本研究論文是利用一高濃度抗生素 chloramphenical 篩選到一突變株 KJ09C，並由抗生素感受性試驗得知此 KJ09C 菌株具有多重抗藥性之現 象，能夠抗 chloramphenicol、quinolone 類及 tetracycline 類抗生素，但對 aminoglycoside 類抗生素之感受性卻上升。利用 qRT-PCR、刪除突變菌株 (deletion mutant) 及抗生素感受性試驗證明此突變株 KJ09C 是過度表現 SmeVWX 這 套 多 重 藥 物 輸 出 幫 浦 （ multidrug efflux pump ）。
SmeVWX 多重藥物輸出幫浦是由 5 個基因 smeU1、 smeV、 smeW、smeU2 及 smeX 基因組成一 operon，且此幫浦之表現受上游一屬於 LysR 家族之 轉錄調控基因 smeRv 正向調控。
分析突變菌株 KJ09C 之 smeU1-V-W-U2-X operon 的基因在抗生素性 中扮演之角色，結果發現：(i) smeU1 基因和 KJ09C 菌株多重抗藥性表型 的關係不大；(ii) smeVW 基因的過度表現與 chloramphenicol、quinolone 及 tetracycline 類抗生素的抗性上升有關，但與 aminoglycoside 類抗生 素的感受性上升沒有明顯的相關性；(iii) smeX 基因過度表現會導致 KJ09C 對 aminoglycoside 類抗生素抗性下降；(iv) smeU2 基因過度表現 可能會增強 SmeVWX 多重藥物輸出幫浦的活性，將 chloramphenicol、
quinolone 及 tetracycline 類抗生素排出，使得 KJ09C 對 chloramphenicol、
57
quninolone 類及 tetracycline 類抗生素抗性上升。另外，smeU2 基因過度 表現可能會增進 SmeX 將 aminoglycoside 送入細菌體內的效率，使得 KJ09C 對 aminoglycoside 類抗生素感受性上升。
58
TABLE 1. Bacterial strains and plasmids used in this study
Strain or plasmid Genotype or properties Source or reference S. maltophilia
KJ Wild type, a clinical isolate from Taiwan 34
KJΔ5 S. maltophilia KJ smeU1-V-W-U2-X operon deletion mutant
This study
KJ09C S. maltophilia KJ MDR mutant This study
KJ09CΔL1ΔL2 S. maltophilia KJ09C L1 and L2 genes double mutant This study KJ09CΔ5 S. maltophilia KJ09C smeU1-V-W-U2-X operon
deletion mutant
This study KJ09CΔRv S. maltophilia KJ09C smeRv deletion mutant This study KJ09CΔU1 S. maltophilia KJ09C smeU1 deletion mutant This study KJ09CΔVW S. maltophilia KJ09C smeV-smeW deletion mutant This study KJ09CΔU2 S. maltophilia KJ09C smeU2 deletion mutant This study KJ09CΔX S. maltophilia KJ09C smeX deletion mutant This study
Escherichia coli
DH5α F- φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rk- mk+)phoA supE44λ- thi-1 gyrA96 relA1
Invitrogen
S17-1 recA pro thi hsdR with inte grated RP4-2-tc::Mu-kan::Tn7; Tra+TrrSmr
40
plasmids
pEX18Tc sacB oriT, Tcr 41
pRK415 Mobilizable broad-host-range plasmid cloning vector, RK2 origin; Tcr
42
pX1918GT Plasmid containing the xylE-gentamicin resistance cassette; Ampr Gmr
43 pΔ5 pEX18Tc vector with a 1982-bp DNA fragment
of S. maltophilia KJ, containing the partial 5'-terminus of smeU1 gene and a partial 3'-terminus of smeX gene;
Tcr
This study
pKJΔRv pEX18Tc vector with an internal-deletion smeRv gene;
Tcr
This study pKJΔU1 pEX18Tc vector with an internal-deletion smeU1 gene;
Tcr
This study
59
pKJΔVW pEX18Tc vector with a 2426-bp DNA fragment of S. maltophilia KJ, containing the smeU1 gene, partial 5'- terminus of smeV gene, and partial 3'- terminus of smeW gene and smeU2 gene; Tcr
