039 Td
(Software\),)Tj
4.2632 0 Td
(each)Tj
2.0296 0 Td
(data)Tj
1.9162 0 Td
(point)Tj
2.3584 0 Td
(representing)Tj
5.2384 0 Td
(the)Tj
1.4683 0 Td
(mean)Tj
2.4492 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.4683 0 Td
(triplicate)Tj
3.7644 0 Td
(luminescence)Tj
5.6579 0 Td
(reading)Tj
3.2598 0 Td
(minus)Tj
-34.8885 -1.2983 Td
(background.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 595.0488 cm
BT
/F14 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Flow)Tj
2.2441 0 Td
(cytometry)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 578.0409 cm
BT
/F7 1 Tf
1 TL
-0.004 Tc
0 0 Td
[()]TJ
/F8 1 Tf
(.)Tj
/F7 1 Tf
[()11()6()2()12()6()11()18()4( )6()]TJ
/F8 1 Tf
5.7372 0 Td
(strains)Tj
2.863 0 Td
(were)Tj
2.1203 0 Td
(grown)Tj
2.8006 0 Td
(to)Tj
1.0205 0 Td
(OD600nm)Tj
4.5014 0 Td
(0.6)Tj
1.3946 0 Td
(in)Tj
1.0375 0 Td
(5ml)Tj
1.7631 0 Td
(BHI.)Tj
2.1317 0 Td
(Cultures)Tj
3.6396 0 Td
(were)Tj
2.1203 0 Td
(centrifuged)Tj
4.7849 0 Td
(at)Tj
-35.9147 -1.2982 Td
(4000g)Tj
2.5851 0 Td
(for)Tj
1.389 0 Td
(5)Tj
0.6916 0 Td
(minutes)Tj
3.4696 0 Td
(and)Tj
1.7292 0 Td
(fixed)Tj
2.194 0 Td
(in)Tj
1.0318 0 Td
(2%)Tj
1.4513 0 Td
(paraformaldehyde)Tj
7.5288 0 Td
(for)Tj
1.3889 0 Td
(1)Tj
0.6917 0 Td
(hour)Tj
2.1543 0 Td
(at)Tj
0.9524 0 Td
(room)Tj
2.4265 0 Td
(temperature)Tj
5.1306 0 Td
(\(RT\).)Tj
-34.8148 -1.2983 Td
(Cells)Tj
2.1599 0 Td
(were)Tj
2.1203 0 Td
(resuspended)Tj
5.2328 0 Td
(in)Tj
1.0374 0 Td
(FACS)Tj
2.5682 0 Td
(buffer)Tj
2.6249 0 Td
(1)Tj
0.6973 0 Td
(\(1x)Tj
1.5023 0 Td
(PBS,)Tj
2.058 0 Td
(1%)Tj
1.457 0 Td
(BSA\))Tj
2.296 0 Td
(and)Tj
1.7292 0 Td
(blocked)Tj
3.3505 0 Td
(at)Tj
0.9524 0 Td
(4C)Tj
1.7518 0 Td
(overnight.)Tj
4.3087 0 Td
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-35.8467 -1.3039 Td
(antibody)Tj
3.7643 0 Td
(staining)Tj
3.4073 0 Td
(100)Tj
/F15 1 Tf
[()]TJ
/F8 1 Tf
(l)Tj
2.4037 0 Td
(of)Tj
1.0148 0 Td
(fixed)Tj
2.1884 0 Td
(bacterial)Tj
3.6226 0 Td
(suspension)Tj
4.6431 0 Td
(was)Tj
1.7008 0 Td
(centrifuged)Tj
4.7849 0 Td
(and)Tj
1.7291 0 Td
(resuspended)Tj
5.2327 0 Td
(in)Tj
-34.4917 -1.2983 Td
(500)Tj
/F15 1 Tf
[()]TJ
/F8 1 Tf
(l)Tj
2.398 0 Td
(buffer)Tj
2.6249 0 Td
(2)Tj
0.6973 0 Td
(\(1x)Tj
1.5024 0 Td
(PBS,)Tj
2.0579 0 Td
(1%)Tj
1.4514 0 Td
(BSA,)Tj
2.1883 0 Td
(0.05%)Tj
2.6248 0 Td
(Tween)Tj
2.914 0 Td
(20\))Tj
1.5081 0 Td
(and)Tj
1.7348 0 Td
(1)Tj
/F15 1 Tf
[()]TJ
/F8 1 Tf
