Primers Sequences 16S RNA position

1A 5’ CAC AAG CGG TGG AGC ATG TGG TT 3’ 932-955

5 5’ CCT ACG CYT ACC TTG TTA CGA CT 3’ 1514-1492

3 5’ GGA ATT CTG CAA CGC GAA CCT TAC TA 3’ 1168-1194

4 5’ GCG GAT CCT GGT KTG ACG GGC GGT TG TA 3’ 1392-1371

Table 1A. Sequences of nested primers used in amplification of 16S rDNA from IC patient biopsies, Y equals C or T, K equals G or T. (Domingue et al. 1995)

Primers Sequences 140kDa adhesin protein of Mycoplasma genitalium

MgPa-1 5’ AGTTGATGAAACCTTAACCCCTTGG 3’ 176-206

MgPa-3 5’ CCGTTGAGGGGTTTTCCATTTTTGC 3’ 435-460

Table 1B. Sequences of primers used in amplification of adhesin protein of Mycoplasma genitalium, MG, blind control DNA. (Jensen et al. 1991)

Primers Sequences pUC18

Forward-Biotin (FB) Biotin-AAA GGG GGA TGT GCT GCA AGG 399-421

Forward (F) AAA GGG GGA TGT GCT GCA AGG

Reverse-Biotin (RB) Biotin-GCT TCC GGC TCG TAT GTT GTG 511-531

Reverse (R) GCT TCC GGC TCG TAT GTT GTG

Table 1C. Biotin-labeled pUC18 forward and reverse primers used in immobilization single strand template DNA and sequencing reaction. The underlined sequences are biotin sequences.

Clone Organisms P(N) Gene References

and Accession #

MG Mycoplasma genitalium 1.2e-28 attachment protein Inamine, J.M.

M31431 GenBank et al.

P5 Pseudomonas pickettii 7.8e-64 rRNA Zhou. J. et al.

L37367 GenBank

63 Caulobacter subvibroidies 2.8e-63 16S rRNA Stahl,D.A.et al

M83797 GenBank

68 Azospirillum sp 4.3e-62 16S rRNA Xa Y., et al.

Z29622 Embl

125 Azospirillum sp 1.4e-61 16S rRNA Xa Y., et al.

Z29622 Embl

129 Pseudomonas flavescens B62 5.2e-61 16S rRNA Johnson,J.L.

U01916 GenBank

131 E. coli 5.2e-61 rrnH gene for Nayashiki, T.

D12649 GenBank rRNA and tRNAs et al.

83-35* Acidiphilium facilis 3.6e-54 16S rRNA Kishimoto, N.

D30774 DDBJ et al.

182-1,5 Acidiphilium facilis 1.2e-11 16S rRNA Wichlac, Z. P.

M79371 GenBank et al.

Table 2. Comparison of different clones’ sequences to 16S rRNA from database of National Center of Biotechnological Information (NCBI) of NIH.

P(N) smallest sum of probability

* 135-17,135-19,175-2,175-4 have the same sequences

Clone Organisms Percent Similarity (%)**

MG Methanosphaera stadtmanii 0.17

P5 Bradyrhizobium japonicum 0.525

63 Bradyrhizobium japonicum 0.495

68 Pseudomonas syringae 0.435

125 Flavobacterium lutescens 0.555

129 Pseudomonas aeruginosa 0.680

131 Escherichia coli subsp. K-12 0.906

83-35* Acidiphilium facilis 0.548

182-1,5 Acidiphilium facilis 0.319

Table 3. Comparison of top bacterial species of different clones’ sequences to 16S

rRNA from database of RDP of University of Illinois

* 135-17,135-19,175-2,175-4 have the same sequences

** Percent similarity was the number that compared each clone with the RDP sequences

from the organisms yielding the highest score.

The values in the table represent percent similarity multiplied by 0.01.

% Percent similarity was used in all RDP searches of this thesis.