This study
pKJΔU2 pEX18Tc vector with an internal-deletion smeU2 gene;
Tcr
This study pKJΔX pEX18Tc vector with an internal-deletion smeX gene;
Tcr
This study
p371RvxylE pRK415 vector with a 371-bp intergenic region of smeRv and smeU1 genes and a transcriptional fusioned-xylE gene; smeRv::xylE fusion construct
This study
p371U1xylE pRK415 vector with a 371-bp intergenic region of smeRv and smeU1 genes and a transcriptional fusioned-xylE gene; smeU1::xylE fusion construct
This study
p323XxylE pRK415 vector with a 323-bp upstream
of smeX gene and a transcriptional fusioned-xylE gene; smeX::xylE fusion construct
This study
60
TABLE 2. The primers used in this study
Primer name Sequence 5'→3' Length Purpose
SmeRv-F GATGGTACCGCCACGCTGCTGAC
1308 Cloning
SmeRv-R CAGAATAAGCTTGCCGCTGCTTTCC
SmeU1-F CGATTAAGCTTCGGCAATGAAG
1341 Cloning
SmeU1-R ACGTTCTAGAGTGGTATTGGGG
SmeU2-F GATGATATCCATCGCGTTCATCGCC
1074 Cloning
SmeU2-R TGACAGAGCTCGCCAGCACCAG
SmeX-F GGCTCTAGAGAAATCAGCGAAG
1591 Cloning
SmeX-R AGAAGAAAGGTACCGAAGCCAC
SmeBQ-F CGCCATCTCGCTGCTGTTC
198 qRT-PCR SmeBQ-R ATGCCGTTCTTCGCTGCC
SmeCQ-F GCGATGCCAACAGCGAGACC
190 qRT-PCR
SmeCQ-R GTCGCCACTTCAGCCACCAG
SmeEQ-F TCCTGCCCAACGAAGACC
203 qRT-PCR SmeEQ-R CTTGACGAACGCCATGCC
SmeFQ-F CCCGAGCATCTCGCTGAC
207 qRT-PCR
SmeFQ-R AAGCCCACCTGGATCGAC
SmeHQ-F GGCTACTCGGCGATCAAC
207 qRT-PCR
SmeHQ-R CAGGCACAGGAACACCAC
SmeJQ-F GTCAGCCACCAGCAGCAG
192 qRT-PCR
SmeJQ-R CAGCAGCCACACCACGTC
SmeKQ-F AACTCCGACCCCAGCGAC
191 qRT-PCR
SmeKQ-R GCGATCATCGAGATCACCGAC
SmeNQ-F CAAGACCTCCACTGCCAAC
198 qRT-PCR
SmeNQ-R AACAGCCAGATCACCGCC
SmePQ-F GTCAGCCAGTTCCTGTCC
191 qRT-PCR SmePQ-R TACTCCATCGTCGCCACC
SmeZQ-F TGTCCAGCGTCAAGCACC
218 qRT-PCR
SmeZQ-R GCCGACCAGCATCAGGAAG
SmeRvQ-F TCGACGAACGCACGCACC
213 qRT-PCR SmeRvQ-R CCCGCTGATGACCGCCAAC
SmeU1Q-F CGGCGAGACCTCGATCAC
201 qRT-PCR SmeU1Q-R CAACCATCCAGCAGCGAG
SmeVQ-F GCGTGACAGCGAACTGCC
219 qRT-PCR SmeVQ-R TCATCGATCAGCAGCGCC
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SmeWQ-F GCCCACACCATCTCGTTCCC
221 qRT-PCR SmeWQ-R TAGCCGTTGCCGTTGCCC
SmeU2Q-F GGTCGAGCAGGTACGCCAG
153 qRT-PCR SmeU2Q-R ACCGCCACCAGCGCATAG
SmeXQ-F TACGACCGCCGCAAGCAACC
219 qRT-PCR
SmeXQ-R CAGCTCGAAGTAGTTGCGTGCC
rDNA-F GACCTTGCGCGATTGAATG
75 qRT-PCR
rDNA-R CGGATCGTCGCCTTGGT
415-F CGACGACACCCGAAAAAAG
281 qRT-PCR
415-R CATTAGCAACATTATCGCACAG
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TABLE 3. Antibiotics used in this study
Antibiotics Type Solubility Stock
concentration Storage Abbreviation