(l)Tj
1.4456 0 Td
(of)Tj
1.0148 0 Td
(each)Tj
2.0296 0 Td
(primary)Tj
3.4583 0 Td
(antibody)Tj
3.7643 0 Td
(was)Tj
1.6952 0 Td
(added)Tj
-35.1097 -1.2982 Td
(\(Anti-Mouse)Tj
5.4198 0 Td
(RrgB)Tj
2.2563 0 Td
(polyclonal)Tj
4.354 0 Td
(\(in)Tj
1.372 0 Td
(house)Tj
2.5681 0 Td
(production\))Tj
5.057 0 Td
(and)Tj
1.7291 0 Td
(Anti-Rabbit)Tj
5.006 0 Td
(Type)Tj
2.2223 0 Td
(Serum)Tj
2.8403 0 Td
(4)Tj
0.6974 0 Td
(\(Statens)Tj
-33.5223 -1.3039 Td
(Serum)Tj
2.8346 0 Td
(Institute,)Tj
3.8097 0 Td
(Denmark\)\))Tj
4.7282 0 Td
(and)Tj
1.7348 0 Td
(incubated)Tj
4.1725 0 Td
(at)Tj
0.9525 0 Td
(RT)Tj
1.457 0 Td
(for)Tj
1.3833 0 Td
(1)Tj
0.6973 0 Td
(hour.)Tj
2.3811 0 Td
(Cells)Tj
2.16 0 Td
(were)Tj
2.1203 0 Td
(washed)Tj
3.1804 0 Td
(once)Tj
2.1203 0 Td
(\(buffer)Tj
2.965 0 Td
(2\))Tj
-36.697 -1.2983 Td
(and)Tj
1.7291 0 Td
(then)Tj
2.0182 0 Td
(resuspended)Tj
5.2328 0 Td
(in)Tj
1.0374 0 Td
(500)Tj
/F15 1 Tf
[()]TJ
/F8 1 Tf
(l)Tj
2.3981 0 Td
(buffer)Tj
2.6249 0 Td
(2.)Tj
0.9241 0 Td
(1)Tj
/F15 1 Tf
[()]TJ
/F8 1 Tf
(l)Tj
1.4456 0 Td
(of)Tj
1.0148 0 Td
(each)Tj
2.0296 0 Td
(secondary)Tj
4.269 0 Td
(antibody)Tj
3.7644 0 Td
(was)Tj
1.7007 0 Td
(added)Tj
2.6589 0 Td
(\(Goat)Tj
2.5172 0 Td
(Anti-)Tj
-35.3648 -1.2983 Td
(Rabbit)Tj
2.8516 0 Td
(IgG)Tj
1.7404 0 Td
(Allophycocyanin)Tj
7.0129 0 Td
(\(APC\))Tj
2.8063 0 Td
(conjugate)Tj
4.1045 0 Td
(\(Southern)Tj
4.2406 0 Td
(Biotech,)Tj
3.4696 0 Td
(USA\))Tj
2.4605 0 Td
(and)Tj
1.7291 0 Td
(Goat)Tj
2.1713 0 Td
(Anti-Mouse)Tj
-32.5868 -1.3039 Td
(IgG)Tj
1.7404 0 Td
(Fluorescein)Tj
4.8472 0 Td
(\(FITC\))Tj
3.0387 0 Td
(Conjugate)Tj
4.3484 0 Td
(\(Southern)Tj
4.2406 0 Td
(Biotech,)Tj
3.4696 0 Td
(USA\)\))Tj
2.7949 0 Td
(and)Tj
1.7348 0 Td
(incubated)Tj
4.1726 0 Td
(for)Tj
1.3889 0 Td
(1)Tj
0.6973 0 Td
(hour)Tj
2.1487 0 Td
(in)Tj
1.0375 0 Td
(the)Tj
-35.6596 -1.2983 Td
(dark)Tj
2.0466 0 Td
(at)Tj
0.9524 0 Td
(RT.)Tj
1.6781 0 Td
(Cells)Tj
2.16 0 Td
(were)Tj
2.1259 0 Td
(washed)Tj
3.1748 0 Td
(three)Tj
2.2677 0 Td
(times)Tj
2.3868 0 Td
(in)Tj
1.0375 0 Td
(500)Tj
/F15 1 Tf
[()]TJ
/F8 1 Tf
(l)Tj
2.3981 0 Td
(buffer)Tj
2.6248 0 Td
(2)Tj
0.6973 0 Td
(and)Tj
1.7292 0 Td
(then)Tj
2.0182 0 Td
(resuspended)Tj
5.2327 0 Td
(in)Tj
1.0375 0 Td
(500)Tj
/F15 1 Tf
[()]TJ
/F8 1 Tf
(l)Tj
-33.5676 -1.2982 Td
(buffer)Tj
2.6248 0 Td
(1.)Tj
-1.4286 -1.2983 Td
(Samples)Tj
3.4979 0 Td
(were)Tj
2.126 0 Td
(run)Tj
1.6667 0 Td
(on)Tj
