Isolation of unculturable soil bacteria and finding of their antibiotic candidates

Dissertation for Degree of Doctor

Isolation Of Unculturable Soil Bacteria

And Finding Of Their Antibiotic Candidates

by

Nguyen Manh Tuan

Department of Life Science

Graduate School

Kyonggi University

(2017)

Isolation Of Unculturable Soil Bacteria

And Finding Of Their Antibiotic Candidates

by

Nguyen Manh Tuan

A dissertation submitted to the faculty of

the Graduate School in partial fulfillment of the requirements

for the degree of Doctor of Philosophy

Department of Life Science

Graduate School

Kyonggi University

December 2017

Approved the dissertation of Nguyen Manh Tuan

as qualified for the Degree of Doctor

of Philosophy

Chairman Signature

Member Signature

Member Signature

Member Signature

Member Signature

December 2017

Graduate School of Kyonggi University

- i -

Contents

List of Tables ………………………………………………………………………….. vii

List of Figures …………………………………………………………………………. viii

Acknowledgement ............................................................................................................. x

Abstract ........................................................................................................................... xi

Chapter I. Modification Of Soil Extraction Method To Cultuvate Unculturable

Soil Bacteria .......................................................................................................... 1

1.1 Introduction ...................................................................................................... 1

1.2 Aims to Study ................................................................................................... 3

1.3 Material and method ......................................................................................... 4

1.3.1 Information of soil property used for the method ....................................... 4

1.3.2. Preparing new soil extraction (NSE) in 1 litre medium ............................. 4

1.3.3 Medium for isolation of soil bacteria .......................................................... 5

1.3.4 Soil sampling site and preparation of soil sample ....................................... 5

1.3.5 New developed method for isolation of previously unculturable soil

bacteria ....................................................................................................... 6

1.3.6 Traditional soil extract method ................................................................... 6

1.3.7 Modified transwell culture system method ................................................. 7

1.3.8 Bacterial genomic extraction and small-subunit rRNA (SSU rRNA)

amplification .............................................................................................. 7

1.3.9 Identify 16S rRNA gene sequences and accession numbers of 16S rRNA

gene sequenses ........................................................................................... 8

- ii -

1.3.10 Parameter tools/software programs ........................................................... 8

1.3.11 Role of complex isolate medium compositions to grow previously

unculturable soil bacteria .......................................................................... 9

1.3.12 DNA extract from soil ............................................................................. 9

1.3.13 PCR amplification and pyrosequencing .................................................. 10

1.3.14 Analysis of pyrosequencing data ............................................................. 10

1.3.15 Determining and comparison of soil extract ingredients investigated .... 11

1.3.16 Sample preparation for simultaneous profiling analysis of amino acids,

organic acids and fatty acids in soil extract .............................................. 12

1.3.17 Gas chromatography-mass spectrometry ................................................ 12

1.3.18 Inorganic ingredients .............................................................................. 13

1.4 Results ............................................................................................................ 14

1.4.1 Shortage of previous methods and new method development .................. 14

1.4.2 Method validation through comparative analysis of organic/inorganic

compounds ............................................................................................... 19

1.4.3 Method validation through cultivability of unculturable bacteria ............. 21

1.4.4 Method validation through isolation rate of unculturable or new taxonomic

bacteria ..................................................................................................... 24

1.4.5 Method validation through taxonomic analysis ......................................... 29

1.5 Discussion ...................................................................................................... 33

Chapter II. Taxonomical Characterization Of Novel Taxa ............................ 37

2.1 A Rapid And Simple Method For Identifying Bacterial Polar Lipid

Components In Wet Biomass ............................................................................ 37

2.1.1 Introduction .............................................................................................. 37

- iii -

2.1.2 Aim to study .............................................................................................. 38

2.1.3 Material and method ................................................................................. 39

2.1.3.1 Organisms and growth conditions ....................................................... 39

2.1.3.2 Polar lipids extraction using the improved method .............................. 40

2.1.3.3 Polar lipids extraction using the the method of Minnikin et al.

(1984) .. ............................................................................................... 42