Chloramphenicol ethanol 25 mg/ml -20℃ CHL
Nalidixic acid Fluoroquinolones water 100 mg/ml -20℃ NAL
moxifloxacin Fluoroquinolones -20℃ MXF
Tetracycline Tetracycline ethanol 5 mg/ml -20℃ TET Doxycyline Tetracycline water 50 mg/ml -20℃ DOX Kanamycin Aminoglycosides water 25 mg/ml -20℃ KAN Gentamycin Aminoglycosides water 15 mg/ml -20℃ GEN Tobramycin Aminoglycosides water 50 mg/ml -20℃ TOB Erythromycin Macrolides ethanol 10 mg/ml -20℃ ERY
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TABLE 4. Antimicrobial susceptibilities of S. maltophilia KJ, its chloramphenicol-selected mutant KJ09C and their derived deletion mutants
MIC（μg ml-1）
Quinolone Tetracycline Aminoglycoside
strain CHL NAL MXF TET DOX KAN GEN TOB ERY
KJ 8 8 0.094 16 1 256 1024 512 64
KJ09C 128 256 0.25 64 2 64 256 256 64
KJ△5 8 8 0.094 16 1 256 1024 512 64
KJ09C△5 8 16 0.094 16 2 128 512 512 64
KJ09C△Rv 8 8 0.094 16 1 256 512 512 64
KJ09C△U1 128 256 0.38 32 2 64 512 128 64
KJ09C△VW 4 4 0.047 16 0.5 64 256 256 64
KJ09C△U2 32 64 0.19 32 2 32 128 128 64
KJ09C△X 32 32 0.064 32 2 512 1024 512 64
KJ09C△5a 8 8 0.094 16 2 128 512 512 64
KJ09C△5L2::U1a 8 8 0.094 16 2 128 512 512 64
KJ09C△5L2::U2a 8 8 0.125 16 2 256 512 512 64
KJ09C△5L2::SmeXa 4 4 0.064 8 1 8 32 8 64
CHL, Chloramphenicol; NAL, Nalidixic acid; MXF, moxifloxacin ; TET, Tetracycline; DOX, Doxycyline; KAN, Kanamycin; GEN, Gentamycin; TOB, Tobramycin; ERY, Erythromycin;
aThe Mueller-Hinton agar contains 30 μg/ml cefoxitin in addition to the antibiotic indicated.
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TABLE 5. The △Ct value of the genes of RND-efflux pumps for strains KJ, KJ09C and its derived mutant, determined by qRT-PCR
.
△Ct value
Gene KJ KJ09C KJ09C△Rv KJ09C△U1 KJ09C△VW KJ09C△U2 KJ09C△X smeB 16.97±0.5 15.28±0.8
smeC 12.88±0.7 14.2±0.7 smeE 20.17±0.6 18.16±0.9 smeF 12.73±0.5 11.04±0.8 smeH 14.34±0.4 14.13±0.6 smeJ 17.10±0.6 17.63±1.1 smeK 14.54±0.5 14.28±0.6 smeN 17.64±0.6 16.85±0.8 smeP 22.20±0.9 21.88±1.4 smeZ 11.76±0.5 12.57±0.8
smeRv 17.44±0.8 11.14±0.5 18.56±0.6 11.08±0.8 10.01±0.5 11.75±0.7 11.88±0.5 smeU1 17.11±0.9 12.01±0.4 17.35±0.8 16.01±0.5 12.51±0.6 11.54±0.5 11.51±0.4 smeV 17.56±0.6 13.07±0.8 18.56±0.3 14.18±0.9 16.78±0.7 13.13±0.6 13.97±0.6 smeW 18.21±0.8 12.33±0.6 18.07±0.7 12.82±0.6 18.07±0.7 13.94±0.5 13.83±0.7 smeU2 18.53±1.0 10.24±0.7 17.85±0.7 11.05±0.8 11.04±0.9 20.95±1.1 11.77±0.5 smeX 18.00±0.9 13.36±0.5 17.57±0.9 12.88±0.5 13.94±0.4 15.99±0.9 18.68±0.9
aThe differene of Ct（critical threshold cycle）values between the gene assayed and 16S rDNA.