1.2756 0 Td
(a)Tj
0.652 0 Td
(FACScalibur)Tj
5.3461 0 Td
(flow)Tj
1.9502 0 Td
(cytometer)Tj
4.2349 0 Td
(\(BD)Tj
1.8766 0 Td
(biosciences,)Tj
4.9606 0 Td
(USA\),)Tj
2.6815 0 Td
(samples)Tj
3.3846 0 Td
(were)Tj
-34.8489 -1.3039 Td
(acquired)Tj
3.702 0 Td
(in)Tj
1.0318 0 Td
(CellQuest-Pro)Tj
5.947 0 Td
(software)Tj
3.5887 0 Td
(\(BD)Tj
1.8765 0 Td
(biosciences,)Tj
4.9549 0 Td
(USA\).)Tj
2.6816 0 Td
(FACS)Tj
2.5738 0 Td
(analysis)Tj
3.3222 0 Td
(was)Tj
1.7008 0 Td
(performed)Tj
4.4674 0 Td
(in)Tj
-35.8467 -1.2983 Td
(FlowJo)Tj
3.0217 0 Td
(9.4.10)Tj
2.5738 0 Td
(for)Tj
1.3833 0 Td
(Macintosh)Tj
4.4787 0 Td
(\(Tree)Tj
2.3924 0 Td
(Star,)Tj
2.024 0 Td
(USA\).)Tj
2.6815 0 Td
(Cells)Tj
2.16 0 Td
(were)Tj
2.1203 0 Td
(gated)Tj
2.3698 0 Td
(initially)Tj
3.2484 0 Td
(on)Tj
1.2756 0 Td
(being)Tj
2.4321 0 Td
(capsule)Tj
3.1521 0 Td
(posi-)Tj
-35.3137 -1.2982 Td
(tive)Tj
1.661 0 Td
(\(APC+\))Tj
3.3903 0 Td
(and)Tj
1.7291 0 Td
(then)Tj
2.0182 0 Td
(gated)Tj
2.3698 0 Td
(on)Tj
1.2756 0 Td
(selecting)Tj
3.6736 0 Td
(RrgB)Tj
2.2564 0 Td
(positive)Tj
3.3165 0 Td
(\(FITC+\))Tj
3.6227 0 Td
(and)Tj
1.7291 0 Td
(RrgB)Tj
2.262 0 Td
(negative)Tj
3.532 0 Td
(popula-)Tj
-32.8363 -1.3039 Td
(tions.)Tj
2.4264 0 Td
(Percentage)Tj
4.5921 0 Td
(RrgB)Tj
2.2564 0 Td
(positive)Tj
3.3165 0 Td
(and)Tj
1.7291 0 Td
(negative)Tj
3.5376 0 Td
(populations)Tj
4.9493 0 Td
(were)Tj
2.1203 0 Td
(compared)Tj
4.2462 0 Td
(between)Tj
3.5263 0 Td
(parent)Tj
2.812 0 Td
(and)Tj
-35.5122 -1.2983 Td
(mutant)Tj
3.1464 0 Td
(strains.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 328.0251 cm
BT
/F14 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Fluorescence)Tj
5.6031 0 Td
(microscopy)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 311.0173 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(10)Tj
/F15 1 Tf
[()]TJ
/F8 1 Tf
(l)Tj
1.9218 0 Td
(of)Tj
1.0148 0 Td
(antibody)Tj
3.7644 0 Td
(stained)Tj
3.0841 0 Td
(bacteria)Tj
3.3732 0 Td
(preparation)Tj
4.9096 0 Td
(was)Tj
1.7007 0 Td
(pippetted)Tj
4.0139 0 Td
(onto)Tj
2.0806 0 Td
(a)Tj
0.652 0 Td
(76x22mm)Tj
4.2292 0 Td
(glass)Tj
2.0863 0 Td
(slide)Tj
2.0466 0 Td
(\(Men-)Tj
-34.8772 -1.2982 Td
(zel)Tj
1.3039 0 Td
(Gl00E4ser,)Tj
4.5581 0 Td
(Germany\))Tj
4.3256 0 Td
(and)Tj
1.7291 0 Td
(a)Tj
0.6577 0 Td
(22x22mm)Tj
4.2236 0 Td
(glass)Tj
2.0862 0 Td
(cover)Tj
2.4038 0 Td
(slip)Tj
1.6101 0 Td
(\(Menzel)Tj
3.5092 0 Td
