Genetic studies on the biosynthesis of the major aminoglycoside antibiotics

188 Pages·2005·2.04 MB·English

Genetic studies on the biosynthesis of the major
aminoglycoside antibiotics:
Isolation, analysis and comparison of the biosynthetic gene
clusters for 12 aminoglycoside antibiotics

A dissertation submitted to the Department of Natural Science
(Chemical Microbiology)
Bergische Wuppertal University

For the degree of

Doctor of Natural Sciences (Doctor rerum naturalium)

Presented by

MSc. Khaled Mohamed Anwar Aboshanab
Assistant Lecturer in the Microbiology and Immunology Department
Faculty of Pharmacy-Ain Shams University
Cairo-Egypt

Wuppertal, July 2005
Diese Dissertation kann wie folgt zitiert werden:

urn:nbn:de:hbz:468-20070029

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Acknowledgment

Firstly, I thank “Allah” for granting me the power to accomplish this work.

I am beholden to a number of people and organisations, who supported me to carry out this
work. First I would like to express my deepest thanks, gratitude and appreciation to my advisor
Prof. Dr. Wolfgang Piepersberg for giving me the opportunity to pursue my doctoral degree
under his supervision as well as suggesting the point, his encouragement, help and kind support.
His invaluable advice, suggestions, discussions and guidance were a real support to complete this
dissertation.

I am also indebted and grateful to Prof. Dr. Hans-Josef Altenbach for his acceptance and
kindness to co-examine this thesis.

It is my great pleasure to express my deep everlasting gratitude and thanks to my co-advisor
Dr. Udo Wehmeier for his valuable discussion through out the work, critical reading and
discussion and also the useful suggestions to my dissertation.

I am also very grateful and thankful to Dr. Heike-Schmidt Beißner for her unfailing help,
valuable discussion through out the work as well as her help in final revision of the thesis.

Furthermore, I would like to express my deepest gratitude and thanks to all staff members of
“Arbeitskreis Chemische Mikrobiologie” for contributing to such an inspiring and pleasant
atmosphere.

I am also indebted and thankful to the staff members of Combinature Biopharm Company in
Berlin for construction and screening the cosmid banks.

As well, my thanks to Dr. M. Podeschwa from the organic chemistry department for his kind
supply of the chemically synthesized 2-deoxy-scyllo-inosose.

Very special thanks to my family, especially my parents and my wife, for their patience,
understanding and encouragement during the different phases of my work. They spared no effort
until this work comes to existence.

Last but not least, I wish to acknowledge the financial support of the Missions Department-
Egypt and the Faculty of Pharmacy, Ain Shams University for giving me the opportunity to
pursue my doctoral degree in Germany.

Khaled M. A. Aboshanab
Wuppertal, 2005
CONTENTS__________________________________________ i
Contents

Page
Contents i
Abbreviations vi
List of tables ix
List of figures x
Summary xii
Zusammenfassung xiii

1 Introduction 1
1.1 Aminoglycoside antibiotics (AGAs) 1
1.1.1 Definition 1
1.1.2 Classes of aminoglycoside antibiotics 1
1.1.3 Mode of action 8
1.1.4 AGAs in medical treatments 8
1.1.5 Biosynthesis and regulation of ACAGAs 9
1.1.5.1 Biosynthesis of 2DOS-containing ACAGAs 10
1.1.5.2 Biosynthesis of fortamine and 2-deoxyfortamine-containing ACAGAs 12
1.1.6 Resistance mechanisms 14
1.1.7 Toxicity 15
1.2 ACAGAs producers 15
1.2.1 Actinomycetales 16
1.2.1 Bacillus 18
1.3 Aim of the work 20