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TABLE6. Comparison of the SmeU1 of S. maltophilia K279a with other homologues
Strain No.of Similarity Identity
/ protein aa （%） （%）
Stenotrophomonas maltophilia K279a
/ putative short chain dehydrogenase 256 100 100%
Stenotrophomonas sp. SKA14
/ aklaviketone reductase 256 99 98%
Stenotrophomonas maltophilia R551-3
/ short-chain dehydrogenase/reductase SDR 256 99 95%
Pseudoxanthomonas suwonensis 11-1
/ short-chain dehydrogenase/reductase SDR 262 63 50%
Xanthomonas gardneri ATCC 19865
/ short-chain dehydrogenase of unknown substrate specificity 255 68 51%
Xanthomonas campestris pv. campestris str. ATCC 33913
/ aklaviketone reductase 272 69 52%
Streptomyces violaceusniger Tu 4113
/ short-chain dehydrogenase/reductase SDR 254 45 27%
Haliangium ochraceum DSM 14365
/ short-chain dehydrogenase/reductase SDR 225 46 33%
Arthrobacter chlorophenolicus A6
/ short-chain dehydrogenase/reductase SDR 244 49 33%
Bordetella petrii DSM 12804
/ short chain dehydrogenase 251 42 32%
Roseiflexus sp. RS-1
/ short-chain dehydrogenase/reductase SDR 292 47 33%
Streptomyces roseosporus NRRL 15998
/ short-chain dehydrogenase/reductase SDR 293 48 39%
Streptomyces roseosporus NRRL 15998
/ putative oxidoreductase 282 45 27%
Streptomyces pristinaespiralis ATCC 25486
/ short-chain dehydrogenase/reductase SDR 276 49 39%
Streptomyces roseosporus NRRL 15998
/ short-chain dehydrogenase/reductase SDR 272 49 40%
Streptomyces roseosporus NRRL 11379
/ short-chain dehydrogenase/reductase SDR 272 48 40%
Mycobacterium smegmatis str. MC2 155
/ short-chain dehydrogenase/reductase SDR 244 40 27%
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TABLE7. Comparison of SmeV of S. maltophilia K279a with other homologues
Strain No.of Similarity Identity
/ protein aa （%） （%）
Stenotrophomonas maltophilia K279a
/ putative multidrug efflux protein, HlyD family 391 100 100 Stenotrophomonas sp. SKA14
/ RND multidrug efflux membrane fusion protein 410 99 99 Stenotrophomonas maltophilia R551-3
/ RND family efflux transporter MFP subunit 408 99 99
Xanthomonas gardneri ATCC 19865
/ RND family efflux transporter, MFP subunit 396 89 78 Xanthomonas campestris pv. campestris str. ATCC 33913
/ RND multidrug efflux membrane fusion protein 396 90 80 Klebsiella variicola At-22
/ efflux transporter RND family, MFP subunit 391 82 68 Klebsiella pneumoniae
/ RND multidrug efflux membrane fusion protein OqxA 391 81 66 Pseudoxanthomonas suwonensis 11-1
/ efflux transporter, RND family, MFP subunit 399 85 73 Enterobacter sp. 638
/ RND family efflux transporter MFP subunit 391 83 70
Pseudomonas stutzeri A1501
/ RND multidrug efflux membrane fusion protein MexE precursor 409 70 50 Pseudomonas aeruginosa PAO1
/ RND multidrug efflux membrane fusion protein MexE precursor 414 69 51
Pseudomonas aeruginosa 39016
/ RND multidrug efflux membrane fusion protein MexE precursor 414 69 51 Burkholderia sp. 383
/ HlyD family secretion protein 414 69 52
Burkholderia sp. TJI49
/ RND family efflux transporter MFP subunit 388 68 53
Pantoea vagans C9-1
/ RND efflux system, membrane-fusion protein 384 82 69 Klebsiella sp. 1_1_55
/ RND multidrug efflux membrane fusion protein OqxA 391 82 68
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TABLE8. Comparison of SmeW of S. maltophilia K279a with other homologues
Strain No.of Similarity Identity
/ protein aa （%） （%）
Stenotrophomonas maltophilia K279a
/ putative drug resistance membrane fusion protein 1056 100 100%
Stenotrophomonas sp. SKA14
/ RND transporter, HAE1 family 1056 99 99%
Stenotrophomonas maltophilia R551-3
/ hydrophobe/amphiphile efflux-1 (HAE1) family transporter 1056 99 98%
Halomonas elongata DSM 2581
/ transporter, hydrophobe/amphiphile efflux-1 (HAE1) family 1052 90 82%
Xanthomonas campestris pv. campestris str. ATCC 33913
/ RND multidrug efflux transporter MexF 1056 90 82%
Xanthomonas gardneri ATCC 19865
/ hydrophobe/amphiphile efflux-1 (HAE1) family transporter 1057 90 81%
Alcanivorax sp. DG881
/ RND transporter, HAE1 family 1064 89 81%
Marinobacter algicola DG893
/ Hydrophobe/amphiphile efflux-1 HAE1 1067 90 80%
Pseudoxanthomonas suwonensis 11-1