(Gla)Tj
0 Tc
0.9808 0 Td
()Tj
-0.004 Tc
0.4139 0 Td
(ser,)Tj
1.5987 0 Td
(Germany\))Tj
4.3313 0 Td
(placed)Tj
-33.732 -1.2983 Td
(over)Tj
1.9729 0 Td
(the)Tj
1.474 0 Td
(sample.)Tj
3.2428 0 Td
(Samples)Tj
3.4979 0 Td
(were)Tj
2.126 0 Td
(heated)Tj
2.8516 0 Td
(for)Tj
1.389 0 Td
(60)Tj
1.1678 0 Td
(minutes)Tj
3.4696 0 Td
(at)Tj
0.9525 0 Td
(50C,)Tj
2.4491 0 Td
(and)Tj
1.7291 0 Td
(observed)Tj
3.7984 0 Td
(at)Tj
0.9524 0 Td
(X40)Tj
1.8255 0 Td
(and)Tj
1.7292 0 Td
(X100)Tj
-34.6278 -1.3039 Td
(magnification)Tj
5.7883 0 Td
(using)Tj
2.3867 0 Td
(a)Tj
0.6577 0 Td
(Zeiss)Tj
2.228 0 Td
(Axioscope)Tj
4.3766 0 Td
(M1)Tj
1.5874 0 Td
(fluorescence)Tj
5.1874 0 Td
(microscope)Tj
4.8415 0 Td
(with)Tj
2.0013 0 Td
(the)Tj
1.4683 0 Td
(correct)Tj
3.0387 0 Td
(filters.)Tj
-33.5619 -1.2983 Td
(Images)Tj
3.0613 0 Td
(were)Tj
2.1203 0 Td
(analysed)Tj
3.6511 0 Td
(in)Tj
1.0374 0 Td
(Volocity)Tj
3.617 0 Td
(software)Tj
3.5886 0 Td
(6.0.1)Tj
2.0977 0 Td
(\(Perkin)Tj
3.2258 0 Td
(Elmer\).)Tj
3.2144 0 Td
(Images)Tj
3.0614 0 Td
(were)Tj
2.126 0 Td
(arranged)Tj
3.7927 0 Td
(in)Tj
-34.5937 -1.2982 Td
(Adobe)Tj
2.8686 0 Td
(Photoshop)Tj
4.5297 0 Td
(elements)Tj
3.7644 0 Td
(10.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 217.0204 cm
BT
/F14 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Hydrogen)Tj
4.4079 0 Td
(peroxide)Tj
3.8646 0 Td
(assay)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 200.0125 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Hydrogen)Tj
4.2859 0 Td
(peroxide)Tj
3.719 0 Td
(assay)Tj
2.2677 0 Td
(was)Tj
1.6951 0 Td
(performed)Tj
4.4731 0 Td
(to)Tj
1.0261 0 Td
(assess)Tj
2.5172 0 Td
(the)Tj
1.474 0 Td
(ability)Tj
2.6929 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.4683 0 Td
(different)Tj
3.6567 0 Td
(strains)Tj
2.863 0 Td
(to)Tj
1.0204 0 Td
(produce)Tj
-34.1742 -1.3039 Td
(hydrogen)Tj
4.0421 0 Td
(peroxide.)Tj
/F7 1 Tf
3.9515 0 Td
[()]TJ
/F8 1 Tf
(.)Tj
/F7 1 Tf
[()11()6()2()12()6()11()18()4( )6()]TJ
/F8 1 Tf
5.7373 0 Td
(cultures)Tj
3.4015 0 Td
(were)Tj
2.1203 0 Td
(grown)Tj
2.795 0 Td
(to)Tj
1.0261 0 Td
(OD600nm)Tj
4.5014 0 Td
(0.6)Tj
1.3946 0 Td
(in)Tj
1.0375 0 Td
(BHI,)Tj
2.1317 0 Td
(180)Tj
/F15 1 Tf
[()]TJ
/F8 1 Tf
(l)Tj
2.3981 0 Td
(of)Tj
1.0148 0 Td
(cul-)Tj
-35.5519 -1.2982 Td
(ture)Tj
1.8425 0 Td
(were)Tj
2.1203 0 Td
(added)Tj
2.6588 0 Td
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