2 Materials and Methods 21
2.1 Chemicals and enzymes 21
2.2 Media 22
2.2.1 Media for culturing Escherichia coli and Bacillus strains 22
2.2.2 Media for culturing Streptomyces and Micromonospora strains 23
2.2.3 Aminoglycoside production medium 24
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Dissertation, 2005 Aboshanab KM, BU Wuppertal
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2.3 Buffers and solutions 24
2.3.1 For the preparation of plasmid DNA from E. coli 24
2.3.2 For the preparation of competent E. coli cells 25
2.3.3 For the preparation of genomic DNA 25
2.3.4 For the preparation of protoplasts from Streptomyces 25
2.3.5 For the transformation of Streptomyces 26
2.3.6 For agarose gel electrophoresis of DNA fragments 26
2.3.7 For harvesting and sonification of cells 26
2.3.8 For Western blotting and detection of proteins 26
2.3.9 For SDS polyacrylamide gel electrophoresis 27
2.3.10 For visualization of spots on TLC 28
2.3.11. X-Gal solution 29
2.3.12 IPTG stock solution 29
2.4 Bacterial strains, vectors and recombinant plasmids 29
2.4.1 Bacterial strains 29
2.4.2 Vectors 30
2.4.3 Recombinant plasmids 31
2.5 Oligonucleotides 34
2.6 Probes and cosmid banks 37
2.7 Antibiotics 39
2.8 Growth and maintenance of bacterial strains 39
2.8.1 Growth and maintenance of E. coli and Bacillus strains 39
2.8.2 Growth and maintenance of Streptomyces sp. and Micromonospora sp. strains 39
2.9 In vitro manipulation of DNA 40
2.9.1 Preparation of plasmid DNA from E. coli 40
2.9.2 Preparation of cosmid DNA from E. coli 40
2.9.3 Extraction of genomic DNA from actinomycetes and bacilli 40
2.9.4 Restriction endonuclease digestion of DNA 41
2.9.5 Blunt end generation using the Klenow fragment of DNA polymerase I 41
2.9.6 Recovery of DNA fragments from agarose gels 42
2.9.7 Removal of phosphate groups from DNA 42
2.9.8 Ligation of DNA fragments 42
2.9.9 Quantitation of DNA 42
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Dissertation, 2005 Aboshanab KM, BU Wuppertal
CONTENTS__________________________________________ iii
2.9.10 Construction of cosmid banks 42
2.9.11 Preparation and screening of the cosmid banks 43
2.9.12 DNA sequencing 43
2.10 Preparation of competent E. coli cells 43
2.11 Transformation of E. coli strains 44
2.12 Preparation of protoplasts from Streptomyces strains 44
2.13 Transformation of streptomycete protoplasts with plasmid DNA 44
2.14 Agarose gel electrophoresis 45
2.15 Polymerase chain reaction (PCR) 45
2.16 SDS polyacrylamide gel electrophoresis (SDS-PAGE) 45
2.17 Heterologous gene expression 46
2.17.1 Protein production in E. coli using the T7 RNA polymerase system 46
2.17.2 Gene expression in S. lividans TK23 under the control of ermE-up promoter 47
2.18 Cell-free extracts of E. coli and Streptomyces 47
2.19 Determination of protein concentration 47
2.20 Western blotting and immuno-detection of proteins 47
2.21 Thin-layer chromatography (TLC) 48
2.22 High performance liquid chromatography (HPLC) 48
2.23 Determination of the activity of KanC 48
2.24 Determination of the activity of KanS1 49
2.25 Determination of the activity of KanE 50
2.26 Computer programs 51

3 Results 52
A Gene clusters for the production of 2-deoxystreptamine (2DOS) 52
and related ACAGAs
3.1 Isolation and sequence analysis of the biosynthetic gene clusters 52
3.2 Gene clusters for the individual classes of 2DOS- and related ACAGAs 54
3.2.1 4,5-glycosylated 2DOS-ACAGAs (NM group) 54
3.2.2 4,6-glycosylated 2DOS-ACAGAs (KM group) 58
3.2.3 4-glycosylated 2DOS-ACAGAs (Apr group) 61
3.2.4 5-glycosylated 2DOS-ACAGAs (HM-B group) 63
3.2.5 ACAGAs related to 2DOS-ACAGAs (FTM and IM group) 64
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CONTENTS__________________________________________ iv
3.3 Newly developed heterologous primers 67