/ transporter, hydrophobe/amphiphile efflux-1 (HAE1) family 1059 89 79%
Klebsiella variicola At-22
/ transporter hydrophobe/amphiphile efflux-1 (HAE1) family 1050 89 79%
Klebsiella pneumoniae 342
/ RND family multidrug efflux permease protein OqxB 1050 88 78%
Escherichia coli
/ OqxB integral membrane protein 1050 88 78%
Burkholderia ambifaria IOP40-10
/ transporter, hydrophobe/amphiphile efflux-1 (HAE1) family 1057 84 71%
Burkholderia cenocepacia J2315
/ putative quinoxaline efflux system transporter protein 1057 83 71%
Klebsiella pneumoniae
/ RND family multidrug efflux permease protein OqxB 1050 88 78%
Acidobacterium sp. MP5ACTX8
/ transporter, hydrophobe/amphiphile efflux-1 (HAE1) family 1058 84 71%
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TABLE 9. Comparison of the SmeU2 of S. maltophilia K279a with other homologues
Strain No.of Similarity Identity
/ protein aa （%） （%）
Stenotrophomonas maltophilia K279a
/ putative short-chain dehydrogenase/reductase 258 100 100%
Stenotrophomonas sp. SKA14
/ short chain dehydrogenase 245 99 98%
Stenotrophomonas maltophilia R551-3
/ short-chain dehydrogenase/reductase SDR 245 98 93%
Xanthomonas campestris pv. vasculorum NCPPB702
/ short chain dehydrogenase 243 87 77%
Xanthomonas axonopodis pv. citri str. 306
/ short chain dehydrogenase 243 88 76%
Xanthomonas gardneri ATCC 19865
/ short-chain alcohol dehydrogenase like protein 243 87 75%
Pseudoxanthomonas suwonensis 11-1
/ short-chain dehydrogenase/reductase SDR 243 82 68%
Streptomyces sp. AA4
/2-hydroxycyclohexanecarboxyl-CoA dehydrogenase 245 74 57%
Streptomyces ambofaciens
/ putative ketoacyl reductase 237 58 44%
Streptomyces ambofaciens ATCC 23877
/ putative ketoacyl reductase 237 58 43%
Burkholderia xenovorans LB400
/ putative short-chain dehydrogenase/oxidoreductase 245 66 50%
Burkholderia gladioli BSR3
/ short chain oxidoreductase 241 56 43%
Burkholderia xenovorans LB400
/ putative short-chain dehydrogenase/reductase 257 62 43%
Candidatus Solibacter usitatus Ellin6076
/ short-chain dehydrogenase/reductase SDR 250 70 53%
Gluconacetobacter diazotrophicus PAl 5
/ putative short-chain dehydrogenase 265 70 46%
Caulobacter segnis ATCC 21756
/ short-chain dehydrogenase/reductase SDR 243 65 49%
Beutenbergia cavernae DSM 12333
/ short-chain dehydrogenase/reductase SDR 244 67 50%
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TABLE 10. Comparison of SmeX of S. maltophilia K279a with other homologues
Strain No.of Similarity Identity
/ protein aa （%） （%）
Stenotrophomonas maltophilia K279a
/ putative outer membrane efflux protein 472 100 100%
Stenotrophomonas maltophilia R551-3
/ NodT family RND efflux system outer membrane lipoprotein 473 99 98%
Stenotrophomonas sp. SKA14
/ outer membrane efflux protein 473 99 98%
Xanthomonas axonopodis pv. citri str. 306
/ outer membrane protein 474 83 74%
Xanthomonas fuscans subsp. aurantifolii str. ICPB 10535
/ outer membrane protein 474 83 74%
Burkholderia sp. H160
/ RND efflux system, outer membrane lipoprotein, NodT family 489 53 37%
Burkholderia sp. 383
/ RND efflux system outer membrane lipoprotein 507 54 39%
Pseudoxanthomonas suwonensis 11-1
/ RND efflux system, outer membrane lipoprotein, NodT family 482 75 66%
Caulobacter segnis ATCC 21756
/ NodT family RND efflux system outer membrane lipoprotein 469 71 55%
Pseudomonas syringae pv. aesculi str. 2250
/ outer membrane efflux protein 465 67 51%
Pseudomonas syringae pv. syringae 642
/ RND efflux system, outer membrane lipoprotein, NodT 465 67 50%
Pseudomonas entomophila L48
/ multidrug efflux RND outer membrane protein OprN 471 66 49%
Achromobacter piechaudii ATCC 43553
/ multidrug efflux RND outer membrane protein OprN 513 65 46%
Bordetella bronchiseptica RB50
/ outer membrane component of multidrug efflux system 487 61 44%
Bordetella parapertussis 12822
/ outer membrane component of multidrug efflux system 487 61 44%
Delftia acidovorans SPH-1
/ RND efflux system outer membrane lipoprotein 477 61 45%
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TABLE11. The determination of C23O activities of the KJ, KJ09C, and their derived mutants containing different transcriptional fusion constructs.