2.1.3.4 Polar lipids analysis ............................................................................. 42

2.1.4 Results and Discussions ............................................................................ 43

2.1.4.1 Polar lipid profiles of Microbacterium lacticum compared ................. 43

2.1.4.2 Polar lipid profiles of Rhodococcus koreensis compared .................... 44

2.1.4.3 Polar lipid profiles of Pseudomonas aeruginosa compared ................. 48

2.1.4.4 Polar lipid profiles of Streptomyces longwoodensis compared ........... 48

2.1.4.5 Polar lipid profiles of Novosphingobium capsulatum compared ......... 48

2.2 Description of Flavobacterium fulvus sp. nov., Flavobacterium pedocola sp.

nov., and Flavobacterium humicola sp. nov., novel members belong to the

family Flaviobacteriaceae, isolated from soil .................................................... 51

2.2.1 Introduction .............................................................................................. 51

2.2.2 Materials and method ................................................................................ 52

2.2.2.1 Sample and isolation of strains ............................................................ 52

2.2.2.2 Phenotypic characterisation ................................................................. 53

2.2.2.3 Genomic DNA isolate .......................................................................... 54

2.2.2.4 Phylogenetic analyses .......................................................................... 55

2.2.2.5 DNA–DNA hybridization (DDH) ....................................................... 56

2.2.2.6 G+C mol content ................................................................................. 58

2.2.2.7 Determining fatty acids components ................................................... 58

- iv -

2.2.2.8 Components of menaquinone and polar lipids .................................... 59

2.2.3 Results and discussion .............................................................................. 61

2.2.3.1 Results of comparision between new isolates and their relative

species ................................................................................................... 61

2.2.3.2 Description of novel isolates of genus Flavobacterium ....................... 72

Chapter III. Isolation And Characterization Of Novel Antibiotic

Candidates ......................................................................................................... 78

3.1 General Information ................................................................................... 78

3.1.1 Introduction ............................................................................................... 78

3.1.2 General background .................................................................................. 79

3.1.2.1 Term of antibiotic ................................................................................ 79

3.1.2.2 Antibiotic history ................................................................................. 80

3.1.2.3 How antibiotics work to kill bacteria ................................................... 82

3.1.2.4 Current problems with known drugs .................................................... 86

3.1.2.5 Mechanisms of drug resistance ............................................................ 88

3.1.3 Goal of this study ...................................................................................... 91

3.2 Screening of Antibiotic-Producing Bacteria Isolated From Soils ........... 92

3.2.1 Materials and methods .............................................................................. 92

3.2.1.1 Soil sample .......................................................................................... 92

3.2.1.2 Isolation of microorganisms ................................................................ 92

3.2.1.3 For members of Actinobacteria ........................................................... 92

3.2.1.4 Modified culture method ..................................................................... 93

3.2.2 Screening antibiotic-producing bacteria .................................................. 93

- v -

3.2.2.1 Microorganisms used to test ................................................................ 93

3.2.2.2 Assay of antimicrooganisms activities ................................................. 94

3.2.2.3 Identification of 16S rRNA gene analysis ............................................ 94

3.2.3 Results and discussion .............................................................................. 94

3.2.3.1 Isolation of anti-microoganisms .......................................................... 94

3.2.3.2 Finding of the best strain to extract antibiotics .................................. 101

3.3 Characteristic Of Antibiotic Derived A Novel Species of Genus

Sphingobium ..................................................................................................... 104

3.3.1 Material and method ............................................................................... 104

3.3.1.1 Fermentation and extraction of antibiotic .......................................... 104

3.3.1.2 Purification of antibiotic extraction by silica gel column

chromatography ................................................................................... 105