Organisms Organisms

in Ribosomal Database Project % in NCBI of NIH P(N)

MG Methanosphaera stadtmanii 0.170 Mycoplasma genitalium M31431* 1.2e-28 Zea mays 0.159 Mycoplasma genitalium X61511** 2.1e-16 Triticum aestivum 0.159 Cowpea mosaic virus bottom

component X00206** 0.67 Clostridium acidiurici 0.159 Yersinia pestis L22495* 0.81 Secale cereale 0.159 Hevea brasiliensis M74798* 0.81 Flavobacterium gondwanense0.154 Yersinia pseudotuberculosis L25667* 0.81

Bacteroides splanchnicus 0.148 Red Clover Mottle Virus X64886** 0.95

Mycoplasma sp. 0.148

Mycoplasma sp. 0.143

Anaeroplasma intermedium 0.143

Table 4. Comparison of top 10 bacterial species of clone MG from database of NCBI of

NIH and RDP of University of Illinois.

P(N) smallest sum of probability

% percent similarity

* genbank

** embl

*** ddbj

Organisms Organisms

in Ribosomal Database Project % in NCBI of NIH P(N)

P5 Bradyrhizobium japonicum 0.525 Pseudomonas pickettii L37367* 7.8e-64

Bradyrhizobium japonicum 0.525 Pseudomonas pickettii gp1X70348**2.8e-63

Bradyrhizobium japonicum 0.471 Pseudomonas pickettii gp2X70349**2.8e-63

Pseudomonas pickettii group 0.466 Pseudomonas solanacearum group1 X70344** 4.3e-62

Burkholderia pickettii 0.460 Pseudomonas syzygii X70347** 1.4e-61

Afipla felis 0.457 Pseudomonas solanacearum

group2 X70345** 5.2e-61

Afipla clevelandensis 0.457 Pseudomonas solanacearum

group 4 X0346** 5.2e-61

Rhodopseudomonas palustris 0.457 Pseudomonas solanacearum

group3 X70350** 5.6e-60

Alcaligenes eutrophus 0.452 Pseudomonas solanacearum

X67041** 6.9e-55

Bradvrhizobium japonicum 0.452 Pseudomonas solanacearum

X67035** 7.3e-55

Table 5. Comparison of top 10 bacterial species of clone P5 from database of NCBI of NIH and RDP of University of Illinois.

P(N) smallest sum of probability

% percent similarity

* genbank

** embl

*** ddbj

Organisms Organisms

in Ribosomal Database Project % in NCBI of NIH P(N)

63 Bradyrhizobium japonicum 0.497 Caulobacter subvibroidies M83797* 3.0e-98

Ancylobacter aquaticus 0.495 S. adhaseiva X72720** 2.0e-96

Blastobacter denitrificans 0.495 Rhizobium leguminosarum X67227** 1.9e-93

Sphingomonas capsulata 0.495 Rhizobium leguminosarum D14513*** 3.1e-93

Rhodopseudomonas palustris 0.492 S. parapaucimobilis X72721** 4.1e-93

Zoogloea ramigera 0.490 Rhizobium leguminosarum D12782*** 1.9e-92

Afipla clevelandensis 0.490 A. aquaticus M62790* 4.9e-92

Bradyrhizobium japonicum 0.486 Thiobacillus novellus D32247*** 6.5e-92

Azorhizobium caulinodans 0.486 Bradyrhizobium sp.D14508*** 7.2e-92

Aquabacter spiritensis 0.484 Rhizobium sp. D14515*** 1.2e-91

Table 6. Comparison of top 10 bacterial species of clone 63 from database of NCBI of NIH and RDP of University of Illinois.

