建議

本論文中能確定 L2-ampH 基因組裝會被 AmpR 蛋白調控，但 是否有其他的調控機制尚未能確定。不過能確定的是 AmpH 蛋白會 影響 β-lactamase 的表現量，或許將 ampH 基因的調控完全解開可 以提供另一個新藥開發的面向。

51

AmpHQ-F/AmpHQ-R HpQ-F/HpQ-R

(B) Recombinant plasmid pKJ567AmpH

xylE

(C) Mutants KJL2

xylE

KJAmpH

xylE

KJ△R

Fig1. Restriction enzyme maps of the S. maltophilia KJ chromosome surrounding the L2-ampH operon. The empty box indicates the deleted region. The directed of xylE gene is indicated by the arrow.

ampR L2 ampH hp

52

53

Fig3. Construction of pRK567AmpHxylE

ampH hp L2

xylE Tc

^r

HindIII BglII

ampH hp L2

xylE

Tc

^r

LacZ

BamHI/HindII

54

0 10 20 30

R a tio of the m -RNA transcript

0 20 40 60 80 100 120

L2 ampH hp

Basal Induced Basal Induced Basal Induced KJ KJΔDI KJΔR

Fig4. The relative mRNA transcript in L2, ampH, and hp of the basal and induced S. maltophilia KJ, KJΔDI, and KJΔR. The bar indicates the ratio of the mRNA transcript of a specific gene to that of the strain KJ basal L2, as measured by QRT-PCR. The induced

condition is 30 μg/ml cefuroxime for 2 h. The mean value is based on three independent experiments. The line indicates the standard deviation.

55

Fig5. The amplicons of reverse transcriptase-PCR (RT-PCR) of the S.

maltophilia KJ. The template is the mRNA transcript of the induced S. maltophilia KJ. The cefuroxime of 30 μg/ml was added as the inducter. Primer sets for lane 1: L2-ampH-F / L2-ampH-R ; for lane 2: AmpH-Hp-F / AmpH-Hp-R.

56

Fig6. The predicted transmembrane domains of AmpH protein.

Prediction by tool TMHMM-2.0.

(http://www.cbs.dtu.dk/services/TMHMM-2.0/ )

57

Fig7. The growth curves of strains KJ, KJAmpH_xylE, and KJΔDI in XOLN containing 10% glucose and 0 M, 0.2 M, and 0.5 M NaCl. Cell growth was measured by recording O.D.450 at an interval of 1 h. Each experiment was repeated three times.

58

(A)

(B)

Fig8. The proposed model for the β-lactamase expression in S.

maltophilia. (A) Repression condition (B) Derepression condition.

59

TABLE 1. Bacterial strains, plasmids, and primers used in this study Strain or plasmid Genotype or properties

S. maltophilia

KJ Wild type, a clinical isolate from Taiwan KJΔR S. maltophilia KJ ampR isogenic mutant KJΔDI S. maltophilia KJ ampDI isogenic mutant KJAmpH_xylE S. maltophilia KJ ampH isogenic mutant,

inserting a xylE gene cassette into the ampH gene

Escherichia coli

DH5α

F- Φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rk

^-

mk

⁺

)

S17-1 phoA supE44λ- thi-1 gyrA96 relA1 λ pir + mating strain

Plasmids

pEX18Tc sacB oriT, Tc^r

pRK415 oriV, oriT, Tc^r

pKJAmpH pEX18Tc vector with a 2280-bp DNA fragment, containing the partial 3’ terminus of L2 gene, 1215-bp ampH gene and partial 5’ terminus of hp gene of S. maltophilia KJ ; Tcr

pKJAmpH_xylE pEX18Tc vector with a 4271-bp DNA fragment, inserting an xylE cassetteinto the SphI site of ampH gene of pKJAmpH plasmid

Primers

AmpH-F 5’ - ACAACACCGCCGCCAACC - 3’

AmpH-R 5’ -TTCAGCCGCAGCCCGTTC - 3’

L2Q-F 5’ - AACGCACCCACCGATGCC - 3’

L2Q-R 5’ - CGCCTGTCCAGCAATGCC - 3’

NaQ-F 5’ - CCCCCATGAGCCATGAAC - 3’

NaQ-R 5’ - GCGAACAGGAACAGCGAG - 3’

60

3720-F 5’ - ATTGCCGTTGCCACTACC - 3’

3720-R 5’ - GTCCTCCACCACCTTGCC - 3’

L2-Nah-F 5’ - GCGATCAGCACGAGCAAC -3’

L2-Nah-R 5’ - ATGCCGATGATGCCGAAC - 3’

Nah-Hp-F 5’- CTGACCACGCTGTTGCCG - 3’

16rDNAQ-F 5’- GACCTTGCGCGATTGAATG - 3’

16rDNAQ-R 5’- CGGATCGTCGCCTTGGT - 3’

61

TABLE 2. The basal and induced C23O activities of KJL2xylE and KJAmpHxylE

aOne unit of catechol 2,3-dioxygenase is defined as one nanomole of catechol hydrolyzed per minute. Results are geometric means of three independent determinations. Standard derivations were within 10% of the means in all cases.

