Identification of Genes Required to Synthesize an Antibiotic-like Compound from the Soil

125 Pages·2017·22.36 MB·English

East Tennessee State University
Digital Commons @ East
Tennessee State University
Electronic Theses and Dissertations Student Works
8-2015
Identification of Genes Required to Synthesize an
Antibiotic-like Compound from the Soil Bacterium
Rhodococcus sp. MTM3W5.2
Amber L. Ward
East Tennessee State University
Follow this and additional works at:https://dc.etsu.edu/etd
Part of theBacteriology Commons, and theOther Microbiology Commons
Recommended Citation
Ward, Amber L., "Identification of Genes Required to Synthesize an Antibiotic-like Compound from the Soil Bacterium Rhodococcus
sp. MTM3W5.2" (2015).Electronic Theses and Dissertations.Paper 2558. https://dc.etsu.edu/etd/2558
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Identification
 of
 Genes
 Required
 to
 Synthesize
 an
 Antibiotic-­‐like
 Compound
 from
 the
 Soil

Bacterium
 Rhodococcus
 sp.
 MTM3W5.2

A
 thesis

presented
 to

the
 faculty
 of
 the
 Department
 of
 Health
 Sciences

East
 Tennessee
 State
 University

In
 partial
 fulfillment

of
 the
 requirements
 for
 the
 degree

Master
 of
 Science
 in
 Biology

by

Amber
 L.
 Ward

August
 2015

Dr.
 Bert
 C.
 Lampson,
 Chair

Dr.
 Eric
 L.
 Mustain

Dr.
 Christopher
 L.
 Pritchett

Dr.
 Abbas
 Shilabin

Keywords:
 Rhodococcus,
 polyketide
 synthase,
 antibiotic,
 natural
 product
ABSTRACT

Identification
 of
 Genes
 Required
 to
 Synthesize
 an
 Antibiotic-­‐like
 Compound
 from
 the
 Soil

Bacterium
 Rhodococcus
 sp.
 MTM3W5.2

by

Amber
 L.
 Ward

Rhodococcus
 is
 a
 soil
 bacterium,
 member
 of
 the
 Actinobacteria,
 and
 a
 close
 relative
 of
 the

prolific
 small
 molecule
 producer
 Streptomyces.
 Recent
 interest
 in
 Rhodococcus
 as
 an
 under

investigated
 source
 of
 possible
 bioactive
 secondary
 metabolites
 is
 sparked
 by
 the
 discovery

of
 many
 polyketide
 synthase
 and
 non-­‐ribosomal
 peptide
 synthetase
 genes
 of
 unknown

function
 from
 sequenced
 Rhodococcus
 genomes.
 Rhodococcus
 species
 strain
 MTM3W5.2

was
 recently
 shown
 to
 produce
 a
 strong
 inhibitory
 compound
 with
 activity
 against
 most

strains
 of
 Rhodococcus
 and
 closely
 related
 genera.
 A
 goal
 of
 this
 investigation
 is
 to
 discover

the
 gene(s)
 required
 to
 synthesize
 this
 inhibitory
 molecule.
 The
 engineered
 Rhodococcus

transposon,
 pTNR,
 was
 used
 to
 generate
 random
 insertional
 mutations
 in
 the
 genome
 of

MTM3W5.2.
 The
 transposon
 insertion
 sites
 for
 8
 non-­‐producing
 mutants
 were
 cloned
 and

sequenced.
 Genes
 that
 encode
 polyketide
 synthases
 usually
 form
 parts
 of
 large
 biosynthetic

gene
 clusters
 responsible
 for
 the
 production
 of
 small
 polyketide
 molecules.

  2
DEDICATION

To
 mom
 and
 dad

  3
ACKNOLWEDGEMENTS

I
 would
 like
 to
 sincerely
 thank
 Dr.
 Bert
 Lampson,
 my
 advisor
 and
 committee
 chair,

for
 being
 an
 excellent
 mentor.
 Thank
 you
 for
 your
 patience,
 infinite
 support
 and
 guidance

and
 allowing
 me
 to
 both
 succeed
 and
 fail.
 Thank
 you
 for
 allowing
 me
 to
 work
 on
 an

amazing
 project
 and
 giving
 me
 the
 opportunity
 to
 continually
 bring
 ideas
 to
 you
 to
 further

the
 project.

 To
 my
 committee
 members,
 Dr.
 Christopher
 Pritchett,
 Dr.
 Eric
 Mustain
 and
 Dr.