P(N) smallest sum of probability

% percent similarity

* genbank

** embl

*** ddbj

Organisms Organisms

in Ribosomal Database Project % in NCBI of NIH P(N)

68 Psuedomonas syringae 0.435 Azospirillum sp Z29622** 1.3e-55 Pseudomonas mendocina 0.432 Pseudomonas syringae L24787* 4.3e-55 Pseudomonas putida 0.432 Unclassified gamma proteobacterium

U12222* 4.2e-54 Pseudomonas flavescens 0.426 Hydrothermal vent eubacterium

U15111* 7.2e-52

Pseudomonas aeruginosa 0.421 X. fastidiosa M26601* 1.5e-49 Pseudomonas aeruginosa 0.412 Flavobacrterium lutescens M59156* 2.5e-49 Azospirillum sp 0.409 Pseudomonas mendocina L37366* 4.6e-49 Flavobacterium lutescens 0.398 Pseudomonas flavescens U01916* 7.4e-49 Pseudomonas fluorescens 0.398 Pseudomonas fluorescens L24790* 1.5e-48 Xanthomonas fragariae 0.393 Pseudomonas putida L37365* 3.9e-48

Table 7. Comparison of top 10 bacterial species of clone 68 from database of NCBI of NIH and RDP of University of Illinois.

P(N) smallest sum of probability

% percent similarity

* genbank

** embl

Organisms Organisms

in Ribosomal Database Project % in NCBI of NIH P(N)

125 Flavobacterium lutescens 0.555 Azospirillum spZ29622** 9.8e-104

Thyasira flexuosa gill symbiont 0.552 Pseudomonas aeruginosa M34133* 3.8e-94

Pseudomonas mendocina 0.546 Pseudomonas aeruginosa X06684** 5.3e-94

Pseudomonas flavescens 0.545 Pseudomonas flavescens

B62 U01916* 6.1e-94

Pseudomonas aeruginosa 0.541 Pseudomonas putida L37365* 4.6e-90

Pseudomonas aeruginosa 0.536 Flavobacterium lutescens M59156* 1.6e-89

symbiont of Solemya velum 0.520 Pseudomonas mendocina L37366* 3.1e-89

Oceanospirillum jannsschii 0.485 Unclassified gamma U12217* 1.0e-85

proteobacterium isolate

Thiomicrospira thyasiris 0.480 Thyasira flexuosa L01575* 4.4e-85

gill symbiont part

Chromatium vincsum 0.478 Unclassified gamma U12213* 7.7e-85

proteobacterium isolate

Table 8. Comparison of top 10 bacterial species of clone 125 from database of NCBI of NIH and RDP of University of Illinois.

P(N) smallest sum of probability

% percent similarity

* genbank

** embl

*** ddbj

Organisms Organisms

in Ribosomal Database Project % in NCBI of NIH P(N)

129 Pseudomonas aeruginosa 0.680 Pseudomonas flavescens B62 U01916* 2.8e-132

Pseudomonas flavescen 0.676 Flavobacterium lutescens M59156* 2.9e-130

Pseudomonas aeruginosa 0.664 Pseudomonas putida L37365* 1.9e-129

Pseudomonas mendocina 0.658 Pseudomonas aeruginosa M34133* 3.4e-129

Flavobacterium lutescens 0.633 Pseudomonas mendocina L37366* 1.3e-128

Thyasira flexuosa 0.617 Pseudomonas aeruginosa X06684** 3.2e-128

symbiont of Solemya velum 0.603 Azospirillum spZ29622** 1.6e-127

Oceanospirillum jannsschii 0.592 Thyasira flexuosa gill symbiont part

L01575* 3.5e-123

Legionella brunensis 0.571 Pseudomonas mendocina M59154* 8.9e-123

Legionella steigerwaltii 0.571 Pseudomonas syringae L24787* 1.0e-121

Table 9. Comparison of top 10 bacterial species of clone 129 from database of NCBI of NIH and RDP of University of Illinois.