B Studies on some selected ACAGAs biosynthetic enzymes 75
3.4 Identification of the enzymes involved in the biosynthesis of 2DOS 75
3.5 Heterologous expression of the KanC, KanS1 and KanE proteins 76
3.5.1 The general strategy for the overexpression of the selected Kan-proteins 76
3.5.2 Heterologous expression in E. coli under T7 promoter 77
3.5.3 Heterologous expression of KanE S. lividans TK23 79
3.6 Biochemical characterization of the KanC, KanS1 and KanE proteins 80
3.6.1 Biochemical analysis of the KanC protein 80
3.6.2 Biochemical analysis of the KanS1 protein 83
3.6.3 Biochemical analysis of the KanE protein 87

4 Discussion 89
4.1 General structure and genomic location of the ACAGA biosynthetic gene 89
clusters
4.2 Proposed biosynthetic pathways for the major groups of 2DOS and related 90
ACAGAs
4.2.1 Biosynthesis of 2DOS and related diaminocyclitols 90
4.2.2 Biosynthesis of paromamine 91
4.2.3 4,5-glycosylated 2DOS-ACAGAs (NM group) 94
4.2.3.1 Biosynthesis of the NM-related ACAGAs 94
4.2.3.2 Resistance mechanisms for the NM-related ACAGAs 98
4.2.3.3 Possible sensor/response regulatory system 99
4.2.4 4,6-glycosylated 2DOS-ACAGAs (KM group) 100
4.2.5 4-glycosylated 2DOS-ACAGAs (Apr group) 104
4.2.6 5-glycosylated 2DOS-ACAGAs (HM-B group) 106
4.2.7 Related 2DOS-ACAGAs (FTM and IM group) 108
4.3 Reconstitution of the 2DOS pathway in vitro 112
4.3.1 Heterologous expression of KanC, KanS1 and KanE proteins 112
4.3.2 Biochemical characterization of KanC, KanS1 and KanE proteins 112
4.4 Prospective of this work 114

5 References 116
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Dissertation, 2005 Aboshanab KM, BU Wuppertal
CONTENTS__________________________________________ v
6 Appendix 131
6.1 Tables of proteins encoded in the genomic region covering the biosynthetic 131
gene clusters of the ACAGAs analysed
A.1 neo-cluster of S. fradiae DSM 40063 (accession code: AJ629247) 131
A.2 rib-cluster of S. ribosidificus NRRL B-11466 (accession code: AJ744850) 133
A.3 par-cluster of S. rimosus NRRL 2455 (accession code: AJ749845) 135
A.4 aacC7 of S. rimosus NRRL 2455 (accession code: AJ628955) 137
A.5 liv-cluster of S. lividus ATCC 31603 (accession code: AJ748832) 138
A.6 btr-cluster of B. circulans ATCC 21558 (accession code: AJ781030) 140
A.7 kan-cluster of S. kanamyceticus DSM 40500 (accession code: AJ628422) 141
A.8 tob-cluster of S. tenebrarius DSM 40477 (accession code: AJ810851) 143
A.9 gen-cluster of M. echinospora DSM 43036 (accession code: AJ628149) 145
A.10 for-cluster of M. olivasterospora DSM 43868 (accession code: AJ628421) 148
A.11 apr-cluster of S. tenebrarius DSM 40477 (accession code: AJ629123) 150
A.12 apr-cluster of St. hindustanus DSM 44523 (accession code AJ875019) 152
A.13 hyg-cluster of S. hygroscopicus DSM 40578 (accession code: AJ628642) 154
A.14 ist-cluster of S. tenjimariensis ATCC 31603 (accession code: AJ845083) 156
6.2 Figures for alignments and phylograms of various proteins encoded by 159
conserved genes among the ACAGA clusters analysed
A.1 Alignment of BtrC homologous 160
A.2 Phylogram of BtrC homologous 160
A.3 Alignment of BtrS homologous 162
A.4 Phylogram of BtrS homologous 163
A.5 Alignment of BtrE homologous 164
A.6 Phylogram of BtrE homologous 165
A.7 Phylogram of BtrB homologous 165
A.8 Alignment of BtrB homologous 167
A.9 Alignment of LivY homologous 168
A.10 Alignment of LivW and AprD4 proteins 169
A.11 Alignment of NeoI homologous 169
A.12 Alignment of NeoH homologous 170
A.13 Alignment of NeoG homologous 171
A.14 Structural comparison of GM-C1, KM-B and FTM-A 172
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Dissertation, 2005 Aboshanab KM, BU Wuppertal
ABBREVIATIONS vi
Abbreviations