Strain C23O activity (Uc^a/OD_450nm)

Basal Induced

KJL2xylE 3 411

KJAmpH_xylE 65 183

62

TABLE 3. The basal and induced C23O activities of KJ(pRK415) and KJ(pRK567NaHxylE)

aOne unit of catechol 2,3-dioxygenase is defined as one nanomole of catechol hydrolyzed per minute. Results are geometric means of three independent determinations. Standard derivations were within 10% of the means in all cases.

Strain C23O activity (Uc^a/OD_450nm)

Basal Induced KJ(pRK415) 0 0

KJ(pRK567NaH_xylE) 40 35

63

TABLE 4. The homologus protein of AmpH protein

Protein source S. maltophilia K279a

/putative transmembrane Na+/H+

antiporter (Smlt_3721)

404 100 100 S. maltophilia R551-3

/ sodium/hydrogen exchanger (Smal_3135)

404 99 98 Xylella fastidiosa 9a5c

/ Na+/H+ exchange protein (NP_298687)

406 80 70 Pseudomonas aeruginosa PAO1

/ NhaP (NP_252576) 424 65 65

Anaeromyxobacter sp. K

/ sodium/hydrogen exchanger (AnaeK_0544)

407 64 47 Anaeromyxobacter dehalogenans 2CP-C

/Sodium/hydrogen exchanger (Adeh_0511)

410 62 46 Synechococcus sp. PCC 7335

/ transporter, CPA2 family (S7335_1904)

683 46 28 Escherichia coli str. K-12 substr. MG1655

/ sodium-proton antiporter (NP_414560) 388 39 30 Vibrio cholerae O1

/ hypothetical protein (VC1510) 140 54 41 Xanthomonas campestris pv. campestris

str. ATCC 33913

/NdvB protein (XCC4077)

798 40 26

64

TABLE 5. The basal and induced C23O activities of KJL2xylE and KJAmpHxylE

aOne unit of catechol 2,3-dioxygenase is defined as one nanomole of catechol hydrolyzed per minute. Results are geometric means of three independent determinations. Standard derivations were within 10% of the means in all cases.

Strain

C23O activity (Uc^a/OD450)

0 M NaCl 0.2 M NaCl

Basal Induced Basal Induced

KJL2_xylE 5 153 3 120

KJAmpH_xylE 21 109 14 69

65

TABLE 6. The basal and induced β-latamasa activities of KJ, KJAmpHxylE, KJ(pRKAmpH)

aOne unit of β-lactamase is defined as one nanomole of nitrocefin

hydrolyzed per minute. Results are geometric means of three independent determinations. Standard derivations were within 10% of the means in all cases.

b30 μg/ml cefuroxime as the inducer

cThe total (L1 and L2), L1, and L2 β-lactamase activity is determined by nitrocefin-EDTA method (Hu, R.M., et al., 2008) ^[53].

Strain(plasmid) Basal

β-lactamase activity (Un^a/mg) Induced^b

L1+L2^c L1^c L2^c

KJ 6 1076 241 835

KJAmpH_xylE 6 888 100 788

KJ(pRKAmpH) 11 665 236 429

66

TABLE. 7. Bioinformatics analysis of the possible homologue of the β-lactamase

Stains β-lactamase homolog

Downstream of the β-lactamase

Xanthomonas oryzae pv.

oryzae KACC10331 YP_200009.1 (bla)

YP_200010.1

( xynB) 69 54

Xanthomonas campestris

pv. vesicatoria str. 85-10 YP_365024.1

(penP) YP_365023.1 69 54

Burkholderia dolosa

AUO158 YP_002100403.1 - 67 53

Acidobacterium

capsulatum ATCC 51196 YP_002756419.1 YP_002756420.1 66 53

Rhodopseudomonas

palustris BisB18 YP_532595.1 YP_532596.1 62 48 Caulobacter sp. K31 YP_001682625.1 YP_001682624.1 63 49 Yersinia enterocolitica

hamburgensis X14 YP_575796.1 YP_575797.1

(Arginase) 62 46

Rhodopseudomonas

palustris CGA009 NP_945715.1 (penP)