3.3.1.3 Collection of individual compound by Prep-HPLC ........................... 105

3.3.1.4 Minimum inhibitory concentration (MIC) test .................................. 107

3.3.2 Results and discussion ............................................................................ 108

3.3.2.1 Major descriptions of strain UCM-25 ................................................ 108

3.3.2.2 Extraction, purification, separation of antibiotics ............................. 110

3.3.2.3 Thin-Layer Chromatography (TLC) assay ........................................ 110

3.3.2.4 Collecting individual compound ........................................................ 112

3.3.2.5 Minimal Inhibitory Concentration (M.I.C) test .................................. 116

3.4 An Antibiotic Producing Streptomyces Species Kills Drug Resistant

Pathogens ......................................................................................................... 118

3.4.1 Material and methods .............................................................................. 118

3.4.2 Results and Discussion ............................................................................ 119

- vi -

3.4.2.1 Major information for strain T1317-0309 ......................................... 119

3.4.2.2 Screening of fractions collected by Prep-HPLC ................................ 126

3.4.2.3 Minimal Inhibitory Concentration (M.I.C) test ................................. 127

Chapter IV. General Conclusion ................................................................... 131

4.1 Modification Of Soil Extraction Method ...................................................... 131

4.2 Taxonomical Characterization Of Novel Taxa ............................................. 132

4.3 Isolation And Characterization Of Novel Antibiotic Candidates ................. 133

Reference .......................................................................................................... 135

Appendix ........................................................................................................... 167

Abstract in Korean ........................................................................................... 202

- vii -

List of Table

Table 1. Brief summary comparision of results by methods determined ................... 15

Table 2. List of soil extract components defined a new way and general

approaches .................................................................................................... 17

Table 3. List of bacterial isolates used for evaluating new soil extract (NSE) ........... 21

Table 4. Comparision of convenient methods for extracting bacterial polar lipids .... 49

Table 5. Comparision of distinct phenotypic characteristics between four isolate strains

and their type strains of the most related species of the genus

Flavobacterium ............................................................................................ 63

Table 6. Cellular fatty acid composition (%) of four isolate strains and the type strains

of related species of the genus Flavobacterium ............................................ 67

Table 7. Antibiotic pathways to inhibit microoganisms ............................................. 83

Table 8. List of isolating bacterial soils that showed target microoganisms .............. 95

Table 9. Antibiotic extract activity of inhibition of target microoganisms using paper

disc ............................................................................................................. 102

Table 10. Activities of each fraction that are collected by Prep-HPLC, which inhibit

target pathogens (Inhibition zone in mm) ................................................... 113

Table 11. Minimal inhibitory concentration (MIC in μg ml-1

) values of Fr-1 and Fr￾2 ................................................................................................................. 117

Table 12. Cultural characteristics of Streptomyces sp. T1317-0309 on various media

tested at 28oC for 21 days ........................................................................... 120

Table 13. Result of identification of strain T1317-0309 based on 16S rRNA gene

sequencing using the eztaxon-e .................................................................. 123

Table 14. Antibacterial activities of the Fr-5 collected from Streptomyces sp. T1317-

0309 .......................................................................................................... 129

- viii -

List of Figure

Fig 1. An impact of various factors on cultivation of uncultured soil bacteria ....... 20

Fig 2. Achievement of phenotypic diversity of species isolated through soil

samples ........................................................................................................... 23

Fig 3. Abundance of bacteria in the soil samples defined by pyrosequencing and a new

method ............................................................................................................ 28

Fig 4. Identifing phenotypic diversity of isolates through analysis of 16S rRNA gene

sequence ......................................................................................................... 32

Fig 5. Brief proceduces for extraction and purification of polar lipids ..................... 41

Fig 6. Polar lipid profiles determined using the two methods ................................. 46

Fig 7. Neighbour-joiningtree, based on 16S rRNA gene sequencing, showing the

position of strains UCM-R15T

, UCM-R21, UCM-R36T

and UCM-46T

, which

are closely related species of the genus Flavobacterium .............................. 62