Abbas
 Shilabin,
 thank
 you
 for
 your
 exceptional
 guidance
 and
 going
 beyond
 your
 duties
 as
 a

committee
 member.
 Thank
 you
 Dr.
 Shilabin
 for
 allowing
 us
 to
 create
 a
 collaborative
 project

with
 you
 and
 your
 lab.
 Thank
 you
 Dr.
 Sean
 Fox,
 Robin
 Grindstaff,
 Sean
 Stacey
 and
 Valeria

Barisic
 for
 the
 technical
 support,
 letting
 me
 borrow
 lab
 equipment
 and
 lending
 an
 ear
 when

I
 was
 frustrated
 and
 couldn’t
 see
 the
 light
 at
 the
 end
 of
 the
 tunnel.
 To
 Danielle
 Williams
 and

Spencer
 Cate,
 thank
 you
 for
 washing
 an
 endless
 number
 of
 beakers
 for
 me
 and
 happily

completing
 other
 lab
 duties
 to
 relieve
 some
 of
 my
 stress.
 I
 would
 like
 to
 thank
 Rhesa
 Dykes

at
 the
 ETSU
 Molecular
 Biology
 Core
 for
 your
 kindness
 and
 patience
 with
 me
 while
 I
 was

learning
 how
 to
 sequence
 and
 use
 your
 facilities.

  To
 my
 parents,
 thank
 you
 for
 your
 endless
 love
 and
 support.
 Thank
 you
 for
 always

encouraging
 me
 to
 push
 through
 when
 I
 thought
 that
 I
 couldn’t
 go
 any
 further
 and
 teaching

me
 that
 there
 is
 never
 a
 dream
 that
 is
 too
 big.
 To
 my
 sister,
 Tiffany,
 and
 brother,
 Michael,

thank
 you
 for
 being
 great
 siblings
 who
 always
 support
 me
 and
 being
 two
 of
 my
 best

friends.
 Thank
 you,
 God,
 for
 your
 infinite
 blessings
 and
 for
 guiding
 me
 to
 a
 career
 that
 I
 can

be
 passionate
 about.

  4
TABLE
 OF
 CONTENTS

 Page

ABSTRACT
 ............................................................................................................................................................
 2

DEDICATION
 ........................................................................................................................................................
 3

ACKNOWLEDGEMENTS

 ................................................................................................................................
 4

LIST
 OF
 TABLES
 .................................................................................................................................................
 9

LIST
 OF
 FIGURES

 ...........................................................................................................................................
 11

 Chapter

1.
 INTRODUCTION
 .........................................................................................................................................
 12

Antibiotic
 Resistance
 .........................................................................................................................
 12

Natural
 Products
 ..................................................................................................................................
 15

Polyketide
 Synthases
 (PKS)
 ...................................................................................................
 19

The
 Genus
 Rhodococcus
 ...................................................................................................................
 21

Industrial
 Importance
 ..............................................................................................................
 23

Secondary
 Metabolites
 Derived
 from
 Rhodococcus
 .....................................................
 24

Rhodococcus
 sp.
 MTM3W5.2
 ...........................................................................................................
 31

Current
 Work
 ........................................................................................................................................
 32

2.
 MATERIALS
 AND
 METHODS
 ................................................................................................................
 34

Bacterial
 Growth
 Media
 ....................................................................................................................
 34

Rich
 Medium
 (RM)
 .....................................................................................................................
 34

M3
 Medium
 (M3)
 ........................................................................................................................
 35

  5
Mueller-­‐Hinton
 Medium
 (MH)
 ..............................................................................................
 36

Luria-­‐Bertani
 Medium
 (LB)
 ...................................................................................................
 36

Bacterial
 Strains
 and
 Growth
 Conditions
 ..................................................................................
 37

Creation
 of
 Bacterial
 Seeds
 .............................................................................................................
 38

Plasmid
 Isolation
 .................................................................................................................................
 38

 Solutions
 for
 Plasmid
 Isolation
 ......................................................................................................
 40

Preparation
 of
 Electro-­‐Competent
 Cells
 ....................................................................................
 40

Transposon
 Mutagenesis
 .................................................................................................................
 41

Mutant
 Wheels
 ......................................................................................................................................
 42

Preparation
 of
 Agar
 Extracts
 from
 RM
 Plates
 .........................................................................
 43

Disk
 Diffusion
 Assay
 ...........................................................................................................................
 44

Auxotrophic
 Mutant
 Screen
 ............................................................................................................
 45

Genomic
 DNA
 Isolation
 .....................................................................................................................
 45

 Solutions
 for
 Genomic
 Isolation
 ....................................................................................................
 47

Southern
 Blot
 Analysis
 ......................................................................................................................
 47