Nb-255 YP_316970.1 YP_316971.1

(Arginase) 64 51

67

TABLE. 8. Bioinformatics analysis of the predicted operon.

aConfidence is an estimation of the lower boundary of the probability that the two corresponding genes are located in the same operon

bn is a number of other genomes that have the same pair of genes located in the same directon

Upstream Downstream confidence^a & n^b YP_001973421

hypothetical protein

YP_001973422

putative transmembrane Na+/H+ antiporter

confidence=56 n=1 YP_001973421

hypothetical protein

YP_001973423

putative beta-lactamase confidence=56 n=1 YP_001973422

putative transmembrane Na+/H+ antiporter

YP_001973423

putative beta-lactamase confidence=56 n=1

68

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2a A penicillin + - Penicillinase from Gram-positive bacteria

Inducible cephalosporinase from P. vulgaris, SFO-1, L2 from S.

L1 from S. maltophilia, CfiA/CcrA from B. gragilis, VIM-1, IMP-1…. IMP-9 4 penicillin - - Penicillinase from B. cepacia

76

附錄二、 β-lactam 藥物的分型與種類

β-lactam Penicillins

Extended spectrum Aminopenicillins:

Amoxicillin, Ampicillin (Pivampicillin, Hetacillin, Bacampicillin, Metampicillin, Talampicillin), Epicillin Carboxypenicillins:

Carbenicillin (Carindacillin) · Ticarcillin · Temocillin Ureidopenicillins:

Azlocillin , Piperacillin, Mezlocillin other:

Mecillinam (Pivmecillinam) · Sulbenicillin Narrow spectrum Beta-lactamase sensitive

Benzylpenicillin :

Azidocillin , Penamecillin Clometocillin, Benzathine benzylpenicillin , Procaine benzylpenicillin

Phenoxymethylpenicillin :

Propicillin , Benzathine phenoxymethylpenicillin , Pheneticillin

Beta-lactamase resistant

Oxacillin, Cloxacillin , (Dicloxacillin, Flucloxacillin) , Meticillin, Nafcillin

Carbapenems Biapenem , Doripenem , Ertapenem , Imipenem · Meropenem , Panipenem

Cephalosporins

1th generation (PEcK)

Cefacetrile , Cefadroxil , Cefalexin , Cefaloglycin , Cefalonium · Cefaloridine , Cefalotin , Cefapirin , Cefatrizine , Cefazedone · Cefazaflur, Cefazolin , Cefradine , Cefroxadine , Ceftezole

2th generation Cefaclor , Cefamandole , Cefminox , Cefonicid ·

77

(HEN) Ceforanide , Cefotiam, Cefprozil , Cefbuperazone · Cefuroxime , Cefuzonam , cephamycin (Cefoxirtin, Cefotetan, Cefmetazole) , carbacephem (Loracarbef) 3th generation Cefcapene , Cefdaloxime , Cefdinir , Cefditoren ,

Cefetamet , Cefixime, Cefmenoxime , Cefodizime, Cefoperazone , Cefotaxime , Cefpimizole ,

Cefpiramide, Cefpodoxime , Cefsulodin , Ceftazidime, Cefteram , Ceftibuten , Ceftiolene , Ceftizoxime , Ceftriaxone, oxacephem (Flomoxef, Latamoxef) 4th generation Cefepime , Cefozopran , Cefpirome , Cefquinome 5th generation Ceftobiprole

Monobactams Aztreonam · Tigemonam

78

附錄三、S. maltophilia 的 L1 和 L2 β-lactamase 的特性比較

L1^a L2^b

Sequence analysis 873 bp (G+C content: 68.4%) 909 bp (G+C content: 71.6%) Structural analysis

Tetramer

molecular mass of ca. 115 kDa bound two Zn²⁺ per monomer

Dimer

molecular mass of 63 kDa/L2 STXK active-site, SDN motif,

KTG loop motif Substrate Penicillin; cephalosporin;

carbapenem

Cephalosporin; monobactam

Inhibitor EDTA Clavulanic acid

BJM classification Group 3a enzyme Group 2e enzyme Ambler

classification Ambler class B Ambler class A

aThis enzyme from S. maltophilia ULA-511 (Michael et al., 1998)

bThis enzyme from S. maltophilia 1275 IID (Walsh TR et al.,1997)

在文檔中 Stenotrophomonas maltophilia 之 AmpH 蛋白以 Dose-Dependent 方式調控L1 及 L2 β-lactamases 之表現; AmpH Regulates the Expression of the Chromosomal L1 and L2 β-lactamases of Stenotrophomonas maltophilia in a Does-Dependent Manner (頁 62-90)