strain C23O activity (Uc/OD450nm) KJ(p371smeRvxylE) 5±0.7
KJ△Rv(p371smeRvxylE) 58±6.1 KJ09C(p371smeRvxylE) 305±41 KJ09C△Rv(p371smeRvxylE) 21±1.9 KJ(p371smeU1xylE) 1±0.4 KJ△Rv(p371smeU1xylE) 3±0.7 KJ09C(p371smeU1xylE) 25±3.1 KJ09C△Rv(p371smeU1xylE) 2±0.5 KJ(p323smeXxylE) 4±0.4 KJ△Rv(p323smeXxylE) 6±0.7 KJ09C(p323smeXxylE) 5±0.5 KJ09C△Rv(p323smeXxylE) 5±0.4
Fig.1. The organisation and operation of antimicrobial efflux pumps of Gram-negative bacteria.
OM, outer membrane; PP, periplasmic space; CM, cytoplasmic membrane; MFS, major facilitator superfamily; ABC, ATP-binding cassette family; RND, resistance-nodulation division; SMR, small multi-drug resistance; MATE, multi-drug and toxic compound extrusion
71
S. maltophilia
smeRv smeU1 smeV smeW smeU2 smeX P. aeruginosa mexT mexE mexF oprN
21％ 51％ 56％ 48％
Xcc1437 dauE mexE mexF Xcc1441 oprN X.campestris pv. campestris
81％ 45％ 74％ 80％ 70％ 71％
Fig.2. Comparison between smeRv-smeU1-V-W-U2-X operon of S. maltophilia and its homologues.
protein identity
protein identity
72
( PCR size：1359 bp ) Ligation
( PCR size：1591 bp )
Fig. 3. Construction of pKJΔ5.
The 1359-bp DNA fragment containing smeU1 and partial smeRv genes gene was obtained by PCR amplification using primers smeU1-F/smeU1-R. HindIII and XbaI restriction sites were used to facilitate the cloning into vector pEX18Tc to yield plasmid pEXSmeU1. The 1591-bp DNA fragment containing smeX gene was obtained by PCR amplification using primers smeX-F/smeX-R.
XbaI and KpnI restriction sites were used to facilitate the cloning into vector pEX18Tc to yield plasmid pEXSmeX. A 1330-bp partial smeX gene was retrieved from pEXSmeX, and inserted into the PstI and EcoRI site of pEXSmeU1, generating plasmid pKJΔ5 .
smeU1-R
HindIII XbaI XbaI KpnI
pEXSmeX
PstI EcoRI PstI EcoRI
HindIII
XbaI
XbaI KpnI
Ligation
73
( PCR size：1308 bp )
Fig. 4. Construction of pKJΔSmeRv.
The 1308-bp DNA fragment containing smeRv gene was obtained by PCR amplification using primers smeRv-F/smeRv-R. HindIII and KpnI restriction sites were used to facilitate the cloning into vector pEX18Tc to yield plasmid pEXSmeRv. Plasmid pEXSmeRv was digested by PstI to delete a 294-bp internal fragment of the smeRv gene, generating plasmid pKJΔsmeRv.
HindIII KpnI pEX18Tc
SmeX
Delect 173 bp pKJΔSmeU1
7535 bp
Tcr sacB
ΔsmeU1
Fig. 5. Construction of pKJΔSmeU1.