Digestion
 of
 Chromosomal
 DNA
 ..........................................................................................
 47

Preparation
 of
 the
 Agarose
 Gel
 ............................................................................................
 48

Loading
 ...........................................................................................................................................
 48

Staining
 ...........................................................................................................................................
 48

Photograph
 the
 Gel
 ....................................................................................................................
 48

Southern
 Blot
 Transfer
 ............................................................................................................
 49

Solutions
 for
 Southern
 Blot
 ....................................................................................................
 51

Preparation
 of
 Labeled
 Probe
 DNA
 ....................................................................................
 51

  6
Hybridization
 of
 Southern
 Transferred
 DNA
 .................................................................
 52

 Hybridization
 Solutions
 ...................................................................................................................
 53

Detection
 of
 the
 DNA
 ................................................................................................................
 53

Recovery
 of
 pTNR
 Insertion
 Sites
 from
 Mutants
 ...................................................................
 54

Sequencing
 of
 pTNR
 Insertion
 Sites
 ............................................................................................
 55

Analysis
 of
 Sequenced
 Insertion
 Sites
 ........................................................................................
 56

Scale-­‐Up
 Production
 of
 Antimicrobial
 Compound
 ................................................................
 56

Sephadex
 LH-­‐20
 Column
 Chromatography
 .............................................................................
 58

High
 Pressure
 Liquid
 Chromatography
 (HPLC)
 .....................................................................
 58

3.
 RESULTS
 ........................................................................................................................................................
 60

Generation
 of
 Mutant
 Strains
 Using
 pTNR
 ...............................................................................
 60

Auxotrophic
 Mutant
 Screen
 ............................................................................................................
 62

Screening
 for
 Non-­‐producing
 Mutants
 .......................................................................................
 64

Disk
 Diffusion
 Assay
 ...........................................................................................................................
 65

Southern
 Blot
 Analysis
 of
 8
 Non-­‐producing
 Mutants
 ..........................................................
 67

Recovering
 pTNR
 Insertion
 Site
 of
 Mutants
 ............................................................................
 70

DNA
 Sequence
 Analysis
 of
 Cloned
 Transposon
 Insertion
 Sites
 ......................................
 72

HPLC
 Analysis
 of
 Producer
 and
 Non-­‐producer
 ......................................................................
 78

Sephadex
 LH-­‐20
 Column
 Chromatography
 ....................................................................
 78

4.
 DISCUSSION
 .................................................................................................................................................
 82

Agar
 Extraction
 Assay
 .......................................................................................................................
 82

Auxotrophic
 Mutant
 Screen
 ............................................................................................................
 84

Southern
 Blot
 Analysis
 of
 8
 Non-­‐producing
 Mutants
 ..........................................................
 85

  7
Sequencing
 Non-­‐Producing
 Mutants
 ..........................................................................................
 86

HPLC
 Analysis
 of
 MTM3W5.2
 and
 Two
 Non-­‐producers
 .....................................................
 91

Future
 Work
 ..........................................................................................................................................
 92

 REFERENCES

 ..................................................................................................................................................
 95

APPENDICES
 ..................................................................................................................................................
 103

Appendix
 A:
 pTNR
 Transposon
 Sequence
 ........................................................................................
 103

 Appendix
 B:
 Primers
 for
 Mutant
 Sequencing

 .................................................................................
 108

Appendix
 C:
 Non-­‐producing
 Mutant
 Raw
 Sequencing
 Data
 .....................................................
 109

 Appendix
 D:
 DNA
 Sequence
 Analysis
 of
 Cloned
 Transposon
 Insertion
 Sites

 ...................
 116

VITA
 ...................................................................................................................................................................
 125

  8
LIST
 OF
 TABLES

Table

 Page

1.
 Modes
 of
 action
 and
 resistance
 mechanisms
 of
 commonly
 used
 antibiotics

 ........................
 14

2.
 Proposed
 biosynthetic
 gene
 clusters
 (genome
 annotation)
 in
 Rhodococcus

 .........................
 30

3.
 The
 sensitivity
 of
 organisms
 to
 the
 inhibitory
 compound
 produced
 by
 strain

MTM3W5.2

 ....................................................................................................................................................
 32

4.
 Number
 of
 pTNR
 mutants
 screened
 .
 .......................................................................................................
 63

5.
 Size
 of
 restriction
 fragments
 of
 the
 8
 non-­‐producing
 mutants
 that
 hybridize
 to
 the

pTNR
 probe

 ...................................................................................................................................................
 68

6.
 Identification
 of
 the
 interrupted
 gene
 in
 each
 non-­‐producing
 mutant
 .....................................
 91

  9