Fig 8. Polar lipid profile of strains UCM-R15T

, UCM-R36T

, and UCM-46T

after two￾dimensional chromatography and staining with 5 % molybdatophosphoric acid

at 180 oC for 30 min ....................................................................................... 69

Fig 9. Scanning electron micrograph showing the cells morphology of strains UCM￾R15T

, UCM-R36T

, and UCM-46T grown on R2A medium. Bars, 0.5 µm ..... 71

Fig 10. Fleming and his penicillin discovery on the original agar plate ..................... 81

Fig 11. Antibiotics and its antibiotic resistance reported ........................................... 87

Fig 12. Machanism of antibiotic resistance through pathway of efflux pump ........... 89

Fig 13. Distribution (number among total 44 strains) of the strains showing activities

against the targets .......................................................................................... 99

Fig 14. Relative distribution of the active strains in the level of bacterial genus, showing

antimicrobial and antifugal activities against the target test ......................... 101

Fig 15. Main steps for extracting antibiotic and further analysis ............................. 106

- ix -

Fig 16. Neighbor-joining phylogeny of strain UCM-25 based on near full-length 16S

rRNA gene sequences .................................................................................. 109

Fig 17. Thin layer chromatography for compound analysis .................................... 111

Fig 18. Collecting individual fraction via a Prep-HPLC system ............................. 112

Fig 19. Inhibition zone of these fractions collected killing microoganisms test ...... 114

Fig 20. Thin layer chromatography for compound analysis of T1317-0309 ........... 121

Fig 21. A neighbour-joining phylogenetic tree of strain T1317-0309 with related species

in genus Streptomyces ................................................................................. 125

Fig 22. Profile of secondary metabolism extracted from the fermentation

supernatant ................................................................................................... 126

Fig 23. Zone of inhibtion of these fractions collected showing activity to kill

microoganisms tested ................................................................................... 126

- x -

Acknowledgement

First, I’m really pleased to Prof. Jaisoo Kim, who gave me an especial opportunity to

study and research in South Korea, as well as great supports for researches and my family

during living in Republic of Korea.

I would like to thank you all of Professors at the Department of Life Science, Kyonggi

University: Prof. Byung-Sun Yoon, Prof. Sang Seob Lee, Prof. Dong-Soo Kong, Prof.

Byung-Sun Yoo, Prof. Ok-Min Lee, and Prof. Seong-Ho Ghil for these suggestions to

improve my researches. I would be glad to Dr. Seung Jae Won, who suggested solving my

problems during study, as well as his interest in my family life in Korea. I am also grateful

to the doctoral communitte members: Prof. Seung-Woo Jeong (Kunsan National University)

and Prof. Song-Bae Kim (Seoul National University) for their valuable suggestions to

improve my dissertation. An especial thank you to Prof. Seung-Woon Myung (Department

of Chemistry, Kyonggi University) and Prof. Man-Jeong Paik (College of Pharmacy and

Research Institute of Life and Pharmaceutical Sciences, Sunchon National University) for

their support during my study.

To complete my doctoral degree, I received special encouragement from my family

and friends that helped me through difficult time, without them my life is certainly very

difficult. I’m thankful to all of lab members, classmates and Korean students always help

me in study and research.

December 2017, Republic of Korea

Nguyen Manh Tuan, Ph.D Student

- xi -

Abstract

Surely, there are so many unknown metabolic potentials of uncultured soil bacteria. A

lot of methods have been established by various approaches to explore unculturable soil

bacteria. But all of these methods have a common obstacle which is limitation of the solid

medium to obtain large groups of not-yet cultivated species. This study is simply designed

to discover more unculturable soil bacteria in aerobic conditions through a new soil extract

(NSE) derived from soil, the "bacterial home". NSE devloped in this study is different from

the existing methods since this method uses a mixuture of water and methanol instead of

water only to extract soil chemical compounds. Moreover, this new method makes easier

to perform and reduces working hours than the existing cultivation methods for

unculturable soil bacteria, and also this method can be working well without enrichment

and even combining with traditional media.