The 1359-bp DNA fragment containing smeU1 and partial smeRv
genes was obtained by PCR amplification using primers
smeU1-F/smeU1-R. HindIII and XbaI restriction sites were used to
facilitate the cloning into vector pEX18Tc to yield plasmid
pEXSmeU1. Plasmid pEXSmeU1 was digested by PstI to delete a
173-bp fragment internal to the smeU1 gene, generating plasmid
pKJΔsmeU1.
pOK12
Fig. 6. Construction of pKJΔSmeVW.
The 1359-bp DNA fragment containing smeU1 and partial smeRv genes was obtained by PCR amplification using primers smeU1-F/smeU1-R. HindIII and XbaI restriction sites were used to facilitate the cloning into vector pEX18Tc to yield plasmid pEXSmeU1. The 1132-bp DNA fragment containing smeU2 gene was obtained by PCR amplification using primers smeU2-F/smeU2-R. EcoRV and SacI restriction sites were used to facilitate the cloning into vector pOK12 to yield plasmid pOKSmeU2. A 1132-bp smeU2 gene cassette was retrieved from pOKSmeU2, and inserted into the XbaI and SacI site of pEXSmeU1, generating plasmid pKJΔSmeVW .
( PCR size：1359 bp ) HindIII XbaI
76
SmeX
Fig. 7. Construction of pKJΔSmeU2.
The 1132-bp DNA fragment containing smeU2 gene was obtained by PCR amplification using primers smeU2-F/smeU2-R. EcoRV and SacI restriction sites were used to facilitate the cloning into vector pOK12 to yield plasmid pOKSmeU2. Plasmid pOKSmeU2 was digested by StuI and HincII to delete a 103-bp internal fragment of the smeU2 gene, generating plasmid pOKΔsmeU2. The 1029-bp DNA fragment retrieved from plasmid pOKΔSmeU2 was used to clone into vector pEX18Tc to yield plasmid pKJΔSmeU2.
( PCR size：1132 bp )
SmeX
Fig. 8. Construction of pKJΔSmeX.
The 1591-bp DNA fragment containing smeX gene was obtained by
PCR amplification using primers smeX-F/smeX-R. XbaI and KpnI
restriction sites were used to facilitate the cloning into vector
pEX18Tc to yield plasmid pEXSmeX. The 722-bp DNA fragment
retrieved from plasmid pOKSmeU2 was cloned into vector
pEX18Tc to yield plasmid pEXSmeU2c. A 770-bp smeX-containing
DNA fragment was retrieved from pEXSmeX ,and inserted into the
SmaI and EcoRI site of pEXSmeU2c, generating plasmid
pKJΔSmeX
pKJ△5 8331 bp
Tc
rsacB
△smeX
△smeU1
SmeX
smeU2
SmeV SmeW
smeU1
smeRv
KJ09C
Double cross-over recombination
△smeX
△smeU1
smeRv
Mutant
Fig. 9. Double cross-over recombination between exotic plasmid and the chromosome of host bacteria ( KJ09C△5 as a representative).
Conjugation was carried out between E. coli S17-1(pKJΔ5) and S.
maltophilia KJ09C. Transconjugants was firstly selected on the LA medium containing 30 g/ml tetracycline and 2.5 g/ml norfloxacin. The double cross-over recombinant was obtained by further selection on the LA medium containing 10% sucrose.
79
Fig. 10. The possible role of SmeU2 in SmeVWX pump module.
In presence of
SmeU2
aminoglycoside81
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Abstract
Stenotrophomonas maltophilia is an important opportunistic pathogen characterized by the phenotype of multidrug resistance (MDR). Overexpression of the resistance nodulation division (RND) efflux systems is a critical cause of the MDR phenotype in gram-negative bacteria. Whole genome analysis revealed that S. maltophilia harbors as many as eight possible RND efflux systems, including SmeABC, SmeDEF, SmeGH,
Stenotrophomonas maltophilia is an important opportunistic pathogen characterized by the phenotype of multidrug resistance (MDR). Overexpression of the resistance nodulation division (RND) efflux systems is a critical cause of the MDR phenotype in gram-negative bacteria. Whole genome analysis revealed that S. maltophilia harbors as many as eight possible RND efflux systems, including SmeABC, SmeDEF, SmeGH,