P(N) smallest sum of probability

% percent similarity

* genbank

** embl

*** ddbj

Organisms Organisms

in Ribosomal Database Project % in NCBI of NIH P(N)

131 Escherichia coli subsp. K-12 0.906 E. coli rrnH geneD12649*** 1.2e-160 Escherichia coli subsp. K-12 0.854 16s rRNA A14565** 1.1e-155 Escherichia coli 0.854 Escherichia coli J01859* 1.1e-155 Escherichia coli subsp. K-12 0.854 Escherichia coli rRNA

operon (rrnB) J01695* 2.6e-154 Escherichia coli subsp. K-12 0.839 Escherichia coli rRNA

operon (rrnB) V00348** 2.6e-154 Escherichia coli subsp. K-12 0.809 Escherichia coli

genomic region L10328* 6.1e-153

Serratia marcescens 0.765 Escherichia coli

chromosomal region U00006* 7.9e-153

Citrobacter freundii 0.747 Escherichia coli K-12

chromosomal region U18997* 1.7e-152

Glossina pallidipes 0.713 Escherichia coli

genomic sequence region M87049* 1.9e-152 Erwinia carotovora subsp 0.713 Escherichia coli 16S ribosomal

RNA partial X72324** 2.8e-152

Table 10. Comparison of top 10 bacterial species of clone 131 from database of NCBI of NIH and RDP of University of Illinois.

P(N) smallest sum of probability

% percent similarity

* genbank

** embl

*** ddbj

Organisms Organisms

in Ribosomal Database Project % in NCBI of NIH P(N)

83-35

Acidiphilium facilis 0.548 Acidiphilium facilis D30774*** 3.6e-54

Acidiphilium aminolytica 0.475 Acidiphilium aminolyticaD30771*** 2.3e-51

Magnetospirillum gryphiswaldense 0.401 Acidiphilium liquefacien X75617** 1.0e-45

Rhodospirillum molischianum0.389 Acidiphilium diazotrophicusX75618** 1.0e-45

Phyllobacterium myrsinacearum 0.376 Acidiphilium methanolicusX77468** 3.1e-45

Agrobacterium tumefaciens 0.376 Acidiphiliumx europaeus Z21936** 1.1e-43

Thiobacillus sp. 0.373 B. doshiae Z31351** 4.0e-41

Rhizobium tropici 0.373 Acidiphilium facilis M79371* 2.8e-40

Magnetospirillum magnetotactium 0.373 Acidiphilium amazonense X79735** 2.9e-40

Agrobacterium tumefaciens 0.369 Acidiphilium tumefaciens X67223** 3.0e-40

Table 11. Comparison of top 10 bacterial species of clone 83-35,135-17,135-19,175-2,175-4 from database of NCBI of NIH and RDP of University of Illinois.

P(N) smallest sum of probability

% percent similarity

* genbank

** embl

*** ddbj

Organisms Organisms

in Ribosomal Database Project % in NCBI of NIH P(N)

182-1,182-5

Acidiphilium facilis 0.319 Acidiphilium facilis M79371* 1.2e-11

Acidiphilium facilis 0.311 Acidiphilium facilis D30774*** 1.2e-10

Zoogloea ramigera 0.272 Acidiphilium aminolytica D30771*** 7.1e-10

Rhizobium loti 0.264 Rhizobiumleguminosarum M63183** 4.2e-07

Agrobacterium rubi 0.264 Rochalimaea henselae M59459** 7.6e-07

Zoogloea ramigera 0.264 G. asaii X80156** 1.6e-06

Rhizobium huaku2 0.260 G. cerinus X80775** 1.6e-06

Rhizobium huakui 0.260 G. frateurii X82290** 1.6e-06

Sinorhizobium xinjiangensis 0.260 G. oxydan X873802** 1.6e-06

Rhizobium fredii 0.255 Acidiphilium amazonense X79742** 2.6e-06

Table 12. Comparison of top 10 bacterial species of clone 182-1,182-5 from database of NCBI of NIH and RDP of University of Illinois.