NSE contains essential ingredients required for soil microorganisms including

chemoorganotrophic, chemolithotrophic, and heterotrophic bacteria based on our anlytical

results. By combination with several active components we chose, a new medium, called

ISEM (Intentive Soil Extraction Medium for unculturable soil bacteria), was created and

showed successful cultivation of bacterial strains involved in 7 phyla: approximately 49%

and 55% of the total isolates (n=258) was belonging to the previously uncultured bacteria

and new taxonomic candidates including species, genus and even family level, repectively.

Therefore, we expect that this discovery could open up a new avenue to explore unknown

mechanisms and new knowledge through sussessful cultivation of a number of bacteria

hidden in various soil environments.

Although the use of freeze-dried cells to determine polar lipid components have

successfully been used, several methods have been developed based on wet cells. However,

wet cell methods showed limitation with narrow scope in applications due to non-polarity

of extracted phospholipids. In this study, wet biomasses of five species of Gram-positive

- xii -

and Gam-negative bacteria including Microbacterium lacticum, Rhodococcus koreensis,

Pseudomonas aeruginosa, Streptomyces longwoodensis and Novosphingobium capsulatum

were used to analyze lipid components, revealing equal quality compared to the most

applied procedure with wet biomasses except a few minor spots. Moreover, the improved

method also ensures simplicity of lipid extraction.

Four Gram-staining negative, non-endospore-forming, non- non-motile strains were

found soil in Yuseong-gu, Daejeon, South Korea. Through analysis of these 16S rRNA gene

sequences suggested they should be belonged the genus Flavobacterium; strain UCM-R15T

and UCM-R21 most closely related to F. enshiense DK69T

(97.38-97.46 %), F.

saliperosum S13T

(96.28-96.35 %), F. suncheonense GH29-5

T

(96.28-96.35 %), F.

limnosediminis JC2902T

(96.14-96.22 %), F. cauense R2A-7

T

(95.17-95.67 %), and F.

columnare IFO 15943T

(94-88-95.25 %); for strain UCM-R36T

showed 97.53 % to F.

suncheonense GH29-5

T

, 96.57 % to F. enshiense DK69T

, 96.43 % to F. limnosediminis

JC2902T

, 96.29 % to F. saliperosum S13T

, 95.74 % to F. cauense R2A-7

T

; UCM-46T

shared

closely related to F. suncheonense GH29-5

T

(98.27 %), F. cauense R2A-7

T

(96.06 %), F.

enshiense DK69T

(95.5 %), F. saliperosum S13T

(95.44 %), and F. limnosediminis JC2902T

(95.44 %). All four strain cannot reduce/digest nitrate or urea. Major menaquinone MK-6

was detected in all isolate strains. Fatty acid as C15:0 iso, C17:0 iso 3-OH, C15:1 iso G, summed

feature 9 (C17:1 iso ω7c and/or C16:0 10-methyl, C15:0 iso 3-OH, C15:0 anteiso, and C16:0 iso

were found in all the strains. Phosphatidylethanolamine was found in three strains as major

polar lipid; phosphatidylserine in strain UCM-R15T

, UCM-R36T

, not UCM-46T

; and

phosphatidylmonomethylethanolamine was only occurred in strain UCM-R15T

; moreover

unidentified polars (L2 or L3) were appeared at 160-180 °C after 15 min, while these

remaining spots were fast appeared. Genomic DNA G+C content of strains UCM-R15T

,

UCM-R21, UCM-R36T

, UCM-46T was 35.3, 36.2, 39.0, and 38.4 mol%, respectively. The

results of DNA-DNA hybridizations between our strains and comparison strains were lower

than 70 % limit off; combining with their physiological and biochemical characteristics,

we suggest that three strains represent novel members within the genus Flavobacterium,