A adenine
aa amino acid
AAC aminoglycoside 3-acetyltransferase
ACAGA(s) aminocyclitol-aminoglycoside antibiotic(s)
AHB (cid:1)-hydroxy-(cid:2)-aminobutyryl residue
AHP (cid:1)-hydroxy-(cid:3)-aminopropionyl residue
ALF automatic laser fluorescence DNA sequencer
APH aminoglycoside phosphotransferase
apr-cluster apramycin gene cluster
Apr(s) apramycin(s)
APS ammonium persulfate
AT aminotransferase
ATCC American Type Culture Collection, Maryland
ATP adenosine 5’-triphosphate
B. Bacillus
bp base pair
BSA bovine serum albumin
btr-cluster butirosin gene cluster
BU(s) butirosin(s)
C cytosine
CB cosmid bank
cf. confers to
(c)HP (conserved) hypothetical protein
COG cluster of orthologous groups
CTAB cetyltrimethylammonium bromide
DH dehydrogenase
DMF dimethylformamide
DMSO dimethylsulfoxide
DNA deoxyribonuclic acid
dNTP deoxyribonucleosidetriphosphate
2DOS 2-deoxystreptamine
2DOS-ACAGAs 2-deoxystreptamine containing aminocyclitol aminoglycoside antibiotics
DSM Deutsche Stammsammlung vom Mikroorganismen und Zellkulturen
DTT dithiotheritol
E. coli Escherichia coli
EDTA ethylene diaminetetraacetic acid
EMBL European Molecular Biology Laboratory
FAD flavin adenine dinucleotide
Fig. Figure
for-cluster fortimicin gene cluster
FTM(s) fortimicin(s)
G guanidine
g gram
gen-cluster gentamicin gene cluster
GM(s) gentamicin(s)
G-6-P D-glucose 6-phosphate
GT gylcosyltransferase
HM-B hygromycin B
HPLC high performance liquid chromatography
hr(s) hour(s)
hyg-cluster hygromycin B gene cluster
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Dissertation, 2005 Aboshanab KM, BU Wuppertal
ABBREVIATIONS vii
IM(s) istamycin(s)
IPTG isopropyl-(cid:3)-D-thiogalactopyranoside
ist-cluster istamycin gene cluster
kan-cluter kanamycin gene cluster
kb kilobase
kDa kilodalton
KM(s) kanamycin(s)
l liter
liv-cluster lividomycin gene cluster
LM lividomycin
M. Micromonospora
MBP2 maltose binding protein 2
min minute
Mr relative molecular weight
mRNA messenger RNA
MT methyltransferase
NAD(H) nicotinamide adenine dinucleotide (reduced form)
NADP(H) nicotinamide adenine dinucleotide phosphate (reduced form)
neo-cluster neomycin gene cluster
NM(s) neomycin(s)
NRPS none ribosomal peptide synthetase
NRRL Northern Regional Research Laboratory
Nt nucleotide
OD optical density at x nm and 1 cm depth
x
ORF(s) open reading frame(s)
P phosphate
PAA polyacrylamide
PAGE polyacrylamide gel electrophoresis
par-cluster paromomycin gene cluster
PCR polymerase chain reaction
PEG polyethylene glycol
pfam protein family
PKS polyketide synthase
PLP pyrodixal phosphate
PM(s) paromomycin(s)
PMSF phenylmethylsulfonyl fluoride
PP inorganic pyrophosphate
PPP pentose phosphate pathway
PVDF polyvinyl difluoride
RBS ribosome binding site
rib-cluster ribostamycin gene cluster
RM ribostamycin
RNA ribonuclic acid
RNase ribonuclease
rpm revolution per minute
rRNA ribosomal ribonuclic acid
RT room temperature
S Svedberg unit (centrifugation unit)
S. Streptomyces
sec second
Sect. section
SDS sodium dodecylsulfate
SMART simple modular architecture research tool
Spc spectinomycin

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Dissertation, 2005 Aboshanab KM, BU Wuppertal