P(N) smallest sum of probability

% percent similarity

* genbank

** embl

*** ddbj

Organisms in Computer Search

Clone NCBI RDP Common

MG Mycoplasma genitalium Methanosphaera stadtmanii Mycoplasma genitalium

P5 Pseudomonas pickettii Bradyrhizobium japonicum Pseudomonas pickettii

63 Caulobacter subvibroidies Bradyrhizobium japonicum Bradyrhizobium japonicum

68 Azospirillum sp Pseudomonas syringae Pseudomonas syringae

125 Azospirillum sp Flavobacterium lutescens Flavobacterium lutescens

129 Pseudomonas flavescens Pseudomonas aeruginosa Pseudomonas aeruginosa

131 Escherichia coli Escherichia coli subsp. K-12 Escherichia coli

83-35*Acidiphilium facilis Acidiphilium facilis Acidiphilium facilis

182-1,5 Acidiphilium facilis Acidiphilium facilis Acidiphilium facilis

Table 13. Summary results of top and common species of each clone by analyzing two databases of NCBI of NIH and RDP of University of Illinois.

* clones 135-17,135-19,175-2,175-4 have the same sequences

NCBI searches of NCBI searches of

overlapped sequences P(N) total sequences P(N)

63 Sphingomonas sp.Z23157** 1.2e-101 Caulobacter subvirbroidies M83797* 3.0e-98

Unidentified activated U45691*2.2E-99 S. adhaseiva X72720** 2.0e-96

Caulobacter subvibroidies Rhizobium leguminosarum 1.9e-93

M83797* 2.9e-98 X67227**

S. Adhaesive X67227** 1.9e-96 Rhizobium leguminosarum D14513*** 3.1e-93

Rhizobium leguminosarum 1.7e-93 S. parapaucimobilis X72721** 4.1e-93

U29368*

Rhizobium leguminosarum 1.8e-93 Rhizobium leguminosarum D12782*** 1.9e-92

Rhizobium etli U28916* 1.8e-93 A. aquaticus M62790* 4.9e-92

Rhizobium etli U47303* 1.9e-93 Thiobacillus novellus D32247*** 6.5e-92

Rhizobium leguminosarum 3.7e-93 Bradyrhizobium sp.D14508*** 7.2e-92

S. Parapaucimobilis X72721** 3.9e-93 Rhizobium sp. D14515*** 1.2e-91

Table 14. Comparison of NCBI search results of overlapped sequences with that of total sequences of clone 63.

P(N) smallest sum of probability

* genbank

** embl

*** ddbj

Adherence to human uroepithelial cells

Capacity to cause D-mannose-sensitive and D-mannose-resistant hemagglutination

Resistance to serum bactericidal activity

Possession of certain K antigens (K1,K5, K12,K13)

Amount of K antigen (extracellular capsular polysaccharide)

Production of hemolysin and colicin

Possession of certain O serotypes

Opsonization requirements

Short bacterial generation time in urine

Production of siderophores

Table 15. Proposed virulence factors of uropathogenic Escherichia coli.

Table 16. Classification of Psuedomonas species on the computer search lists.

Working Day Steps of Diagnostic protocols

Day 1 Receiving biopsy from IC patient

Day 2 DNA extraction and PCR amplification by 16S rRNA universal primers

Day 3 Purification and phosphate kinasing of PCR products and ligation into ready-to-go pUC18 vector with SmaI cloning site

Day 4 Transformation of ligation DNA into E. coli and culture on agar plates

Day 5 Pick-up vector containing colons and overnight culture

Day 6 Plasmid DNA extraction and PCR amplification by biotin-labeling primers

Day 7 Magnetic separation of biotin-labeling single-strand DNA, sequencing

reaction, sequencing gel running and autoradiography

Day 8 Reading sequences of clones and array by PC/Gene program and send to

NCBI and RDP by E-mail and gaining the results of bacteria species

Table 17. The ideal timetable of IC diagnostic protocols which are developed in this thesis.

Sequence 1. Sequences of Clone MG

Sequence 2. Sequences of Clone P5

Sequence 3. Sequences of Clone 63

Sequence 4. Sequences of Clone 68

Sequence 5. Sequences of Clone 125

Sequence 6. Sequences of Clone 129

Sequence 7. Sequences of Clone 131

Sequence 8. Sequences of Clone 83-35,135-17,135-19,175-2,175-4

Sequence 9. Sequences of Clone 182-1,182-5